Datasets:

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sandy37

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elife

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

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

[ [ "G-protein-coupled receptors ( GPCRs ) are important therapeutic targets for numerous human diseases .", "Our understanding of GPCR functional mechanisms has evolved from a simple demarcation of single active and inactive states to the appreciation and detection of multiple active states responsible for partial or biased agonism ( Latorraca et al . , 2017; Venkatakrishnan et al . , 2013; Weis and Kobilka , 2018 ) .", "High-resolution crystal structures of these proteins are vital for structure-based ( rational ) drug discovery ( RDD ) efforts designed to tailor selectivity and efficacy ( Congreve et al . , 2014; Michino et al . , 2015a ) .", "While considerable efforts have been directed at the development of biased agonists that couple preferentially to a particular effector pathway ( Free et al . , 2014; Manglik et al . , 2016; McCorvy et al . , 2018 ) , less attention has been dedicated to the possibility that different antagonist scaffolds with differing efficacy of inverse agonism might lead to different receptor conformations and hence different ‘inactive’ states .", "Such a possibility could have a major impact on RDD for antagonists , since a GPCR crystal structure stabilized by a particular antagonist might represent an invalid docking target for an antagonist that prefers a different inactive conformation .", "Although substantial differences in antagonist binding mode and position of the binding pockets have been revealed among different aminergic receptors , no conformational differences has been detected for the inactive state in any individual aminergic receptor ( Michino et al . , 2015a ) .", "In particular , although a number of antagonists derived from different scaffolds have been co-crystallized with the β2 adrenergic receptor , conformational differences among these crystal structures are minimal ( Michino et al . , 2015a ) .", "Curiously , the inactive state structures of the highly homologous dopamine D2 and D3 receptors ( D2R and D3R ) revealed substantial differences on the extracellular side of the transmembrane domain , especially in TM6 ( Figure 1 ) , when bound with antagonists derived from different scaffolds ( Chien et al . , 2010; Wang et al . , 2018 ) .", "Specifically , the D3R structure is in complex with eticlopride , a substituted benzamide ( PDB: 3PBL ) ( Chien et al . , 2010 ) , while the D2R structure is bound with risperidone , a benzisoxazole derivative ( PDB: 6CM4 ) ( Wang et al . , 2018 ) .", "The binding poses of the two ligands differ substantially .", "Risperidone is oriented relatively perpendicular to the membrane plane with its benzisoxazole ring penetrating into a hydrophobic pocket beneath the orthosteric binding site ( OBS ) of D2R; in contrast , eticlopride is oriented relatively parallel to the membrane plane and contacts the extracellular portion of TM5 in D3R , a sub-pocket that risperidone does not occupy in D2R ( Sibley and Shi , 2018; Wang et al . , 2018 ) .", "Nemonapride , another substituted benzamide , binds in the OBS of the slightly divergent D4R ( PDB: 5WIV ) ( Wang et al . , 2017 ) in a manner very similar to that of eticlopride in the D3R ( Sibley and Shi , 2018 ) .", "Importantly , the co-crystalized ligands ( risperidone , eticlopride , and nemonapride ) display little subtype selectivity across D2R , D3R , and D4R ( Chien et al . , 2010; Hirose and Kikuchi , 2005; Silvestre and Prous , 2005; Wang et al . , 2017 ) ( also see PDSP database; Roth et al . , 2000 ) .", "Given the high homology among these D2-like receptors , especially between D2R and D3R , the drastic conformational differences between the inactive state structures of these receptors may be better explained by different binding poses of antagonists bearing different scaffolds rather than inherent differences in the receptors .", "Thus , we hypothesized that different antagonist scaffolds may favor distinct inactive conformations of D2R .", "To test this hypothesis , we carried out extensive molecular dynamics ( MD ) simulations of D2R in complex with non-selective antagonists derived from different scaffolds to characterize the plasticity of the OBS and the extracellular loop dynamics in the inactive conformational state ." ], [ "Compared to eticlopride bound in the D3R structure , risperidone in the D2R structure penetrates deeper into the binding site , with its benzisoxazole moiety occupying a sub-pocket that eticlopride does not reach .", "By examining the D2R/risperidone structure , we found that the benzisoxazole moiety is enclosed by eight residues in D2R , which are identical among all D2-like receptors ( i . e . D2R , D3R , and D4R ) : Cys1183 . 36 ( superscripts denote Ballesteros-Weinstein numbering Ballesteros and Weinstein , 1995 ) , Thr1193 . 37 , Ile1223 . 40 , Ser1975 . 46 , Phe1985 . 47 , Phe3826 . 44 , Trp3866 . 48 , and Phe3906 . 52 .", "Notably , three of these residues ( Ile1223 . 40 , Phe1985 . 47 , and Phe3826 . 44 ) on the intracellular side of the OBS that we previously defined ( Michino et al . , 2015a ) , accommodate the F-substitution at the tip of the benzisoxazole ring in a small cavity ( termed herein as the Ile3 . 40 sub-pocket ) ( Figure 2a ) .", "Both Ile1223 . 40 and Phe3826 . 44 of this Ile3 . 40 sub-pocket are part of the conserved Pro5 . 50-Ile3 . 40-Phe6 . 44 motif that undergoes rearrangement upon receptor activation ( Rasmussen et al . , 2011 ) , and we have found that the I1223 . 40A mutation renders D2R non-functional ( Klein Herenbrink et al . , 2019; Wang et al . , 2018 ) .", "Interestingly , this Ile3 . 40 sub-pocket is collapsed in both the D3R and D4R structures ( Sibley and Shi , 2018; Figure 2b , c ) .", "We noted that this collapse is associated with rotation of the sidechain of Cys3 . 36: In the D2R/risperidone structure , the sidechain of Cys3 . 36 faces the OBS , whereas in the D3R/eticlopride and D4R/nemonapride structures , it rotates downwards to partially fill the Ile3 . 40 sub-pocket ( Figure 2a–c ) .", "To test our hypothesis that these observed differences in the crystal structures are due to the binding of antagonists bearing different scaffolds but not intrinsic divergence of D2-like receptors , we compared the binding modes of three non-selective antagonists in D2R .", "We reverted three thermostabilizing mutations introduced for crystallography ( I1223 . 40A , L3756 . 37A , and L3796 . 41A ) back to their WT residues , established WT D2R models in complex with risperidone , spiperone , or eticlopride , and carried out extensive MD simulations ( see Materials and methods , Figure 1—figure supplement 1 and Table 1 ) .", "In our prolonged MD simulations of the WT D2R/risperidone complex ( >65 µs , Table 1 ) , we observed that risperidone stably maintains the binding pose captured in the crystal structure , even without the thermostabilizing mutations ( Figure 2d ) .", "Thus , the I1223 . 40A mutation has minimal impact on the binding pose of risperidone .", "Interestingly , in the simulations of the WT D2R model in complex with spiperone , a butyrophenone derivative , the F-substitution on the butyrophenone ring similarly occupies the Ile3 . 40 sub-pocket as risperidone ( Figure 2e ) .", "Note that the F-substitutions in risperidone and spiperone are located at similar distances to the protonated N atoms that interact with Asp3 . 32 ( measured by the number of carbon atoms between them , Figure 1—figure supplement 1 ) and these two ligands appear to be optimized to occupy the Ile3 . 40 sub-pocket .", "In contrast , in our simulations of the D2R/eticlopride complex , the eticlopride pose revealed in the D3R structure ( PDB: 3PBL ) is stable throughout the simulations and does not protrude into the Ile3 . 40 sub-pocket ( Figure 2f ) .", "Consistent with the difference in the crystal structures noted above ( Figure 2a , b ) , when risperidone and spiperone occupy the Ile3 . 40 sub-pocket , the sidechain of Cys1183 . 36 rotates away with its χ1 rotamer in gauche- , while in the presence of the bound eticlopride , this rotamer is stable in trans ( Figure 2—figure supplement 1 ) .", "To validate these computational findings regarding the occupation of the Ile3 . 40 sub-pocket , we mutated Ile1223 . 40 of WT D2R to both Trp and Ala and characterized how these mutations affect the binding affinities for spiperone , risperidone , and eticlopride ( Table 2 ) .", "We hypothesized that the bulkier sidechain of Trp at position 3 . 40 would hamper the binding of spiperone and risperidone since they occupy the Ile3 . 40 sub-pocket but have no effect on eticlopride binding , while the smaller Ala should not affect the binding of spiperone or risperidone .", "Consistent with this hypothesis , the I122W mutation decreased the binding affinities of risperidone ( 13-fold ) and spiperone ( 6-fold ) compared to WT but had no effect on that of eticlopride .", "In contrast , the I122A mutation did not affect the affinities of spiperone or risperidone , which is consistent with our simulation results that show the I122A mutation has minimal impact on risperidone binding .", "In contrast , I122A caused a threefold increase in the affinity of eticlopride , suggesting that the I122A mutation may promote an inactive conformation of D2R that favors eticlopride binding .", "Together these results support our proposal that different antagonist scaffolds may favor distinct inactive conformations of D2R .", "Ligand binding in D2-like receptors can be modulated by Na+ bound in a conserved allosteric binding pocket coordinated by Asp2 . 50 and Ser3 . 39 ( Michino et al . , 2015b; Neve , 1991; Wang et al . , 2017 ) .", "Note that the aforementioned Cys3 . 36 and Ile3 . 40 are adjacent to the Na+ coordinating Ser3 . 39; thus , we further hypothesized that the occupation of the Ile3 . 40 sub-pocket by spiperone or risperidone makes them insensitive to Na+ .", "To test this hypothesis , we simulated D2R/risperidone , D2R/spiperone , D2R/eticlopride , and D2R/ ( - ) -sulpiride complexes in the presence versus absence of bound Na+ ( Table 1 ) .", "Interestingly , the occupancy of the Ile3 . 40 sub-pocket by either spiperone or risperidone was unaffected by the presence or absence of bound Na+ ( Figure 2—figure supplement 1 ) .", "In contrast , while the poses of eticlopride and ( - ) -sulpiride are highly stable in the presence of bound Na+ , they oscillated between different poses in the absence of Na+ .", "These oscillations are associated with the sidechain of Cys3 . 36 swinging back and forth between the two rotamers , suggesting an important role of Na+ binding in stabilizing the poses of eticlopride and ( - ) -sulpiride and the configuration of the Ile3 . 40 sub-pocket ( Figure 2—figure supplement 1 ) .", "Interestingly , the previous MD simulations described by Wang et al . indicated that nemonapride’s binding pose in D4R is more stable in the presence of bound Na+ as well ( Wang et al . , 2017 ) .", "Consistent with these computational results , we have previously shown that spiperone binding is insensitive to the presence of Na+ , while the affinities of eticlopride and sulpiride are increased in the presence of Na+ ( Michino et al . , 2015b ) .", "In this study , we performed binding experiments in the absence or presence of Na+ and found the affinity of risperidone to be unaffected , in accordance with this hypothesis ( Table 2 ) .", "Together these findings support our hypothesis that the ability of a ligand to bind the Ile3 . 40 sub-pocket relates with its sensitivity to Na+ in binding , due to allosteric connections between the sub-pocket and the Na+ binding site .", "To further investigate the functional impact of these conformational differences surrounding the OBS , we used a bioluminescence resonance energy transfer ( BRET ) assay , which measures conformational changes of the Go protein heterotrimer following activation by D2R ( Michino et al . , 2017 ) , to evaluate the inverse agonism activities of several representative D2R ligands .", "These ligands can be categorized into two groups according to their sensitivities to Na+ in binding at D2R , which have been characterized either in our current study or in previous studies ( Michino et al . , 2015b; Neve , 1991; Newton et al . , 2016 ) .", "While risperidone , spiperone , and ( + ) -butaclamol have been found to be insensitive to Na+ in binding , ( - ) -sulpiride , eticlopride , and raclopride show enhanced binding affinities in the presence of Na+ .", "Using quinpirole as a reference full agonist , we found that the Na+ insensitive ligands display significantly greater inverse agonism ( < −30% that of the maximal response of quinpirole ) relative to the Na+-sensitive ligands ( > −15% that of the maximal response of quinpirole , Figure 3 ) .", "These observations are consistent with findings from earlier [35S]GTPγS binding experiments of Roberts and Strange in which ( + ) -butaclamol , risperidone , and spiperone were found to inhibit significantly more [35S]GTPγS binding than raclopride and ( - ) -sulpiride ( Roberts and Strange , 2005 ) .", "Of note , these [35S]GTPγS-binding experiments were performed in the absence of Na+ .", "Based on these functional data together with the different binding modes revealed by our computational simulations , we propose that ligands that occupy the Ile3 . 40 sub-pocket exhibit a greater level of inverse agonism as compared to those that do not .", "Therefore , across the tested inverse agonists there is a negative relation between ligand sensitivity to Na+ and the extent of inverse agonism at D2R .", "The differential occupation of the Ile3 . 40 sub-pocket is the structural basis for the Na+ sensitivity , which contributes significantly to the extent of inverse agonism of the tested ligands .", "By occupying the Ile3 . 40 sub-pocket , the benzisoxazole moiety of risperidone pushes the conserved Phe6 . 52 away from the binding site in the D2R/risperidone structure compared to its position in the D3R/eticlopride structure .", "This interaction is responsible for positioning the aromatic cluster of TM6 and TM7 ( Trp6 . 48 , Phe6 . 51 , Phe6 . 52 , His6 . 55 , and Tyr7 . 35 ) in D2R differently from its configurations in the D3R and D4R structures , resulting in an overall outward positioning of the extracellular portion of TM6 in D2R ( Figure 4—figure supplement 1 ) .", "On the extracellular side of the OBS , the space near Ser5 . 42 and Ser5 . 43 that accommodates the bulky substitutions of the benzamide rings of the bound eticlopride and nemonapride in the D3R and D4R structures is not occupied by risperidone in D2R , which is likely associated with the inward movement of the extracellular portion of TM5 in D2R relative to those in the D3R and D4R structures ( Figure 1 ) .", "To evaluate whether these conformational rearrangements are due to the minor divergence in these regions of the receptors or to the ligand-binding site plasticity that accommodates ligands bearing different scaffolds , we compared the resulting conformations of D2R bound with risperidone or eticlopride .", "We observed the same trend of rearrangements of the transmembrane segments surrounding the OBS in the resulting receptor conformations from our D2R/risperidone and D2R/eticlopride simulations ( Figure 4a ) , that is , an inward movement of TM6 and outward movement of TM5 in the presence of the bound eticlopride ( Figure 4b , c ) .", "Without such movements in D2R/eticlopride , Ser1935 . 42 and Ser1945 . 43 would clash with the bound eticlopride ( Figure 4a ) .", "These findings further support our inference that differences between the D2R and D3R inactive structures are largely due to the different scaffolds of the bound non-selective ligands .", "In addition to differences in the transmembrane segments surrounding the OBS , there are also substantial differences in the configuration of EL2 in the D2R and D3R structures .", "EL2 between TM4 and TM5 is connected to TM3 via a disulfide bond formed between CysEL2 . 50 ( see Materials and methods and Figure 5—figure supplement 1 for the indices of EL1 and EL2 residues ) and Cys3 . 25 .", "The conformation of EL2 , the sequence of which is not conserved among aminergic GPCRs , is expected to be dynamic .", "Indeed , in the D2R/risperidone structure , the sidechains of residues 176EL2 . 40 , 178EL2 . 46 , 179EL2 . 47 , and 180EL2 . 48 , which are distal to the OBS were not solved , likely due to their dynamic nature .", "Curiously , the portion of EL2 C-terminal to Cys182EL2 . 50 ( residues 182EL2 . 50-186EL2 . 54 ) , which forms the upper portion of the OBS that is in contact with ligand , is in a helical conformation in the D2R/risperidone structure .", "Strikingly , in our MD simulations of D2R complexes , we found that this helical region showed a tendency to unwind ( Video 1 ) .", "The unwinding of EL2 involves a drastic rearrangement of the sidechain of Ile183EL2 . 51 , which dissociates from a hydrophobic pocket formed by the sidechains of Val1113 . 29 , Leu1704 . 60 , Leu174EL2 . 38 , and Phe1895 . 38 .", "Specifically , the unwinding process is initiated by the loss of a hydrogen-bond ( H-bond ) interaction between the sidechain of Asp1083 . 26 and the backbone amine group of Ile183EL2 . 51 formed in the D2R/risperidone structure ( Figure 5—figure supplement 2b , step", "( i ) .", "When this interaction is broken , the orientation of residues 182EL2 . 50-186EL2 . 54 deviates markedly from that of the crystal structure , losing its helical conformation ( see below ) .", "Subsequently , the sidechain of Ile183EL2 . 51 rotates outwards and passes a small steric barrier of Gly173EL2 . 37 ( Figure 5—figure supplement 2b , step", "( ii ) , and in some trajectories makes a favorable hydrophobic interaction with the sidechain of Ala177EL2 . 45 .", "In a few long trajectories , Ile183EL2 . 51 rotates further toward the extracellular vestibule where it can make favorable interactions with hydrophobic or aromatic residues from the N terminus , or the bound risperidone ( Video 1 ) .", "Consequently , residues 182EL2 . 50-186EL2 . 54 are in a fully extended loop conformation while Ile184EL2 . 52 tilts under EL2 ( Figure 5—figure supplement 2b , step", "( iii ) .", "In the D3R structure , the aligned residue for Asp1083 . 26 of D2R is conserved as Asp1043 . 26; its sidechain forms an interaction not with Ile182EL2 . 51 but rather with the sidechain of Asn173EL2 . 39 , which is also conserved in D2R as Asn175EL2 . 39 .", "In the D4R , the aligned two residues ( Asp1093 . 26 and Asn175EL2 . 39 ) are conserved as well , their sidechains are only 4 . 3 Å away in the D4R structure , a distance slightly larger than the 3 . 2 Å in the D3R structure .", "Even though these residues are conserved in D2R , the interaction in D3R ( and potentially in D4R ) , between Asp3 . 26-AsnEL2 . 39 , is not present in the D2R structure in which the aligned Asn175EL2 . 39 faces lipid ( Figure 5—figure supplement 2a ) .", "However , in a few of our long D2R simulations , Asn175EL2 . 39 gradually moves inwards and approaches Asp1083 . 26 ( Figure 5—figure supplement 2b , step", "( iv ) .", "At this point , the EL2 conformation of D2R is highly similar to that of D3R ( Figure 5—figure supplement 2c ) , suggesting that EL2 is dynamic and can exist in both conformations .", "We evaluated the tendency of the EL2 helix to unwind in each of the simulated D2R complexes by measuring the stability of the backbone H-bond between Ile183EL2 . 51 and Asn186EL2 . 54 , a key stabilizing force of the helix ( Figure 5a ) .", "When we plotted the Ile183EL2 . 51-Asn186EL2 . 54 distance against the Asp1083 . 26-Ile183EL2 . 51 distance for each D2R complex ( Figure 5b ) , we found that the loss of the Asp1083 . 26-Ile183EL2 . 51 interaction increases the probability of breaking the Ile183EL2 . 51-Asn186EL2 . 54 H-bond , that is the unwinding of EL2 .", "Interestingly , in all our simulated D2R complexes , EL2 has a clear tendency to unwind , regardless of the scaffold of the bound ligand ( Figure 5c , d , Videos 1–3 ) .", "Note that in the D3R/eticlopride simulations , the aligned residues Ser182EL2 . 51 and Asn185EL2 . 54 do not form such a H-bond , and EL2 is always in an extended conformation ( Figure 5b–d ) .", "This tendency of EL2 to transition toward the extended conformation is also present in our simulations of D2R in complex with a partial agonist , aripiprazole , whereas EL2 in the D3R complexes with partial agonists ( R22 and S22 ) remains in the extended conformation ( Table 1 and Figure 5—figure supplement 3 ) .", "Interestingly , Asp1043 . 26 and Ser182EL2 . 51 can move into interacting range in the D3R/eticlopride simulations , and the Ser182EL2 . 51-Asn185EL2 . 54 interaction can sporadically form in the D3R/R22 simulations – both raise the possibility that the extended conformation of D3R EL2 may transition to a helical conformation .", "Interestingly , in one of our long MD trajectories of the D2R/risperidone complex , EL2 evolved into a conformation that has a helical N-terminal portion and an extended C-terminal portion ( Video 4 and Figure 5—figure supplement 4 ) .", "This conformation is not observed in either of the D2R/risperidone and D3R/eticlopride structures but is similar to that of the 5-HT2AR/risperidone structure , further demonstrating the dynamics of this loop region ( Figure 5—figure supplement 4 ) .", "In marked contrast to the obvious trend toward unwinding of EL2 in all our simulated D2R complexes , in our recent simulations of MhsT , a transporter protein with a region found by crystallography to alternate between helical and unwound conformations ( Malinauskaite et al . , 2014 ) , we failed to observe any spontaneous unwinding over a similar simulation timescale ( with the longest simulations being ~5–6 µs ) when the region was started from the helical conformation ( Abramyan et al . , 2018; Stolzenberg et al . , 2017 ) .", "This shows how difficult it can be to capture known dynamics in simulations and suggests that the C-terminal helical conformation of EL2 in D2R represents a higher energy state than the extended conformation , which allows for observation of the transitions in a simulation timescale not usually adequate to sample folding/unfolding events ( Piana et al . , 2011 ) .", "We have previously shown that the divergence in both the length and number of charged residues in EL1 among D2R , D3R , and D4R is responsible for the selectivity of more extended ligands ( Michino et al . , 2013; Newman et al . , 2012 ) .", "Another striking difference in the D2R , D3R , and D4R structures is the position of the conserved TrpEL1 . 50 in EL1 .", "Trp100EL1 . 50 is in a much more inward position in the D2R structure , making a direct contact with the bound risperidone ( Figure 6a ) , Trp101EL1 . 50 in D4R interacts with the bound nemonapride that has an extended structure , whereas Trp96EL1 . 50 in D3R is not in contact with eticlopride ( Figure 6b ) .", "Thus , we asked whether these distinct positions of TrpEL1 . 50 are due to the divergence in EL1 among these receptors ( Michino et al . , 2013 ) or due to the multiple inactive conformations that differentially accommodate the binding of non-selective ligands of divergent scaffolds .", "When residues 182EL2 . 50-186EL2 . 54 of EL2 are in a helical conformation , in the D2R/risperidone simulations , we found that there is more room in the extracellular vestibule and the position of Trp100EL1 . 50 is flexible and can adopt several positions and orientations ( Figure 6c , e , f ) .", "In the D2R/eticlopride simulations , Trp100EL1 . 50 , which cannot interact with eticlopride , shows more flexibility than that observed in the presence of risperidone and can move to a similar position like that of Trp96EL1 . 50 in the D3R structure ( Figure 6—figure supplement 1 and Video 2 ) .", "Interestingly , in this position , the conformation of TrpEL1 . 50 can be stabilized by the disulfide bond of EL2 ( Ioerger et al . , 1999 ) ( as shown in Video 2 ) or by interaction with the N terminus , which was truncated in the receptor construct used in the determination of the crystal structure .", "In the D2R/spiperone simulations , the phenyl substitution on the triazaspiro[4 . 5]decane moiety protrudes toward the interface between TM2 and TM3 , and contacts Trp100EL1 . 50 , which is flexible as well and can adopt a position that is even further away from the OBS than that of Trp96EL1 . 50 in the D3R structure ( Figure 6—figure supplement 1 ) .", "In contrast , when EL2 is in an extended conformation like that in D3R , it restricts the flexibility of Trp100EL1 . 50 ( Video 3 ) .", "This trend is consistent with the D3R/eticlopride simulations in which we do not observe any significant rearrangement of Trp96EL1 . 50 ( Figure 6d , e , f ) .", "Thus , we infer that the distinct conformation of Trp100EL1 . 50 in the D2R structure is a combined effect of the helical EL2 conformation and the favored interaction that Trp100EL1 . 50 can form with the bound risperidone in the crystal structure , the latter of which however , has a limited influence on the binding affinity of risperidone ( Wang et al . , 2018 ) , consistent with the unstable interaction between risperidone and Trp100EL1 . 50 in our simulations ( Figure 6 , Video 2 ) .", "Indeed , in the fully extended EL2 conformation in which Ile183EL2 . 51 rotates to face the extracellular vestibule , Ile183EL2 . 51 makes a direct contact with the bound risperidone , whereas Trp100EL1 . 50 loses its interaction with the ligand entirely ( Video 1 ) .", "Nevertheless , risperidone retains all other contacts in the OBS .", "In the recently reported 5-HT2AR/risperidone structure ( PDB: 6A93 ) Kimura et al . ( 2019 ) , risperidone has a very similar pose in the OBS as that in the D2R structure , occupying the Ile3 . 40 sub-pocket as well .", "However , on the extracellular side of the OBS , EL2 in the 5-HT2AR/risperidone complex is in an extended conformation and the EL2 residue Leu228EL2 . 51 contacting risperidone aligns to Ile183EL2 . 51 of D2R , whereas the conserved Trp141EL1 . 50 does not interact with risperidone in the 5-HT2AR .", "It is tempting to speculate that the EL2 and EL1 dynamics we observe in the D2R/risperidone simulations represents a more comprehensive picture , as the divergent interactions shown in the extracellular loops of the 5-HT2AR/risperidone and D2R/risperidone structures may not result from differences in the protein sequences of this dynamic region between these two receptors but rather two different static snapshots due to differences in the crystallographic conditions ( Note risperidone has similarly high affinities for both D2R and 5HT2AR; Kimura et al . , 2019; Wang et al . , 2018 ) .", "Thus , the plasticity of the OBS and the dynamics of the extracellular loops appear to be two relatively separated modules in ligand recognition .", "To the extent of our simulations , we did not detect strong ligand-dependent bias in the EL2 dynamics as we did for the OBS .", "However , when EL2 is helical , the EL1 dynamics are sensitive to the bound ligand ( compare Figure 6 and Figure 6—figure supplement 1 ) ; when EL2 is extended , it restricts EL1 dynamics ( Figure 6 ) .", "To further investigate the dynamics and coordination of EL2 and EL1 loops , we mutated Leu942 . 64 , Trp100EL1 . 50 , and Ile184EL2 . 52 , and evaluated the effects of the L94A , W100A , and I184A , mutations on the binding affinities of eticlopride , risperidone , and spiperone .", "As shown in Figure 6—figure supplement 2 , Leu942 . 64 and Trp100EL1 . 50 are closely associated in both the D2R and D3R structures , while Ile184EL2 . 52 interacts with Trp100EL1 . 50 only in the D2R structure .", "In our time-resolved energy transfer ( Tr-FRET ) binding experiments , using a fluorescently labeled spiperone derivate ( spiperone-d2 ) as a tracer ligand , we found that both L94A and W100A significantly reduced the affinities of all tested antagonists , whereas I184A only reduced the affinity of eticlopride while it improved that of risperidone ( Table 3 ) .", "Thus , the effects of the L94A and W100A mutations have similar trends , which appear independent of the effect of I184A .", "Indeed , for Trp100 to switch between the positions in the D2R and D3R structures , it must pass the steric hinderance of the sidechain of Leu94; thus , some effects of the L94A mutation may reflect its perturbation of the positioning of Trp100 , and vice versa .", "These findings support our conclusions that the close interaction between Ile184EL2 . 52 and Trp100EL1 . 50 revealed by the D2R/risperidone crystal structure is not necessary for the stabilization of the risperidone pose .", "Indeed , in our simulations , EL2 has significant intrinsic dynamics and transitions from the helical to unwound conformation independent of the bound ligands ( see above ) .", "When it is in an extended conformation , Ile184 is dissociated from Trp100 .", "Virtual screening has been widely used as an initial step in drug discovery for novel ligand scaffolds .", "To this end , we found that D2R can significantly change its binding site shape to accommodate antagonists bearing different scaffolds , while EL2 is intrinsically dynamic .", "Thus , it is necessary to comprehensively consider the binding site conformations in virtual screening campaigns against D2R , because limiting the screening to only a single conformation will miss relevant ligands .", "Indeed , the strategy of ensemble docking , in which each ligand is docked to a set of receptor conformers , has been adapted in recent virtual screening efforts ( Amaro et al . , 2018 ) .", "To characterize the OBS conformational ensemble sampled by D2R in complex with ligands bearing different scaffolds in the context of EL2 dynamics , we clustered the OBS conformations in our representative D2R/eticlopride and D2R/risperidone MD trajectories in which EL2 transitioned from helical to unwound conformations ( see Materials and methods ) .", "As expected , the OBS conformations in these two complexes are significantly different and can be easily separated into distinct clusters .", "For the clustering results shown in Table 4 , the average pairwise RMSDs of the OBS residues ( apRMSDs , see Materials and methods ) between the D2R/eticlopride and D2R/risperidone clusters are >1 . 1 Å , which are similar to that between the D2R and D3R structures ( 1 . 2 Å ) , while the apRMSDs within each cluster is smaller than those between any two clusters ( Figure 7 ) .", "Interestingly , at this level of clustering , when the two clusters for each complex are ~0 . 8–0 . 9 Å apRMSD away from each other , the extended and helical conformations of EL2 are always mixed in a cluster ( Table 4 ) .", "This observation suggests that the helical versus extended EL2 conformations are not closely associated with the OBS conformations .", "Thus , while the centroid frames from each cluster can form an ensemble for future virtual screening for the primary scaffold occupying the OBS , in order to discover novel extended ligands that protrude out of the OBS to interact with EL2 and EL1 residues ( Michino et al . , 2015a ) , additional frames that cover both helical and extended EL2 conformations from each cluster will have to be used to screen for the optimal extensions of the primary scaffold ." ], [ "Our results highlight unappreciated conformational complexity of the inactive state of GPCRs and suggest that the risperidone bound D2R structure represents only one of a number of possible inactive conformations of D2R .", "Critically , this conformation is incompatible with the binding of other high-affinity D2R ligands such as eticlopride .", "While distinct conformational states responsible for functional selectivity have garnered great attention , the potential existence of divergent inactive conformations is of critical importance as well .", "By combining in silico and in vitro findings , we propose that occupation of the Ile3 . 40 sub-pocket by antagonists confers a distinct D2R conformation that is associated with both a greater degree of inverse agonism and Na+ insensitivity in binding , such that Na+ sensitivity is negatively related with the extent of inverse agonism for the tested ligands .", "However , other structural elements may also contribute to the extent of inverse agonism ( Zhang et al . , 2014 ) .", "Regardless , the distinct inactive conformations stabilized by antagonists with different scaffolds may reflect different degrees of inactivation .", "In addition to advancing our mechanistic understanding of receptor function , our findings have implications for high-throughput virtual screening campaigns , as important hits would be missed by focusing on a single inactive state captured in a crystal structure that is stabilized by an antagonist bearing a specific scaffold .", "Moreover , rational lead optimization requires rigorous physical description of molecular recognition ( Beuming and Shi , 2017 ) , which depends on adequate understanding of the conformational boundary and flexibility of the targeted state .", "We have shown previously that both dopamine receptor subtype selectivity and modulation of agonist efficacy can be achieved through the design of ligands that extend from the OBS into an extracellular secondary binding pocket ( SBP ) ( Michino et al . , 2015a; Newman et al . , 2012 ) .", "We now show that one might consider the occupation of the Ile3 . 40 sub-pocket in the process of decorating an D2R antagonist scaffold to attain a desired level of inverse agonism .", "Our findings also reveal allosteric communication between the IIe3 . 40 sub-pocket and the Na+-binding site .", "Thus , Na+ sensitivity in antagonist binding may provide useful mechanistic insights as part of such efforts .", "The mutation of Trp100EL1 . 50 in D2R to alanine , leucine or phenylalanine cause substantial increases in both the association and dissociation rate of risperidone ( Wang et al . , 2018 ) .", "Curiously , both the dissociation and association rates of D2R antagonists used as antipsychotics have been proposed to determine their propensity to cause extrapyramidal side-effects and hyperprolactinaemia ( Seeman , 2014; Sykes et al . , 2017 ) .", "Our results indicate that both the EL2 conformation and antagonist scaffolds may influence the dynamics of Trp100EL1 . 50 , which in turn controls ligand access and egress to and from the OBS .", "Thus , understanding the relationship between the distinct inactive D2R conformations stabilized by different antagonist scaffolds and these kinetic parameters will likely be important to facilitate the design of D2R antagonists with an optimal kinetic profile that minimizes the risk of side effects .", "Previously , using the substituted-cysteine accessibility method ( SCAM ) in D2R ( Javitch et al . , 2000; Shi and Javitch , 2004 ) , we found that G173EL2 . 37C , N175 EL2 . 39C , and I184EL2 . 52C were accessible to charged MTS reagents and that this accessibility could be blocked by the bound Na+-sensitive antagonist sulpiride , consistent with their water accessibility and involvement in ligand binding and not with a static orientation facing lipid , whereas A177EL2 . 45C and I183EL2 . 51C were accessible but not protected by sulpiride .", "Curiously , in the D2R/risperidone structure , Ile184EL2 . 52 is only marginally in contact with the ligand , Ile183EL2 . 51 blocks the accessibility of Gly173EL2 . 37 to the OBS and is itself buried in a hydrophobic pocket , whereas Asn175EL2 . 39 faces lipid , where it would be much less reactive .", "In the D3R/eticlopride structure , Ile183EL2 . 52 is in close contact with the bound ligand , Ser182EL2 . 51 faces the extracellular vestibule , whereas the sidechain of Asn173EL2 . 39 is oriented toward the OBS ( Figure 5—figure supplement 5 ) .", "Thus , our analysis shows that the accessibility pattern of EL2 revealed by previous SCAM studies in D2R are more consistent with the extended EL2 conformation revealed by the D3R/eticlopride structure but not with the D2R/risperidone structure .", "Indeed , we observed spontaneous transitions of EL2 from a helical to extended conformation in our D2R simulations , which suggests that EL2 of D2R exists in an ensemble of structured and unwound conformations , with substantial occupation of the configuration found in the D3R structure .", "Such dynamics of EL2 suggest that the drastically different conformations between the D2R and D3R structures near EL2 are not related to the divergence of the receptors .", "Thus , the D2R EL2 appears to have quite dramatic dynamics that are not captured by the crystal structure .", "Taken together , our findings reveal that both the plasticity of the transmembrane domain in accommodating different scaffolds and the dynamics of EL2 and EL1 are important considerations in RDD targeting the inactive conformation of D2R ." ], [ "Based on a systematic analysis of aminergic receptors , we found a Trp in the middle of EL1 and the disulfide-bonded Cys in the middle of EL2 are the most conserved residues in each segment , and defined their residue indices as EL1 . 50 and EL2 . 50 , respectively ( Michino et al . , 2015a ) , In this study , for the convenience of comparisons among D2R , D3R , and D4R , and 5-HT2AR , based on the alignments of EL1 And EL2 shown in Figure 5—figure supplement 1 , we index the EL1 and EL2 residues of each receptor in the same way as the Ballesteros-Weinstein numbering , for example the residues before and after the EL2 . 50 are EL2 . 49 and EL2 . 51 , respectively .", "Note the indices for the shorter sequences are not necessarily be consecutive , given the gaps in the alignment .", "The D2R models in this study are based on the corrected crystal structure of D2R bound to risperidone ( PDB: 6CM4 ) ( Wang et al . , 2018 ) .", "We omitted T4 Lysozyme fused into intracellular loop", "3 . Three thermostabilizing mutations ( Ile1223 . 40A , L3756 . 37A , and L3796 . 41A ) were reverted to their WT residues .", "The missing N terminus in the crystal structure was built de novo using Rosetta ( Bradley et al . , 2005 ) , and then integrated with the rest of the D2R model using Modeller ( John and Sali , 2003 ) .", "Using Modeller , we also extended two helical turns at the TM5 C terminus and three residues at the TM6 N terminus of the structure and connected these two ends with a 9 Gly loop , similar to our experimentally validated treatment of D3R models ( Michino et al . , 2017 ) .", "The position of the Na+ bound in the canonical Na+-binding site near the negatively charged Asp2 . 50 was acquired by superimposing the Na+-bound structure of adenosine A2A receptor ( Liu et al . , 2012 ) to our D2R models .", "The binding poses of risperidone and eticlopride were taken according to their poses in the D2R ( Wang et al . , 2018 ) and D3R ( Chien et al . , 2010 ) structures , respectively .", "Docking of spiperone in our D2R model was performed using the induced-fit docking ( IFD ) protocol ( Sherman et al . , 2006 ) in the Schrodinger software ( release 2017–2; Schrodinger , LLC: New York NY ) .", "Based on our hypothesis regarding the role of the Ile3 . 40 sub-pocket in the Na+ sensitivity ( see text ) , from the resulting poses of IFD , we choose the spiperone pose with the F-substitution on the butyrophenone ring occupying the Ile3 . 40 sub-pocket .", "Note that in risperidone and spiperone the F-substitutions have similar distances to the protonated N atoms that interact with Asp3 . 32 ( measured by the number of carbon atoms between them , Figure 1—figure supplement 1 ) .", "MD simulations of the D2R and D3R complexes were performed in the explicit water and 1-palmitoyl-2-oleoylphosphatidylcholine ( POPC ) lipid bilayer environment using Desmond MD System ( version 4 . 5; D . E . Shaw Research , New York , NY ) with either the OPLS3e force field ( Roos et al . , 2019 ) or the CHARMM36 force field ( Best et al . , 2012; Klauda et al . , 2010; MacKerell et al . , 1998; MacKerell et al . , 2004 ) and TIP3P water model .", "For CHARMM36 runs , the eticlopride parameters were obtained through the GAAMP server ( Huang and Roux , 2013 ) , with the initial force field based on CGenFF assigned by ParamChem ( Vanommeslaeghe et al . , 2010 ) .", "The system charges were neutralized , and 150 mM NaCl was added .", "Each system was first minimized and then equilibrated with restraints on the ligand heavy atoms and protein backbone atoms , followed by production runs in an isothermal–isobaric ( NPT ) ensemble at 310 K and one atm with all atoms unrestrained , as described previously ( Michino et al . , 2017; Michino et al . , 2015b ) .", "We used Langevin constant pressure and temperature dynamical system ( Feller et al . , 1995 ) to maintain the pressure and the temperature , on an anisotropic flexible periodic cell with a constant-ratio constraint applied on the lipid bilayer in the X-Y plane .", "For each condition , we collected multiple trajectories , the aggregated simulation length is ~392 μs ( Table 1 ) .", "While the majority of our D2R simulations in this study used the OPLS3e force field , to compare with the D3R simulations using CHARMM36 that have been continued from the previously reported shorter trajectories ( Michino et al . , 2017; Michino et al . , 2015b ) , we carried out the D2R/eticlopride simulations using both the OPLS3e and CHARMM36 force fields ( see Table 1 ) .", "We did not observe significant differences and pooled their results together for the analysis .", "Distances and dihedral angles of MD simulation results were calculated with MDTraj ( version 1 . 8 . 2 ) ( McGibbon et al . , 2015 ) in combination with in-house Python scripts .", "To characterize the structural changes in the receptor upon ligand binding , we quantified differences of structural elements between the D2R/eticlopride and D2R/risperidone conditions ( using last 600 ns from a representative trajectory for each condition ) , by applying the previously described pairwise interaction analyzer for GPCR ( PIA-GPCR ) ( Michino et al . , 2017 ) .", "The subsegments on the extracellular side of D2R were defined as following: TM1e ( the extracellular subsegment ( e ) of TM1 , residues 31–38 ) , TM2e ( residues 92–96 ) , TM3e ( residues 104–113 ) , TM4e ( residues 166–172 ) , TM5e ( residues 187–195 ) , TM6e ( residues 364–369 ) , and TM7e ( residues 376–382 ) .", "For the PIA-GPCR analysis in Figure 4 and the distance analysis in Figure 6 , we used the set of ligand-binding residues previously identified by our systematic analysis of GPCR structures .", "Specifically , for D2R , they are residues 91 , 94 , 95 , 100 , 110 , 111 , 114 , 115 , 118 , 119 , 122 , 167 , 184 , 189 , 190 , 193 , 194 , 197 , 198 , 353 , 357 , 360 , 361 , 364 , 365 , 367 , 368 , 376 , 379 , 380 , 383 , 384 , 386 , and 387; for D3R , they are residues 86 , 89 , 90 , 96 , 106 , 107 , 110 , 111 , 114 , 115 , 118 , 165 , 183 , 188 , 189 , 192 , 193 , 196 , 197 , 338 , 342 , 345 , 346 , 349 , 350 , 352 , 353 , 362 , 365 , 366 , 369 , 370 , 372 , and 373 .", "For the clustering of the OBS conformations , we used representative D2R/eticlopride and D2R/risperidone MD trajectories in which EL2 transitioned from the helical to unwound conformations .", "For each complex , using the Ile183-Asn186 distance as a criterion to differentiate the EL2 conformation ( Figure 5 ) , 1000 MD frames with helical EL2 conformations and another 1000 frames with extended EL2 conformations were randomly selected .", "For these 4000 frames , the pairwise RMSD of the backbone heavy atoms of the OBS residues defined in Michino et al . ( 2015a ) , except for Ile184EL2 . 52 , were calculated .", "The resulting 4000 × 4000 matrix was used to cluster these frames using the k-mean algorithm implemented in R . We chose nstart to be 20 to assure the convergence of cluster centroids and boundaries .", "We chose the clustering level to be 4 , so that the average pairwise RMSDs ( apRMSDs ) between the D2R/eticlopride and D2R/risperidone clusters are similar to that between D2R and D3R structures ( 1 . 2 Å ) , while all the apRMSDs within a cluster are smaller than those between any given two clusters .", "The same frame selection and clustering procedure was repeated to 20 times .", "The averages of these 20 runs for the compositions of each cluster were reported in Table", "4 . The MSM analysis was performed using the pyEMMA program ( version 2 . 5 . 5 ) ( Scherer et al . , 2015 ) .", "To characterize the dynamics of EL2 of D2R , specifically the transitions between helical and extended conformations of its C-terminal portion , we focused on a key hydrogen bond formed in the helical conformation between the backbone carbonyl group of Ile183 and the backbone amine group of Asn186 .", "Thus , for each of the simulated conditions , the distance of Ile183-Asn186 ( Ser182-Asn185 in D3R ) was used as an input feature for the MSM analysis .", "We discretized this feature into two clusters – distances below and above 4 Å ( i . e . EL2 forming a helical conformation and unwinding ) .", "Implied relaxation timescale ( ITS ) ( Swope et al . , 2004 ) for the transition between these clusters was obtained as a function of various lag times .", "Convergences of ITS for the MSMs for all conditions was achieved at a lag time of 300 ns ( Figure 5—figure supplement 6 ) , which we further used to estimate Bayesian Markov models with 500 transition matrix samples ( Trendelkamp-Schroer and Noé , 2013 ) .", "The maximum likelihood transition matrix was used to calculate the transition and equilibrium probabilities ( π ) shown in Figure 5 and Figure 5—figure supplement", "3 . Site-directed mutagenesis was performed using the Quickchange method using pEF5/DEST/FRT plasmid encoding FLAG-SNAP-D2SR as the DNA template .", "The mutagenesis was confirmed , and the full coding region was checked using Sanger sequencing at the DNA Sequencing Laboratory ( University of Nottingham ) .", "Stable cell lines were generated using the Flp-In recombination system ( Invitrogen ) .", "FlpIn CHO cells ( Invitrogen ) stably expressing WT or mutant SNAP-D2s cells were cultured before the preparation of cell membrane as described before ( Klein Herenbrink et al . , 2019 ) .", "All stable cell lines were confirmed to be mycoplasma free .", "For saturating binding assays cell membranes ( Mutant or WT SNAP-D2s-FlpIn CHO , 2 . 5 µg ) were incubated with varying concentrations of [3H]spiperone and 10 µM haloperidol as a non-specific control , in binding buffer ( 20 mM HEPES , 100 mM NaCl , 6 mM MgCl2 , 1 mM EGTA , and 1 mM EDTA , pH 7 . 4 ) to a final volume of 200 μL and were incubated at 37°C for 3 hr .", "For competition binding assays , cell membranes ( SNAP-D2s-FlpIn CHO , 2 . 5 µg ) were incubated with varying concentrations of test compound in binding buffer containing 0 . 2 nM of [3H]spiperone to a final volume of 200 µL and were incubated at 37°C for 3 hr .", "Binding was terminated by fast-flow filtration using a Uniplate 96-well harvester ( PerkinElmer ) followed by five washes with ice-cold 0 . 9% NaCl .", "Bound radioactivity was measured in a MicroBeta2 LumiJET MicroBeta counter ( PerkinElmer ) .", "Data were collected from at least three separate experiments performed in triplicate and analysed using non-linear regression ( Prism 7 , Graphpad software ) .", "For radioligand saturation binding data , the following equation was globally fitted to nonspecific and total binding data: ( 1 ) Y=BmaxAA+KA+NSAwhere Y is radioligand binding , Bmax is the total receptor density , [A] is the free radioligand concentration , KA is the equilibrium dissociation constant of the radioligand , and NS is the fraction of nonspecific radioligand binding .", "The Bmax of the SNAP-tagged D2SRs we as follows; WT = 7 . 95 ± 1 . 63 pmol . mg−1 , 6 . 39 ± 1 . 04 pmol . mg−1 , 4 . 37 ± 0 . 92 pmol . mg−1 , 2 . 61 ± 0 . 50 pmol . mg−1 .", "For competition binding assays , the concentration of ligand that inhibited half of the [3H]spiperone binding ( IC50 ) was determined by fitting the data to the following equation: ( 2 ) Y=Bottom+ ( Top-Bottom ) 1+10X-LogIC50nHwhere Y denotes the percentage specific binding , Top and Bottom denote the maximal and minimal asymptotes , respectively , IC50 denotes the X-value when the response is midway between Bottom and Top , and nH denotes the Hill slope factor .", "IC50 values obtained from the inhibition curves were converted to Ki values using the Cheng and Prusoff equation .", "No statistical methods were used to predetermine sample size .", "The Go-protein activation assay uses a set of BRET-based constructs previously described ( Michino et al . , 2017 ) .", "Briefly , HEK293T cells were transiently co-transfected with pcDNA3 . 1 vectors encoding", "( i ) D2R ,", "( ii ) GαoA fused to Renilla luciferase 8 ( Rluc8; provided by Dr . S . Gambhir , Stanford University , Stanford , CA ) at residue 91 ,", "( iii ) untagged Gβ1 , and", "( iv ) Gγ2 fused to mVenus .", "Transfections were performed using polyethyleneimine ( PEI ) at a ratio of 2:1 ( PEI:total DNA; weight:weight ) , and cell culture was maintained as described previously ( Bonifazi et al . , 2019 ) .", "After ~48 hr of transfection , cells were washed with PBS and resuspended in PBS + 0 . 1% glucose + 200 µM Na Bisulfite buffer .", "Approximately 200 , 000 cells were then distributed in each well of the 96-well plates ( White Lumitrac 200 , Greiner bio-one ) .", "5 μM Coelenterazine H , a luciferase substrate for BRET , was then added followed by addition of vehicle and test compounds using an automated stamp transfer protocol ( Nimbus , Hamilton Robotics ) from an aliquoted 96-well compound plate .", "Following ligands were used – quinpirole , eticlopride , raclopride , and ( - ) -sulpiride ( Tocris Bioscience ) , ( + ) -butaclamol , dopamine , and risperidone ( Sigma Aldrich ) , and Spiperone ( Cayman chemicals ) .", "mVenus emission ( 530 nm ) over RLuc 8 emission ( 485 nm ) were then measured after 30 min of ligand incubation at 37°C using a PHERAstar FSX plate reader ( BMG Labtech ) .", "BRET ratio was then determined by calculating the ratio of mVenus emission over RLuc eight emission .", "Data were collected from at least nine independent experiments and analyzed using Prism 7 ( GraphPad Software ) .", "Drug-induced BRET , defined by BRET ratio difference in the presence and absence of compounds , was calculated .", "Concentration response curves ( CRCs ) were generated using a non-linear sigmoidal dose-response analyses using Prism 7 ( GraphPad Software ) .", "CRCs are presented as mean drug-induced BRET ± SEM .", "Emax bar graphs are plotted as the percentage of maximal drug-induced BRET by quinpirole ± SEM .", "Materials: Spiperone-d2 , SNAP-Lumi4-Tb and 5x SNAP/CLIP labeling medium were purchased from Cisbio Bioassays .", "Eticlopride hydrochloride was purchased from Tocris Bioscience .", "Saponin was purchased from Fluka/Sigma-Aldrich .", "Bromocriptine , haloperidol , risperidone , spiperone , pluronic-F127 , Gpp ( NH ) p , DNA primers , Hanks Balanced Salt Solution H8264 ( HBSS ) and phosphate buffered saline ( PBS ) was purchased from Sigma-Aldrich ." ] ]

[ "By analyzing and simulating inactive conformations of the highly homologous dopamine D2 and D3 receptors ( D2R and D3R ) , we find that eticlopride binds D2R in a pose very similar to that in the D3R/eticlopride structure but incompatible with the D2R/risperidone structure .", "In addition , risperidone occupies a sub-pocket near the Na+ binding site , whereas eticlopride does not .", "Based on these findings and our experimental results , we propose that the divergent receptor conformations stabilized by Na+-sensitive eticlopride and Na+-insensitive risperidone correspond to different degrees of inverse agonism .", "Moreover , our simulations reveal that the extracellular loops are highly dynamic , with spontaneous transitions of extracellular loop 2 from the helical conformation in the D2R/risperidone structure to an extended conformation similar to that in the D3R/eticlopride structure .", "Our results reveal previously unappreciated diversity and dynamics in the inactive conformations of D2R .", "These findings are critical for rational drug discovery , as limiting a virtual screen to a single conformation will miss relevant ligands ." ]

[ "Almost a third of prescribed drugs work by acting on a group of proteins known as GPCRs ( short for G-protein coupled receptors ) , which help to transmit messages across the cell’s outer barrier .", "The neurotransmitter dopamine , for instance , can act in the brain and body by attaching to dopamine receptors , a sub-family of GPCRs .", "The binding process changes the three-dimensional structure ( or conformation ) of the receptor from an inactive to active state , triggering a series of molecular events in the cell .", "However , GPCRs do not have a single ‘on’ or ‘off’ state; they can adopt different active shapes depending on the activating molecule they bind to , and this influences the type of molecular cascade that will take place in the cell .", "Some evidence also shows that classes of GPCRs can have different inactive structures; whether this is also the case for the dopamine D2 and D3 receptors remained unclear .", "Mapping out inactive conformations of receptors is important for drug discovery , as compounds called antagonists can bind to inactive receptors and interfere with their activation .", "Lane et al . proposed that different types of antagonists could prefer specific types of inactive conformations of the dopamine D2 and D3 receptors .", "Based on the structures of these two receptors , the conformations of D2 bound with the drugs risperidone and eticlopride ( two dopamine antagonists ) were simulated and compared .", "The results show that the inactive conformations of D2 were very different when it was bound to eticlopride as opposed to risperidone .", "In addition D2 and D3 showed a very similar conformation when attached to eticlopride .", "The two drugs also bound to the inactive receptors in overlapping but different locations .", "These computational findings , together with experimental validations , suggest that D2 and D3 exist in several inactive states that only allow the binding of specific drugs; these states could also reflect different degrees of inactivation .", "Overall , the work by Lane et al . contributes to a more refined understanding of the complex conformations of GPCRs , which could be helpful to screen and develop better drugs ." ]

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

[ "stem cells and regenerative medicine" ]

[ [ "Adult stem cells ( ASCs ) are ultimately responsible for all tissue turnover in humans , which has been estimated to be approximately 1010 cells per day ( Reed , 1999 ) .", "This feat is achieved through a delicate balance of proliferation and differentiation , in order to maintain a stable stem cell population while replacing the exact number and type of cells lost to cell turnover or injury .", "This requires inherent asymmetry in stem cell lineages , with some daughter cells retaining stem cell identity while others become committed to differentiate ( Rambhatla et al . , 2001; Sherley , 2002; Simons and Clevers , 2011 ) .", "Asymmetry in cell fate outcomes in stem cell lineages is known to happen in several ways .", "Asymmetry can be largely intrinsic , driven by the asymmetric distribution of RNA and proteins that drive different fates ( Bossing et al . , 1996; Doe , 1996 , 2008; Doe and Bowerman , 2001; Bayraktar et al . , 2010 ) .", "For example , in Drosophila neuroblasts , the cell fate determinant Prospero is physically segregated into the daughter cell of a neuroblast division , where it drives differentiation and suppresses stem cell identity ( Doe et al . , 1991 ) .", "In contrast , the size of the stem cell population can be controlled almost entirely by extrinsic means , such as in the mammalian intestinal crypt where paneth cells use WNT/Lgr5 signaling to maintain stem cell identity ( Snippert et al . , 2010; Sato et al . , 2011 ) .", "As the paneth cell niche expands in colon cancer , so too does the stem cell population ( de Lau et al . , 2007 ) .", "Other stem cell types can use a combination of mechanisms , such as in the mammalian postnatal cortex of the brain where Hedgehog signaling maintains stem cell identity , and asymmetric segregation of RNA-binding protein complexes and cellular processes determines cell fate choice ( Machold et al . , 2003; Miller and Gauthier-Fisher , 2009; Vessey et al . , 2012 ) .", "For both regenerative medicine and cancer biology , elucidating how non-stem cell fates are specified is a fundamental aspect of understanding the mechanisms of stem cell lineage development .", "The freshwater planarian Schmidtea mediterranea ( a Lophotrochozoan flatworm ) is quickly becoming a powerful model system to study gene function , regeneration , and ASC biology ( Salo and Baguna , 2002; Sánchez Alvarado , 2003 , 2006; Cebria , 2007; Gurley and Sánchez Alvarado , 2008; Rossi et al . , 2008; Salo et al . , 2009; Aboobaker , 2011; Gentile et al . , 2011; Tanaka and Reddien , 2011; Baguna , 2012; Elliott and Sánchez Alvarado , 2013; Rink , 2013 ) .", "Asexual planarians are constitutive adult animals and their well-known regenerative abilities are dependent on neoblasts , which possess stem cell activity and express the piwi homolog smedwi-1 ( piwi-1 ) ( Sánchez Alvarado and Kang , 2005; Reddien et al . , 2005b; van Wolfswinkel et al . , 2014 ) .", "Neoblasts are broadly distributed throughout the mesenchyme where they constitute approximately 20–30% of all the cells in the animal , and serve to replenish all the differentiated cell types during normal tissue turnover and regeneration after injury ( Krichinskaya and Martynova , 1975; Hayashi et al . , 2006; Wagner et al . , 2011 ) .", "At steady state , the population of neoblasts is relatively constant in number despite high levels of tissue turnover , indicating that proliferation , self-renewal , and differentiation are finely balanced ( Pellettieri and Sánchez Alvarado , 2007; Pearson and Sánchez Alvarado , 2008; Pellettieri et al . , 2010 ) .", "Currently , how neoblasts make the choice to adopt non-stem cell fates remains largely unknown; physical asymmetric segregation of cell fate components has not been demonstrated , and descriptions of any permissive niche-like signal remain elusive ( Reddien , 2013; Rink , 2013 ) .", "Although all planarian stem cells are piwi-1+ , not all piwi-1+ cells are necessarily stem cells .", "Evidence increasingly shows commitment to particular lineages can occur at the piwi-1+ level as small numbers of these cells also express tissue-specific genes , though it remains unknown whether these cells are self-renewing or will directly differentiate ( Lapan and Reddien , 2012; Cowles et al . , 2013; Currie and Pearson , 2013; Scimone et al . , 2014 ) .", "Furthermore , recently neoblasts were divided into three major subclasses ( sigma , zeta , gamma ) based on transcriptional analyses , one of which generates non-dividing progenitors/progeny for the epithelium ( zeta ) and another postulated to be an intestinal-restricted subclass ( gamma ) ( van Wolfswinkel et al . , 2014 ) .", "As neoblasts are the only mitotic cell type in planarians , they can be selectively ablated by irradiation , and over time , the immediate progeny of neoblasts eventually disappear as well ( Reddien et al . , 2005b; Eisenhoffer et al . , 2008 ) .", "Using fluorescence-activated cell sorting ( FACS ) analysis of irradiated animals stained with Hoechst , two cell populations can be discerned that are lost compared to non-irradiated animals: the ‘X1’ gate , which represents cells in the cell cycle with >2C DNA; and the ‘X2’ gate , which registers as a <2C DNA population due to Hoechst efflux .", "The remaining irradiation-insensitive ( Xins ) cells are assumed to be postmitotic differentiated cell types possessing 2C DNA ( Reddien et al . , 2005b; Hayashi et al . , 2006 ) .", "Studies have shown that the X1 population is >90% piwi-1+ , and this gate has been used as the stem cell fraction in comparative transcriptomic studies ( Reddien et al . , 2005b; Eisenhoffer et al . , 2008; Abril et al . , 2010; Sandmann et al . , 2011; Labbe et al . , 2012; Onal et al . , 2012; Resch et al . , 2012; Solana et al . , 2012 ) .", "The X2 cell population is only 10–20% piwi-1+ and is thought to be enriched with immediate postmitotic progeny of neoblasts .", "This notion is supported by the finding that the five known markers of postmitotic progeny ( prog-1 , prog-2 , and AGAT-1/2/3 ) , predicted to label epithelial progenitors , are most highly expressed in the X2 cell fraction based on RNA-deep sequencing ( RNAseq ) ( Eisenhoffer et al . , 2008; Labbe et al . , 2012; Onal et al . , 2012; van Wolfswinkel et al . , 2014 ) .", "Additionally , other markers that associate with putative committed progenitor cells of the gut , brain , and eyes are also enriched in this cell fraction ( hnf4 , chat , and sp6-9 , respectively ) ( Wagner et al . , 2011; Lapan and Reddien , 2012; Cowles et al . , 2013; Scimone et al . , 2014 ) .", "No study has comprehensively investigated the cells and transcripts specific to the X2 cell fraction , and the cell types within it remain an enigma .", "Here , we hypothesized that regulators that drive stem cells toward postmitotic fates will be highly enriched in the stem cell progeny-associated X2 FACS fraction .", "Thus , we selected the top 100 transcripts enriched in this FACS gate , as well as 20 that were irradiation-sensitive with no X1 or X2 enrichment , to explore as putative markers or regulators .", "We found that while X2-enriched genes represented a heterogeneous mixture of cell types , transcripts expressed in epithelial progenitors comprised the predominant gene signature in the X2 fraction .", "We identified 32 new progeny markers and demonstrated that they are expressed predominantly in either prog-1/2+ or AGAT-1+ epithelial progenitors .", "In addition , prog-1 and prog-2 represent members of a larger gene family of unknown function , expressed throughout this epithelial lineage .", "Through RNA interference ( RNAi ) screening of the 120 candidate transcripts , we identified a homolog to the RNA-binding protein MEX3 ( Smed-mex3-1 ) as a critical regulator of postmitotic stem cell progeny .", "Knockdown of mex3-1 completely abolishes regenerative ability and halts the production of prog-1/2+ and AGAT-1+ postmitotic progeny populations .", "piwi-1+ stem cells concomitantly increase in number , an increase that was observed in all three neoblast subclasses .", "Finally , mex3-1 ( RNAi ) worms have impaired contribution to tissue turnover , as evidenced by drastically reduced production of lineage-restricted neoblast descendants and diminished cell addition towards multiple tissue types , in addition to the epithelium .", "These results suggest that mex3-1 functions to maintain asymmetry in stem cell lineage progression by promoting postmitotic fates and suppressing self-renewal .", "Due to the well-known function of MEX3 in mediating asymmetric cell fates during Caenorhabditis elegans embryogenesis ( Draper et al . , 1996 ) , we propose that Smed-mex3-1 mediates a similar process in planarian stem cell lineages ." ], [ "Previously , we published two replicates of Illumina RNAseq of the X2 cell fraction to a depth of 206 million reads ( Labbe et al . , 2012 ) .", "Here , we sequenced a third replicate to 63 million reads .", "We found a very high correlation across all of our sequencing replicates , as well as with two irradiated samples from a previous study , which we subsequently analyzed along with our irradiated sequencing ( Figure 1—figure supplement 1 , Supplementary file", "1 ) ( Onal et al . , 2012; Resch et al . , 2012; Solana et al . , 2012 ) .", "To identify transcripts enriched in the X2 cell fraction , we used the program DESeq ( Anders and Huber , 2010 ) to compare RNAseq from purified X1 and X2 cells vs whole irradiated animals at 7 days after exposure to 60–100 Gray ( Gy ) of γ-irradiation ( Anders and Huber , 2010; Solana et al . , 2012; Fernandes et al . , 2014 ) .", "This identified 2839 X1 and 1512 X2 transcripts with a p-value ≤ 0 . 01 ( Figure 1A , B , Supplementary file 1 ) .", "It is important to note that X1 and X2 cells shared the majority of their transcriptional profiles ( Figure 1C ) , and bona fide progeny markers can be highly expressed in the X1 fraction , while bona fide stem cell markers can be highly expressed in the X2 fraction ( Figure 1A , B ) .", "Therefore , to be considered X2-enriched , we imposed the additional criterion that the expression ratio of X2/X1 was >1 , to exclude transcripts jointly expressed in both irradiation-sensitive populations .", "This eliminated previously known stem cell genes , such as piwi-1 and -2 , PCNA ( proliferating cell nuclear antigen ) , and cyclinB ( Figure 1A; 8th , 45th , 57th , and 80th highest enriched X2 genes , respectively ) , and reduced the total number of enriched X2 genes to 735 ( Figure 1D , Supplementary file", "1 ) ( Orii et al . , 2005; Reddien et al . , 2005b; Eisenhoffer et al . , 2008 ) .", "Finally , we observed 66 transcripts that were highly expressed in wild-type animals , yet exhibited low counts in irradiated worms and in both X1 and X2 cell fractions ( WThighXlow , Figure 1D , Supplementary file 1 ) .", "From the remaining 735 X2-specific genes as well as these 66 other irradiation-sensitive transcripts , we hypothesized that these represented multiple types of irradiation-sensitive progenitor cells .", "We next cloned the top 100 X2-specific and 20 WThighXlow transcripts for expression and functional analyses ( Figure 1D , Supplementary file 2 ) .", "Genes were annotated based on the top BLAST hit in mouse , when the Expect value passed the threshold of e−5 . 10 . 7554/eLife . 07025 . 003Figure 1 . Transcriptional analysis of irradiation-sensitive cell populations in Schmidtea mediterranea . Irradiation-sensitive cell populations ( X1 , X2 ) isolated by fluorescence-activated cell sorting ( FACS ) , lethally irradiated whole worms ( Irrad ) , and intact control worms ( WT ) were analyzed by RNA-deep sequencing ( RNAseq ) .", "( A and B )", "MA plots comparing enrichment in the X2 ( A ) or X1 ( B ) cell populations over Irrad , with average expression level .", "Each gray dot represents one transcript .", "Blue dots represent transcripts found to be significantly enriched in the respective cell population through DESeq analysis ( X2 , p < 0 . 01; X1 , p < 0 . 001 ) .", "Previously established postmitotic lineage markers are indicated by orange triangles .", "Previously established stem cell-specific transcripts are indicated as green triangles .", "Candidate genes identified in this study as markers of epithelial progenitors are indicated as red circles ( X2-enriched ) or purple boxes ( WThighXlow ) .", "( C ) Hierarchical clustering of RNAseq data identifies transcripts enriched in the X2 population ( red box ) , as well as a group of irradiation-sensitive transcripts , which have high expression in intact control worms but low expression in X1 , X2 , and Irrad populations ( purple box , WThighXlow ) .", "To improve visualization , the heatmap depicts z-scores scaled to the range of −0 . 5 to +0 . 5 .", "( D ) Selection of candidate progeny genes for analysis was determined by enriched expression in X2 fraction compared to both Irrad and X1 fractions .", "The pool of candidate genes validated in this study to be expressed in epithelial progenitors is indicated in orange .", ">> denotes more than fivefold enrichment . DOI: http://dx . doi . org/10 . 7554/eLife . 07025 . 00310 . 7554/eLife . 07025 . 004Figure 1—figure supplement 1 . Statistical correlations between new and published data sets . Scatter plots of transcripts ( gray circles ) with reads per million values of 1000 or less , displayed as log-transformed values .", "The black diagonal line represents a Pearson correlation coefficient of 1 .", "The corresponding Pearson correlation coefficients are shown at the top of each panel .", "All correlation-test p-values were found to be equal to 0 ( Student's t-test ) .", "As expected , the replicates in each data are highly correlated , and the Pearson correlation coefficients between the replicates in X1 or X2 are greater than 0 . 95 .", "Furthermore , sequencing from whole irradiated planarians is highly correlated . DOI: http://dx . doi . org/10 . 7554/eLife . 07025 . 00410 . 7554/eLife . 07025 . 005Figure 1—figure supplement 2 . Predicted protein alignments of the PROG family that are irradiation-sensitive . An alignment of the 17 PROG family genes used in this study is shown using the tool MUSCLE .", "Blue shading of residues reflects conservation , which is also plotted below the alignment in the ‘conservation’ plot . DOI: http://dx . doi . org/10 . 7554/eLife . 07025 . 005 Interestingly , the predicted proteins encoded by prog-1 and prog-2 do not have clear homology to genes in other animals , including the genomes of other sequenced flatworms ( Echinococcus multilocularis , Schistosoma mansoni , Macrostomum lignano—www . macgenome . org ) ( Berriman et al . , 2009; Zheng et al . , 2013 ) .", "However , we observed that they had low similarity to each other and represented a family of at least 24 distinct members across multiple transcriptomes in S . mediterranea , 15 of which were represented in the top 100 X2-enriched gene set ( Sandmann et al . , 2011; Solana et al . , 2012; Currie and Pearson , 2013; Vogg et al . , 2014 ) .", "Translations of the predicted open reading frames ( average size 179 amino acids ) for these 15 prog-related genes were aligned and analyzed by protein domain prediction software SMART ( Figure 1—figure supplement", "2 ) ( Schultz et al . , 1998; Letunic et al . , 2014 ) .", "The only motif that could be detected was a signal sequence at the N-terminal end of the predicted proteins , suggesting that these proteins are secreted .", "These PROG-1/2 homologous genes were then named in a numbered sequence based on their closest homolog ( e . g . , prog-1-1 , prog-2-1 ) .", "To begin our analysis of the top 100 X2-enriched transcripts and the top 20 WThighXlow transcripts , we performed whole-mount in situ hybridization ( WISH ) to elucidate gene expression patterns .", "We observed that 40/120 transcripts were either not detectable or not specific , and the remaining 80/120 genes could be binned into one of four categories ( Figure 2A , Figure 2—figure supplement 1 ) .", "One category ( 13/120 ) contained genes with the most intense expression in the bi-lobed brain and nervous system , with low levels of expression elsewhere in the body .", "A second group of genes ( 18/120 ) exhibited a predominantly stem cell-like expression pattern , with or without brain expression , which was confirmed by high expression in the X1 cell fraction ( Figure 2A , Figure 2—figure supplement 1A , Supplementary file 2 ) .", "A third subset of genes was expressed in a variety of distinct patterns including the gut , pharynx , peri-pharyngeal region , and neck ( 17/120 ) ( Figure 2A , Figure 2—figure supplement 1A ) .", "Finally , the fourth and largest group ( 32/120 ) consisted of genes with an expression pattern highly similar to those of the known early and late progeny markers prog-1 , prog-2 , and AGAT-1 , which are sub-epithelial across the entire animal with expression anterior to the photoreceptors ( Figure 2A , Figure 2—figure supplement", "2 ) ( Eisenhoffer et al . , 2008 ) .", "This prog-like subset exhibited varying degrees of X2-enrichment , and some were highly expressed in the X1 population ( Figure 1A , Supplementary file 2 ) .", "Transcripts that displayed a prog-like pattern where BLAST did not identify significant similarity were named as postmitotic progeny ( pmp's ) with ascending numerals ( e . g . , pmp-3 , pmp-4 ) . 10 . 7554/eLife . 07025 . 006Figure 2 . Expression analyses of candidate progeny genes .", "( A ) Whole-mount in situ hybridization ( WISH ) analysis of X2-enriched and WThighXlow candidate progeny genes in control and lethally irradiated worms .", "Examples of genes expressed in a stem cell-like pattern , strong in the bi-lobed brain , distinct and unique patterns , or in a prog-like sub-epithelial pattern are shown .", "prog-like genes were categorized as early progeny markers when they displayed similar post-irradiation down-regulation kinetics as prog-1 and prog-2 , or late progeny markers when they displayed similar loss kinetics as AGAT-1 .", "Established lineage markers prog-1 , prog-2 , and AGAT-1 are included as comparisons and highlighted in blue text .", "Candidate epithelial progenitor genes with WThighXlow expression are indicated in purple text .", "Anterior , left; scale bar , 200 μm .", "( B ) Combinatorial double fluorescent WISH ( dFISH ) between lineage markers and identified X2-enriched and WThighXlow postmitotic progeny markers was performed to assess co-localization with stem cell , early progeny , and late progeny cell populations .", "Percent co-localization is shown at the top of each panel and is averaged from 3 to 4 animals .", "Magenta , percent co-expression in [row gene]+ cells; green , percent co-expression in [column gene]+ cells .", "Images from the head , trunk , and tail regions were used for all cell counts , with a minimum of 300 cells counted .", "Dispersions of data are in Supplementary file 3 .", "Representative confocal projections spanning 4–6 μm of the head region are shown .", "Eyespots are marked by asterisks .", "Anterior , left; scale bar , 100 μm . DOI: http://dx . doi . org/10 . 7554/eLife . 07025 . 00610 . 7554/eLife . 07025 . 007Figure 2—figure supplement 1 . Gene expression analysis of candidate progeny genes .", "( A ) WISH was used to determine the gene expression patterns of X2-enriched and WThighXlow genes in intact control worms .", "Genes for which a reliable and discrete pattern could be obtained are shown .", "Genes were named based on the best BLASTx result in mouse ( when Expect value < 1 × e−5 ) or based on transcript identity if no homology is found .", "Genes were categorized as either predominant in the brain ( Brain ) , predominant in a stem cell pattern with or without expression elsewhere ( Stem cell-like ) , or possessing unique patterns ( Various ) .", "( B ) A subset of candidate progeny genes was assessed by whole-mount ISH after irradiation with 60 Gys , up to 7 days after exposure .", "Scale bars , 200 μm . DOI: http://dx . doi . org/10 . 7554/eLife . 07025 . 00710 . 7554/eLife . 07025 . 008Figure 2—figure supplement 2 . Gene expression analysis of candidate progeny genes with a prog-like expression pattern . Candidate progeny genes , which exhibited a prog-like expression pattern , were all assessed by WISH in irradiated worms ( 60 Gy ) at the indicated time points .", "Genes were categorized as either early progeny or late progeny depending on whether they exhibited similar kinetics of down-regulation to prog-1/2 or AGAT-1/2/3 , respectively .", "Genes , which are WThighXlow , are indicated in purple text .", "Scale bar , 200 μm . DOI: http://dx . doi . org/10 . 7554/eLife . 07025 . 00810 . 7554/eLife . 07025 . 009Figure 2—figure supplement 3 . Co-localization of progeny markers with dFISH . Co-expression of X2-enriched and WThighXlow early and late progeny markers with established lineage markers ( prog-1 , prog-2 , and AGAT-1 ) or with each other was determined using dFISH .", "Confocal projections spanning 6–10 μm are shown .", "Smaller panels on the left show individual channels with expression of one gene; larger panels on the right show merged channels .", "Numbers indicate the percent of magenta-labeled cells , which co-express the green-labeled transcript .", "200–2000 cells were counted for each dFISH combination .", "Location of eyespots is marked by asterisks; anterior , up . DOI: http://dx . doi . org/10 . 7554/eLife . 07025 . 00910 . 7554/eLife . 07025 . 010Figure 2—figure supplement 4 . Analysis of new progeny markers in zfp-1 ( RNAi ) animals . X2-enriched and WThighXlow early and late progeny transcripts identified as potential markers of epithelial progenitors were assessed by WISH in zfp-1 ( RNAi ) animals .", "Knockdown of zfp-1 has previously been shown to selectively ablate zeta-neoblasts and output of epithelial progenitors , and down-regulate prog-1 and prog-2 . DOI: http://dx . doi . org/10 . 7554/eLife . 07025 . 010 Using WISH , we confirmed that transcripts from each of these expression categories were irradiation-sensitive ( Figure 2A , Figure 2—figure supplements 1B , 2 ) .", "Genes with a stem cell-like component to the expression pattern exhibited down-regulation after 24 hr , as anticipated for bona fide stem cell markers .", "However , we observed that multiple genes with distinct tissue expression patterns did not change appreciably following irradiation ( Figure 2—figure supplement 1B ) and were deemed unlikely to be candidates of progenitor cell types .", "We subsequently focused on the group of genes exhibiting an epithelial progenitor-like pattern , given their prevalence , and sought to determine whether they were co-expressed in previously described prog-1+ and AGAT-1+ populations or specified towards other unknown lineages .", "The postmitotic progeny markers prog-1/2 and AGAT-1/2/3 not only have largely non-overlapping expression patterns , but the kinetics of down-regulation following irradiation substantially differ as well ( Figure 2A ) ( Eisenhoffer et al . , 2008 ) .", "prog-1/2 expression is lost within 48 hr post-irradiation , while AGAT-1 expression is lost between 48 hr and 7 days , leading to these two cell populations being termed ‘early’ and ‘late’ progeny , respectively .", "Combined with evidence from BrdU-labeling studies , the early and late progeny populations have been proposed to reflect a spatiotemporal progression along one lineage of ASC differentiation destined for the epithelium , such that prog-1/2 expression represents the transition state between neoblasts ( potentially zeta-neoblasts ) and AGAT-1+ cells ( Eisenhoffer et al . , 2008; Pearson and Sánchez Alvarado , 2010; van Wolfswinkel et al . , 2014 ) .", "To demonstrate that our newly identified prog-like markers were irradiation-sensitive and to determine their kinetics of down-regulation , animals were lethally irradiated ( 60 Gy ) and analyzed for each marker for 7 days .", "We observed that 13/32 transcripts showed similar kinetics of down-regulation as the early progeny markers prog-1 and prog-2 and were almost completely lost by 48 hr post-irradiation ( Figure 2A , Figure 2—figure supplement 2 ) .", "The remaining 19/32 transcripts showed the majority of loss beyond 48 hr post-irradiation , consistent with being late progeny markers ( Figure 2A , Figure 2—figure supplement 2 ) .", "Interestingly , all WThighXlow genes grouped as late progeny , and moreover , were the only genes with detectable expression at 7 days post-irradiation .", "These highly similar expression patterns and irradiation-sensitivity kinetics were strong indicators that these new progeny markers were also expressed in epithelial progenitors .", "Through co-localization analyses , we next investigated whether that was the case , or whether these markers represented novel irradiation-sensitive stem cell progeny .", "Prior to determining how the new progeny markers fit into this putative lineage using double fluorescent WISH ( dFISH ) , we first duplicated previous experiments with piwi-1 , prog-1 , and AGAT-1 in order to establish baseline values of overlap .", "Congruent with previous studies , we found a small amount of overlap between piwi-1 with prog-1 , and minimal with AGAT-1: 7 . 2% ± 2 . 1 of prog-1+ cells were piwi-1+ , while merely 0 . 29% ± 0 . 50 of AGAT-1+ cells were piwi-1+ ( Supplementary file 3 ) .", "In contrast to a previous study that found nearly 45% overlap between the progeny populations ( Eisenhoffer et al . , 2008 ) , we found that only 5 . 4% ± 2 . 4 of prog-1+ cells co-expressed low levels of AGAT-1 , while 5 . 2% ± 3 . 2 of AGAT-1+ cells had prog-1 expression .", "dFISH with new progeny markers also showed little overlap between early and late progeny , confirming that these progeny transcripts mark two mainly non-overlapping populations ( Figure 2B , Figure 2—figure supplement 3 ) , and that previous overlap was likely an dFISH artifact .", "It has been shown that PIWI-1 protein has a wider expression domain than piwi-1 mRNA , reflecting the perdurance of PIWI-1 into postmitotic progeny ( Guo et al . , 2006; Wenemoser and Reddien , 2010 ) .", "It is unknown how differentiated these piwi-1−PIWI-1+ cells are , but they are clearly in a transition from a stem cell gene expression state to various postmitotic fates .", "In support of these data , we found that 17 . 8% ± 12 . 2 of prog-1+ cells to be PIWI-1+ , while 1 . 6% ± 1 . 2 of AGAT-1+ cells had detectable PIWI-1 expression ( Figure 2B , Supplementary file 3 ) .", "To determine whether our newly identified early and late progeny markers represented distinct population ( s ) of descendent cells outside of the putative prog-1/prog-2/AGAT-1 lineage , pairwise dFISH was performed and quantified .", "Consistent with the kinetics of loss post-irradiation , dFISH uncovered extensive overlap between the new early progeny markers with prog-1 and prog-2 ( Figure 2B , Figure 2—figure supplement 3 , Supplementary file 3 ) .", "Some genes , such as prog-1-1 and prog-2-3 , exhibited nearly 100% co-localization with prog-1 and prog-2 , respectively , whereas the lowest percentage overlap was observed with egr-1 and sox9-1 , with approximately 50–70% of these cells co-expressing prog-1 .", "sox9-1 and egr-1 were previously identified to be expressed in zeta-class piwi-1+ neoblasts ( referred to as soxP-3 and egr-1 [Wagner et al . , 2012] ) , and we confirmed that a sizeable percentage of sox9-1+ and egr-1+ cells co-expressed piwi-1 ( 49 . 1% ± 5 . 2 and 29 . 4% ± 11 . 9 , respectively , Supplementary file 3 ) .", "Almost all newly identified late progeny transcripts examined were found to exhibit substantial co-expression with AGAT-1 , with overlap ranging from 100% with prog-1-7 to 55 . 5% with prog-1-4 ( Figure 2—figure supplement 3 , Supplementary file 3 ) .", "The exception was the late progeny marker prog-1-2 , where only 20 . 4% of prog-1-2+ cells co-expressed AGAT-1 ( Figure 2—figure supplement 3 ) .", "Given that prog-1-2 is expressed in both subepithelial and epithelial cells , we propose that prog-1-2+ cells represent a more differentiated state along the epithelial lineage and are possibly the subsequent transition for AGAT-1+ cells .", "We also performed dFISH between newly identified early and late progeny markers , which similarly showed that genes within each category exhibit highly overlapping expression ( Figure 2—figure supplement 3 , Supplementary file 3 ) .", "Finally , to validate that expression of these genes marked epithelial progenitors , we examined their expression by WISH in zfp-1 ( RNAi ) worms .", "Knockdown of zfp-1 has been demonstrated to specifically ablate epithelial progenitors and epithelial differentiation , but not the differentiation of other tissue types ( van Wolfswinkel et al . , 2014 ) .", "We observed that every new epithelial progenitor marker assessed was down-regulated after zfp-1 RNAi , confirming that these genes were indeed expressed in epithelial progenitors ( Figure 2—figure supplement 4 ) .", "Together , these findings demonstrated that our newly identified transcripts represent markers of the early and late progeny produced by zeta neoblasts and comprise the primary two progenitor populations en route to the epithelium ( van Wolfswinkel et al . , 2014 ) .", "The self-renewal of ASCs and the appropriate differentiation of postmitotic progeny are the driving force behind homeostatic cell turnover and regeneration of all tissues in planarians ( Newmark and Sánchez Alvarado , 2000; Rossi et al . , 2008; Baguna , 2012; van Wolfswinkel et al . , 2014 ) .", "RNAi against genes required for differentiation , such as p53 , CHD4 , zfp-1 , and vasa-1 , results in the decline of postmitotic progeny without depletion of ASCs , and subsequent defects in tissue homeostasis and regeneration ( Pearson and SánchezAlvarado , 2010; Scimone et al . , 2010; Wagner et al . , 2012 ) .", "To determine whether progeny-enriched genes were regulators of postmitotic fates , we used RNAi knockdown against our set of 100 X2-enriched and 20 WThighXlow genes and screened for the above phenotypes .", "In agreement with previous data , RNAi against prog-1 and prog-2 separately or together did not yield any detectable phenotype ( Eisenhoffer et al . , 2008 ) .", "Unexpectedly , none of the new epithelial progenitor markers yielded phenotypes upon knockdown either , suggestive of considerable functional redundancy among these genes .", "In contrast , knockdown of a gene encoding a homolog to the RNA-binding protein MEX3 , Smed-mex3-1 ( mex3-1 ) produced phenotypes highly suggestive of defective stem cell lineage progression .", "mex3-1 ( RNAi ) was completely penetrant and lethal , resulting in ventral curling , head regression , and dorsal lesioning during homeostasis , as well as loss of regenerative ability after amputation ( Figure 3A–C ) , indicative of epithelial homeostasis defects as well as overall stem cell impairment ( Bardeen and Baetjer , 1904; Reddien et al . , 2005a ) .", "Using reciprocal BLAST , we identified two other MEX3 homologs in S . mediterranea ( mex3-2 and mex3-3 , transcripts SmedASXL_000637 and SmedASXL_01505 , respectively ) , which both contained two KH RNA-binding domains and had top reciprocal BLAST hits in mouse , fly , and C . elegans .", "These additional MEX3 homologs were neither irradiation-sensitive nor produced observable phenotypes after knockdown and thus not investigated further ( Figure 3—figure supplement 1 ) . 10 . 7554/eLife . 07025 . 011Figure 3 . mex3-1 is required for tissue homeostasis and regeneration .", "( A ) Survival curves of mex3-1 ( RNAi ) animals to determine the optimal RNAi dosage .", "One RNAi feed was sufficient to produce completely penetrant lethality by 27 days .", "( B ) RNAi worms were observed for homeostatic abnormalities after 1–3 feeds .", "Time point at which animals were imaged is indicated as x days after z feeds ( zfdx ) .", "( C ) Regenerative ability after injury was tested according to the experimental timeline shown .", "Trunk fragments after pre- and post-pharyngeal amputations are shown .", "( D ) Expression levels of mex3-1 in different FACS populations by RNAseq .", "Error bars show standard deviation .", "( E ) WISH analysis of mex3-1 in intact worms after 60 Gray ( Gy ) irradiation .", "Scale bar , 200 μm .", "( F ) dFISH was performed to examine mex3-1 expression in stem cells and postmitotic progeny .", "Numbers indicate the percentage of stem cells , early progeny , or late progeny co-expressing mex3-1 ( n > 400 cells per dFISH , ± standard error ) .", "Scale bar , 10 μm . DOI: http://dx . doi . org/10 . 7554/eLife . 07025 . 01110 . 7554/eLife . 07025 . 012Figure 3—figure supplement 1 . Identification and analysis of MEX3 homologs in S . mediterranea .", "( A ) Phylogenetic analysis of the mex3 gene family in planarians shows that they are likely the results of planarian-specific duplications .", "A Bayesian phylogeny was run as outlined in the ‘Materials and methods’ .", "Only posterior probabilities 50% and above are shown .", "S . mediterranea sequences are in red .", "Multiple mex3 homologs could not be found in individual species of other flatworms .", "( B ) Expression levels of mex3-2 and mex3-3 in RNAseq of FACS-isolated populations and control intact worms .", "Expression of mex3 homologs by WISH after lethal irradiation ( 60 Gy ) is shown below .", "( C ) Phenotypes of mex3-2 and mex3-3 RNAi animals during homeostasis ( upper panel ) and during regeneration after amputation ( lower panel ) .", "Species sequences used in the phylogeny: Smed = Schmidtea mediterranea; S . mansoni = Schistosoma mansoni; Echinococcus = Echinococcus multilocularis; Aplysia = Aplysia californica; Ci = Ciona intestinalis; Lg = Lottia gigantea; Dm = Drosophila melanogaster; Tc = Tribolium castaneum; Nvit = Nasonia vitripennis; Mm = Mus musculus; Xt = Xenopus tropicalis; Nvect = Nematostella vectensis; Sp = Strongylocentrotus purpuratus . DOI: http://dx . doi . org/10 . 7554/eLife . 07025 . 012 To begin elucidating the mechanism by which mex3-1 potentially regulates stem cell lineage progression , we examined where the gene was expressed in the epithelial lineage .", "By RNAseq , mex3-1 showed >fivefold enrichment in the X2 fraction over irradiated worms but also exhibited significant expression in the X1 stem cell fraction ( Figure 3D ) .", "WISH of wild-type worms revealed a stem cell-like expression pattern with substantial expression peripheral to the stem cell compartment ( Figure 3E ) .", "Following irradiation , progressively more of the pattern was lost over 7 days ( Figure 3E ) , consistent with expression in both stem cell and immediate postmitotic progeny populations , and also consistent with previous irradiation data for mex3-1 ( Solana et al . , 2012 ) .", "Analysis of mex3-1 using dFISH with lineage markers confirmed that mex3-1 was widely expressed in stem cells , early progeny , and late progeny ( Figure 3F ) .", "Therefore , the homeostasis and regeneration defects described above could result from defects in any one or all of these cell populations , which was subsequently tested .", "To ascertain which step ( s ) in stem cell lineage progression were aberrant after mex3-1 knockdown , we performed WISH analysis to follow stem cell and postmitotic prog-1/2+ and AGAT-1+ progeny population dynamics after RNAi was initiated .", "Knockdown of mex3-1 leads to a rapid decline of the two progeny populations but not of stem cells ( Figure 4A , Figure 4—figure supplement 1A ) , with the majority of progeny gene expression lost by 6 days after RNAi ( Figure 4—figure supplement 1B ) .", "We assessed the expression of additional newly identified epithelial progenitor markers as well and observed that all were similarly abolished , supporting the loss of these two progeny types ( Figure 4B ) . 10 . 7554/eLife . 07025 . 013Figure 4 . RNAi against mex3-1 selectively affects progeny markers and causes hyper-proliferation .", "( A ) Lineage markers labeling stem cells ( piwi-1 ) , early ( prog-1 , prog-2 ) , and late ( AGAT-1 ) progeny were assessed by WISH after RNAi .", "The day 12 time point reflects the maximal perturbation to progeny markers prior to obvious health decline in the animal .", "( B ) Representative newly identified early and late progeny transcripts were assessed by WISH after RNAi .", "( C ) Whole-animal quantification of TUNEL was performed to measure cell death in RNAi animals .", "Representative stains and time points are shown to the right .", "( D ) Whole-animal quantification of H3P immunolabeling was performed to assess cell proliferation after RNAi .", "Representative stains and time points are shown to the right .", "Unless otherwise noted , all experimental time points are indicated as after a single RNAi feeding .", "Scale bars , 200 μm .", "Error bars are standard deviations .", "**p < 0 . 01 , ***p < 0 . 001 ( Student's t-test ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 07025 . 01310 . 7554/eLife . 07025 . 014Figure 4—figure supplement 1 . mex3-1 RNAi depletes progeny without impairing stem cell proliferation .", "( A ) Lineage markers labeling stem cells ( piwi-1 ) , early ( prog-1 , prog-2 ) , and late ( AGAT-1 ) progeny were assessed by WISH after RNAi .", "Shown are a late time point after one RNAi feeding and a late time point with multiple RNAi feeds , when health decline is evident .", "( B ) Detailed time course analysis of early ( prog-2 ) and late ( AGAT-1 , pmp-11 ) progeny marker down-regulation after mex3-1 RNAi .", "Scale bars , 200 μm .", "( C ) FACS analysis with Hoechst staining of mex3-1 ( RNAi ) animals 9 days after RNAi .", "FACS plots from one run are shown on the left; right table indicates proportion of X1 and X2 populations of all runs . DOI: http://dx . doi . org/10 . 7554/eLife . 07025 . 014 The down-regulation of progeny gene expression without corresponding decreases in the stem cell population suggested a selective defect in specifying committed progeny .", "However , these results could similarly arise from defective maintenance and survival of progeny , thus , we examined levels of apoptosis with TUNEL ( terminal deoxynucleotidyl transferase dUTP nick end labeling ) during phenotypic progression .", "Quantification of TUNEL showed that mex3-1 ( RNAi ) animals had comparable levels of cell death to control worms up until 9 days after RNAi , but significantly higher levels from 12 days and onward ( Figure 4C ) .", "Given that progeny gene expression was mostly ablated by 6 days after RNAi , we concluded that the loss of progeny populations was not attributed to progeny cell death .", "The cause of the late rise in cell death remains unclear but is coincident with when morphological homeostatic phenotypes begin to manifest and may be a secondary effect of declining animal health .", "We next investigated whether impaired stem cell proliferation was the underlying cause of reduced progeny production and performed time-course analysis of phosphorylated histone H3 immunolabeling ( H3P ) .", "We observed significantly increased levels of proliferation in mex3-1 ( RNAi ) worms at every time point examined ( Figure 4D ) , which suggested that stem cell division was not impeded by mex3-1 knockdown .", "To exclude the possibility that the heightened H3P+ levels in mex3-1 ( RNAi ) worms reflected an accumulation of cells arrested in G2/M phase and an inability to complete the cell cycle , we performed labeling with the thymidine analog F-ara-EdU to measure S-phase progression .", "Worms pulsed at either 6 or 9 days after RNAi and fixed 24 hr later showed significantly increased numbers of total EdU-labeled cells in mex3-1 ( RNAi ) animals compared to controls ( Figure 5A ) , demonstrating that during phenotypic progression , a greater number of cells were entering the cell cycle .", "FACS profiles of mex3-1 ( RNAi ) animals at day 9 revealed relatively normal proportions of cells in both the X1 and X2 gates ( Figure 4—figure supplement 1C ) , further evidence that stem cells were progressing through the cell cycle and not arrested at 4C DNA in the X1 gate .", "Worms exposed to sublethal doses of irradiation initially lose the vast majority of their stem cells and immediate postmitotic descendants; over time , the stem cell and progeny populations gradually recover , offering an easily quantifiable approach to assess both self-renewal and differentiation ( Wagner et al . , 2012 ) .", "Sublethally irradiated mex3-1 ( RNAi ) worms expanded piwi-1+ stem cell numbers over time but produced disproportionately fewer prog-1+ progeny than control worms ( Figure 5B ) .", "These data demonstrated that the loss of early and late progeny marker expression after mex3-1 knockdown could not be attributed to cell cycle arrest or increased cell death , but rather result from a failure in cell fate specification . 10 . 7554/eLife . 07025 . 015Figure 5 . mex3-1 ( RNAi ) animals exhibit expansion of the stem cell compartment .", "( A ) RNAi worms were administered EdU at 6 or 9 days after RNAi and quantified after a 24-hr period .", "Counts were performed on whole-animal single confocal planes .", "( B ) mex3-1 ( RNAi ) animals were irradiated with a sublethal dose ( 16 . 5 Gy ) , and stem cells ( piwi-1 ) and progeny ( prog-1 ) were quantified by dFISH at 7 and 9 days after irradiation .", "The proportion of progeny to stem cells between mex3-1 ( RNAi ) and control worms differed significantly ( p < 0 . 001 , analysis of covariance ) .", "Whole-animal confocal projections are shown .", "Each point on the graph represents one animal .", "( C ) Stem cells were quantified in pre-pharyngeal cross sections of intact worms after RNAi by piwi-1 fluorescent WISH ( FISH ) during phenotypic progression .", "Single confocal planes at day 9 after RNAi are shown; dorsal , top .", "( D ) Quantification of stem cell subclasses in intact worms 12 days after RNAi .", "Stem cell subclass was determined by piwi-1 labeling and expression of soxP-1 pooled with soxP-2 ( sigma subclass ) , hnf4 ( gamma ) , or zfp-1 ( zeta ) .", "Diagrams indicate areas of worms quantified , and arrows indicate example double-positive cells .", "Scale bars , 200 μm in ( A–C ) and 50 μm in ( D ) .", "Error bars represent standard deviation .", "*p < 0 . 05 , **p < 0 . 01 , ***p < 0 . 001 ( Student's t-test ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 07025 . 01510 . 7554/eLife . 07025 . 016Figure 5—figure supplement 1 . Quantification of stem cells in mex3-1 ( RNAi ) animals . Stem cells were quantified in mid-pharyngeal and tail cross sections of intact worms at 6 and 9 days after RNAi by piwi-1 FISH .", "Single confocal planes are shown; dorsal , top .", "Scale bar , 200 μm .", "*p < 0 . 05; **p < 0 . 01 ( Student's t-test ) .", "10 animals were counted per RNAi treatment . DOI: http://dx . doi . org/10 . 7554/eLife . 07025 . 016 Given that early and late progeny cell fates failed to be specified and adopted in mex3-1 ( RNAi ) animals despite ongoing stem cell divisions , we hypothesized that there was a loss in stem cell lineage asymmetry in favor of stem cell self-renewal .", "We sought to determine whether this was the case by quantifying piwi-1+ stem cells in transverse cross sections after RNAi .", "By 6 days after RNAi , we observed highly significant increases in the number of piwi-1+ stem cells in mex3-1 ( RNAi ) animals compared to controls , which rose to a 50% increase by day 12 ( Figure 5C , Figure 5—figure supplement 1 ) .", "The expansion of piwi-1+ stem cells after mex3-1 knockdown could either reflect a global increase in all stem cell subclasses or reflect an increase in a specific subclass ( zeta- , sigma- , and gamma-neoblasts ) ( van Wolfswinkel et al . , 2014 ) .", "To ascertain whether subclasses were selectively affected , we quantified the number of piwi-1+ stem cells belonging to each subclass using the following probes: zfp-1 for the zeta subclass , hnf4 for the gamma subclass , and a pooled mix of soxP-1 and soxP-2 for the sigma subclass .", "Assessment at 12 days after RNAi revealed that all three subclasses were significantly increased by approximately 50% in mex3-1 ( RNAi ) animals compared to controls ( Figure 5D ) .", "Together , these results demonstrated that the failure to specify epithelial progenitors in mex3-1 ( RNAi ) animals was not due to loss of zeta-neoblasts , and suggested that the expansion of the stem cell pools was due to an imbalance in cell fates favoring stem cell self-renewal over differentiation .", "The ventral curling defect during homeostasis and ablation of all early and late progeny markers in mex3-1 ( RNAi ) worms suggested that epithelial turnover may be severely compromised .", "We performed EdU labeling in intact mex3-1 ( RNAi ) animals to measure the entry of new cells into the epithelium under normal homeostatic conditions .", "7 days following EdU administration , numerous EdU+ cells were present in the epithelium of control ( RNAi ) worms , but virtually none had been incorporated into the epithelium in mex3-1 ( RNAi ) animals ( Figure 6A ) .", "Additionally , following amputation , mex3-1 ( RNAi ) worms failed to re-establish the expression of epithelial genes at wounding sites , demonstrating that during both homeostasis and regeneration , mex3-1 is required for specification and differentiation of epithelial cell types ( van Wolfswinkel et al . , 2014 ) ( Figure 6B ) . 10 . 7554/eLife . 07025 . 017Figure 6 . mex3-1 is required for epithelial turnover and regeneration as well as differentiation toward multiple tissues .", "( A ) RNAi animals were administered EdU 6 days after RNAi , and EdU+ labeling in the epithelium was quantified after a 7-day period .", "Single confocal planes are shown , with EdU+ cells in the epithelium indicated by arrows .", "Top panel scale bar , 100 μm; bottom panel scale bar , 50 μm .", "( B ) RNAi animals amputated 7 days after RNAi were analyzed after 7 days regeneration by dFISH for stem cells and epithelial-associated genes .", "Single confocal planes or projections of head blastemas are shown , with anterior to the left .", "Dotted lines indicate animal boundary .", "Scale bar , 100 μm .", "( C ) Diagram indicating regions of animal imaged and examined for quantification of lineage-restricted neoblast progeny .", "( D ) Lineage-restricted progeny populations were quantified in intact worms 12 days after RNAi , using PIWI-1 immunolabeling and FISH for ovo ( eyes ) , chat or coe ( brain ) , FoxA ( pharynx ) , and six1/2-2 ( protonephridia ) .", "Single confocal planes are shown .", "Arrows indicate example double-positive cells .", "Magnified areas are indicated by dashed boxes and inset to the right of each image .", "Scale bar , 50 μm .", "( E ) RNAi animals were administered BrdU 6 days after RNAi , and BrdU+ labeling in differentiated tissues ( chat , brain; mat , gut; laminin , pharynx ) was examined after a 5-day period .", "Single confocal planes are shown .", "Arrowheads indicate example double-positive cells .", "Scale bar , 50 μm .", "Error bars represent standard deviation .", "*p < 0 . 05 , **p < 0 . 01 , ***p < 0 . 001 ( Student's t-test ) .", "( F ) Quantification of stem cells ( piwi-1+PIWI-1+ ) and immediate postmitotic stem cell descendants ( piwi-1−PIWI-1+ ) were performed 6 days after RNAi , in head , pre-pharyngeal , and tail regions .", "Shown are single confocal planes from the tail region .", "Magnified areas are indicated by dashed boxes .", "The proportion of postmitotic descendants to stem cells between mex3-1 ( RNAi ) and control worms differed significantly ( p < 0 . 001 , non-linear regression analysis ) .", "Scale bar , 50 μm on left panels , 10 μm on right panels .", "All counts were performed in 5–10 animals . DOI: http://dx . doi . org/10 . 7554/eLife . 07025 . 01710 . 7554/eLife . 07025 . 018Figure 6—figure supplement 1 . mex3-1 RNAi impairs brain differentiation .", "( A ) Expression of mex3-1 in neural-restricted neoblast progeny by mex3-1 and chat dFISH with PIWI-1 immunolabeling .", "Percentage of chat+PIWI-1+ cells which express mex3-1 is indicated at top-right of panel .", "Arrows indicate example triple-labeled cells .", "Dashed box indicates area of magnified panels .", "Left scale bar , 50 μm; right scale bar , 10 μm .", "( B ) Animals were administered BrdU at 6 days after RNAi and assessed at 5 days post-BrdU .", "Scale bar , 100 μm .", "All images shown are single confocal planes . DOI: http://dx . doi . org/10 . 7554/eLife . 07025 . 01810 . 7554/eLife . 07025 . 019Figure 6—figure supplement 2 . Differentiation in p53 ( RNAi ) animals .", "( A ) Stem cell ( piwi-1 ) and epithelial progenitor ( prog-1 ) populations were examined by WISH in RNAi animals .", "Scale bar , 200 μm .", "( B ) Eye and neural lineage-restricted neoblast progeny were quantified in RNAi animals by FISH ( ovo , eye; chat , brain ) with PIWI-1 immunolabeling .", "Arrows indicate example double-positive cells .", "Scale bars , 50 μm . DOI: http://dx . doi . org/10 . 7554/eLife . 07025 . 019 Previously , it was shown that even by selectively abolishing zeta-neoblasts and epithelial turnover , a regenerative blastema can still form with differentiated tissues such as brain , protonephridia , intestine , and muscle ( van Wolfswinkel et al . , 2014 ) .", "Given that mex3-1 ( RNAi ) animals were unable to produce any regenerative blastema , we hypothesized that mex3-1 may have a crucial role in the broad specification of multiple lineages .", "To examine this possibility , we assessed mex3-1 ( RNAi ) worms for changes in the number of lineage-specified neoblast progeny of other tissue types in various body regions ( Figure 6C ) .", "As PIWI-1 protein persists in immediate postmitotic stem cell descendants for up to 72 hr ( Guo et al . , 2006 ) , co-localization of PIWI-1 with tissue-specific markers was used to identify lineage-restricted neoblast descendants , encompassing undifferentiated progenitors and newly differentiating cells .", "The neural genes , chat and coe , eye-specific transcription factor ovo , pharyngeal marker FoxA , and protonephridial marker six1/2-2 were used as markers to indicate differentiation toward their respective tissues ( Scimone et al . , 2011; Wagner et al . , 2011; Lapan and Reddien , 2012; Cowles et al . , 2013; Adler et al . , 2014 ) .", "Quantification of lineage-restricted neoblast progeny in intact mex3-1 ( RNAi ) worms 12 days after RNAi showed significant reductions in all examined cell types ( Figure 6D ) .", "We observed that virtually all chat+PIWI-1+ cells expressed mex3-1 ( Figure 6—figure supplement 1A ) , suggesting a direct role for mex3-1 in regulating differentiation outside the epithelial lineage .", "Concordant with decreased production of lineage-restricted neoblast progeny , we also observed that 5 days following labeling with BrdU , the entry of new cells into brain , intestine , and pharynx was significantly decreased in mex3-1 ( RNAi ) animals ( Figure 6E , Figure 6—figure supplement 1B ) .", "These data demonstrate that the diminished capacity of mex3-1 ( RNAi ) animals to produce differentiated progeny is not restricted to the epidermal lineage but is characteristic of multiple lineages in planarians .", "We examined whether impaired differentiation toward multiple tissues could be observed in p53 ( RNAi ) animals as well , as p53 knockdown has previously been shown to deplete prog-1+ progeny and increase stem cell proliferation ( Pearson and Sánchez Alvarado , 2010 ) .", "We found that knockdown of p53 did not alter the numbers of PIWI-1+ovo+ eye- or PIWI-1+chat+ brain-specified neoblast progeny ( Figure 6—figure supplement 2 ) , demonstrating a broader role for mex3-1 in differentiation .", "To determine whether mex3-1 knockdown resulted in a global impediment in generating postmitotic cells , we quantified the proportion of piwi-1−PIWI-1+ cells , which are thought to represent immediate stem cell progeny that have permanently exited the cell cycle .", "We found that mex3-1 RNAi resulted in a significant increase in the number of piwi-1+PIWI-1+ cells and simultaneous decrease in piwi-1−PIWI-1+ cells compared to controls ( Figure 6F ) , supporting a general reduction in the ability of stem cells to progress to a postmitotic state .", "From these data demonstrating abrogated production of lineage-restricted stem cell progeny , impaired contribution to the turnover of multiple tissue types , and concomitant increases in all known stem cell subclasses , we propose that mex3-1 is a critical regulator for all differentiating progeny , mediating the adoption of a non-stem cell fate .", "RNAseq of mex3-1 ( RNAi ) whole animals 12 days after RNAi was performed to provide a broad and comprehensive overview of gene expression changes .", "Given that mex3-1 RNAi specifically eliminates postmitotic progeny fates , we reasoned that this approach may offer a more selective method than X2-FACS enrichment in order to identify additional progenitor-specific transcripts both within and outside the epithelial lineage .", "In agreement with our data demonstrating hyper-proliferation and expansion of the entire stem cell compartment after mex3-1 knockdown , 13/59 known stem cell-specific transcripts were significantly upregulated upon knockdown of mex3-1 ( p < 0 . 01 , Figure 7—figure supplement 1A , Supplementary file 4 ) .", "These included cell cycle genes , two of which were further confirmed by WISH ( PCNA and H2B; Figure 7—figure supplement 1B ) .", "Importantly , we also observed an approximately 1 . 5-fold increase in soxP-1 , zfp-1 , and piwi-1 transcripts in mex3-1 ( RNAi ) animals ( Figure 7—figure supplement 1A; Supplementary file 4 ) , concordant with the increase in stem cell subclasses quantified by cell counting ( Figure 5C , D ) .", "As anticipated from the effects of mex3-1 ( RNAi ) on the loss of all epithelial progenitor markers by WISH analyses , all but two of our new early and late progeny markers were severely down-regulated in the RNAseq data set for mex3-1 ( RNAi ) animals compared to controls ( Figure 7A; Supplementary file 4 ) .", "The two transcripts that did not appreciably change ( sox9-1 and RPC-2 ) have very high X1 expression in addition to labeling epithelial progenitors , which was not expected to change in mex3-1 RNAi .", "Together , the RNAseq data corroborate the in vivo cell type analyses , where mex3-1 was required to restrict the stem cell compartment and promote differentiation of progenitor fates .", "We next tested whether transcripts down-regulated following mex3-1 RNAi mark novel neoblast progeny . 10 . 7554/eLife . 07025 . 020Figure 7 . Transcriptional analysis after mex3-1 RNAi identifies novel progenitor transcripts .", "( A ) RNAseq was performed on mex3-1 ( RNAi ) animals 12 days after RNAi .", "Each gray dot represents one transcript .", "Established and newly identified progeny markers are indicated .", "Top mex3-1 down-regulated transcripts cloned out for validation by WISH are indicated as well .", "( B ) The blue-circled transcripts from ( A ) all require mex3-1 for expression .", "Scale bar , 200 μm .", "( C ) Assessment of the mex3-1 ( RNAi ) -down-regulated transcript Smed_ASXL059179 as a marker for a novel pharynx progenitor cell type using FISH and PIWI-1 immunolabeling .", "Confocal projections in control and mex3-1 RNAi animals are shown .", "Error bars represent standard deviation .", "Scale bar , 50 μm .", "**p < 0 . 01 ( Student's t-test ) .", "( D ) Model of lineage specification in planarian stem cells .", "mex3-1 is a key determinant in balancing stem cell self-renewal and differentiation , acting as a promoter of postmitotic cell fates and commitment toward multiple lineages . DOI: http://dx . doi . org/10 . 7554/eLife . 07025 . 02010 . 7554/eLife . 07025 . 021Figure 7—figure supplement 1 . RNAseq analysis of mex3-1 ( RNAi ) animals .", "( A ) RNAseq was performed on mex3-1 ( RNAi ) animals 12 days after RNAi , and X1 enrichment is compared to fold change after mex3-1 knockdown .", "Each gray dot represents one transcript .", "Established stem cell genes ( from Figure 1B ) , which were found to be significantly upregulated in mex3-1 ( RNAi ) , are highlighted in blue ( p < 0 . 01 ) .", "The fold changes of a subset of known stem cell genes ( involved in proliferation , pan-stem cell gene , or subclass identity ) are shown on the right .", "( B ) Upregulation of the cell cycle genes PCNA and H2B in RNAseq analysis was confirmed by WISH of mex3-1 ( RNAi ) worms 6 days after RNAi .", "( C ) From the top down-regulated genes after mex3-1 knockdown , 21 uncharacterized genes were chosen for cloning and expression analysis by WISH ( 11/21 genes are shown ) .", "mex3-1 ( RNAi ) animals were stained 12 days after RNAi to confirm that target genes were down-regulated .", "Genes were named based on the best BLASTx result to mouse when the Expect value <1 × e−5 , or based on transcript number if no homology was found .", "Scale bars , 200 μm . DOI: http://dx . doi . org/10 . 7554/eLife . 07025 . 02110 . 7554/eLife . 07025 . 022Figure 7—figure supplement 2 . Characterization of down-regulated genes in mex3-1 ( RNAi ) animals .", "( A ) Expression of three genes down-regulated after mex3-1 RNAi was assessed in lethally irradiated worms by WISH .", "Scale bar , 200 μm .", "( B ) Expression of two mex3-1 ( RNAi ) -down-regulated genes in late progeny was examined by dFISH with AGAT-1 .", "Percentages of cells that express AGAT-1 are indicated in the top-right of each panel .", "Images shown are confocal projections spanning 4 μm in depth .", "Scale bar , 100 μm .", "( C ) Functional analysis by RNAi knockdown of the new pharyngeal progenitor marker SmedASXL_059179 .", "Expression of SmedASXL_059179 after knockdown was examined by WISH .", "Scale bar , 200 μm .", "( D ) Diagram outlining schedule of RNAi feeds and amputation for assessment of pharynx regeneration .", "Shown is WISH of the pharynx marker laminin in regenerating tail fragments .", "Scale bar , 200 μm . DOI: http://dx . doi . org/10 . 7554/eLife . 07025 . 022 We chose 21 down-regulated and uncharacterized transcripts for validation by WISH .", "By RNAseq , all were irradiation-sensitive but not X2-enriched ( Figure 7A , Supplementary file 4 ) and thus could be classified as WThighXlow .", "We found that 20/21 transcripts produced a prog-like pattern in control worms and exhibited severely reduced expression in mex3-1 ( RNAi ) worms , as anticipated from RNAseq ( Figure 7B , Figure 7—figure supplement 1C ) .", "The remaining transcript , SmedASXL_059179 , was highly expressed near the proximal end of the pharynx , throughout the pharynx proper , and was similarly dependent on mex3-1 for its expression ( Figure 7B ) .", "Three transcripts were confirmed to be irradiation-sensitive by WISH ( Figure 7—figure supplement 2A ) , and two of these were verified to be additional late progeny markers based on their high degree of co-localization with AGAT-1 expression ( Figure 7—figure supplement 2B ) .", "We predicted that SmedASXL_059179+ cells may represent a pharyngeal progenitor cell type , and indeed , we observed PIWI-1+SmedASXL_059179+ cells near the proximal base of the pharynx ( Figure 7C ) .", "However , no regulatory roles were uncovered for this gene , as RNAi against SmedASXL_059179 did not result in apparent perturbations to pharynx function during homeostasis or regeneration ( Figure 7—figure supplement 2C , D ) .", "Overall , these results further support a role for mex3-1 as a critical regulator of differentiation toward multiple lineages and also demonstrate the utility of transcriptional analysis of mex3-1 ( RNAi ) in identifying additional markers of tissue-specific progenitor populations , as an alternative to the criteria of irradiation-sensitivity and FACS localization ." ], [ "At the outset of this study , most efforts had been put forth to understand stem cell ( X1 ) transcriptomes , heterogeneity , and genetic regulators .", "Relatively little was known about the X2 FACS cell fraction , with the exception that it expresses a disproportionate number of transcription factors and shares most of its gene signature with the X1 fraction , and the few known stem cell progeny markers are most highly expressed in this fraction ( Labbe et al . , 2012 ) .", "We reasoned here that investigating more genes specific to the X2 fraction could yield many markers and regulators for other tissue-specific progeny types .", "We found that transcripts enriched in the progeny-associated X2 FACS gate were expressed in a variety of cell types and not necessarily in stem cell progeny .", "Although transcripts with unique tissue-specific and non-stem cell expression patterns were uncovered and appeared to be potential candidates for novel progenitor types , it is now clear that the epithelial lineage has the strongest bias in terms of sequencing numbers .", "In total , we describe 32 new markers for early and late epithelial progenitors .", "Combined with the revelation that zeta-neoblasts are the largest known lineage-restricted stem cell subclass in planarians , we conclude that epithelial progenitors are the predominant output of stem cells , most likely reflecting highest turnover needs as well as molecular complexity of this organ .", "Through RNAi screening of the 120 candidate progeny transcripts , we identified mex3-1 as a critical factor in cell fate decision-making .", "mex3-1 was required not only for all epithelial progenitor fates but also all other tested lineage-restricted stem cell progeny , and to restrict the expansion of the stem cell compartment .", "MEX3 is an RNA-binding translational repressor discovered in C . elegans with embryonic and post-embryonic developmental roles ( Draper et al . , 1996 ) .", "MEX3 is a fundamental regulator of asymmetry , mediating one of the earliest steps of cell fate determination in C . elegans by restricting expression of the cell fate determinant pal-1 transcription factor to the posterior blastomere of the early embryo ( Draper et al . , 1996; Huang et al . , 2002 ) .", "In adult C . elegans , MEX3 participates in the maintenance of germline stem cells ( GSCs ) by promoting proliferation , and interestingly , is not required for GSCs to differentiate and undergo meiosis ( Ariz et al . , 2009 ) .", "Four mex3 homologs ( MexA-D ) are present in vertebrates , which remain poorly characterized ( Buchet-Poyau et al . , 2007 ) .", "Recent work on MEX3A in murine intestinal cell culture showed that the master intestinal differentiation factor Cdx2 , one of the vertebrate homologs of pal-1 , is a target of MEX3A translational inhibition , and furthermore , MEX3A overexpression increased levels of intestinal stem cell markers such as Bmi1 and Lgr5 ( Pereira et al . , 2013 ) .", "Though the role of MEX3A in intestinal stem cell fate in vivo remains to be fully explored , these findings combined with our results in planarians suggest a conserved ancestral function for mex3 genes in cell fate choice .", "The precise mechanisms by which mex3-1 exerts its effects in planarians are unresolved .", "Although both zfp-1 and mex3-1 are required for specification of epithelial-fated progeny , unlike zfp-1 ( RNAi ) worms , mex3-1 ( RNAi ) worms completely lose the ability to form any regenerative blastema .", "mex3-1 was further needed to prevent the expansion of the stem cell compartment , including sigma- , gamma- , and zeta-class neoblasts , which suggests that mex3-1 acts to mediate stem cell lineage asymmetry in the general stem cell pool and regulate all postmitotic lineages .", "As both C . elegans and vertebrate MEX3 proteins have been shown to be translational repressors , it is likely that this molecular function has been conserved in planarians .", "Thus , we propose a model of stem cell lineage progression with mex3-1 acting as a repressor of stem cell identity and self-renewal genes in postmitotic progenitors to promote differentiation ( Figure 7D ) .", "During early C . elegans embryogenesis , the asymmetric distribution of both mex3 mRNA and protein underlies the asymmetric expression of pal-1 ( Draper et al . , 1996 ) .", "While Smed-mex3-1 mRNA shows no such asymmetry at the current resolution of our tools in planarians , it is possible that asymmetric distribution or activation of MEX3-1 leads to the execution of its function in progeny only .", "An example of such a mechanism is Prospero in Drosophila neuroblasts , where it is transcribed and translated in stem cells and daughter cells , but is segregated to and functions in progeny in a classic example of intrinsic asymmetric cell division ( Doe et al . , 1991 ) .", "Alternatively , MEX3-1 may have multiple cell type-specific roles and cell type-specific mRNA targets .", "No planarian homolog to the conserved MEX3 target Cdx2/pal-1 could be found in existing transcriptomes or genomic sequence ( Sánchez Alvarado et al . , 2003; Robb et al . , 2008; Abril et al . , 2010; Sandmann et al . , 2011; Labbe et al . , 2012; Onal et al . , 2012; Resch et al . , 2012; Solana et al . , 2012; Fernandes et al . , 2014 ) .", "Therefore , elucidating the RNA targets of MEX3-1 will provide an opportunity to uncover novel RNA targets in other organisms and ASC systems .", "Now that we have identified additional epithelial progenitor markers and progeny regulators , the question then becomes , what it is the best method to discover non-epithelial progenitors of other tissue types in planarians ?", "With the evidence described in this study implicating mex3-1 as a regulator of multiple neoblast progeny for other tissues , transcriptional analysis of mex3-1 ( RNAi ) animals may be a promising avenue to discover rare and novel progeny markers .", "For instance , comparing the wild-type X2 fraction with X2 cells isolated from mex3-1 ( RNAi ) worms could provide insight into other cell types lost in this FACS gate due to specific cessation of differentiation , without confounding effects of irradiation or general ablation of the stem cell pool .", "In addition to the assumption that differentiating progeny cells exist in the X2 fraction , it is clear that there are irradiation-sensitive transcripts and cells outside of the X1 and X2 FACS gates .", "At this juncture , isolating these populations from irradiation-insensitive cells is not achievable as there are no clear boundaries on where these cells would lie in FACS plots .", "Numerous transcripts , down-regulated after mex3-1 RNAi , were observed to belong to this WThighXlow category with little expression in the X1 or X2 fractions .", "The identification of one of these transcripts as a pharyngeal marker with expression in PIWI-1+ cells demonstrates that lineage-restricted progeny are present outside the X1 and X2 gates and merits the investment of future efforts toward screening further WThighXlow genes that are regulated by mex3-1 in order to uncover novel progenitors .", "This knowledge is necessary in order to achieve a comprehensive understanding of the asymmetries specific to ASC lineage organization .", "Due to the conserved role of MEX3 in regulating cell fate determination across multiple phyla , understanding how mex3-1 achieves lineage asymmetry in planarians will contribute to informing ASC biology and regeneration in other organisms ." ], [ "We previously performed RNAseq of the planarian X1 , X2 , and irradiation-insensitive compartments where the X2 cell fraction was sequenced to a depth of 206 million reads in two biological replicates ( Labbe et al . , 2012 ) .", "Here , we performed an additional replicate by using flow cytometry to obtain cell populations as previously described ( Hayashi et al . , 2006; Pearson and Sánchez Alvarado , 2010 ) .", "Approximately , 1 million X2 cells from 100 animals were isolated on a Becton–Dickinson FACSaria over multiple sorts .", "Total RNA was purified and poly-A-selected cDNA libraries were prepped using the TruSeq kits from Illumina .", "This new X2 sample was multiplexed together with a new X1 and Irradiated control and each was sequenced to a depth of >63 million single-end 50 base pair reads on an Illumina HiSeq2500 with v4 chemistry .", "Raw sequence data were uploaded to NCBI GEO under accession number GSE68581 .", "Each sample was aligned to the transcriptome under NCBI BioProject PRJNA215411 using Bowtie2 with no sequence trimming .", "Mapped reads per million reads ( CPM ) of each transcript was calculated .", "Note that kilobase-length of each transcript was not taken into account because in any expression ratio , the length scaling factor cancels out and no inter-transcript comparisons were performed .", "To ensure a well-defined statistic in the calculation of fold-change , pseudocounts of +1 were added to every numerator and denominator as a way to not bias differentially expressed genes toward lowly expressed transcripts ( Klattenhoff et al . , 2013 ) .", "The transcripts listed in Supplementary files 1–4 can be found in the same transcriptome database .", "The heatmap in Figure 1C was created using the Partek Genomics Suite of software ( www . partek . com ) with the unsupervised hierarchical clustering algorithm: Pearson's Absolute Value Dissimilarity .", "Intact unirradiated control ( WT ) transcript levels were averaged from 12 replicates of unfed and control ( RNAi ) experiments and time points using over 700 million sequencing reads ( Labbe et al . , 2012; Solana et al . , 2012; Currie and Pearson , 2013; Zhu and Pearson , 2013; Lin and Pearson , 2014 ) .", "In order to validate the consistency of our previous and new deep sequencing replicates , Pearson correlations were performed with our own data as well as all previously published RNAseq relevant to the current study using CPM for each transcript with CPM <1000 ( Figure 1—figure supplement 1 ) ( Labbe et al . , 2012; Onal et al . , 2012; Resch et al . , 2012; Solana et al . , 2012 ) .", "The program DESeq was used to determine significantly enriched transcripts in the X2 ( FDR < 0 . 01 ) or X1 ( FDR < 0 . 001 ) cell fractions vs irradiated whole animals using the three biological replicates for each tissue type .", "MA plots , Pearson correlations , and log fold change plots were made using R . Transcripts identified by differential expression were cloned using forward and reverse primers into a double-stranded RNA expression vector as previously described ( Rink et al . , 2009 ) .", "Riboprobes were made from PCR templates from the same vector ( pT4P ) ( Pearson et al . , 2009 ) .", "The three MEX3 homologs in S . mediterranea were identified with tBLASTn searches of the planarian genome/transcriptomes using MEX3 protein sequences from C . elegans and mouse .", "Candidate planarian MEX3 homologs were validated by reciprocal BLASTx against the nr database ( NCBI ) .", "The predicted proteins of the planarian MEX3 homologs were aligned using the program T-coffee along with MEX3 homologs from other species ( Notredame et al . , 2000 ) .", "The program Geneious ( www . geneious . com ) was used to run a Bayesian phylogeny using the MrBayes plugin with the following settings: a WAG substitution model , 25% burnin , subsample frequency of 1000 , 1 million replicates , and four heated chains ( Ronquist and Huelsenbeck , 2003 ) .", "The transcripts for mex3-1 and all new progeny markers from this manuscript are listed in Supplementary file 2 .", "Asexual S . mediterranea CIW4 strain was reared as previously described ( Sánchez Alvarado et al . , 2002 ) .", "RNAi experiments were performed using previously described expression constructs and HT115 bacteria ( Newmark et al . , 2003 ) .", "Briefly , bacteria were grown to an O . D . 600 of 0 . 8 and induced with 1 mM isopropyl β-D-1-thiogalactopyranoside ( IPTG ) for 2 hr .", "Bacteria were pelleted and mixed with liver paste at a ratio of 500 μl of liver per 100 ml of original culture volume .", "Bacterial pellets were thoroughly mixed into the liver paste and frozen as aliquots .", "The negative control RNAi was the unc22 sequence from C . elegans as previously described ( Reddien et al . , 2005a ) .", "For the screening of all genes in this study , RNAi food was fed to 7-day starved experimental worms every third day for five feedings .", "Subsequent functional analyses for mex3-1 were performed with one feed unless noted otherwise .", "Time points in figures denote the number of feeds for each gene as well as the number of days after the last feed .", "For example , 1fd12 corresponds to one RNAi feeding and 12 days after that feeding .", "Amputations were performed 6 days after the final feeding unless noted otherwise .", "All animals used for immunostaining were 3–4 mm in length and size-matched between experimental and control worms .", "WISH , dFISH , and immunostaining were performed as previously described ( Pearson et al . , 2009; Lauter et al . , 2011; Cowles et al . , 2013; Currie and Pearson , 2013 ) .", "Colorimetric WISH and fluorescent phospho-histone H3 ( H3P ) stains were imaged on a Leica M165 fluorescent dissecting microscope .", "The rabbit monoclonal antibody to H3ser10p from Millipore ( 04–817 ) was used for all cell division assays ( Newmark and Sánchez Alvarado , 2000 ) .", "TUNEL was performed as previously described ( Pellettieri and Sánchez Alvarado , 2007 ) .", "Mouse anti-PIWI-1 ( gift of Dr Jochen Rink [Wagner et al . , 2011] ) was used at 1:1000 .", "H3ser10p and TUNEL were quantified using freely available ImageJ software ( http://rsb . info . nih . gov/ij/ ) .", "Significance was determined by a 2-tailed Student's t-test unless otherwise noted .", "All experiments were , at minimum , performed in triplicate with at least 10 worms per stain and per time point ( i . e . , n > 30 ) .", "For irradiation experiments , planarians were exposed to 16 . 5 or 60 Gy of γ-irradiation from a 137Cs source .", "F-ara-EdU was fed to worms in liver paste at a concentration of 0 . 05 mg/ml for a 7-day chase ( fed at 1fd6 ) or 0 . 5 mg/ml for a 24-hr chase ( fed at 1fd6 and 1fd9 ) and stained as previously described following the normal fixation for ISH ( Pearson et al . , 2009; Neef and Luedtke , 2011 ) .", "BrdU was fed ( at 1fd6 ) in liver paste at a concentration of 10 mg/ml and stained as previously described ( van Wolfswinkel et al . , 2014 ) .", "Confocal images were acquired on a Leica DMIRE2 inverted fluorescence microscope with a Hamamatsu Back-Thinned EM-CCD camera and spinning disc confocal scan head , and stitched together for whole-animal images .", "Images were post-processed in Adobe Photoshop and figures assembled in Macromedia Freehand ." ] ]

[ "Neoblasts are adult stem cells ( ASCs ) in planarians that sustain cell replacement during homeostasis and regeneration of any missing tissue .", "While numerous studies have examined genes underlying neoblast pluripotency , molecular pathways driving postmitotic fates remain poorly defined .", "In this study , we used transcriptional profiling of irradiation-sensitive and irradiation-insensitive cell populations and RNA interference ( RNAi ) functional screening to uncover markers and regulators of postmitotic progeny .", "We identified 32 new markers distinguishing two main epithelial progenitor populations and a planarian homolog to the MEX3 RNA-binding protein ( Smed-mex3-1 ) as a key regulator of lineage progression .", "mex3-1 was required for generating differentiated cells of multiple lineages , while restricting the size of the stem cell compartment .", "We also demonstrated the utility of using mex3-1 ( RNAi ) animals to identify additional progenitor markers .", "These results identified mex3-1 as a cell fate regulator , broadly required for differentiation , and suggest that mex3-1 helps to mediate the balance between ASC self-renewal and commitment ." ]

[ "Adult tissues constantly replace the millions of cells they lose on a daily basis .", "This is made possible by adult stem cells .", "But how is a stable population of stem cells maintained throughout the life of the organism with constant cell division ?", "One way this can be accomplished is if at every stem cell division , only one of the daughter cells remains a stem cell , while the other becomes specialized .", "For humans , if this balance is disturbed , cancers may result from too many stem cells , and early aging may result from too few stem cells .", "A freshwater flatworm called Schmidtea mediterranea is known for its ability to regenerate nearly every part of its body after injury .", "This flatworm possesses stem cells called neoblasts that can form all of the flatworm's different cell types both during regeneration and during normal tissue turnover .", "Evidence suggests that the number of neoblasts and the number of specialized cells that neoblasts produce are finely balanced , similar to adult human tissues .", "However , little is known about the mechanism that controls whether a neoblast takes on a more specialized form .", "To express a gene , it must first be copied or ‘transcribed’ into an RNA molecule .", "Identifying the RNA molecules that are enriched in the non-stem cells that develop from neoblasts could therefore indicate which genes regulate the cell specialization process .", "These RNA molecules could also be used as markers that identify which cells have taken on a more specialized form .", "Using techniques called transcriptional profiling and RNA interference , Zhu et al . identified 32 new markers that indicate that the neoblasts have started to specialize into epithelial cells: cells that line the surfaces of many structures in the body .", "Further investigation revealed that one gene , called mex3-1 , is needed for many specialized cell types—not just epithelial cells—to mature from neoblasts in the flatworms .", "In doing so , mex3-1 also limits the size of the stem cell population .", "Equivalents of mex3-1 are found in many different species including humans , and so Zhu et al . 's results may help us to understand how other animals regenerate and control the size of their stem cell populations .", "Mutant flatworms that cannot express mex3-1 could also be used to study other genes that help neoblasts to specialize ." ]

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

[ "cancer biology" ]

[ [ "Angiogenesis , the process of forming new blood vessels from pre-existing ones is essential for embryonic development , tissue growth , wound healing , and regeneration .", "However , angiogenesis also substantially contributes to the pathogenesis of several diseases , most notably tumor progression ( Potente et al . , 2011 ) .", "Angiogenesis is stimulated by vascular endothelial growth factor ( VEGF ) , which activates quiescent endothelial cells ( EC ) .", "These cells subsequently degrade the extracellular matrix and migrate toward the VEGF gradient .", "A new vessel sprout is guided by the tip cell , which expresses high amounts of VEGF receptors ( Siekmann et al . , 2013 ) .", "VEGF signaling induces the expression of the Notch Delta-like ligand 4 ( DLL4 ) on activated ECs .", "This transmembrane protein activates Notch receptors on adjacent cells , which adopt the stalk cell phenotype and form the new vessel lumen .", "Notch signaling requires ligand binding in trans that triggers Notch receptor cleavage to release the intracellular domain ( NICD ) .", "NICD translocates to the nucleus where it acts as a transcriptional regulator .", "Prototypical Notch target genes are the HES and HEY transcriptional repressors .", "These inhibit VEGF receptor expression which limits responsiveness to VEGF , tip cell formation and vessel branching ( Bray , 2016; Fischer et al . , 2004 ) .", "Inhibition of DLL4/Notch signaling is a powerful tool to interfere with angiogenesis as it results in the formation of excessive tip cell numbers and vessel branches .", "This chaotic vessel network precludes proper blood perfusion leading to severe tumor hypoxia .", "Interestingly , inactivation of the Notch ligand Jagged1 ( JAG1 ) results in a reduced sprouting angiogenesis , indicating that JAG1 and DLL4 have opposing roles during blood vessel formation ( Benedito et al . , 2009; Kangsamaksin et al . , 2015; Kofler et al . , 2011 ) .", "It remains poorly understood which factors modulate and control Notch activity during angiogenesis .", "To become fully competent for Notch receptor activation , Notch ligands need to gain several posttranslational modifications , for example ubiquitinylation ( D'Souza et al . , 2008 ) .", "In addition , Notch ligands need to be presented on the cell surface at an area , that is likely to be in contact with Notch receptors on adjacent cells ( Shaya et al . , 2017 ) .", "Delta-like and Jagged proteins contain different PDZ binding motifs at their intracellular carboxyterminus , which enable binding to certain PDZ domain containing proteins .", "There are some indications that binding of Notch ligands to PDZ domain proteins , for example DLL1 to MAGI1 and SYN2BP as well as JAG1 to AF6 control their cellular localization or their protein stability ( Adam et al . , 2013; Ascano et al . , 2003; Mizuhara et al . , 2005 ) .", "Interestingly , these proteins are associated with either adherens or tight junctions .", "Notch receptors are also localized at adherens junctions in several cell types ( Batchuluun et al . , 2017; Benhra et al . , 2011; Hatakeyama et al . , 2014; Sasaki et al . , 2007 ) .", "Therefore , clustering ligands and receptors at cellular junctions might increase the rate of physical binding events and subsequent Notch signaling activity .", "In yeast , two-hybrid screening approaches the Notch ligands DLL1 and DLL4 , but not JAG1 , interacted with the multiple PDZ domain protein ( MPDZ ) also known as MUPP1 ( Adam et al . , 2013; Estrach et al . , 2007 ) .", "Also a synthetic DLL1 peptide containing the 27 carboxyterminal amino acids interacted with PDZ proteins including MPDZ ( Wright et al . , 2004 ) .", "However , this interaction had not yet been confirmed by independent methods and any potential functional consequences are elusive .", "MPDZ contains 13 PDZ domains and a single L27 domain ( Ullmer et al . , 1998 ) .", "It lacks an intrinsic catalytic function , and it is assumed that its function is to cluster proteins at the cell membrane or at adherens and tight junctions ( Adachi et al . , 2009 ) .", "Such clustering of proteins was shown to affect the strength of melatonin or the AMPA transmembrane receptor signaling ( Guillaume et al . , 2008; Krapivinsky et al . , 2004 ) .", "As Notch receptor expression is often enriched at cellular junctions ( Batchuluun et al . , 2017; Benhra et al . , 2011; Hatakeyama et al . , 2014; Sasaki et al . , 2007 ) , we analyzed how the protein interaction of MPDZ with the Notch ligands DLL1 and DLL4 affects Notch signaling during angiogenesis ." ], [ "MPDZ has been identified in screening approaches as a putative binding partner of the Notch ligands DLL1 and DLL4 ( Adam et al . , 2013; Estrach et al . , 2007; Wright et al . , 2004 ) .", "To verify this , we performed co-immunoprecipitation studies in HEK293T cells .", "Full-length DLL1 and DLL4 or mutants thereof lacking the carboxyterminal PDZ-binding site ( amino acids IATEV ) were co-expressed with MPDZ fused with an amino-terminal fluorescent Citrine .", "Co-immunoprecipitation revealed that MPDZ associated with DLL1 and DLL4 proteins .", "However , DLL1 or DLL4 lacking their PDZ-binding site did not interact with MPDZ ( Figure 1A and B ) , indicating that the carboxyterminus of Delta-like ligands binds to MPDZ .", "This protein-protein interaction could also be detected in primary human umbilical venous endothelial cells ( HUVEC ) as well as in whole murine kidney lysates using a co-immunoprecipitation approach ( Figure 1C and D , Figure 1—figure supplement 1A and B ) .", "Since SYNJ2BP binds also to DLL1 and DLL4 via the PDZ-binding motif and induces Notch signaling , a competition between SYNJ2BP and MPDZ might be possible .", "However , pull-down studies showed that the absence of MPDZ did not overtly affect the binding of SYNJ2BP to DLL1 or DLL4 ( Figure 1—figure supplement 1C and D ) .", "The activity of Notch signaling depends critically on the amount of active DLL1/4 molecules on the cell surface .", "We tested whether the MPDZ-DLL1/4 protein interaction could alter Notch signaling activity .", "Forced expression of MPDZ promoted Notch signaling in HUVEC as indicated by higher expression levels of the Notch target genes HEY1 , HEY2 and HES1 ( Figure 1E ) .", "To test if MPDZ would alter the ability of DLL4 to activate Notch receptors in trans , IMCD3 cells expressing DLL4 ( sender cells ) were transfected with plasmids encoding MPDZ cDNA or empty vector control .", "A Notch luciferase reporter CHO cell line ( receiver cells ) was co-cultured with the IMCD3 sender cells ( Figure 1F ) .", "This showed that higher amounts of MPDZ in the DLL4 expressing sender cells resulted in increased Notch signaling activity in receiver cells ( Figure 1F ) .", "To test if MPDZ contributes to basal Notch signaling in ECs , we silenced MPDZ expression in HUVEC using established lentiviruses expressing independent shRNAs ( Feldner et al . , 2017 ) .", "The reduction of MPDZ expression ( 93 ± 3% , n = 4 , p<0 . 001 ) resulted in a significant reduction of mRNA expression of the Notch target genes HEY1 , HEY2 and HES1 ( Figure 2A ) , indicating diminished Notch activity .", "This could also be observed in cardiac CD31-positive ECs derived from EC-specific Mpdz-deficient mice ( Tek-Cre;Mpdzfl/fl referred to as MpdzΔEC ) ( Feldner et al . , 2017 ) .", "qPCR analysis revealed that the reduction of Mpdz expression ( 69 ± 12% , n = 4 , p<0 , 001 ) resulted in a diminished Notch signaling activity as the relative Hey1 and Hey2 mRNA amounts were lower compared to Cre-negative littermate controls ( Figure 2B ) .", "Next , we aimed at elucidating the mechanism of how MPDZ alters the activity of Notch ligands .", "The total expression levels of DLL1 and DLL4 proteins in HUVEC lysates were not altered upon silencing of MPDZ ( Figure 2C ) .", "The same was observed after adenoviral MPDZ overexpression ( Figure 2D ) .", "Lung ECs derived from MpdzΔEC mice also did not show changes in total Dll1 and Dll4 protein expression levels compared to controls ( Figure 2E ) .", "MPDZ is able to cluster several transmembrane proteins at tight and adherens junctions .", "For instance , MPDZ facilitates RhoA signaling by recruiting Syx to endothelial junctions ( Ngok et al . , 2012 ) .", "The activation of Notch receptors requires the interaction with Notch ligands presented on the cell surface .", "MPDZ is expressed in ECs derived of arteries , veins and microvessels with pronounced localization at the cell membrane ( Figure 3—figure supplement 1 ) .", "To address whether MPDZ affects the cell surface expression of the Notch ligands DLL1 and DLL4 , we first tested if the carboxyterminal PDZ-binding sites of these Notch ligands are important for cell surface presentation .", "Constructs in which either full length DLL1 and DLL4 or versions lacking the PDZ-binding motifs were fused to a mCherry tag were generated .", "Those constructs were expressed in HUVEC and cell surface expression was analyzed by flow cytometry .", "Only mCherry-positive HUVEC were gated and the cell surface presentation of the Notch ligands was analyzed using specific antibodies against the extracellular domains of DLL1 and DLL4 , respectively .", "Cell surface expression levels of full length DLL1 and DLL4 were higher than those of their mutants lacking the PDZ-binding site ( Figure 3A ) .", "This indicates that protein-protein interactions via the PDZ-binding site could influence the localization of Delta-like proteins .", "To test whether indeed MPDZ is involved in regulating DLL4 cell surface localization , we analyzed cell surface presentation on isolated mouse embryonic EC at the developmental stage E11 . 5 by flow cytometry .", "This revealed that the Dll4 cell surface expression of CD34-positive ECs was lower in Mpdz-deficient cells compared to wild type littermate controls ( Figure 3B ) .", "MPDZ binds to the intracellular domain of the single-pass type I membrane glycoprotein Nectin-2 , which is a component of adherens junctions ( Adachi et al . , 2009 ) and is expressed in the endothelium ( Rehm et al . , 2013; Wallez and Huber , 2008 ) .", "Thus , MPDZ could be involved in recruiting DLL1 and DLL4 to Nectin-2 containing adherens junctions .", "To test this , we performed co-immunoprecipitation studies in HEK293 cells .", "This revealed that DLL1 and DLL4 could be co-immunoprecipitated with Nectin-2 .", "However , silencing of MPDZ expression abolished the interaction of DLL1 and DLL4 with Nectin-2 ( Figure 3C and D ) .", "Interestingly , loss of Mpdz in mice did not affect the overall formation of vascular tight junctions ( e . g . staining patterns of Claudin-5 and Occludin were unremarkable ) , as well as adherens junctions ( VE-Cadherin ) and Nectin-2-containing junctions ( Figure 4 , Figure 4—figure supplement 1 ) , similar as observed before ( Feldner et al . , 2017 ) .", "In vitro experiments indicated that the co-localization of DLL1 and DLL4 with the adherens junction protein Nectin-2 at the cell membrane was diminished upon silencing MPDZ expression in ECs ( Figure 4—figure supplement 2A , B ) , whereas silencing of Nectin-2 did not affect localization of DLL1 and DLL4 ( Figure 4—figure supplement 2C ) .", "Taken together , the data suggest that MPDZ stabilizes the cell surface presentation of DLL1 and DLL4 through recruitment to the adherens junction protein Nectin-2 .", "DLL4/Notch signaling restricts sprouting angiogenesis and cells with high Notch signaling activity adopt the stalk cell phenotype in a growing vessel sprout ( Blanco and Gerhardt , 2013; Eilken and Adams , 2010; Pitulescu et al . , 2017 ) .", "As such , the protein interaction of MPDZ with the Notch ligand DLL4 and its promotion of Notch signaling activity could potentially be important to control angiogenesis .", "To address this , MPDZ was silenced or over-expressed in HUVEC , which were embedded as spheroids into a collagen matrix to analyze endothelial sprout formation .", "HUVEC silenced for MPDZ expression had increased angiogenic potential and formed more capillary-like sprouts compared to control cells , both under basal conditions and after VEGF stimulation ( Figure 5A ) .", "Oppositely , forced MPDZ expression resulted in impaired sprout formation under both conditions ( Figure 5B ) .", "Furthermore , MPDZ-expressing cells , which exhibit increased Notch signaling activity , preferred the stalk cell position in the sprouting angiogenesis assay .", "In line with this result , MPDZ silenced cells , which showed less Notch signaling activity , adopted preferentially the tip cell position in a growing sprout ( Figure 5C ) .", "The increased angiogenic potential of Mpdz-deficient cells could also be shown in the aortic ring assay , in which slices of the mouse aorta from MpdzΔEC or littermate control mice ( Mpdzfl/fl ) were embedded into Matrigel .", "Aortic ECs from MpdzΔEC mice showed a significantly higher outgrowth rate compared to control ( Figure 6A ) .", "Based on the in vitro and ex vivo data , we tested whether MPDZ also affects angiogenesis during mouse development .", "Vascularization of the hindbrain occurs in a stereotypic manner and is ideally suited to examine sprouting angiogenesis ( Fantin et al . , 2013 ) .", "Hindbrains were resected at developmental stage E12 . 5 to analyze the vasculature .", "This revealed a much denser vessel network in Mpdz-/- embryos compared to wild-type littermates ( Figure 6B ) .", "Mpdz-/- embryos had a significant higher number of vessel junctions and branches compared to littermate controls ( Figure 6C ) .", "This indicates that Mpdz is needed to limit developmental angiogenesis .", "Angiogenesis is a hallmark of cancer and therefore we examined how genetic inactivation of Mpdz specifically in the endothelium would affect tumor growth and tumor angiogenesis .", "B16F10 melanoma and Lewis lung carcinoma ( LLC ) cells were injected subcutaneously into syngeneic MpdzΔEC and control mice .", "No significant differences in the tumor growth rates were observed between Mpdzfl/fl and MpdzΔEC mice ( Figure 7A and B ) .", "The resected tumors were stained against the EC marker CD31 and α-SMA ( smooth muscle cells , myofibroblasts ) ( Figure 7C ) .", "Microvessel density was significantly higher in MpdzΔEC compared to Mpdzfl/fl mice ( Figure 7D ) , whereas vessel coverage was not altered ( Figure 7D ) , similar as observed after blocking Dll4-induced Notch signaling in the tumor vasculature ( Kangsamaksin et al . , 2015 ) .", "Increased numbers of blood vessels can support tumor growth , whereas too many vessel branches disturb the functionality of the vessel network , impair proper perfusion , inhibit tumor growth and can lead to tissue hypoxia .", "For instance , this was observed after inhibition of endothelial Dll4/Notch signaling in tumors ( Kangsamaksin et al . , 2015; Noguera-Troise et al . , 2006; Ridgway et al . , 2006 ) .", "Indeed , both B16F10 and LLC tumors grown in MpdzΔEC mice contained larger hypoxic areas compared to controls , as indicated by Glut1 expression ( Figure 7E and F ) .", "To elucidate whether tumor perfusion is impaired , we injected Hoechst 33342 and FITC-labeled Lycopersicon Esculentum lectin into a tail vein and resected the tumors 5 min later .", "This revealed that B16F10 as well as LLC tumors were less well perfused in MpdzΔEC mice compared to control littermates ( Figure 7—figure supplement 1A and B ) .", "In the melanoma model , the percentage of Lectin-positive tumor blood vessels was reduced in MpdzΔEC mice ( Figure 7—figure supplement 1C ) , whereas in the LLC model , which contains a better structured vasculature than the melanoma model , the Hoechst dye was delivered to a lesser extend into the tumor mass in MpdzΔEC mice compared to control littermates ( Figure 7—figure supplement 1D ) ." ], [ "Notch signaling is of utmost importance to control numerous cell differentiation steps during development .", "The activation of Notch receptors depends on physical contact with Notch ligands expressed on the adjacent cell .", "This study describes a novel mechanism that improves presentation of Delta-like ligands on the cell surface .", "MPDZ could be identified as a protein that mediates intracellular protein interactions with DLL1 , DLL4 and Nectin-2 to facilitate presentation of DLL1 and DLL4 at the EC surface and to strengthen Notch signaling activity .", "Upon posttranslational modifications , Notch ligands are transported to the plasma membrane .", "It is not yet clear how Notch ligands are recruited to the plasma membrane or to cellular junctions .", "Previous studies have shown that PDZ domain proteins interact with Notch ligands ( Adam et al . , 2013; Ascano et al . , 2003; Mizuhara et al . , 2005; Pfister et al . , 2003; Six et al . , 2004; Wright et al . , 2004 ) .", "However , the functional consequences of these are mostly elusive .", "The interaction of DLL1 with SYNJ2BP acts via a different mechanism on Notch signaling strength .", "SYNJ2BP prevents lysosomal degradation of DLL1 ( Adam et al . , 2013 ) .", "The interaction with MPDZ however promotes cell surface presentation .", "Based on this , one can assume that changes in expression levels of Notch ligand-interacting PDZ proteins influence strongly the behavior of Notch ligands .", "Here , we demonstrated that MPDZ interacts with Delta-like ligands and the transmembrane protein Nectin-2 , a component of adherens junctions .", "This is interesting as also Notch receptors can be enriched at adherens junctions ( Batchuluun et al . , 2017; Benhra et al . , 2011; Hatakeyama et al . , 2014; Sasaki et al . , 2007 ) to facilitate the physical interactions with ligands .", "Indeed , we found evidence that MPDZ mediates Notch signaling in cultured cells and isolated ECs derived from Mpdz-deficient mice .", "Whereas forced MPDZ expression enhanced Notch target gene expression , inactivation of the MPDZ gene resulted in a lower Notch target gene expression which might be due to the reduced DLL1 and DLL4 localization at the cell surface .", "The reduction of endothelial Notch signaling activity in MpdzΔEC mice was only moderate .", "One possible explanation is the redundancy between PDZ proteins that bind the same motif .", "For example , MPDZ shares large structural homology with the INADL protein ( Adachi et al . , 2009 ) and the Notch ligands can be bound also by several other PDZ domain proteins ( Adam et al . , 2013; Estrach et al . , 2007; Pfister et al . , 2003; Six et al . , 2004; Wright et al . , 2004 ) .", "As such it is not surprising that Mpdz-deficient mice did not exhibit major angiogenesis defects , which would cause embryonic lethality .", "The vascular phenotype is similar to Notch1 heterozygous mice .", "These mutants develop normally and show only slight vascular abnormalities ( higher microvessel density and vessel branching ) during phases of rapid vessel growth ( Huppert et al . , 2000 ) .", "Pharmacological Notch inhibition also leads to excessive tumor vessel sprouting and the formation of a poorly functional vessel network due to too many branches ( Funahashi et al . , 2008; Noguera-Troise et al . , 2006; Ridgway et al . , 2006 ) .", "This is in particular achieved by DLL4-specific , but not JAG1-specific inhibition of Notch signaling ( Kangsamaksin et al . , 2015 ) .", "We could also observe an increased tumor vessel sprouting in mice lacking Mpdz expression in the endothelium .", "Tumor vessel density was significantly increased , tumor perfusion impaired and this led to larger hypoxic tumor areas .", "Again , similar as after DLL4 blockade , vessel coverage with mural cells was not altered in tumors grown in MpdzΔEC mice ( Funahashi et al . , 2008; Kangsamaksin et al . , 2015 ) .", "Taken together , this work shows that MPDZ is a novel modulator of DLL4-induced Notch signaling in the vasculature by recruiting DLL1 and DLL4 to Nectin-2 on the cell surface ." ], [ "Mice were kept under pathogen-free barrier conditions .", "All animal procedures were performed in accordance with the institutional and national regulations and approved by the local committees for animal experimentation and the local government ( reference number: 35–9185 . 81/G-30/14 and 35–9185 . 81/G-259/12 ) .", "Generation of global Mpdz-/- mice and conditional Tek-Cre;Mpdzfl/fl mice was previously described ( Feldner et al . , 2017 ) .", "Mice had been backcrossed on a C57Bl/6 background for 10 generations .", "For tumor experiments , 500 , 000 syngeneic tumor cells ( B16F10 or LLC ) in 100 µl PBS were injected subcutaneously in the abdominal flanks of mice .", "To analyze tumor perfusion , 100 µl of fluorescein-labeled lectin ( 1 mg/ml , FL-1171 , Vector Laboratories , Burlington , CA ) and Hoechst 33342 ( 5 mg/ml , H3570 , Life Technologies , Carlsbad , CA ) was injected into the tail vein 5 min prior to sacrifice .", "B16F10 , LLC and HEK293T cells were cultured in DMEM containing 10% fetal calf serum , 100 units/ml penicillin and 100 μg/ml streptomycin .", "Primary human umbilical cord endothelial cells ( HUAEC and HUVEC ) were freshly isolated and cultured in Endopan-3 medium with supplements ( P04-0010K , PAN Biotech , Aidenbach , Germany ) and used until passage five .", "HUVEC of three donors were pooled .", "Human brain microvascular endothelial cells were also cultured in Endopan-3 medium with supplements ( P04-0010K , PAN Biotech ) .", "Standardized multiplex cell contamination and cell line authentication testing ( Multiplexion , Heidelberg , Germany ) were conducted on a regular basis .", "HUVECs were transduced with lentivirus and adenovirus as described ( Brütsch et al . , 2010 ) .", "For MPDZ silencing three different lentiviral shRNA vectors ( Biocat V2LHS_3656 , 16945 16946 ) were used ( Feldner et al . , 2017 ) .", "Forced MPDZ expression was achieved by lentiviral or adenoviral transduction expressing MPDZ cDNA ( BioCat clone BC140793 ) .", "Forced expression of Nectin-2 was achieved by transient over-expression of Nectin-2 cDNA in HEK293T cells ( DKFZ clone BC003091 ) .", "SYNJ2BP , DLL1 and DLL4 expression constructs were described ( Adam et al . , 2013; Diez et al . , 2007 ) .", "Mutations in cDNAs were introduced by site-directed mutagenesis using the QuickChange XL Kit ( Agilent , Santa Clara , CA ) .", "Mutagenesis primers for the deletion of the DLL1-PDZ-binding site were as follows: 5'-gagaaggatgagtgcgtctaagcaactgaggtgtaagg-3' , 5'-ccttacacctcagttgcttagacgcactcatccttctc-3' .", "Mutagenesis primers for the deletion of the DLL4-PDZ-binding site were as follows: 5'-gaggagaggaatgaatgtgtctatgccacggaggtataagg-3' , 5'-ccttatacctccgtggcatagacacattcattcctctcctc-3' , 5'-ggagaggaatgaatgtgtctaagccacggaggtat-3' , 5'-atacctccgtggcttagacacattcattcctctcc-3' .", "HUVECs were transfected with siRNA using RNAiMAX transfection reagent ( Life Technologies ) .", "For Nectin-2 silencing , three different siRNAs were used ( SR321541 , Origene , Rockville , MD ) .", "Cells were lysed with Cell Lysis Buffer ( 9803S , Cell Signaling Technology , Danvers , MA ) complemented with 1 mM PMSF .", "Proteins were separated by SDS-PAGE and blotted on nitrocellulose membranes .", "Membranes were blocked with 5% skim milk in PBS containing 0 . 05% Tween-20 .", "Following primary antibodies were used: anti-HA ( #3724 , Cell Signaling Technology , 1:1000 ) , anti-FLAG ( F3165 , Sigma-Aldrich , St . Louis , MO , 1:1000 ) , anti-DLL1 ( ab85346 , abcam , Cambridge , UK , 1:1000 ) , anti-DLL4 ( #2589S , Cell Signaling Technology , 1:1000 ) , anti-beta-actin ( A5441- . 2ML , Sigma-Aldrich , 1:2500 ) , anti-DLL1 ( AF3970 , R & D Systems , Minneapolis , MN , 1:500 ) , anti-DLL4 ( AF1389 , R & D Systems , 1:250 ) , anti-MPDZ ( HPA020255 , Sigma-Aldrich , 1:500 ) , anti-Mpdz ( 42–2700 , Invitrogen , Carlsbad , CA , 1:500 ) , anti-GFP ( ab290 , abcam , 1:1000 ) , anti-SYNJ2BP ( ab69431 , abcam , 1:500 ) , anti-Nectin-2 ( sc-32804 , Santa Cruz Biotechnology , Dallas , TX , 1:500 ) .", "Membranes were incubated overnight at 4°C with primary antibodies .", "HRP-conjugated secondary antibodies ( DAKO , Santa Clara , CA ) were added for 1 hr at room temperature .", "Chemoluminescence was detected by Aceglow ECL Western Blotting Substrate ( PEQL37-3420 , VWR International , Darmstadt , Germany ) using a ChemiDoc imaging system ( Bio-Rad Laboratories , Hercules , CA ) .", "Western Blots were quantified with Image Lab 3 . 0 software ( Bio-Rad Laboratories ) .", "For immunoprecipitation , primary antibodies were added to protein lysates and incubated overnight at 4°C .", "Protein-G-coupled dynabeads ( 10003D , Invitrogen ) were added to the protein lysates at the next day and were incubated for 30 min at 4°C .", "Precipitated dynabeads were washed three times with ice-cold PBS and denatured in Laemmli sample buffer at 95°C for 5 min .", "Samples were then subjected to Western blotting .", "Quantitative Real-Time-PCR mRNA was isolated with the innuPrep RNA Mini Kit ( 845-KS-2040250 , Jena Analytics , Jena , Germany ) and transcribed into cDNA ( High Capacity cDNA Reverse Transcription Kit; 4368814 , Applied Biosystems , Foster City , CA ) .", "POWER SYBR Green Master Mix ( 4368708 , Applied Biosystems ) was used to perform qPCR on an ABI StepOnePlus cycler ( Applied Biosystems ) .", "Rpl32 and OAZ1 were used for normalization .", "Primers: hOAZ ( fw ) : 5’-gagccgaccatgtcttcatt-3’ , hOAZ ( rev ) : 5’-ctcctcctctcccgaagact-3’; mRpl32 ( fw ) : 5’-aggcattgacaacagggttc-3’ , mRpl32 ( fw ) : 5’-gttgcacatcagcagcactt-3’; hHEY1 ( fw ) : 5’-gagaaggctggtacccagtg-3’ , hHEY1 ( rev ) : 5’-cgaaatcccaaactccgata-3’; mHey1 ( fw ) : 5’-gaaaagacggagaggcatca-3’ , mHey1 ( rev ) : 5’-gtgcgcgtcaaaataacctt-3’; hHEY2 ( fw ) : 5’-cttgtgccaactgcttttga-3’ , hHEY2 ( rev ) : 5’-gcactctcggaatcctatgc-3’; mHey2 ( fw ) : 5’- tgagaagactagtgccaacagc-3’ , mHey2 ( rev ) : 5’-tgggcatcaaagtagccttta-3’; hHES1 ( fw ) : 5’-tcaacacgacaccggataaa-3’ , hHES1 ( rev ) : 5’-ccgcgagctatctttcttca-3’ .", "All experiments included two technical and three biological replicates .", "A Notch expressing cell line , CHO-N1-CIT , was transfected in 24-well dishes using TransIT-LT1 ( Mirus Bio , Madison , WI ) with a TP1-firefly Notch luciferase reporter ( 800 ng ) together with SV-40 Renilla luciferase ( 10 ng ) ( Shaya et al . , 2017 ) .", "IMCD3 cells were transfected under similar conditions with either an MPDZ construct or empty vector ( pORI ) as control .", "24 hr after transfection , cells were trypsinized and the CHO-N1-CIT cells were co-cultured with either IMCD3 cells transfected with MPDZ or pORI .", "The cells were plated in a ratio of 40:60 ( IMCD3: CHO-N1-CIT ) and were incubated for 48 hr , after which the cells were lysed with lysis buffer ( E1960 , Promega , Madison , WI ) .", "The light emission of the luciferin and the Renilla luciferase activities were measured from cell lysates using a dual luciferase kit ( E1960 , Promega ) and a Veritas luminometer ( Promega ) .", "The assay was repeated five independent times .", "HUVECs were suspended in growth medium containing 20% methocel ( Sigma-Aldrich ) .", "Endothelial cells were cultured as hanging drops for 24 hr to form spheroids .", "Each spheroid contained approximately 400 cells .", "Spheroids were suspended in 2 ml methocel containing 20% FCS and 2 ml rat collagen at neutral pH . The collagen matrix polymerized for 30 min and hereon 0 . 1 ml basal culture medium or 0 . 1 ml basal culture medium containing VEGF-A ( final concentration 25 ng/ml; Peprotech , Hamburg , Germany ) was added .", "After 24 hr , cells were fixed with 10% formaldehyde .", "The lengths of all sprouts of at least 10 spheroids per condition were measured using an inverted microscope ( Leica DM IRB , Leica Microsystems , Wetzlar , Germany ) .", "Image analysis was done by using Fiji software .", "The assay is described in more detail at Bio-protocol ( Tetzlaff and Fischer , 2018 ) .", "To determine which cells prefer the tip or stalk cell position , a sprouting assay with a co-culture of endothelial cells was performed .", "Cells expressed either a fluorophore ( GFP or mCherry ) or were labeled with Cell Tracker Red ( C34552 , Life Technologies ) .", "Equal amount of each cell type were cultured as hanging drops .", "Sprouting assay was performed as described above .", "Using the inverted microscope ( Leica DM IRB ) , the number of green or red fluorescent cells in the tip cell position was determined .", "Per condition at least 10 spheroids were analyzed .", "Embryonic hindbrains were isolated as described previously ( Fantin et al . , 2013 ) .", "Samples were fixed with 4% PFA overnight at 4°C and hereon permeabilized in blocking buffer ( 0 . 3% Triton X-100% and 1% BSA in PBS ) overnight at 4°C .", "Samples were washed three times for 20 min with Pblec buffer at room temperature and stained with FITC-IsolectinB4 ( 1:100; L2895 , Sigma-Aldrich ) in Pblec overnight at 4°C .", "After washing , samples were mounted using fluorescence mounting medium ( S3023 , DAKO ) .", "Z-stack images were acquired using a confocal microscope ( Zeiss LSM 700 , Zeiss , Oberkochen , Germany ) and image analysis was done by Fiji software .", "Aortae were isolated from mice ( 8 weeks old ) and cut into ~25 rings each .", "Aortic rings were embedded in matrigel ( 356234 , BD Biosciences , Franklin Lakes , NJ ) , and stimulated with 30 ng/ml VEGF-A165 ( 450–32 , Peprotech ) .", "Images were taken after 24 hr with a Nikon SMZ800 microscope .", "Freshly dissected tumors were embedded in Tissue-Tek ( 4583 , Sakura ) , frozen and stored at −80°C .", "Sections ( 7 µm ) were cut and fixed in methanol for 20 min at −20°C .", "The primary antibodies against CD31 ( 550274 , BD Biosciences , 1:50 ) , α-SMA ( C6198 , Sigma-Aldrich , 1:100 ) and Glut1 ( ab40084 , abcam , 1:200 ) were incubated over night at 4°C and secondary antibodies ( Thermo Fisher Scientific , 1:400 ) for 1 hr at room temperature .", "Sections were washed three times with TBS-T and mounted with Fluoromount ( S3023 , Dako ) .", "Confocal images were obtained using an LSM 700 microscope ( Zeiss ) and analyzed using Fiji software .", "Retinae were isolated , fixed and processed as previously described ( Yang et al . , 2015 ) .", "Specimens were stained for IsolectinB4 ( 1:100; L2895 , Sigma-Aldrich ) , Claudin 5 ( 1:100 , ab53765 , abcam ) , VE-Cadherin ( 1:100 , 555289 , BD Biosciences ) , Nectin-2 ( 1:50 , ab16912 , abcam ) .", "HUVEC were seeded on glass slides coated with 0 . 5% gelantine .", "Cells were washed twice with PBS , fixed with 4% PFA for 10 min , washed three times with PBS , permeabilized with PBS-T ( containing 0 . 1% TritonX ) and washed again three times with PBS .", "Alternative to PFA fixation , cells were fixed with ice-cold methanol for 20 min at −20°C and then washed three times with PBS .", "After blocking with 3% BSA in PBS , cells were incubated with the primary antibodies against MPDZ ( HPA020255 , Sigma-Aldrich , 1:50 ) , DLL1 ( ab85346 , abcam , 1:100 ) , DLL4 ( WH0054567M4 , Sigma-Aldrich , 1:100 ) , Nectin-2 ( ab135246 , abcam , 1:50 ) , Nectin-2 ( sc-32804 , Santa Cruz Biotechnology , 1:50 ) over night at 4°C and secondary antibodies ( Thermo Fisher Scientific , 1:400 ) for 1 hr at room temperature .", "Sections were counterstained with DAPI , washed three times with PBS and mounted with Fluoromount ( S3023 , Dako ) .", "Confocal images were obtained using an LSM 700 microscope ( Zeiss ) and analyzed using Fiji software .", "HUVECs were detached from cell culture plates using trypsin-EDTA ( 25300054 , Thermo Fisher Scientific , Waltham , MA ) .", "Mouse embryos were minced and incubated with 0 . 5 mg/ml collagenase type II ( LS004177 , Worthington , Lakewood , CA ) for 45 min at 37°C .", "Tissue suspensions were mashed twice through cell strainers ( BD Biosciences; 100 and 40 μm ) .", "Endothelial cells were enriched by CD31 magnetic beads .", "Cells were suspended ( 106 cells/ml ) and incubated with different fluorophores coupled to primary antibodies against DLL1 ( FAB1818A , R & D Systems ) , DLL4 ( FAB1506A , R & D Systems ) , Dll4 ( 563802 , BD Biosciences ) , CD34 ( 553733 , BD Biosciences ) for 20 min on ice .", "Concentration of the different antibodies was determined by titration , in order to get optimal compensation during acquisition .", "Statistical analysis was performed with SigmaPlot software 12 . 5 ( Systat Software Inc . , San Jose , CA ) .", "Statistical significance was calculated using unpaired Student’s t-test and one-way ANOVA , as adequate .", "p-values<0 . 05 were considered as significant .", "All animal works were approved by the local committees for animal experimentation and the local government ( reference number: 35–9185 . 81/G-30/14 and 35–9185 . 81/G-259/12 ) .", "This work is not considered ‘Human Subjects Research’ ." ] ]

[ "Angiogenesis is coordinated by VEGF and Notch signaling .", "DLL4-induced Notch signaling inhibits tip cell formation and vessel branching .", "To ensure proper Notch signaling , receptors and ligands are clustered at adherens junctions .", "However , little is known about factors that control Notch activity by influencing the cellular localization of Notch ligands .", "Here , we show that the multiple PDZ domain protein ( MPDZ ) enhances Notch signaling activity .", "MPDZ physically interacts with the intracellular carboxyterminus of DLL1 and DLL4 and enables their interaction with the adherens junction protein Nectin-2 .", "Inactivation of the MPDZ gene leads to impaired Notch signaling activity and increased blood vessel sprouting in cellular models and the embryonic mouse hindbrain .", "Tumor angiogenesis was enhanced upon endothelial-specific inactivation of MPDZ leading to an excessively branched and poorly functional vessel network resulting in tumor hypoxia .", "As such , we identified MPDZ as a novel modulator of Notch signaling by controlling ligand recruitment to adherens junctions ." ]

[ "Blood vessels transport oxygen and nutrients to all our organs and also remove waste products .", "New blood vessels form – in a process called angiogenesis – when a tissue is not receiving enough oxygen .", "This happens during normal development and wound healing , but also during tumor growth .", "Cells at the tip of a branching blood vessel sense when a tissue lacks oxygen and use proteins on their cell surfaces to help new vessels to grow .", "During this process , the tip cells of an existing vessel relay the signal from the tissue to other cells ‘behind’ them , in the so-called stalk of the vessel .", "It is known that tip- and stalk cells communicate by using specific proteins at their interfaces .", "The tip cells activate proteins called Notch ligands , such as DLL4 , while stalk cells express the Notch receptor .", "During a process called Notch signaling , the ligands bind to the receptor , which becomes active and helps to control angiogenesis .", "It also hinders excessive vessel branching and so prevents the blood vessels from becoming leaky and inefficient .", "However , it was not known exactly how Notch ligands interact with their receptors on neighboring cells , and Notch signaling is regulated .", "Here , Tetzlaff et al . sought to answer these questions by using blood vessel cells from the human umbilical cord grown in the laboratory and blood vessel cells in mice .", "The results showed that the proteins DLL1 and DLL4 interacted with a protein called MPDZ .", "This interaction stabilized the DLL proteins at the cell membrane , which increased the Notch-signaling activity .", "When Tetzlaff et al . experimentally reduced the amount of MPDZ in the laboratory-grown cells , the Notch signaling decreased .", "Furthermore , the cells with less MPDZ formed more branching structures .", "And when MPDZ was genetically removed in mice , the embryos had more branched blood vessels in their developing brains .", "Lastly , when mice without MPDZ were transplanted with tumor cells , the tumors contained more , but leakier , blood vessels and were not supplied with enough oxygen .", "This suggests that MPDZ is an important factor that helps to regulate angiogenesis by enhancing Notch signaling between tip and branch cells in a new blood vessel .", "The increased activity of the Notch limits new blood vessels from branching too much .", "A better understanding of how blood vessels form or become leaky may help to find ways to prevent tumors from growing ." ]

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

[ "structural biology and molecular biophysics" ]

[ [ "The success of mitosis depends on the intricate timing , precise localisation and regulated interactions between multiple spindle components .", "MKLP2 ( Kif20A ) , a kinesin-6 family motor , is involved at several key stages of cell division , and in particular is essential for cytokinesis in mammals ( Hill et al . , 2000 ) .", "MKLP2 interacts with and is regulated by a number of mitotic kinases including Cdk1/cyclin B prior to anaphase onset ( Hümmer and Mayer , 2009; Kitagawa et al . , 2014 ) .", "MKLP2 is required for localisation of Chromosome Passenger Complex ( CPC ) components , including Aurora B kinase , to the midzone at anaphase ( Gruneberg et al . , 2004 ) , which is in turn required for Kif4A and PRC1 function ( Nunes Bastos et al . , 2013 ) .", "MKLP2 also participates in myosin II recruitment for cleavage furrow ingression ( Kitagawa et al . , 2013 ) and interacts with the cell membrane to promote abscission ( Fung et al . , 2017 ) .", "In addition , it is a substrate for polo-like kinase 1 ( Plk1 ) , and the co-association at the midzone of MKLP2 and Plk1 is also required for cytokinesis ( Neef et al . , 2003 ) .", "In the light of these activities and because MKLP2 is found to be highly expressed in tumour tissue ( Gasnereau et al . , 2012 ) , inhibition of MKLP2 by antimitotics has been explored , and small molecule inhibitors of MKLP2 have been shown to perturb mammalian cytokinesis ( Tcherniuk et al . , 2010; Labrière et al . , 2016 ) .", "While there has been significant focus on dissecting cell cycle dependent function and regulation of MKLP2 , little is known about the molecular mechanisms that drive this function .", "MKLP2 shares only approximately 35% sequence identity with kinesin-1 ( Kin1 ) , which suggests that much of what is known about kinesin molecular function may not necessarily apply to this motor ( Hizlan et al . , 2006 ) .", "Little is known about the molecular and mechanistic properties of MKLP2 .", "A related kinesin family member , the kinesin-6 MKLP1 - part of the centralspindlin complex that is also essential for cytokinesis - is a plus-end directed motor ( Nislow et al . , 1992 ) , as is Kif20B , another kinesin-6 ( Abaza et al . , 2003 ) .", "The centralspindlin holocomplex has anti-parallel microtubule ( MT ) bundling capability ( Davies et al . , 2015 ) consistent with its localisation and function in the spindle midzone .", "Functional diversification within the kinesin superfamily is derived in part from variation within kinesin motor domain itself .", "Kinesin-6s are particularly intriguing in this context because they are up to ~40% larger than the canonical Kin1 motor domain ( Figure 1A ) due to a number of unique loop insertions .", "Within the mammalian MKLP2 motor domain sequence , these unique features include a ~ 60 residue N-terminal extension , insertions in loop 2 ( 18 aa ) , loop 6 ( 99 aa ) , loop 8 ( 5 aa ) and loop 12 ( 6 aa ) , along with an extended non-canonical neck-linker; there is also a ~ 40 aa insert between this neck linker region and MKLP2’s coiled coil .", "However , the effects of these modifications on motor structure and function are unknown .", "Visualisation of the kinesin-MT interaction and its sensitivity to nucleotide provides a structural framework for understanding motor functional output .", "Cryo-EM is an essential structural method for characterising kinesin-MT complexes and has , to date , revealed a mechanistic consensus for plus-end directed kinesins ( Atherton et al . , 2014; Shang et al . , 2014; Goulet et al . , 2014 ) .", "To address the deficit in molecular understanding of the MKLP2 mechanism , we have determined cryo-EM reconstructions and calculated atomic models of MT-bound MKLP2 in different nucleotide states .", "These structures provide a visualisation of the conformations that functional MKLP2 undergoes , and which are blocked by small molecule inhibitors ( Tcherniuk et al . , 2010; Labrière et al . , 2016 ) .", "These structures , supported by biochemical and biophysical characterisations , reveal many atypical properties and behaviours of the MKLP2 motor domain .", "Strikingly , these noncanonical properties are superimposed on more generally conserved ATP-dependent subdomain movements within the motor domain .", "MKLP2 motor domain binding on the MT surface is shifted compared with other kinesins , and additional contacts between the MT and MKLP2 insertions in loops 2 , 8 and 12 are formed .", "Additionally , while the MKLP2 neck-linker is directed towards the MT plus end in an ATP-like state enabling formation of the cover-neck bundle , the neck-linker does not fully dock against the motor core and exhibits more flexibility than is seen in transport motors such as Kin1 and kinesin-3 ( Kin3 ) .", "Moreover , the characteristic kinesin-6 large insertion in loop6 forms a discrete additional subdomain protruding from the MKLP2 motor core and away from the MT . Our characterisation provides a context in which the functional contribution of this motor in mitosis can be evaluated and offers a mechanistic framework in which other atypical members of the kinesin superfamily can be considered ." ], [ "We expressed monomeric mammalian MKLP2 motor domain constructs 1–520 and 25–520 and measured their steady-state MT-stimulated ATPase activity ( Figure 1B ) .", "Fits of the data to a Michaelis Menten function reveal values of kcat = 4 . 34 ± 0 . 14 s−1 and K0 . 5 , MT = 0 . 93 ± 0 . 12μM for 1–520 and kcat = 4 . 38 ± 0 . 20 s−1 and K0 . 5 , MT = 1 . 07 ± 0 . 18μM for 25–520 , showing that the presence or absence of the extended MKLP2 N-terminus does not affect steady state ATPase parameters .", "MT binding stimulates kcat >1000 fold ( Woehlke et al . , 1997; Hackney , 1988; Gilbert et al . , 1995 ) to levels similar to the mitotic kinesin-5 ( Kin5 ) and about 10-fold slower than the transport kinesins Kin1 or Kin3 ( Cochran et al . , 2004 ) ( Table 1 ) .", "Since the residues 1–25 are intrinsically disordered we used the 25–520 construct - which has a better yield during recombinant protein expression - for all subsequent assays .", "We refer to this 25–520 construct as MKLP2-MD .", "To further dissect the MKLP2-MD ATPase cycle and its dependence on MTs , the dynamic interaction between MKLP2-MD and either 2’deoxy 3’mant ATP ( 2’dmT ) or 2’deoxy 3’mant ADP ( 2’dmD ) in the presence and absence of MT was measured ( Figure 1C , D ) .", "In each case , the nucleotide binding transients consisted of fast and slow phases .", "The faster phase varies linearly with [nucleotide] , implying that this is a readout of the initial binding of nucleotide to the active site; the slower phase shows little dependence on [nucleotide] , implying that it measures a subsequent isomerization step .", "For 2’dmT ( Figure 1C ) , rates in the presence and absence of MT are nearly identical ( including Y intercept ) , implying that – unlike e . g . Kin-1 or Kin-5 – the kinetics of binding 2’dmT to MKLP2-MD are essentially independent of MTs .", "Binding of 2’dmD produces similar behaviour ( Figure 1D ) , with an approximately two-fold increase in the apparent second order rate constant for 2’dmD binding .", "Extrapolation of the linear fit to the origin defines an apparent 2’dmD dissociation rate constant , and in other kinesins , such plots typically go through the origin in the absence of MTs , implying tight ADP binding within the error of the measurement ( Hackney , 1988; Gilbert et al . , 1995; Cochran et al . , 2004; Ma and Taylor , 1995 ) .", "However , as is evident from Figure 1D , the corresponding plot for MKLP2-MD intercepts the y axis well above the origin .", "Although this is an indirect way of calculating the ADP dissociation rate constant , it implies that the interaction of ADP with MKLP2 is unlike that of other kinesins .", "In order to directly examine the kinetics of ADP release from MKLP2-MD in the absence of MTs , we formed a complex of MKLP2-MD:2’dmD and mixed it in the stopped flow with a large excess of unlabelled ADP .", "Figure 1—figure supplement1 shows that the resulting fluorescence decrease , which reflects 2’dmD dissociation , fits a single exponential process with rate constant of 51 . 9 ± 0 . 1 s−1 , i . e . the rate of ADP release from MKLP2-MD in the absence of MTs is unusually high .", "Taken together , these data suggest that there is a relative uncoupling of allosteric communication between the MT binding and catalytic sites in MKLP2-MD compared to other kinesins that have been described ( Cochran et al . , 2004; Ma and Taylor , 1995 ) .", "The binding affinity of MKLP2-MD to MTs was measured as a function of nucleotide state by a co-sedimentation assay , using a fixed [MKLP2-MD] and a range of [MT] .", "Plots of fraction of bound MKLP2-MD versus [MT] are depicted in Figure 1E .", "These were fit to a quadratic function assuming a 1:1 motor:tubulin dimer stoichiometry to yield apparent dissociation constants ( Figure 1E; Table 1 ) .", "Like other kinesin family members ( Table", "1 ) ( Atherton et al . , 2014; Cochran et al . , 2004; Rosenfeld et al . , 1996; Nitta et al . , 2004; Gigant et al . , 2013; Cochran et al . , 2006 ) , MT affinity of MKLP2-MD is modulated by the state of the catalytic site , with binding in the absence of nucleotide ( NN ) or presence of AMPPNP demonstrating a higher affinity than that with ADP or ADP . AlFx ( Table 1 ) .", "In marked contrast to other kinesins , however , the ADP state of MKLP2-MD - which usually represents the kinesin weak binding state ( Rosenfeld et al . , 1996; Gigant et al . , 2013; Crevel et al . , 1996 ) – demonstrates a relatively high affinity .", "Thus , the difference in affinity between nucleotide states that in other kinesins are ‘strong’ binding ( NN , AMPPNP ) and ‘weak’ binding ( ADP ) is only about 8 . 5-fold .", "Overall , MKLP2-MD associates with MTs relatively strongly throughout its ATPase cycle .", "Together , these results provide additional evidence of the non-canonical properties of the MKLP2-MD mechanism .", "To visualise the MKLP2-MD ( Figure 2A ) and provide insight into these unusual biochemical properties , we calculated MT-bound MKLP2-MD reconstructions with different nucleotides bound: ( 1 ) ADP; ( 2 ) no nucleotide ( NN ) using apyrase treatment; ( 3 , 4 ) ATP-like analogues ( AMPPNP and ADP . AlFx ) ( Figure 2—figure supplement 1 ) .", "Our ability to determine an ADP-bound structure highlights the tighter association of this motor with MTs throughout its ATPase cycle and is consistent with the biochemical data ( Figure 1E ) .", "The asymmetric unit of all these reconstructions is the MKLP2 motor domain bound to an αβ-tubulin dimer within the MT ( Figure 2B ) , which shows a resolution gradient between the MT and bound MKLP2 ( Figure 2—figure supplement 1; Figure 2—figure supplement 2 ) ; for interpretation of MKLP2-MD mechanochemistry , reconstructions were filtered according to the local resolution of the motor domain .", "To facilitate interpretation of the cryo-EM density , we calculated comparative models for each nucleotide state and docked them into the density using flexible fitting ( Table 2 , Figure 2—figure supplement 3 , Figure 2—figure supplement 4 ) .", "In addition , multiple loop conformations were generated and clustered to represent loops with ambiguous fit .", "This yielded a set of models ( Table 2; Figure 2—figure supplement 4 ) that show nucleotide-dependent conformational changes of MT-bound MKLP2 including the conformation and variation of the MKLP2-specific inserts ( Figure 2A ) .", "These structures show that the overall organisation of MKLP2-MD is similar compared to other kinesins , with the major contact site between motor and MT being centred over the tubulin intradimer interface ( Figure 2B ) .", "The coordinated conformational changes that occur in Kin1 in response to MT and nucleotide binding have been described in terms of three independently articulated subdomains that move with respect to each other during the MT-bound ATPase cycle ( Shang et al . , 2014; Cao et al . , 2014 ) : the tubulin-binding subdomain , the Switch I/II subdomain and the P-loop subdomain ( Figure 2C ) .", "This structural simplification is useful in describing global rearrangements in the motor domain .", "Our reconstructions demonstrate that the overall features of nucleotide-dependent subdomain movement described for Kin1 also apply to MKLP2 ( Figure 2D; Video 1 ) , albeit the comparison is incomplete given the lack of a tubulin/MT-bound Kin1 ADP structure .", "In the transition from ADP-bound to NN structures ( Figure 2D , left to middle ) , the MKLP2 Switch I/II and P-loop subdomains both rotate away from each other slightly and relative to the static tubulin-binding domain .", "In the transition from the NN to ADP . AlFx conformations ( Figure 2D , middle to right ) , both domains rotate towards the catalytic site , with the rotation of the P-loop subdomain being greater than the Switch I/II subdomain .", "This supports the idea that these subdomain movements are a conserved facet of kinesin mechanochemistry even amongst motors with highly distinct functions .", "To determine how MKLP2’s divergent mechanochemistry functionally harnesses these subdomain movements , and to further characterise MKLP2’s differences compared to other kinesins , we examined the response of different regions of the MT-bound MKLP2-MD in different nucleotide states .", "At the MKLP2-MD catalytic site , the nucleotide-dependent movement of three conserved , mobile elements that lie at the junction of the three subdomains can be tracked: ( 1 ) the P-loop ( brown ) ; ( 2 ) loop9 ( yellow , contains Switch I ) ; ( 3 ) loop 11 ( red , contains Switch II ) ( Figure 3 , Video 2 ) .", "The N-terminal half of helix-α4 lies at the back of the nucleotide-binding site and provides a structural link to the MT binding interface .", "These elements are sensors of the nucleotide present in the active site and also participate in the conformational response of the motor domain to nucleotide .", "The conformation ( s ) of MT-bound kinesins in the presence of ADP are still relatively poorly described ( Atherton et al . , 2014 ) especially at high resolution , and depend on the MT affinity and kinetic behaviour of individual proteins .", "The properties of MKLP2-MD are such that an MT-bound MKLP2-MD-ADP structure could be captured , in which density corresponding to ADP is coordinated primarily via the P-loop with some connectivity to Switch II/loop11 ( Figure 3 , left; Figure 2—figure supplement 1 ) .", "A portion of loop11 is visible near the P-loop and the N-terminus of helix-α6 , while most is not visible .", "This flexibility in loop11 is also seen in Kin1-ADP ( Kull et al . , 1996 ) and MT-bound Kin3-ADP ( Atherton et al . , 2014 ) .", "However , in MT-bound MKLP2-MD-ADP , loop9 as well as the N-terminus of helix-α4 are more flexible compared to these other kinesin-ADP conformations , reflecting distinct properties of MKLP2-MD .", "Highly unusually , in the MKLP2-MD-NN structure , no major conformational differences are seen compared to MKLP2-MD-ADP ( Figure 3 , middle ) , although nucleotide density is clearly lacking from the active site ( Figure 2—figure supplement 1 ) .", "Local differences due to subtle rotations in both the Switch I/II and the P-loop subdomains are seen ( Figure 2D , middle ) , and helix-α4 is longer by ~1 turn compared to MKLP2-MD-ADP , but its N-terminal end along with most of loop11 are flexible .", "However , unlike in the ADP-bound structure , a region of Switch II density in loop11 now appears connected to helix-α4 .", "The connection to helix-α4 is close to the highly conserved ‘linchpin’ Asn ( Shang et al . , 2014 ) ( N430 in mouse MKLP2 , N255 in Kin1 ) , is also seen in Kin1/3 NN states ( Atherton et al . , 2014; Shang et al . , 2014; Cao et al . , 2014 ) .", "In addition , density corresponding to the P-loop has shifted away from the MT surface , and while density corresponding to loop9 is visible , with part of it contacting the P-loop ( Figure 3 , middle ) , loop9 does not adopt a defined conformation , due to flexibility .", "The overall disordered nature of the N-terminus of helix-α4 , loop 11 and loop 9 is very different from the more ordered conformation of these elements consistently seen in the MT-bound NN state of other plus-end kinesins ( Figure 3—figure supplement 1 ) ( Atherton et al . , 2014; Shang et al . , 2014; Cao et al . , 2014; Goulet et al . , 2012 ) .", "Moreover , the well-defined connectivity of loop11 and the MT surface seen in Kin1/3/5 NN states is not well ordered in MKLP2-MD .", "Thus , the MKLP2-MD-ADP and –NN structures are more similar to each other with respect to flexibility of MT binding elements at the helix-α4 N-terminus and loop11 than is typical for other kinesins .", "Transition to the MKLP2-MD-ADP . AlFx structure causes substantial local rearrangements in the vicinity of the active site ( Figure 3 , right ) , which are in turn linked to large subdomain rearrangements compared to the ADP-to-NN transition ( Figure 2D ) .", "These rearrangements can also be described by the configuration of clefts between the subdomains ( Shang et al . , 2014 ) ; Figure 3—figure supplement 2 , see also Discussion ) .", "First , density connects the P-loop and loop11 in the presence of bound nucleotide ( Figure 3 , right ) .", "However , in the presence of the ADP . Pi-like analogue , stable conformations for all components around the active site are also observed: density corresponding to the N-terminal end of helix-α4 and loop11 are seen and exhibit connectivity to adjacent regions ( Figure 3 , right ) , while the N-terminus of loop11 runs past the P-loop , the γ-phosphate mimic , and is connected to helix-α4 , ( Figure 3 , right ) .", "The C-terminal end of loop11 forms a helical turn and density in this region connects to the N-terminus of helix-α6 .", "There is also connectivity between the N-terminal end of helix-α4 and loop9 , while the proximity of loop9 and loop11 is consistent with a ‘phosphate tube’ structure as seen in other ATP/ADP . Pi-like kinesin structures ( Gigant et al . , 2013; Sindelar and Downing , 2010; Chang et al . , 2013; Parke et al . , 2010 ) .", "Overall , the compactness of the ATPase site is consistent with adoption of a catalytically competent conformation ( Parke et al . , 2010 ) , similar to that seen in other kinesins , highlighting mechanochemical conservation within the superfamily with respect to closure of the active site to enable ATP hydrolysis .", "However , the MKLP2-MD-AMPPNP reconstruction illustrates another interesting divergence of MKLP2-MD mechanochemistry compared to Kin1/3 ( Figure 3—figure supplement 3 ) .", "In Kin1 and Kin3 , the motor conformation in the presence of AMPPNP and ADP . AlFx are essentially indistinguishable ( Atherton et al . , 2014; Gigant et al . , 2013 ) .", "However , in MKLP2-MD , while density corresponding to bound AMPPNP is observed ( Figure 2—figure supplement 1 ) , none of the characteristic features in the motor observed for the catalytically competent ADP . AlFx conformation are seen upon AMPPNP binding .", "Instead , this reconstruction exhibits a similar conformation to the MKLP2-ADP and –NN reconstructions , such that at the nucleotide binding site , helix-α4 shows a partially extended conformation and only some density corresponding to loop9 and loop11 is visible ( Figure 3—figure supplement 3B ) .", "This reconstruction thereby illustrates the large difference in structural response of MKLP2-MD to different ATP-like analogues - AMPPNP compared to ADP . AlFx – and is a further readout of the divergent properties of MKLP2 .", "The divergent properties of MKLP2-MD are further emphasised in its interaction with the MT surface .", "The MKLP2-MD interface with the MT is composed of nucleotide-invariant and nucleotide-sensitive elements .", "The nucleotide-invariant elements are formed by the tubulin-binding subdomain ( Figure 2C , D ) .", "In this subdomain of MKLP2-MD , a 5 aa insertion ( Figure 2A ) both contributes an additional helical turn to the C-terminus of helix-α4 and adds length to loop12 compared to Kin1 ( Figure 4A ) ; however this insert does not contact the MT . This C-terminus extension of helix-α4 is nucleotide-insensitive ( Figure 4—figure supplement 1A , D ) and thus is structurally similar to the helix-α4 extension formed by part of Kin3’s loop12 insert ( Atherton et al . , 2014 ) , although these insertions have no sequence homology .", "MKLP2-MD subdomain rearrangement in response to nucleotide ( Figure 2D ) causes nucleotide-dependent conformational changes in other elements at the MT binding interface .", "This includes movement of loop7 in the Switch I/II subdomain closer to the MT in the ATP-like state ( Figure 4B , Figure 4—figure supplement 1B , E ) , and movement of helix-α6 in the P-loop subdomain relative to the MT surface ( Figure 2D ) .", "As described above , however , in the vicinity of the nucleotide binding site , MKLP2-MD loop11 and helix-α4’s N-terminus are less ordered than in Kin1 in the absence of nucleotide ( Figure 3 , Figure 3—figure supplement 1C , D , E ) .", "MKLP2’s Switch I/II subdomain also contains a 5 aa insertion in loop8 ( Figure 2A ) , which is only well ordered in the presence of ADP . AlFx , and reaches across to contact H10-β9 of the neighbouring protofilament within the MT ( Figure 4B , Figure 4—figure supplement 1B , E ) .", "To our knowledge MKLP2-MD is the first kinesin described where a monomeric motor domain can bridge across two protofilaments .", "MKLP2-MD also has a larger 18 aa insert ( Figure 2A ) in both the β-sheet1 and loop2 of the P-loop subdomain .", "In the NN and ADP states , density for this insert is not visible unless much less conservative thresholds are used , indicating greater flexibility of loop2 in these two states ( Figure 4—figure supplement 1C , F ) .", "In the presence of ADP . AlFx , density for this element extends from the minus end of the MKLP2-MD and connects to α-tubulin ( Figure 4C ) , consistent with the large rotation of the P-loop subdomain in this ADP . AlFx state ( Figure 2D ) .", "Compared to Kin1 , not only does the MKLP2-MD contain extra sequences that contact the MT , but the precise position and orientation of MKLP2-MD as a whole is also noticeably shifted and rotated on the MT surface ( Figure 4D ) .", "A similar shift and rotation is present in both our MT-bound NN and ADP . AlFx MKLP2-MD reconstructions ( Figure 4—figure supplement 2 ) , and is therefore independent of the nucleotide-dependent conformational changes .", "This shift and rotation particularly alters the MT interface of MKLP2-MD near the nucleotide binding pocket .", "For example , in MKLP2-MD , loop7 is closer to β-tubulin ( Figure 4E ) compared to Kin1 , while the ordered single-turn helix in loop11 is closer to H3 than H12 in α-tubulin in the MKLP2-MD-ADP . AlFx state ( Figure 4F ) .", "Altogether , both kinesin-conserved and MKLP2-MD-specific elements contribute to the motor’s interaction with the MT , several of which are nucleotide sensitive .", "The collective effect of MKLP2-specific modifications leads to the shift and rotation of the MKLP2-MD on the MT surface .", "The differences in MKLP-MD compared to Kin1 also modify its footprint on the MT surface , leading to MKLP2-MD contacting a greater MT surface area in all states ( Figure 5; Figure 5—figure supplement 1 ) .", "The enhancing effect of the shift and rotation ( Figure 4D ) is most clearly seen in the increased footprint of loop7 and α4 ( in both NN and ADP . AlFx states ) , and loop11 in the ADP . AlFx state ( Figure 5 , Figure 5—figure supplement 1 ) .", "The transition from NN to ADP . AlFx states and further rotation of the Switch I/II subdomain brings loop7 and loop8 into even more extensive contact with β-tubulin’s H4 , while full ordering of helix-α4 and loop11 adjacent to the active site leads to additional contacts with α-tubulin .", "Moreover , ordering of MKLP2-specfic insertions that are present in the ADP . AlFx reconstruction produce a further enhancement of the MKLP2-MD footprint ( Figure 5 ) .", "In addition to the enlarged footprint of MKLP2-MD on the negatively charged MT surface ( Woehlke et al . , 1997 ) , the electrostatic surface of MKLP2-MD shows a more pronounced positive interaction surface compared to kinesin-1 , especially around the C-terminal end of helix-α4 , a region that is nucleotide invariant .", "Thus , through its ATPase cycle , the footprint of MKLP2-MD on the MT is altered and is most different in its ATP-like state .", "Nucleotide-dependent subdomain rearrangements in MKLP2-MD yield conformational changes at both termini of the motor .", "This includes opening of the cleft between the P-loop and tubulin-binding subdomains that can accommodate neck-linker reorientation and docking upon ATP binding ( Figure 6 ) .", "With ADP bound , as for other kinesins , the C-terminal helix-α6 lies close to the motor-MT interface and terminates adjacent to the C-terminus of helix-α4 ( Figure 6A , arrow ) .", "The density for helix-α6 is less well-defined , with a length of three helical turns at most and is thus shorter than typically seen in other kinesins ( Figure 6A , arrowhead; [Atherton et al . , 2014; Shang et al . , 2014] ) .", "In this configuration , extension of helix-α6 , docking of the neck-linker and formation of the cover-neck bundle ( CNB ) are all prevented .", "Strong density corresponding to only a few residues of each of the N- and C-termini is observed , although additional density alongside β-sheet1 is visible suggesting partial occupancy of neck-linker conformers directed towards the MT minus end ( Figure 6A , dotted line ) .", "In the NN reconstruction , the overall organisation of this region is very similar to that in the ADP state ( Figure 6B ) ( and of the AMPPNP state , Figure 6—figure supplement 1 ) , although density corresponding to helix-α6 is better defined in the NN compared to the ADP structure .", "Following the subdomain rearrangement accompanying the NN/ADP . AlFx transition , the neck-linker cleft opens .", "Helix-α6 of MKLP2-MD extends beneath the core β-sheet , thereby directing the neck-linker towards the MT plus end ( Figure 6C ) .", "This is consistent with the presence of a conserved cluster of hydrophobic residues in helix-α4 , helix-α6 and the N-terminus that form a structural pocket that accommodates the helix-α6 extension and neck-linker reorientation ( Figure 6D ) .", "This is also consistent with orientation of the N-terminus towards the MT plus end and formation of the CNB .", "The density of these N-terminal residues corresponds to S61-V64 and for the neck-linker corresponds to Q505-H508 .", "In support of partial docking and reorientation of the neck-linker , anisotropy decay curves show that the MKLP2-MD neck-linker becomes more ordered in the presence of ADP . AlFx - as shown by the increase in rotational correlation time - compared to the absence of nucleotide , and depends on MT binding ( Figure 6E ) .", "Strikingly , however , density corresponding to C-terminal residues extending from around H508 , which would correspond to a docked neck-linker conformation , is not visible in the ADP . AlFx density map ( Figure 6F ) .", "Residues of the MKLP2-MD extended N-terminus prior to S61 are also not visible .", "Consistent with a non-canonical neck-linker conformation , the neck-linker sequence itself differs markedly from other plus-end kinesins ( Figure 6D ) .", "Most plus-end directed kinesins have a conserved sequence in which two Asn residues make hydrogen bonds against the core , stabilizing neck-linker docking ( Gigant et al . , 2013 ) ( Figure 6D , Figure 6—figure supplement 2 ) .", "In MKLP2 , these two Asn residues have been substituted for basic His residues , which would be predicted to preclude neck-linker docking .", "In addition , conserved core residues contacting this region of the neck-linker in plus-end directed kinesins are not fully conserved in MKLP2; for example Kin1-Y77→MKLP2- Q151 ( Figure 6D ) .", "Thus , although subdomain rearrangement results in reorientation of the MKLP2-MD neck-linker towards the MT plus end – as with other plus end kinesins – the docking of the neck linker that is typically seen does not occur in MKLP2-MD .", "Loop6 is the largest MKLP2-specific insertion ( 99 aa ) and density corresponding to it is present in all nucleotide states ( Figure 7 , Figure 7—figure supplement 1 ) .", "The higher resolution of the ADP . AlFx reconstruction facilitates the characterisation of density corresponding to loop6 ( Figure 7A , B , Video 3 ) , which emerges from the side of the motor domain facing the MT plus end .", "Given its position , connectivity and its apparent movement in the NN/ADP . AlFx transition , it is effectively part of the SwI/II subdomain ( Figure 7C ) .", "Loop6 forms density that spreads across the MT-facing surface of the central β-sheet .", "This density also connects the MT facing surface of the core β-sheet with β-sheet-5a/b of the tubulin-binding subdomain that connects to the MT surface ( Figure 7B ) .", "However , loop6 itself does not appear to contact the MT surface .", "Creating a full model of loop6 was not possible , but secondary structure prediction of loop6 converged on the presence of a 4-turn helical region towards its N-terminus ( Figure 7—figure supplement 1E ) .", "Density that corresponds to a helical structure was observed lying close to helix-α3 and we modelled an α-helix within it ( Figure 7A , B ) .", "However , the rest of loop6 has no predicted secondary structure and may be intrinsically disordered ( Seeger and Rice , 2013 ) .", "As displayed in Figure 7A , B , loop6 is incompletely visualised , partly due to the lack of discrete secondary structural elements and also presumably due to flexibility in this region of the motor .", "This conclusion is reinforced when , with coarser ( low-pass ) filtering of the EM reconstruction , a large and lower resolution cloud of density is revealed ( Figure 7D ) , consistent with what was previously described from a 2 . 5 nm resolution reconstruction of the C . elegans kinesin-6 , MKLP1 ( Zen4 ) ( Hizlan et al . , 2006 ) .", "This density is unconnected to other regions of the motor domain , and does not contact adjacent motor domains or the MT , but likely corresponds to multiple flexible conformations of the remaining loop6 residues ." ], [ "While the cell biological contributions of MKLP2 and other kinesin-6s during mitosis are increasingly well understood , little has been known about the molecular mechanism of these divergent mitotic motors .", "Using cryo-EM to generate structures that guide comparative modelling and flexible fitting , we have been able to visualise sequential states of the MT-bound MKLP2-MD ATPase cycle .", "Our structural dissection of a mammalian MKLP2-MD allows identification of aspects of its mechanism that appear conserved among kinesins along with many properties that differ substantially from other members of the superfamily .", "Our set of structures allows us to characterise the response of the track-bound MKLP2-MD to nucleotide .", "Some aspects of its nucleotide-dependent conformational responses appear to be similar to Kin1/3/5 , and can be framed in terms of relative movements of subdomains within the motor domain and the clefts that open and close between them ( Video 1 , Figure 6 , Figure 3—figure supplement 2 ) ( Shang et al . , 2014; Cao et al . , 2014 ) .", "The so-called nucleotide cleft ( NC ) that lies between the Switch I/II and P-loop subdomains is predicted to close in the presence of bound nucleotide and to open in its absence .", "The polymer cleft ( PC ) lies between the tubulin-binding and the Switch I/II subdomains; it is closed in the presence of a close interaction between helix-α4 and SwII and open in its absence , and is proposed to change at different points in the MT-bound kinesin ATPase cycle .", "The docking cleft ( DC ) lies between the P-loop and tubulin-binding subdomains and when open allows insertion of helix-α6 and reorientation of the neck-linker towards the MT plus end ( Shang et al . , 2014 ) .", "Thus , for MKLP2-MD:", "( i ) in the ADP-bound structure , the PC is open , while the NC is closed and the DC for the neck-linker is also closed;", "( ii ) in the NN state , the PC is closed while the NC is open , such that the DC remains closed;", "( iii ) in the ADP . AlFx state in which the active site is closed in a catalytic-competent conformation , both the PC and NC are closed leading to an opening of the DC and reorientation of the neck-linker towards the MT plus end .", "Our MKLP2-MD structures thus reinforce the logic of cleft opening/closure with respect to kinesin mechanochemistry first described for Kin1 ( Shang et al . , 2014 ) , while demonstrating an elaboration of motor function through family-specific modifications .", "The biochemistry of MKLP2 is highly divergent compared to canonical kinesins .", "The kcat of MKLP2-MD ( 4 . 4 s−1 ) is ~10 fold lower than kinesin-1 and -3 ( Table 1 ) and a number of MKLP2-MD substitutions at otherwise highly conserved regions could influence multiple steps in its catalytic cycle .", "For example , MKLP2-MD has low ADP affinity in solution and this is barely altered on interaction with MTs .", "Indeed , the ADP release rate in the absence of MTs is only 2–3-fold less than that for Kin1/3 s in the presence of saturating MTs .", "Thus , while MT binding activates the MKLP2 ATPase , its basal ADP release rate is higher than kcat .", "The small structural changes observed when comparing our ADP and NN structures – in particular , that the N-terminus of helix-α4 and loop11 remain largely disordered in the NN state – is a distinctive property of these MKLP2/MT complexes compared to other plus end kinesins ( Figure 3—figure supplement 1 [Atherton et al . , 2014; Shang et al . , 2014; Goulet et al . , 2014; Cao et al . , 2014] ) .", "Substitutions at conserved sites around the nucleotide pocket might be predicted to destabilise the nucleotide binding network that is present in other kinesins .", "For example , substitution in loop9 at a residue otherwise completely conserved in all kinesins ( Kin1-R190→MKLP2-Q365 , Figure 3—figure supplement 1A ) could perturb side chain interactions proposed to be part of the so-called ‘Mg2+ stabiliser’ ( Chang et al . , 2013 ) .", "Indeed , mutagenesis of this network in Kin1 ( Cao et al . , 2014 ) or Kin3 ( Nitta et al . , 2008 ) increases ADP release rates in the absence of MTs .", "Similarly , substitutions in and around the P-loop ( Kin1-A83 →MKLP2-T157 and Kin1-Q86 →MKLP2-V160 ) , along with helix-α6 ( Kin1-N308→MKLP2-T489 ) may perturb the stability of loop11/P-loop at the active site through alteration of the so-called ‘anchor’ network ( Chang et al . , 2013 ) .", "Loop11 itself is one residue shorter than in other N-kinesins , adopts a different conformation compared to Kin1 and contains substitutions in residues that contribute to stabilisation of Kin1’s ordered conformation in the NN and ATP-like states ( Kin1-T241→MKLP2-Q417 , and Kin1-A243→MKLP2-S419 , Figure 3—figure supplement 1A ) .", "We conclude that the collective effect of these substitutions – along with possible effects from the shift of MKLP2-MD binding on the MT surface – is likely to be the reason for lower stabilisation of nucleotide binding to MKLP2-MD .", "Given these properties , it is thus not clear what state our ADP-bound reconstruction corresponds to in terms of intermediates in the MKLP2 ATPase cycle .", "It could for example be the MKLP2-MD-ADP complex as it binds the MT from solution , or could represent a MT-bound NN structure captured after ADP binding .", "Given the fast kinetics of ADP release by MKLP2-MD , the latter seems more likely .", "Therefore , previously proposed mechanisms of MT-stimulated ADP release in Kin1/3 s may not be applicable to MKLP2-MD ( Atherton et al . , 2014; Shang et al . , 2014; Cao et al . , 2014 ) .", "Nevertheless , the position of the P-loop is shifted away from the MT in the MKLP2-NN structure compared to the ADP-bound structure and the PC is closed , conformational changes that were described to coincide with ADP release in Kin1 ( Shang et al . , 2014; Cao et al . , 2014 ) .", "While apparently redundant in actively driving ADP release in MKLP2 , these cleft and subdomain transitions when ADP is absent may still be important for subsequent larger scale rearrangements in the motor domain on ATP-binding and its conversion towards a hydrolysis-competent state .", "The structure of the MKLP2-MD-ADP . AlFx reconstruction suggests that MT binding participates in stabilising helix-α4 and loop11 in this structure , supporting the conserved SwitchI/II and P-loop residues in adopting a catalytically relevant conformation .", "The MKLP2-ADP . AlFx reconstruction is overall comparable to hydrolysis-competent conformations seen in Kin1/3/4/5 with a so-called phosphate tube formed from loop9 and 11 supporting ATP hydrolysis .", "However , the reconstruction in the presence of AMPPNP most closely resembles that of ADP – rather than ADP . AlFx as is seen in Kin1/3 – and it may also represent the MKLP2-MD NN conformation after AMPPNP has bound to it .", "This too could be a reflection of MKLP2-specific substitutions within and around the nucleotide binding site ( Figure 3—figure supplement 1 ) that influence the binding kinetics and structural transitions of MT-bound MKLP2-MD in response to different nucleotides , and which also contribute to the overall turnover rate of the motor’s ATPase .", "Precedents for different responses to different nucleotide analogues have also been observed in other kinesins – for example , small differences in motor conformation between AMPPNP and ADP . AlFx were observed for human Kin5 ( Goulet et al . , 2014 ) .", "Future detailed kinetics will yield further insights into the defining transitions of MKLP2 during its ATPase cycle .", "The MKLP2-MD steady state ATPase activity shows that its K0 . 5 , MT is ~1 . 1 μM .", "This agrees well with the co-sedimentation analysis , which also shows that , while the affinity is higher in the NN and AMPPNP states , there is less effect of nucleotide on the overall affinity of the MKLP2-MD for MTs compared to Kin1 .", "Our reconstructions do not provide obvious structural explanations for these subtle nucleotide-dependent differences in the MT affinity of MKLP2-MD .", "Indeed , the additional MKLP2-MD loop contacts with the MT were modelled most easily in the highest resolution reconstruction ( ADP . AlFx ) , which has a slightly lower MT affinity .", "It is thus possible that the flexible loops formed by the MKLP2-MD specific insertions do not directly contribute to differences in affinity in this motor compared to Kin1; rather this is dominated by the differences in the surface charge of the MT interface ( Figure 5 ) .", "Further insight concerning the effects on the kinetics of structural transitions induced by different nucleotides will be required to fully dissect these effects .", "MKLP2-MD K0 . 5 , MT is ~10 fold lower than the Kin1 motor domain ( 13 μM ) ( Atherton et al . , 2014 ) .", "While both motors bind MTs via their conserved tubulin-binding subdomains , strikingly the MKLP2-MD MT interface is overall more positively charged than that of Kin1 , which likely contributes to the overall increased affinity of MKLP2 throughout the ATPase cycle .", "In addition , the MKLP2-specific , nucleotide invariant extension of loop12 is positively charged and may interact with the nearby acidic C-terminal tails ( CTTs ) of β-tubulin , although there is no visible connection in our reconstructions , likely due to its conformational variability .", "A similar interaction between the β-tubulin CTT and loop12 of Kin3 has previously been described ( Okada and Hirokawa , 2000 ) , although Kin3 loop12 has a higher net positive charge ( +4 ) than MKLP2 and is longer and conformationally flexible in all nucleotide states ( Atherton et al . , 2014 ) .", "Sequence insertions within loop2 are more usually associated with depolymerisation activity , for example in Kin13s ( Ogawa et al . , 2004 ) , but here MKLP2-MD loop2 appears to simply form a MT contact , with no current evidence of MKLP2 activity influencing MT dynamics .", "In addition to these loop insertions creating additional MT contacts with the primary tubulin binding site , MKLP2-MD loop8 can reach to contact an adjacent protofilament .", "The collective effects of all the differences between MKLP2-MD and Kin1/3 manifest in the striking observation of bound MKLP2-MD shifted rotationally and translationally relative to the MT surface in all nucleotide states , creating a different footprint on the MT surface .", "This produces altered interactions across the MT surface including in loop11 adjacent to the active site and , together with the MKLP2-specific modifications around the active site , may have a knock-on effect on the catalytic activity of the motor .", "Thus , while not sufficient to perturb the fundamentals of nucleotide-sensitive subdomain rearrangement or formation of a hydrolysis competent conformation at the active site , MKLP2-MD altered position on the MT surface may regulate the transition rates between structural states .", "The altered footprint may also enable MKLP2 to select different populations of MT tracks within the spindle for interaction .", "Thus , our structural characterisation of MKLP2-MD suggests that the binding site of kinesins on the MT surface has some previously unsuspected plasticity that enables tuning of motor function while still supporting conserved conformational changes .", "In Kin1/3/5 , the key conformational change elicited by ATP binding to MT-associated motors is ordering of the neck-linker towards the MT plus end ( Rice et al . , 1999 ) .", "Such neck-linker docking is supported by formation of the CNB between the neck-linker and the motor domain N-terminus ( Khalil et al . , 2008 ) .", "In MKLP2 , reorientation of the neck-linker and CNB formation occur in the presence of ADP . AlFx and the neck-linker is less mobile than in other nucleotide states ( Figure 6E ) .", "This is despite the shortness of helix-α6 in the ADP/NN states and substitution of Kin1-K323→MKLP2-S504 at the transition between helix-α6 and neck linker .", "CNB formation is , however , consistent with conservation across all N-kinesins including MKLP2 of a cluster of hydrophobic residues in this region of the motor domain ( Figure 6 [Hwang et al . , 2008] ) .", "Formation of the CNB also contributes to stable closure of the nucleotide binding pocket and supports ATPase activity ( Cao et al . , 2014 ) .", "Remarkably , however , the distal region of the putative MKLP2-MD neck-linker does not visibly dock along the plus end portion of the motor domain core , and demonstrates residual flexibility despite CNB formation .", "This observation is consistent with the divergent MKLP2 sequences both in the neck-linker and along the core β-sheet where the neck-linker might otherwise dock ( Figure 6D , Figure 6—figure supplement 2 ) [Vale and Fletterick , 1997] ) .", "Furthermore , the MKLP2-MD neck-linker also contains two adjacent proline residues ( P510 and P511 ) that are likely to affect the conformation of the neck-linker .", "Divergence in the distal neck-linker region is a feature of the MKLP2 sequence in a number of organisms ( Figure 6—figure supplement 2 ) .", "Indeed , our structural findings suggest that analysis of sequences immediately C-terminal of kinesin motor domains could be considered in two segments according to ( 1 ) CNB formation by the proximal region and ( 2 ) docking of the distal region , when predicting motor function .", "It is not known if dimers of MKLP2 operate by taking steps in the same way as characterised for transport kinesins , and our structures predict that the molecular properties of full length MKLP2 are likely to be distinct from Kin1 .", "In addition , the MKLP2 neck region – separating the neck-linker and the coiled coil – is substantially longer ( ~40 residues ) than for example Kin1 , making it unlikely that conventional models of processive stepping apply to MKLP2 .", "An extended loop6 insertion is characteristic of kinesin-6s and was visible in our MKLP2 reconstructions , with its location and its partial conformation visualised both at lower and higher resolutions , respectively ( Figure 7 , Video 3 ) .", "In addition , sequence analysis suggests that the N-terminal helical portion of loop6 is also likely to be conserved in MKLP2s in metazoa ( Figure 7—figure supplement 1E ) .", "During preparation of our manuscript , a structural snapshot of the C . elegans kinesin-6 Zen4 motor domain in the absence of its MT/tubulin track was published ( Guan et al . , 2017 ) .", "Intriguingly , although the loop6 insertion of this MKLP1 relative is substantially shorter than in MKLP2 , a helical segment is also observed in this region of Zen4 , consistent with our predictions .", "However , the connectivity of loop6 in the two kinesin-6s is different , with MKLP2 loop6 forming much more extensive contacts with other regions within the SwI/II subdomain .", "This leaves open the question as to this loop’s functional contribution to MKLP2 .", "Given its connectivity with more conserved portions of the motor domain such as loop8/beta-5 , it could have an overall modulatory role on motor mechanochemistry .", "It is also possible that it is the binding site for other regulatory components of the central spindle .", "Sequence comparisons informed by our structural data suggest that the presence of loop6 , other extended insertions including loop2 , and modifications related to neck-linker docking are highly characteristic of this subfamily of motors .", "Thus , our structural data shed light on the unusual mechanochemistry of the MKLP2-MD and could be used to predict the properties of other members of the kinesin-6 subfamily , including the two other kinesin-6 family members within vertebrates .", "Similarly , sequence analysis in the structural context of one highly divergent kinesin subfamily can also be mapped onto other members of the wider superfamily .", "Our structural and biophysical characterisations of the MKLP2 motor domain suggest that this protein and its relatives could act as an organiser or a tension sensor , rather than a classical transport motor , within the MT bundles at the spindle midzone .", "A non-transport role for kinesin-6s was also implied by recent computational analysis of the kinesin superfamily ( Richard et al . , 2016 ) .", "Nevertheless , MKLP2 inhibitor studies highlight the importance of the ATPase activity of MKLP2 for its role within the spindle , suggesting that the nucleotide-dependent conformational changes we have described are important for its mitotic function ( Labrière et al . , 2016 ) .", "The atypical mechanochemistry of MKLP2 could also facilitate the motor’s response to signalling cues throughout cell division , especially as mitosis proceeds towards cytokinesis .", "In combination with the capacity of its C-terminal region also to bind MTs ( Echard et al . , 1998 ) , teams of MKLP2 motors in the spindle midzone could contribute to collective sliding and organisation of the dense collection of MTs found there .", "The altered footprint of MKLP2-MD binding on the MT surface may influence MT bundle organisation in this context .", "The activity of MKLP2 is also subject to numerous points of post-translational modification and regulation by mitotic kinases , including within or near the motor domain .", "For example , a Cdk1 site ( T197 ) is located within loop6 in the connecting region between the body of the motor domain and the α-helix visualised in our reconstruction ( Kitagawa et al . , 2014 ) , while a Plk1 site/binding site is in the neck region beyond the neck linker ( S527 ) where other binding partners are known to interact .", "Such modifications could also fine tune the mechanochemistry of MKLP2 and regulate its precise function .", "Our characterisation of MT-bound mammalian MKLP2 by cryo-EM sets the structural stage for future studies to further dissect the mechanism , function and regulation of this divergent mitotic kinesin ." ], [ "The MKLP2 constructs were cloned into a pET28 vector with C-terminal 6His tag and expressed in BL21 Gold ( DE3 ) E . coli cells by induction with 0 . 2 mM IPTG at 20°C overnight .", "Cells were harvested by centrifugation at 4500 × g for 10 min and lysed by sonication in lysis buffer ( 50 mM HEPES , pH 7; 500 mM NaCl , 40 mM Imidazole , 1 mM TCEP , 0 . 5 mM PMSF , 0 . 1 mM ADP , 5 mM MgCl2 ) .", "The lysate was clarified at 20 , 000 × g for 45 min at 4°C; and the protein was purified by HisTrap column ( GE Healthcare Life Science , Pittsburgh , PA ) .", "Further purification was achieved by size exclusion chromatography in buffer containing 20 mM HEPES pH 7 , 100 mM NaCl , 1 mM TCEP , 5 mM MgCl2 , and 0 . 1 mM ADP .", "Fractions containing purified MKLP2 protein were pooled and concentrated to ~10 mg/ml , flash-frozen in liquid nitrogen and stored in −80°C until usage .", "For anisotropy decay studies , the 25–520 MKLP2-MD construct was modified by inserting the sequence CCPGCC at its C-terminus before the His tag sequence .", "The construct was expressed and purified as above .", "It was labelled with the fluorescein derivative FlAsH ( Molecular Probes , Eugene , OR ) by incubation of a 10-fold molar excess of label over protein in 25 mM HEPES , 50 mM KAc , 5 mM MgAc , pH 7 . 5 for 12 hr followed by removal of unbound label over prepoured Sephadex G25 columns ( GE Healthcare ) .", "The stoichiometry of labelling was typically 0 . 8–0 . 9 FlAsH:MKLP2-MD .", "MKLP2 construct ATPase activities were determined , in duplicate , in a buffer containing 50 mM KAc , 25 mM HEPES , 5 mM MgAc , and 1 mM EGTA , pH 7 . 5 ( RT ) by measuring released phosphate using a commercial kit ( EnzChek Phosphate Assay , Molecular Probes ) .", "Binding of the fluorescent nucleotide analogues 2’dmT ( in duplicate ) and 2’dmD ( once ) to 4:1 complexes of MTs:MD were measured by mixing with an excess of fluorescent nucleotide in the stopped flow at 20°C .", "Samples were rendered nucleotide free prior to mixing by incubating for 20 min with 0 . 2 U/ml apyrase ( Type VII , Sigma Aldrich , St Louis , MO ) .", "Fluorescence enhancement of the mant fluorophor was monitored by energy transfer from vicinal tryptophans by exciting at 295 nm and monitoring 90o from the incident beam through a 450 nm broad bandpass filter ( Omega Optical , Brattleboro , VT ) .", "Data were subjected to linear least squares fitting .", "Dissociation of 2’dmD from MKLP2-MD was measured by adding a 10-fold molar excess of 2’dmD to MKLP2-MD in ATPase buffer and mixing with 2 mM MgADP .", "The resulting fluorescence decrease was monitored by energy transfer from vicinal tryptophans by exciting at 295 nm and monitoring 90° from the incident beam through a 450 nm broad bandpass filter ( Omega Optical ) .", "Tubulin ( Cytoskeleton Inc , Denver , CO ) was resuspended in BRB80 ( 80 mM PIPES , 2 mM MgCl2 , 1 mM EGTA , 1 mM DTT ) to 50 μM .", "Polymerization of tubulin into MTs was carried out by addition of 1 mM GTP and incubated at 37°C for 1 hr , followed by addition of 200 μM paclitaxel ( Cytoskeleton ) and additional incubation for 1 hr at 37°C .", "Paciltaxel-stabilized MTs were kept at room temperature for >24 hr , centrifuged at 15 , 000 × g for 30 min , and resuspended to 100 μM with BRB80 buffer plus 200 μM paclitaxel .", "Equilibrium binding experiments were performed at 25°C in BRB80 buffer plus 25 mM NaCl ( all concentrations reported as final values after mixing ) .", "MKLP2-MD was incubated with ADP or ADP . AlFx for 30 min .", "For NN sample , MKLP2-MD was first treated with apyrase to remove all bound nucleotides .", "2 . 5 μM MKLP2-MD in different nucleotide state was incubated with paclitaxel-stabilized MTs ( 0–10 μM polymerized tubulin; 20 μM paclitaxel ) in a 100 μl reaction with 1 mM corresponding nucleotide .", "The mixture was incubated for 30 min and centrifuged at 100 , 000 × g for 30 min at 25°C .", "The supernatant was removed and the pellet was gently rinse with warm BRB80 buffer plus 20 μM paclitaxel .", "The pellet was resuspended in cold BRB80 buffer plus 10 mM CaCl2 .", "The supernatant and pellet from each sample were analysed on SDS-PAGE gels .", "The concentrations of MKLP2-MD in the supernatant and pellet were quantified using Image J software .", "GraphPad Prism was used for curve fitting using a quadratic function that assumes 1:1 MD:tubulin binding stoichiometry:MDbound= ( MTtotal+MDtotal+KD ) − ( MTtotal+MDtotal+KD ) 2−4 ( MTtotal ) ( MDtotal ) 2 where MDbound is [MKLP2-MD] bound to MTs , MDtotal is total MKLP2-MD in the assay , MTtotal is total MT content in the assay , and KD is the dissociation constant for binding .", "Time-resolved fluorescence anisotropy ( TFA ) data were acquired as described previously ( Muretta et al . , 2013 ) using 9 μM FlAsH labeled MKLP2-MD ( 85% labeled ) in the presence or absence of 15 μM MT . Single exponential functions to the nanosecond FlAsH anisotropy decays of labelled MKLP2-MD were fitted for:", "( i ) bound to MTs in the absence of nucleotide ,", "( ii ) in the presence of ADP . AlFx in solution or", "( iii ) bound to MTs in the presence of ADP . AlFx .", "The buffer used was 25 mM HEPES , 50 mM KAc , 5 mM MgAc , pH 7 . 5 .", "Anisotropy decays were acquired from two independent preparations of protein .", "For each biochemical condition assayed , a minimum of 5 independent samples were acquired .", "Data were subjected to non-linear optimization to determine best fitting single exponential decay parameters .", "Data reported represent the mean of parameters from fits of replicate data ± the standard error for the fit parameter .", "MTs for MKLP2-MD ADP . AlFx , AMPPNP and no nucleotide data sets were polymerized by incubating 50 μM bovine tubulin ( Cytoskeleton Inc , Denver , CO ) in MES polymerization buffer ( 100 mM MES , pH 6 . 5 , 1 mM MgCl2 , 1 mM EGTA , 1 mM DTT , 5 mM GTP ) for 1 hr at 37°C .", "GMPCPP MTs used for the MKLP2-MD ADP dataset were double-cycled by polymerizing 20 μM bovine tubulin in BRB80 buffer with 1 mM GMPCPP for 45 mins at 37°C , depolymerizing on ice and repolymerizing for a further 45 mins with an additional 2 mM GMPCPP .", "1 mM paclitaxel was then added to all MT preps , incubated for a further 1 hr at 37°C , and left at room temperature for 24–48 hr before use .", "MTs were diluted to a final concentration of 2 . 5 μM in BRB80 buffer .", "MKLP2-MD was buffer exchanged into BRB20 ( 20 mM PIPES , 2 mM MgCl2 , 1 mM EGTA , 1 mM DTT ) containing either 2 mM of AMPPNP , ADP , ADP +AlF4 , or apyrase ( 10 units/ml ) , diluted to a final concentration of 60 μM MKLP2-MD and left for 15 min at room temperature before use .", "MT and MKLP2-MD samples were added in a sequential fashion as 4 μl droplets to glow-discharged C-flatTM holey carbon EM grids ( Protochips , Morrisville , NC ) before blotting and plunge-freezing in liquid ethane using a Vitrobot ( FEI Co . , Hillsboro , OR ) .", "Images of MKLP2-MD ADP , ADP . AlFx and NN states were collected on a FEI Tecnai G2 Polara operating in low dose mode at 300 kV , using a DE20 direct electron detector ( Direct Electron , San Diego , CA ) with a final sampling of 1 . 53 Å/pixel .", "A total electron dose of ~50e-/Å2 over a 1 . 5 s exposure at 15 frames/s gave a total of 22 frames , at ~2 . 2e-/Å2 per frame .", "Images of the MKLP2-MD-AMPPNP state were collected on a FEI Tecnai F20 operating in low dose mode at 200 kV using a DE20 direct electron detector ( Direct Electron ) with a final sampling of 1 . 54 Å/pixel .", "A total electron dose of ~40e-/Å2 over a 1 s exposure at 25 frames/s gave a total of 25 frames , at ~1 . 6e-/Å2 per frame .", "To correct for sample drift and local beam induced movement respectively , individual frames were globally aligned using IMOD ( RRID:SCR_003297 ) scripts , then locally aligned using the Optical Flow approach implemented in Xmipp ( de la Rosa-Trevín et al . , 2013 ) .", "The full dose was used for particle picking and CTF determination using CTFFIND3 ( Mindell and Grigorieff , 2003 ) , while 25e-/Å2 or 20e-/Å2 were used for particle alignment , angular assignment and 3D reconstruction on data collected at 300 kV or 200 kV respectively .", "MT segments selected in Eman boxer served as input to a set of custom-designed semi-automated single-particle processing scripts utilizing Spider and Frealign as described previously , with minor modifications ( Atherton et al . , 2014; Shang et al . , 2014; Sindelar and Downing , 2010 ) .", "Poorly aligned particles were excluded from final reconstructions according to Frealign’s reported phase-residual values .", "The final dataset size ( in asymmetric units ) are: MKLP2-MD-ADP – 137 , 788; MKLP2-MD-NN – 120 , 341; MKLP2-MD-ADP . AlFx – 276 , 111; MKLP2-MD-AMPPNP – 141 , 154 .", "The resolutions of symmetrized reconstructions are shown in Figure 2—figure supplement 1A–D .", "The binding surfaces of kinesin/MTs in these final structures were coloured according to the electrostatic potential as calculated with pdb2pqr ( Dolinsky et al . , 2007 ) and APBS ( Baker et al . , 2001 ) .", "As shown in Figure 2—figure supplements 3 , 100 homology models for each nucleotide state were generated with MODELLER v9 . 15 ( RRID:SCR_008395 ) ( Sali and Blundell , 1993 ) using multiple templates ( Table 2 ) .", "Templates were selected based on sequence identity and structural identity ( inferred from secondary structure predictions made using Psipred ( RRID:SCR_010246 ) , Jpred , and RaptorX , [Drozdetskiy et al . , 2015; McGuffin et al . , 2000; Källberg et al . , 2012] ) .", "In addition , the two crystal structures of kinesin-1-α/βtubulin complexes in ADP . AlFx ( Gigant et al . , 2013 ) or no nucleotide states ( Cao et al . , 2014 ) were selected as templates to capture the nucleotide/tubulin-dependent conformations of the motor domain; and were verified as a suitable templates by rigid fitting into MKLP2-MD densities .", "Given the low sequence identity of MKLP2-MD to the identified templates ( 28–34% ) , sequence alignment was initially performed in MUSCLE ( RRID:SCR_011812 ) ( Edgar , 2004 ) , then manually adjusted , based on conserved motor domain residues identified in the Pfam database ( version 29 . 0 ) ( Bateman et al . , 2004 ) .", "For each nucleotide state , the 10 best models from MODELLER were selected using SOAP scoring ( Dong et al . , 2013 ) , and QMEAN ( Benkert et al . , 2009 ) was then used to determine the top model .", "Global QMEAN scores indicated the models were of good quality ( Table 2 ) .", "The fitted MKLP2-MD models were first refined without tubulin ( tubulin density was removed using Segger ( Pintilie et al . , 2010 ) in Chimera ( RRID:SCR_004097 ) ( Pettersen et al . , 2004 ) .", "Owing to the size and likely disordered nature of the most of loop6 , we did not model it .", "To avoid loop6 density biasing modelling , the difference mapping function in the TEMPy software package ( Joseph et al . , 2016; Farabella et al . , 2015 ) was used to remove the density .", "The Fit in Map tool ( Goddard et al . , 2007 ) in Chimera was used to perform a rigid fit of the homology models into EM maps filtered to appropriate resolution ( ADP . AlFx = 5 . 5 Å , no nucleotide = 7 Å , ADP = 7 Å , AMPPNP = 8 Å ) .", "The quality of fit was assessed using SMOC scoring in the TEMPy .", "This identified several MKLP2-MD loop insertions associated with poor fit .", "Where loops had clear corresponding density , 100–500 loop conformers were calculated using MODELLER’s loop model class ( Fiser and Sali , 2003 ) , and were ranked and selected based on improved cross-correlation .", "A hybrid approach to flexible fitting was performed using simulated annealing molecular dynamics with Flex-EM ( Topf et al . , 2008 ) , and normal mode analysis with iMODFIT ( Lopéz-Blanco and Chacón , 2013 ) similar to a previous study ( Pandurangan et al . , 2014 ) .", "Flexible fitting was performed using secondary structure elements ( SSEs ) as rigid bodies ( and loop regions treated as ‘all-atoms’ ) , and the local cross-correlation of SSEs were calculated using the SCCC scoring function in TEMPy .", "The SCCC scores for Flex-EM and iMODFIT models were compared .", "To capture the results of both fitting methods , the best fitting SSEs from iMODFIT were combined with the Flex-EM models .", "Finally , using Flex-EM , an all atom refinement was performed on loops that connected iMODFIT and Flex-EM SSEs .", "Global cross-correlation analysis showed an increase in score after fitting , and QMEAN analysis showed no degradation in model quality ( Table 2 ) .", "In addition , SMOC scores show an increase in local cross-correlation ( Figure 2—figure supplement 4 ) .", "For the ADP , NN , and AMPPNP states , there remained several loops with low local cross-correlation scores ( 7–20% below the mean score ) .", "These loops had clear corresponding density , but which was also ambiguous .", "To represent this , 500–1000 loop conformers were calculated as above .", "Following clustering based on cross-correlation and Cα RMSD , five conformers from the top cluster were selected .", "Finally , tubulin ( PDB ID: 1JFF [Löwe et al . , 2001] ) was rigidly fit into the EM densities .", "The interaction interface between MKLP2-MD and tubulin was calculated via docking driven by HADDOCK ( Dominguez et al . , 2003; van Zundert et al . , 2016 ) .", "50 non-interfacial MKLP2-tubulin residue pairs were selected at random , then the Cα-Cα distances were calculated and used as unambiguous distance restraints to preserve the MKLP2-tubulin rigid fit .", "Phi/psi angles were also restrained .", "In addition , density corresponding to loop6 was calculated by generating synthetic density of each MKLP-MD motor core model ( i . e . modelled elements apart from loop6 ) and subtracting it from the relevant experimental density ." ] ]

[ "MKLP2 , a kinesin-6 , has critical roles during the metaphase-anaphase transition and cytokinesis .", "Its motor domain contains conserved nucleotide binding motifs , but is divergent in sequence ( ~35% identity ) and size ( ~40% larger ) compared to other kinesins .", "Using cryo-electron microscopy and biophysical assays , we have undertaken a mechanochemical dissection of the microtubule-bound MKLP2 motor domain during its ATPase cycle , and show that many facets of its mechanism are distinct from other kinesins .", "While the MKLP2 neck-linker is directed towards the microtubule plus-end in an ATP-like state , it does not fully dock along the motor domain .", "Furthermore , the footprint of the MKLP2 motor domain on the MT surface is altered compared to motile kinesins , and enhanced by kinesin-6-specific sequences .", "The conformation of the highly extended loop6 insertion characteristic of kinesin-6s is nucleotide-independent and does not contact the MT surface .", "Our results emphasize the role of family-specific insertions in modulating kinesin motor function ." ]

[ "Cells constantly replicate to provide new cells for growing tissues , and to replace ageing or defective cells around the body .", "Each new cell needs a copy of the genetic material , and a cellular structure called the mitotic spindle makes sure that this material is shared correctly when a cell divides in two .", "The spindle is built from protein filaments called microtubules , and the protein filaments grow and shrink as the mitotic spindle carries out its role .", "Many of these changes in the spindle are driven by proteins called molecular motors , which break down energy-rich molecules of ATP to power them as they walk along the filaments .", "Kinesins , for example , are molecular motors that can move along microtubules and there are over 40 different kinesins encoded in the human genome .", "More than half of the human kinesins are involved in cell division including one called MKLP2 .", "Little is known about MKLP2 but some earlier findings had suggested that it would behave very differently compared to other kinesins .", "Understanding how a kinesin motor works requires studying it in complex with its microtubule tracks .", "Atherton , Yu et al . have now used a technique called cryo-electron microscopy – which is uniquely suited to looking at large and complicated samples in three dimensions – to observe how the motor in MKLP2 changes shape as it works .", "This revealed that , while MKLP2 works in a fundamentally similar way to other kinesins , many aspects of its molecular mechanism are highly unusual .", "These include how it binds to the microtubule , how it interacts with ATP and how it generates force .", "These findings show that there is much greater diversity in the molecular mechanisms of the kinesins involved in cell division than was previously thought .", "Several anticancer drugs target kinesins to stop cells dividing and so this diversity may make it easier to target only certain kinesins with drugs , which in turn would have fewer side effects .", "First , though , it will be important to find out how the unusual mechanism of MKLP2 coordinates and influences other components of the spindle to reveal a fuller picture of what happens when cells replicate ." ]

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

[ "immunology and inflammation" ]

[ [ "Rheumatoid arthritis ( RA ) is a highly prevalent systemic autoimmune disease with predilection for the joints .", "If left untreated , RA can lead to chronic joint deformity , disability , and increased mortality .", "Despite recent advances towards understanding its pathogenesis ( Mcinnes and Schett , 2011 ) , the etiology of RA remains elusive .", "Many genetic susceptibility risk alleles have been discovered and validated ( Stahl et al . , 2010 ) but are insufficient to explain disease incidence .", "RA is therefore a complex ( multifactorial ) disease requiring both environmental and genetic factors for onset ( Mcinnes and Schett , 2011 ) .", "Among environmental factors , the intestinal microbiota has emerged as a possible candidate responsible for the priming of aberrant systemic immunity in RA ( Scher and Abramson , 2011 ) .", "The microbiota encompasses hundreds of bacterial species whose products represent an enormous antigenic burden that must largely be compartmentalized to prevent immune system activation ( Littman and Pamer , 2011 ) .", "In the healthy state , intestinal lamina propria cells of both innate and adaptive immune systems cooperate to maintain physiological homeostasis .", "In RA , there is increased production of both self-reactive antibodies and pro-inflammatory T lymphocytes .", "Although mechanisms for targeting of synovium by inflammatory cells have not been fully elucidated , studies in animal models suggest that both T cell and antibody responses are involved in arthritogenesis .", "Moreover , an imbalance in the composition of the gut microbiota can alter local T-cell responses and modulate systemic inflammation .", "Mice rendered deficient for the microbiota ( germ-free ) lack pro-inflammatory Th17 cells , and colonization of the gastrointestinal tract with segmented filamentous bacteria ( SFB ) , a commensal microbe commonly found in mammals , is sufficient to induce accumulation of Th17 cells in the lamina propria ( Ivanov et al . , 2009; Sczesnak et al . , 2011 ) .", "In several animal models of arthritis , mice are persistently healthy when raised in germ-free conditions .", "However , the introduction of specific gut bacterial species is sufficient to induce joint inflammation ( Rath et al . , 1996; Abdollahi-Roodsaz et al . , 2008; Wu et al . , 2010 ) , and antibiotic treatment both prevents and abrogates a rheumatoid arthritis-like phenotype in several mouse models .", "Upon mono-colonization of arthritis-prone K/BxN mice with SFB , the induced Th17 cells potentiate inflammatory disease ( Wu et al . , 2010 ) .", "An imbalance in intestinal microbial ecology , in which SFB is dominant , may result in reduced proportions or functions of anti-inflammatory regulatory T cells ( Treg ) and a predisposition towards autoimmunity .", "This appears to affect not only the local immune response , but also systemic inflammatory processes , and may explain , at least in part , reduced Treg cell function in RA patients ( Zanin-Zhorov et al . , 2010 ) .", "Thus , T cells whose functions are dictated by intestinal commensal bacteria can be effectors of pathogenesis in tissue-specific autoimmune disease .", "Although recent studies of the human microbiome ( Arumugam et al . , 2011; Human Microbiome Project Consortium , 2012 ) have characterized the composition and diversity of the healthy gut microbiome , and disease-associated studies revealed correlations between taxonomic abundance and some clinical phenotypes ( Frank et al . , 2011; Morgan et al . , 2012; Qin et al . , 2012 ) , a role for distinct microbial taxa and metagenomic markers in systemic inflammatory disease has not been defined .", "While treatment with antibiotics has been a therapeutic modality in RA for decades , no microbial organism has been shown to be associated with the disease .", "Based on the discovery that SFB-induced Th17 cells directly contribute to the onset of arthritis in gnotobiotic mice ( Wu et al . , 2010 ) , we analyzed the fecal microbiota in patients with RA .", "We used 16S ribosomal RNA gene sequencing to classify the microbiota in patients with new-onset ( untreated ) RA , chronic ( treated ) RA , psoriatic arthritis , and age- and ethnicity-matched healthy controls .", "We found a marked association of Prevotella copri with new-onset RA ( NORA ) patients and not with other patient groups .", "Shotgun sequencing of the microbiome indicated that some P . copri genes are differentially present in NORA-associated and healthy samples .", "Colonization of mice with P . copri enhanced susceptibility to chemical colitis , consistent with a pro-inflammatory potential of this organism .", "Taken together , our results suggest that NORA-associated P . copri may contribute to the pathogenesis of human arthritis ." ], [ "To determine if particular bacterial clades are associated with rheumatoid arthritis , we performed sequencing of the 16S gene ( regions V1–V2 , 454 platform ) on 114 fecal DNA samples—44 samples collected from NORA patients at time of initial diagnosis and prior to immunosuppressive treatment , 26 samples from patients with chronic , treated rheumatoid arthritis ( CRA ) , 16 samples from patients with psoriatic arthritis ( PsA ) , and 28 samples from healthy controls ( HLT ) ( Table 1 ) .", "Sequences were analyzed with MOTHUR ( Schloss et al . , 2009 ) to cluster operational taxonomic units ( OTUs , species level classification ) at a 97% identity threshold , assign taxonomic identifiers , and calculate clade relative abundances .", "Although PsA patients revealed a reduction in sample diversity similar to that of IBD patients ( Morgan et al . , 2012 ) , diversity was comparable between NORA , CRA and healthy groups at 3 . 02 +/− 0 . 66 ( mean , SD ) overall by Shannon Diversity Index ( Figure 1—figure supplement 1A ) .", "However , when applying Simpson’s Dominance Index , the NORA group was less diverse ( Figure 1—figure supplement 1B ) , suggesting that these patients harbored a relatively higher abundance of common taxa .", "Analysis at the major taxonomic hierarchy levels showed no significant differences in either phyla abundance or the ratio of Bacteroidetes/Firmicutes ( Figure 1—figure supplement 1C ) between all groups .", "At the level of family abundances , however , we noted a significant enrichment of Prevotellaceae in NORA subjects ( Figure 1A , Figure 1—figure supplement 1D ) .", "Using the linear discriminant effect size method ( LEfSe , see ‘Materials and methods’ ) ( Segata et al . , 2011 ) to compare detected clades ( 33 families , 177 genera , 996 OTUs ) among all groups , we found a positive association of two specific Prevotella OTUs with NORA and an inverse correlation with Group XIV Clostridia , Lachnospiraceae , and Bacteroides as compared to healthy controls ( Figure 1A ) .", "Of all detected Prevotellaceae OTUs , OTU4 was the most highly represented with 171 , 486 supporting reads at 11 . 49 +/− 17 . 85 ( mean , SD ) percent of reads per sample .", "OTU12 , the next most abundant Prevotellaceae , was supported by 12 , 119 reads at 2 . 00 +/− 5 . 42 ( mean , SD ) percent of reads per sample .", "Other Prevotellaceae OTUs ( including Prevotella OTU934 ) were more scarcely represented with 1 , 232 +/− 2 , 305 ( mean , SD ) total supporting reads at less than 0 . 5% total reads per sample .", "We therefore reasoned that OTU4 was the dominant Prevotella in our cohort with sixfold more supporting reads than the next most abundant OTU .", "Principal coordinate analysis with Bray-Curtis distances demonstrated that subjects form distinct clusters , irrespective of health or disease status ( Figure 1B ) .", "The largest component of microbial variation corresponded to the carriage ( or absence ) of Prevotella , which significantly differentiated NORA subjects from healthy controls and other forms of arthritis .", "Consistent with other reports of either high Prevotella or high Bacteroides relative abundance , but rarely a high relative abundance of both , ( Faust et al . , 2012; Yatsunenko et al . , 2012 ) , we found segregation of Prevotella or Bacteroides dominance in the intestinal microbiome ( Figure 1C ) . 10 . 7554/eLife . 01202 . 003Table 1 . Demographic and clinical data among subjects with new-onset rheumatoid arthritis ( NORA ) , chronic , treated rheumatoid arthritis ( CRA ) , psoriatic arthritis ( PsA ) , and healthy controls ( HLT ) DOI: http://dx . doi . org/10 . 7554/eLife . 01202 . 003NORACRAPsAHealthy ( n = 44 ) ( n = 26 ) ( n = 16 ) ( n = 28 ) Age , years , mean ( median ) 42 . 4 ( 40 . 0 ) 50 . 0 ( 49 . 0 ) 46 . 3 ( 46 . 0 ) 42 . 8 ( 40 . 0 ) Female , %75885675Disease duration , months , mean ( median ) 5 . 4 ( 2 . 0 ) 72 . 3 ( 48 . 0 ) 0 . 8 ( 0 . 0 ) N/ADisease activity parameters ESR , mm/h , mean34 . 633 . 519 . 710 . 2 CRP , mg/l , mean20 . 68 . 27 . 61 . 1 DAS28 , mean ( median ) 5 . 4 ( 5 . 7 ) 4 . 7 ( 5 . 0 ) 4 . 8 ( 4 . 7 ) N/A Patient VAS pain , mm , mean ( median ) 61 . 4 ( 57 . 5 ) 51 . 5 ( 62 . 5 ) 50 . 6 ( 45 . 0 ) N/A TJC-28 , mean ( median ) 11 . 2 ( 8 . 5 ) 7 . 6 ( 7 . 0 ) 8 . 8 ( 6 . 5 ) N/A SJC-28 , mean ( median ) 8 . 3 ( 8 . 0 ) 4 . 6 ( 3 . 0 ) 4 . 8 ( 3 . 0 ) N/AAutoantibody status IgM-RF positive , %95811311 ACPA positive , %1008567 IgM-RF and/or ACPA positive , %100961314 IgM-RF titer , kU/l , mean ( median ) 341 . 3 ( 157 . 0 ) 178 . 2 ( 89 . 0 ) 3 . 6 ( 0 . 0 ) 20 . 5 ( 0 . 0 ) ACPA titer , kAU/l , mean ( median ) 117 . 6 ( 114 . 0 ) 90 . 8 ( 57 . 0 ) 1 . 6 ( 0 . 0 ) 9 . 6 ( 0 . 0 ) Medication use Methotrexate , %04260 Prednisone , %01560 Biological agent , %0120010 . 7554/eLife . 01202 . 004Figure 1 . Differences in the relative abundance of Prevotella and Bacteroides in 114 subjects with and without arthritis , determined by 16S sequencing ( regions V1–V2 , 454 platform ) .", "( A ) LEfSe ( Segata et al . , 2011 ) was used to compare the abundances of all detected clades among all groups , producing an effect size for each comparison ( ‘Materials and methods’ ) .", "All results shown are highly significant ( q<0 . 01 ) by Kruskal-Wallis test adjusted with the Benjamini-Hochberg procedure for multiple testing , except that indicated with an asterisk , which is significant at q<0 . 05 .", "Negative values ( left ) correspond to effect sizes representative of NORA groups , while positive values ( right ) correspond to effect sizes in HLT subjects .", "Prevotella was found to be over-represented in NORA patients , while Bacteroides was over-represented in all other groups .", "( B ) The Bray-Curtis distance between all subjects was calculated and used to generate a principal coordinates plot in MOTHUR ( Schloss et al . , 2009 ) .", "The first two components are shown .", "Subjects with an abundance of Prevotella greater than 10% were colored red .", "Other subjects were colored according to their Bacteroides abundance as shown .", "NORA subjects ( stars ) primarily cluster together according to their Prevotella abundance , and the x-axis is representative of differences in the relative abundance of Prevotella and Bacteroides .", "( C ) The abundances of Prevotella ( red ) and Bacteroides ( blue ) are shown for all subjects , sorted in order of decreasing Prevotella abundance ( >5% ) and increasing Bacteroides abundance . DOI: http://dx . doi . org/10 . 7554/eLife . 01202 . 00410 . 7554/eLife . 01202 . 005Figure 1—source data 1 . Intermediate data and analysis tools for Figure 1 . DOI: http://dx . doi . org/10 . 7554/eLife . 01202 . 00510 . 7554/eLife . 01202 . 006Figure 1—source data 2 . Intermediate data and analysis tools for Figure 1—figure supplement 1 . DOI: http://dx . doi . org/10 . 7554/eLife . 01202 . 00610 . 7554/eLife . 01202 . 007Figure 1—figure supplement 1 . Gut microbiota richness , diversity and relative abundance in NORA patients and controls .", "( A and B )", "Gut microbiota richness and diversity are similar among RA groups and healthy controls .", "( C ) Phyla abundance by group .", "No significant differences were found at this taxonomic level .", "( D ) Family abundance by group .", "NORA subjects have a significant increase in Prevotellaceae ( red ) and a concomitant decrease in Bacteroidaceae ( blue ) by FDR-adjusted Kruskal-Wallis test ( q<0 . 01 ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 01202 . 007 To taxonomically identify Prevotella OTU4 , OTU12 , and OTU934 , we generated a phylogenetic tree using the consensus 16S sequences of these OTUs and matched regions from known Prevotella taxa ( Figure 2—figure supplement 1 ) .", "The analysis revealed these OTUs to cluster tightly with Prevotella copri , a microbe isolated from human feces ( Hayashi et al . , 2007 ) and sequenced as part of the HMP’s reference genome initiative .", "To further characterize Prevotella OTU4 , the most abundant taxon , we selected four high abundance NORA samples ( 028B , 030B , 061B , and 089B ) for shotgun sequencing ( single-end , 454 platform ) .", "The resulting long reads were used to generate metagenomic assemblies ( Table 2 , see ‘Materials and methods’ ) which served as input to PhyloPhlAn ( Segata et al . , 2013 ) .", "Briefly , PhyloPhlAn locates 400 ubiquitous bacterial genes in a given assembly by sequence alignment in amino acid space , then builds a tree by concatenating the most discriminative positions in each gene into a single long sequence and applying FastTree ( Price et al . , 2010 ) , a standard tree reconstruction tool .", "This produced a phylogenomic tree placing the taxon most represented in each sample’s metagenomic contigs ( i . e . , Prevotella OTU4 ) again in close association with Prevotella copri ( Figure 2A ) .", "We therefore chose to filter the resulting metagenomic assemblies by alignment to the P . copri reference genome to generate draft patient-derived genome assemblies ( see ‘Materials and methods’ ) .", "Comparison of these draft assemblies to reference P . copri and to one another revealed a high degree of similarity , with possible genome rearrangements ( Figure 2B ) . 10 . 7554/eLife . 01202 . 008Table 2 . Draft genome assembly statistics of four subjects with a high abundance of Prevotella OTU4DOI: http://dx . doi . org/10 . 7554/eLife . 01202 . 008TotalP .", "copri alignedSubject IDGroupPrevotella OTU4 abundance ( % ) # reads# of contigsSize ( Mb ) N50 ( kb ) Mean depth# of contigsSize ( Mb ) N50 ( kb ) Mean depth028BNORA27 . 71 , 240 , 51519 , 98823 . 241 . 456 . 131153 . 2159 . 8436 . 76030BNORA50 . 91 , 041 , 54621 , 57917 . 351 . 016 . 972322 . 6016 . 1844 . 14061BNORA66 . 51 , 209 , 3929 , 24112 . 81 . 589 . 88743 . 2379 . 98172 . 64089BNORA56 . 31 , 395 , 87212 , 11223 . 474 . 6423 . 121 , 9633 . 963 . 1930 . 39Ref .", "genome–––––––833 . 51131 . 4–10 . 7554/eLife . 01202 . 009Figure 2 . Homology-based classification of patient-associated Prevotella . Four NORA subjects with a high abundance of Prevotella OTU4 were selected for shotgun sequencing and metagenome assembly .", "( A ) The resulting metagenomic contigs were used to generate a phylogenomic tree with PhyloPhlAn ( Segata et al . , 2013 ) .", "( B ) Assemblies were filtered by alignment to the reference Prevotella copri DSM 18205 genome , keeping contigs with at least one 300 bp region aligned at 97% identity or greater .", "The resulting draft patient-derived P . copri assemblies were aligned to one another , the reference P . copri genome , and two distinct Prevotella taxa ( Prevotella buccae and Prevotella buccalis ) .", "Colored arcs represent assemblies as labeled , lines connecting arcs represent regions of >97% identity >1 kb in length , and gray lines dividing colored arcs represent boundaries between contigs .", "These results demonstrate that Prevotella OTU4 , OTU12 , and OTU934 form a clade with P . copri ( left , red highlighted subtree ) that is genetically distinct from more distant Prevotella taxa . DOI: http://dx . doi . org/10 . 7554/eLife . 01202 . 00910 . 7554/eLife . 01202 . 010Figure 2—source data 1 . Intermediate data and analysis tools for Figure 2 . DOI: http://dx . doi . org/10 . 7554/eLife . 01202 . 01010 . 7554/eLife . 01202 . 011Figure 2—source data 2 . Intermediate data and analysis tools for Figure 2—figure supplement 1 . DOI: http://dx . doi . org/10 . 7554/eLife . 01202 . 01110 . 7554/eLife . 01202 . 012Figure 2—figure supplement 1 . The representative 16S sequenced reads for Prevotella OTU4 , OTU12 , and OTU934 were aligned with MUSCLE ( Edgar , 2004 ) and clustered with FastTree ( Price et al . , 2010 ) All three Prevotella OTUs cluster with the full-length reference 16S sequence of P . copri . DOI: http://dx . doi . org/10 . 7554/eLife . 01202 . 012 Overall , 75% ( 33/44 ) of NORA patients and 21 . 4% ( 6/28 ) of healthy controls carried P . copri in their intestinal microbiota compared to 11 . 5% ( 3/26 ) and 37 . 5% ( 6/16 ) in CRA and PsA patients , respectively , at a threshold for presence of >5% relative abundance .", "The prevalence of P . copri in NORA compared to CRA , PsA , and healthy controls was statistically significant by chi-squared test , but was not significant in pairwise comparisons of the latter three cohorts ( Table 3 ) . 10 . 7554/eLife . 01202 . 013Table 3 . Statistical comparisons of Prevotella copri prevalence between cohort groupsDOI: http://dx . doi . org/10 . 7554/eLife . 01202 . 013ComparisonPrevalence #1Prevalence #2Chi-squared p-valueFisher’s exact p-value*NORA vs HLT33/446/282 . 612e-051 . 025e-05*NORA vs CRA33/443/261 . 031e-062 . 551e-07†NORA vs PsA33/446/160 . 016980 . 013HLT vs CRA6/283/260 . 54250 . 4704HLT vs PsA6/286/160 . 42390 . 3032CRA vs PsA3/266/160 . 10870 . 06282*p<0 . 01 . †p<0 . 05 .", "Although initial shotgun sequencing of the patient-derived strains showed their similarity to P . copri , there were notable differences observed in assembled genomes upon comparison with the P . copri reference genome .", "This observation suggested that the presence or absence of particular genes in these strains might correlate with health or disease phenotypes in this cohort .", "To address this question , we performed shotgun sequencing on fecal DNA from NORA and healthy subjects , and chose to compare Prevotella sequences from 18 NORA Prevotella-positive subjects , which allowed for a depth of at least 7 M Prevotella-aligned reads ( paired-end , 100 nt , Illumina platform ) , to those of P . copri from 17 healthy subjects ( including 15 from the HMP database and 2 HLT from our cohort ) ( Supplementary file 1A ) .", "Samples sequenced to a depth of less than 7 M such reads were excluded ( Figure 3—figure supplement 1C ) , having insufficient depth for complete recovery of P . copri ORFs ( see ‘Materials and methods’ ) .", "First , we examined the coverage of the P . copri reference genome by all subjects , as an indicator of inter-individual strain variability ( Human Microbiome Project Consortium , 2012 ) .", "Overall , coverage was similar between healthy and NORA subjects in all but a few regions ( Figure 3A , blue and red horizontal lines ) .", "Eight regions were poorly covered in all subjects with mean coverage below the 25th percentile of 0 . 79 FPKM , while several regions showed substantial variability between individuals ( Figure 3A , gray vertical lines ) .", "To determine if the presence or absence of these regions within individuals was consistent between samplings , we applied MetaPhlAn ( Segata et al . , 2012 ) to Prevotella-positive HMP samples collected over multiple visits ( Figure 3B ) .", "Briefly , MetaPhlAn determines the presence or absence of metagenomic marker genes that are specific to particular bacterial clades by analyzing the coverage of such genes by sequenced reads .", "Genes are called specific for a bacterial clade if they are not found in any reference genomes outside the clade , but are found in all such genomes within the clade .", "In concordance with a previous report ( Schloissnig et al . , 2013 ) documenting the temporal stability of metagenomic SNP patterns in individuals , we found that carriage of P . copri genes within an individual varied little between samplings .", "In addition to a stable set of P . copri core marker genes common to all samples , a subset of variable marker genes was observed to co-occur in islands across the P . copri genome , suggesting genomic rearrangements as a mechanism of variability ( Figure 3A , blue boxes below plot ) .", "Together , these results suggest that P . copri strains vary between individuals and retain their individuality over time . 10 . 7554/eLife . 01202 . 014Figure 3 . Comparison of P . copri genomes from healthy and NORA subjects .", "( A ) Comparative coverage of the draft P . copri DSM 18205 genome between individuals and within healthy and NORA groups .", "Gray points are median fragments per kilobase per million ( FPKM ) for 1-kb windows , gray lines within the plot are the interquartile range for each window , red and blue lines the LOWESS-smoothed average for NORA and healthy groups , respectively .", "Gray lines on the horizontal axis represent boundaries between assembled contigs .", "Regions are variably covered between subjects and groups , with several genomic islands lacking overall or especially variable ( dark blue lines below the plot ) .", "( B ) The presence ( blue ) or absence ( gray ) of previously-reported P . copri-unique marker genes ( Segata et al . , 2012 ) in 11 stool samples from five subjects of the Human Microbiome Project ( HMP ) are shown as a heatmap .", "We report , in columns , only those P . copri-specific markers showing variable presence/absence patterns across the considered HMP samples .", "Each row represents a different sample collection date , groups of rows represent subjects , and groups of columns correspond to different variably covered genomic islands .", "Strains of P . copri are defined by the presence and absence of particular genes , which remain stable for at least 6 months in these individuals .", "All inter- and intra-individual comparisons between rows are highly statistically significant ( p<<0 . 001 , ‘ Materials and methods’ ) .", "( C ) The P . copri pangenome was identified by finding P . copri ORFs in all HMP and NORA cohort subjects , and the presence or absence of these ORFs was calculated for each subject ( ‘Materials and methods’ , Figure 3—figure supplement 1 ) .", "Several ORFs are statistically significant biomarkers between healthy and NORA status ( q<0 . 25 ) ( Supplementary file 1B , ‘Materials and methods’ ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 01202 . 01410 . 7554/eLife . 01202 . 015Figure 3—source data 1 . Intermediate data and analysis tools for Figure 3 . DOI: http://dx . doi . org/10 . 7554/eLife . 01202 . 01510 . 7554/eLife . 01202 . 016Figure 3—source data 2 . Intermediate data and analysis tools for Figure 3—figure supplement 1 . DOI: http://dx . doi . org/10 . 7554/eLife . 01202 . 01610 . 7554/eLife . 01202 . 017Figure 3—figure supplement 1 . Recovery of P . copri pangenome from HMP/RA shotgun reads and determination of presence/absence of P . copri ORFs by alignment of reads to pangenome gene catalog .", "( A ) Genes were called present in a sample if they were covered by aligned reads at an identity threshold of >97% over >97% of their length ( red lines ) .", "( B ) ORFs were called on contigs using MetaGeneMark ( Zhu et al . , 2010 ) and were dereplicated with UCLUST ( Edgar , 2010 ) at an identity threshold of 97% ( red line ) .", "( C ) Recovery of a sample's P . copri pangenome saturated at approximately 7 million reads ( red line ) .", "We therefore excluded samples with less than 7 million P . copri reads , defined as P . copri abundance determined by MetaPhlAn ( Segata et al . , 2012 ) multiplied by the total number of quality-filtered reads .", "Samples with P . copri abundance likely misestimated ( i . e . , those with <3000 ORFs present ) were also excluded ( Supplementary file 1A ) .", "( D ) Contigs were said to have originated from P . copri if they had at least one hit >97% identity over >300 bp ( red lines ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 01202 . 01710 . 7554/eLife . 01202 . 018Figure 3—figure supplement 2 . Metagenomic context of discriminative biomarker ORFs . ORFs found in the P . copri DSM 18205 reference genome are colored red , while those identified as differentially present in healthy and NORA groups are indicated with red asterisks .", "( A ) Two ORFs , 3690 and 3694 , are healthy-specific , occur on the same contig , and encode different components of the same NADH:quinone oxidoreductase .", "( B ) Similarly , ORFs 62 , 568 and 62 , 569 occur on the same contig , are NORA-specific , and encode components of the same iron ABC transporter . DOI: http://dx . doi . org/10 . 7554/eLife . 01202 . 018 Next , we assembled a catalog of P . copri genes present across many individuals ( i . e . , the P . copri pangenome ) , by performing de novo metagenome assembly and gene calling on a per-sample basis ( see ‘Materials and methods’ ) .", "To determine if any ORFs were differentially present in NORA subjects as compared to healthy controls , we first reduced the set of interrogated ORFs by filtering partially assembled ( i . e . , containing gaps , lacking stop codons ) , short ( i . e . , less than 300 bp ) , and low-coverage ( i . e . , present in fewer than five subjects ) ORFs to yield a final set of 3 , 291 high-confidence P . copri ORFs ( Figure 3—figure supplement 1 ) .", "We found two ORFs differentially present in healthy controls , and 17 ORFs differentially present in NORA ( Figure 3C; Supplementary file 1B ) .", "The two healthy-specific ORFs appear on the same metagenomic contig , encoding a nearly-complete nuo operon for NADH:ubiquinone oxidoreductase ( Figure 3—figure supplement 2A ) , adjacent to a Bacteroides conjugative transposon .", "Similarly , two of the NORA-specific ORFs appear together on another metagenomic contig , encoding an ATP-binding cassette iron transporter ( Figure 3—figure supplement 2B ) .", "These ORFs may represent good biomarkers for discrimination between healthy and disease-associated microbiota in the population at risk for RA .", "To determine if the NORA metagenome encodes unique functions compared to healthy subjects , we applied HUMAnN ( Abubucker et al . , 2012 ) to quantitate the coverage and abundances of KEGG ( Kanehisa and Goto , 2000 ) modules ( small sets of genes in well-defined metabolic pathways ) in healthy controls ( n = 5 ) and a representative set of NORA subjects ( n = 14 ) with and without Prevotella .", "We then applied LEfSe ( Segata et al . , 2011 ) to find statistically significant differences between groups .", "This analysis revealed a low abundance of vitamin metabolism ( i . e . , biotin , pyroxidal , and folate ) and pentose phosphate pathway modules in NORA , consistent with a lack of these functions in Prevotella genomes ( Figure 4 ) .", "At the coverage level ( presence or absence ) , the NORA metagenome is defined by an absence of functions present in Bacteroides and Clostridia , clades typically found in low abundance in Prevotella-high NORA subjects . 10 . 7554/eLife . 01202 . 019Figure 4 . Metabolic pathway representation in the microbiome of healthy and NORA subjects . HUMAnN ( Abubucker et al . , 2012 ) was applied to metagenomic reads ( paired-end , 100 nt , Illumina platform ) from NORA subjects ( n = 14 ) and healthy controls ( n = 5 ) to quantitate the abundances of hierarchically related KEGG modules in these samples ( ‘Materials and methods’ and Supplementary file 1A ) .", "LEfSe ( Segata et al . , 2011 ) was used to find statistically significant differences between groups at an alpha cutoff of 0 . 001 and an effect size cutoff of 2 . 0 .", "Results shown here are highly significant ( p<0 . 001 ) and represent large differences between groups .", "Modules highlighted in red are over-abundant in NORA samples while modules highlighted in blue are over-abundant in healthy samples .", "Prevotella-dominated NORA metagenomes have a dearth of genes encoding vitamin and purine metabolizing enzymes , and an excess of cysteine metabolizing enzymes . DOI: http://dx . doi . org/10 . 7554/eLife . 01202 . 01910 . 7554/eLife . 01202 . 020Figure 4—source data 1 . Intermediate data and analysis tools for Figure 4 . DOI: http://dx . doi . org/10 . 7554/eLife . 01202 . 020 Prevotella and Bacteroides are closely related both functionally and phylogenetically , yet , surprisingly , are rarely found together in high relative abundance despite their ability to dominate the gut microbiome individually ( Faust et al . , 2012 ) .", "We hypothesized that there might be a genetic difference in these two clades that could account for their apparent co-exclusionary relationship .", "We therefore sought to find genes differentially present in P . copri but not in any of the most abundant Bacteroides species .", "This revealed K05919 ( superoxide reductase ) , K00390 ( phosphoadenosine phosphosulfate reductase ) , and several transporters as uniquely present in P . copri ( Supplementary file 1C ) , and also a set of genes absent in P . copri but present in Bacteroides ( Supplementary file 1D ) .", "Certain alleles within the human leukocyte-antigen ( HLA ) Class II locus confer higher risk of disease , in particular those belonging to DRB1 ( i . e . , ‘shared epitope’ alleles or SE ) ( du Montcel et al . , 2005; Gregersen et al . , 1987 ) .", "To determine whether a higher abundance of P . copri is associated with the host genotype , we carried out HLA sequencing on DNA from all participants in our study ( Supplementary file 1E ) .", "Consistent with recently published mouse data ( Gomez et al . , 2012 ) , the presence of SE alleles correlated with the composition of the gut microbiota .", "A subgroup analysis of NORA patients and healthy controls according to presence ( or absence ) of SE alleles revealed a significantly higher relative abundance of P . copri in those subjects lacking predisposing genes ( Figure 5 , p<0 . 001 in NORA , p<0 . 05 in HLT , ‘Materials and methods’ ) . 10 . 7554/eLife . 01202 . 021Figure 5 . Relationship of host HLA genotype to abundance of P . copri ( OTU4 , OTU12 , and OTU934 combined relative abundance ) .", "The HLA-class II genotype of all subjects was determined by sequence-based typing methodology ( ‘Materials and methods’ ) .", "Groups were subdivided by the presence or absence of shared-epitope RA risk alleles ( +/− SE as indicated above ) and correlated with relative abundance of intestinal P . copri .", "A statistically significant correlation is seen between P . copri abundance and the genetic risk for rheumatoid arthritis in NORA ( red stars ) and healthy ( blue circles ) subjects by Welch’s two-tailed t test . DOI: http://dx . doi . org/10 . 7554/eLife . 01202 . 02110 . 7554/eLife . 01202 . 022Figure 5—source data 1 . Intermediate data and analysis tools for Figure 5 . DOI: http://dx . doi . org/10 . 7554/eLife . 01202 . 022 To determine if the Prevotella-associated metagenome is sufficient to predispose to increased inflammatory responses , antibiotic-treated C57BL/6 mice were colonized with P . copri by oral gavage .", "Analysis of DNA extracted from fecal samples 2 weeks post-gavage revealed robust colonization with P . copri ( Figure 6A ) .", "Sequencing of the 16S gene ( regions V1–V2 , 454 platform ) in fecal DNA from two representative mice colonized with P . copri revealed the ability of Prevotella to dominate the gut microbiota ( Figure 6B ) .", "In comparison to fecal DNA from mice gavaged with media alone , P . copri-colonized mice had reduced Bacteroidales and Lachnospiraceae , similar to what was observed in this patient cohort ( Figure 1A , Figure 1—figure supplement 1D ) .", "Consistent with a previous report of a Prevotella taxon exacerbating an inflammatory phenotype ( Elinav et al . , 2011 ) , exposure of P . copri-colonized mice to 2% dextran sulfate sodium ( DSS ) in drinking water for 7 days resulted in more severe colitis as assessed by enhanced weight loss ( Figure 6C ) , worse endoscopic score ( Figure 6D ) , and increased epithelial damage on histological analysis ( Figure 6E , F ) when compared to littermate controls gavaged with media alone .", "Furthermore , in contrast to mice colonized with mouse commensal Bacteroides thetaiotamicron ( Figure 6—figure supplement 1A ) , P . copri colonized mice similarly showed significantly decreased weight loss at day 7 following DSS exposure ( Figure 6—figure supplement 1B ) .", "Analysis of the lamina propria CD4+ T-cell response revealed an increase in IFNγ production following DSS induction , although no statistically significant differences were seen in IFNγ ( Th1 ) or IL-17 production ( Th17 ) following P . copri colonization ( Figure 6—figure supplement 1C ) .", "Likewise , no differences in Foxp3+ CD4+ T-cells were observed .", "These data suggest that a Prevotella-defined microbiome may have the propensity to support inflammation in the context of a genetically susceptible host . 10 . 7554/eLife . 01202 . 023Figure 6 . Colonization with P . copri dominates the colonic microbiome and exacerbates local inflammatory responses .", "( A ) DNA was extracted from fecal pellets of media-gavaged mice and P . copri-gavaged mice 2 weeks after colonization and assayed by QPCR with P . copri specific primers compared to universal 16S .", "( B ) Relative abundance of bacterial families in fecal DNA from media-gavaged and P . copri-colonized mice ( shown in duplicate ) by high-throughput 16S sequencing ( regions V1–V2 , 454 platform ) .", "( C ) C57BL/6 mice colonized with P . copri ( n = 15 ) or media alone ( n = 13 ) controls were exposed to DSS for seven days and percent of starting body weight is shown .", "Composite data from three representative experiments are shown .", "( D ) Representative colonoscopic images of mice colonized with P . copri or media gavage following DSS-induced colitis .", "Endoscopic colitis score for five individual animals is displayed .", "( E and F )", "Gross pathology ( E ) and histology ( F ) of colons from mice colonized with P . copri or media gavage following DSS-induced colitis . DOI: http://dx . doi . org/10 . 7554/eLife . 01202 . 02310 . 7554/eLife . 01202 . 024Figure 6—source data 1 . Intermediate data and analysis tools for Figure 6 . DOI: http://dx . doi . org/10 . 7554/eLife . 01202 . 02410 . 7554/eLife . 01202 . 025Figure 6—figure supplement 1 . P .", "copri colonization exacerbates chemically induced colitis .", "( A ) DNA was extracted from fecal pellets of media , P . copri , and B . thetaiotamicron gavaged mice 2 weeks after colonization and assayed by QPCR with P . copri or Bacteroides specific primers compared to universal 16S amplicon .", "( B ) C57BL/6 mice colonized with P . copri ( n = 10 ) or B . theta ( n = 10 ) were exposed to DSS for seven days and percent of starting body weight is shown .", "( C ) Percent of total CD4+ T-cells in the colonic lamina propria expressing IL-17 ( Th17 ) or IFNγ ( Th1 ) following PMA/ionomycin stimulation or expressing Foxp3 ( Treg ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 01202 . 025" ], [ "Multiple lines of investigation have revealed that RA is a multifactorial disease that occurs in sequential phases .", "Notably , there is a prolonged period of autoimmunity ( i . e . , presence of circulating auto-antibodies such as rheumatoid factor and anti-citrullinated peptide antibodies ) in a pre-clinical state that lasts many years , during which time there is no clinical or histologic evidence of inflammatory arthritis ( Deane et al . , 2010 ) .", "Before the onset of clinical disease , there is an increase in autoantibody titers and epitope spreading coupled with elevation in circulating pro-inflammatory cytokines .", "These findings have led to the ‘second-event’ hypothesis in RA , which proposes that an environmental factor triggers systemic joint inflammation in the context of pre-existent autoimmunity .", "Multiple mucosal sites and their residing microbial communities have been implicated , including the airways , the periodontal tissue and the intestinal lamina propria ( Mcinnes and Schett , 2011; Scher et al . , 2012 ) .", "Although a role for the gut microbiota has been clearly established in animal models of arthritis , it is not known if dysbiosis influences human RA .", "The human gut microbiota has been classified into unique enterotypes , one of which is defined by the predominance of Prevotella ( Arumugam et al . , 2011 ) .", "In our cohort , we found the microbiota of many subjects to be defined by a single taxon—P .", "copri—which was associated with the majority of untreated , new-onset rheumatoid arthritis ( NORA ) patients .", "P . copri was also detected in a minority of healthy subjects in cohorts from the Human Microbiome Project ( Human Microbiome Project Consortium , 2012 ) , the European MetaHIT project ( Qin et al . , 2010 ) , and our study .", "Surprisingly , the prevalence of P . copri in chronic rheumatoid arthritis ( CRA ) patients , all of whom had been treated and exhibited reduced disease activity , was similar to that observed in the healthy subjects .", "One hypothesis is that the Prevotella-defined microbiota fail to thrive when there is less inflammation , perhaps due to a lack of inflammation-derived terminal electron acceptors , as seen for E . coli in inflammatory bowel disease ( Winter et al . , 2013 ) .", "Alternatively , the gut microbiota changes observed in newly diagnosed RA patients may be the consequence of a unique , NORA-specific systemic inflammatory response .", "While DAS28 scores were slightly lower in CRA and PsA patients ( Table 1 ) , the most remarkable difference was in levels of C-reactive protein ( CRP ) .", "This raises the question of whether CRP itself may have microbial modulating properties .", "CRP is characteristically high in early and flaring RA , but not in other autoimmune diseases ( e . g . , systemic lupus erythematous , scleroderma , and PsA ) .", "A member of the pentraxin protein family , CRP was first identified in the plasma of patients with Streptococcus pneumoniae infection ( Tillett and Francis , 1930 ) .", "Further , the primary bacterial ligand for CRP is phosphocholine , a component of multiple bacterial cell-wall components , including lipopolysaccharides ( LPS ) .", "CRP binding to bacterial phosphocholine activates the complement system and enhances phagocytosis by macrophages .", "Whether or not CRP itself represents a specific response to the presence of P . copri in NORA is an area of future investigation .", "Interestingly , Prevotella-dominated healthy omnivore individuals were recently reported to have increased basal levels of serum TMAO ( trimethylamine N-oxide ) , a product of inflammation linked to atherogenesis , compared to Bacteroides-dominated healthy individuals ( Koeth et al . , 2013 ) .", "While TMAO could be derived from increased consumption of meat ( Koeth et al . , 2013 ) , Prevotella has been previously associated with a dearth of meat in the diet ( Wu et al . , 2011 ) .", "Additional studies are needed to determine if prevalence of P . copri in the microbiota is associated with changes in specific metabolites .", "Sequence alignment most closely linked NORA-associated Prevotella with the P . copri genome .", "Interestingly , large regions of the P . copri genome were scarcely covered in both our cohort and subjects of the HMP .", "As the reference strain of P . copri was isolated in Japan and all samples analyzed in our study were collected and sequenced in North America , these differences may reflect geographically-associated strain variability , consistent with a report ranking P . copri as the second-most variable member of the human gut microbiota between continents ( Schloissnig et al . , 2013 ) .", "Notably , comparison of sequences in NORA samples with those of P . copri-dominated healthy individuals evaluated in the HMP allowed us to identify ORFs associated with the NORA phenotype .", "Two ORFs , both encoding components of an iron transporter , were specific for NORA-associated P . copri , while two ORFs were specific for HLT-associated P . copri and encode components of a nuo operon .", "Iron transporters are known to be virulence factors in other bacterial clades , while the ubiquinone oxidoreductase pathway encoded by the nuo operon may provide a fitness advantage in the context of a healthy microbiome by allowing use of metabolites available therein .", "While colonization with P . copri increases the pre-test probability of NORA from 1% to approximately 3 . 95% in western cohorts ( by Bayes’ theorem , see ‘Materials and methods’ ) , the presence of one of the aforementioned ORFs may markedly increase the pre-test probability of NORA status .", "The diagnostic application of these biomarkers needs to be confirmed in larger cohorts .", "Analysis of enzymatic functions in the Prevotella-dominated metagenome reveals a significant decrease in purine metabolic pathways , including tetrahydrofolate ( THF ) biosynthesis .", "This may have therapeutic implications since methotrexate ( MTX ) , a folate analogue and a dihydrofolate ( DHF ) reductase inhibitor , remains the anchor drug for the treatment of RA ( Singh et al . , 2012 ) and has inter-individual variability in terms of absorption and bioavailability .", "The THF biosynthetic pathway encoded by the gut metagenome , which includes a DHF reductase enzyme , may compete with host DHF reductase for MTX binding and metabolism .", "If so , an increase in DHF reductase-high microbiota in some RA subjects ( i . e . , Bacteroides overabundant ) may help explain , at least partially , why only about half of RA patients respond adequately to oral MTX , ultimately requiring either parenteral administration or the addition of complementary immunosuppressants .", "Prevotella-high NORA subjects , with a dearth of DHF reductase in the gut , may respond better to oral MTX .", "Prospective human studies should help to clarify these observations .", "RA is a multifactorial autoimmune disease in which certain alleles within the major histocompatibility complex ( MHC ) class II locus , specifically those belonging to DRB1 ( i . e . , shared epitope alleles ) , confer higher risk for disease .", "A recently published study with HLA-DR transgenic mice revealed that the gut microbiota was , at least partially , regulated by the HLA genes ( Gomez et al . , 2012 ) .", "Arthritis-susceptible DRB1*04:01 transgenic mice had a markedly different intestinal microbiota when compared to arthritis-resistant DRB1*04:02 animals , and this was associated with altered mucosal immune function ( i . e . , increased gene transcripts for Th17-related cytokines ) and increased intestinal permeability .", "Our results suggest that , similarly , SE risk-alleles in humans may have an impact on the composition of the gut microbiota .", "Intriguingly , patients in the NORA cohort showed a significant inverse correlation between P . copri relative abundance and presence of SE alleles ( Figure 5 ) .", "It is therefore possible that , as in mice , certain human gut microbial communities are determined by specific MHC alleles that favor the expansion of particular species .", "As in the case of cigarette smoking , this could also represent a gene-environment interaction that contributes to RA pathogenesis .", "It is conceivable that a certain threshold for P . copri abundance may be necessary to overcome the lack of genetic predisposition in RA subjects , while a lower abundance may be sufficient to trigger disease in those carrying risk-alleles .", "Validation in expanded cohorts and mechanistic studies are needed to better understand the significance of these findings .", "Colonization of mice with P . copri recapitulated the differences in relative abundances of Prevotella and Bacteroides previously reported in humans , and confirmed the ability of P . copri to dominate the colonic commensal microbiota in the absence of apparent disease ( Faust et al . , 2012 ) .", "This shift in abundances correlated with a metagenomic shift , which may support and/or perpetuate an inflammatory environment .", "For example , uniquely present superoxide reductase in P . copri may facilitate resistance to or allow the use of host-derived reactive oxygen species ( ROS ) generated during inflammation , perhaps as terminal electron acceptors for respiration ( Winter et al . , 2013 ) .", "Similarly , the P . copri genome encodes phosphoadenosine phosphosulfate reductase ( PAPS ) , an oxidoreductase absent in Bacteroides that participates in sulfur metabolism and leads to the production of thioredoxin .", "Intriguingly , thioredoxin has been widely implicated in the pathogenesis of RA and high levels of this redox protein have been found in both serum and synovial fluid of RA patients ( Maurice et al . , 1999 ) .", "Mice colonized with P . copri displayed increased inflammation in DSS-induced colitis .", "An appealing hypothesis from an evolutionary and ecological perspective is that the P . copri-defined microbiota thrives in a pro-inflammatory environment and may exacerbate inflammation for its own benefit .", "Another key feature of the P . copri-dominated microbiome is a community shift away from Bacteroides , Group XIV Clostridia , Blautia , and Lachnospiraceae clades , previously reported to be associated with an anti-inflammatory state and regulatory T-cell ( Treg ) production ( Atarashi et al . , 2011; Round et al . , 2011 ) .", "This could account , in part , for the observed differences in susceptibility to inflammation ( Tao et al . , 2011 ) .", "Further characterization of changes in the host immune system associated with a Prevotella-dominated microbiota should provide deeper insight into whether expansion of P . copri contributes causally to the development of autoimmunity in early onset RA ." ], [ "Consecutive patients from the New York University rheumatology clinics and offices were screened for the presence of RA .", "After informed consent was signed , each patient’s medical history ( according to chart review and interview/questionnaire ) , diet , and medications were determined .", "A screening musculoskeletal examination and laboratory assessments were also performed or reviewed .", "All RA patients who met the study criteria were offered enrollment .", "The criteria for inclusion in the study required that patients meet the American College of Rheumatology/European League Against Rheumatism 2010 classification criteria for RA ( Aletaha et al . , 2010 ) , including seropositivity for rheumatoid factor ( RF ) and/or anti–citrullinated protein antibodies ( ACPAs ) ( assessed using an anti–cyclic citrullinated peptide ELISA; Euroimmun ) , and that all subjects be age 18 years or older .", "New-onset RA was defined as disease duration of a minimum of 6 weeks and up to 6 months since diagnosis , and absence of any treatment with disease-modifying anti-rheumatic drugs ( DMARDs ) , biologic therapy or steroids ( ever ) .", "Chronic RA was defined as any patient meeting the criteria for RA whose disease duration was a minimum of 6 months since diagnosis .", "Most subjects with chronic RA were receiving DMARDs ( oral and/or biologic agents ) and/or corticosteroids at the time of enrollment .", "Healthy controls were age- , sex- , and ethnicity-matched individuals with no personal history of inflammatory arthritis .", "The exclusion criteria applied to all groups were as follows: recent ( <3 months prior ) use of any antibiotic therapy , current extreme diet ( e . g . , parenteral nutrition or macrobiotic diet ) , known inflammatory bowel disease , known history of malignancy , current consumption of probiotics , any gastrointestinal tract surgery leaving permanent residua ( e . g . , gastrectomy , bariatric surgery , colectomy ) , or significant liver , renal , or peptic ulcer disease .", "This study was approved by the Institutional Review Board of New York University School of Medicine .", "Fecal samples were obtained within 24 hr of production .", "All samples were suspended in MoBio buffer-containing tubes .", "DNA was extracted using a combination of the MoBio Power Soil kit ( Mo Bio Laboratories , Inc , Carlsbad , CA , USA ) and a mechanical disruption ( bead-beater ) method based on a previously described protocol ( Ubeda et al . , 2010 ) .", "Samples were stored at −80°C .", "For each sample , three replicate PCRs were performed to amplify the V1 and V2 regions as previously described ( Ubeda et al . , 2010 ) .", "PCR products were sequenced on the 454 GS FLX Titanium platform ( 454 Life Sciences , Branford , CT , USA ) to a depth of at least 2 , 600 reads per subject .", "Sequences have been deposited in the NCBI Sequence Read Archive under the accession number SRP023463 .", "Sequence data were compiled and processed using MOTHUR ( Schloss et al . , 2009 ) .", "Sequences were converted to standard FASTA format .", "Sequences shorter than 200 bp , containing undetermined bases or homopolymer stretches longer than 8 bp , with no exact match to the forward primer or a barcode , or that did not align with the appropriate 16S rRNA variable region were not included in the analysis .", "Using the 454 base quality scores , which range from 0–40 ( 0 being an ambiguous base ) , sequences were trimmed using a sliding-window technique , such that the minimum average quality score over a window of 50 bases never dropped below 30 .", "Sequences were trimmed from the 3′-end until this criterion was met .", "Sequences were aligned to the 16S rRNA gene , using as template the SILVA reference alignment ( Pruesse et al . , 2007 ) , and the Needleman-Wunsch algorithm with the default scoring options .", "Potentially chimeric sequences were removed using the ChimeraSlayer program ( Haas et al . , 2011 ) .", "To minimize the effect of pyrosequencing errors in overestimating microbial diversity ( Huse et al . , 2010 ) , rare abundance sequences that differ in one or two nucleotides from a high abundance sequence were merged to the high abundance sequence using the pre . cluster option in MOTHUR .", "Sequences were grouped into operational taxonomic units ( OTUs ) using the average neighbor algorithm .", "Sequences with distance-based similarity of 97% or greater were assigned to the same OTU .", "OTU-based microbial diversity was estimated by calculating the Shannon diversity index and Simpson Index using mothur .", "Phylogenetic classification was performed for each sequence using the Bayesian classifier algorithm described by Wang and colleagues with the bootstrap cutoff 60% ( Wang et al . , 2007 ) .", "Briefly , LEfSe pairwise compares abundances of all biomarkers ( e . g . , bacterial clades ) between all groups using the Kruskal-Wallis test , requiring all such tests to be statistically significant .", "Vectors resulting from the comparison of abundances ( e . g . , Prevotella relative abundance ) between groups are used as input to linear discriminant analysis ( LDA ) , which produces an effect size ( Figure 1A ) .", "In analyses performed here , the main utility of LEfSe over traditional statistical tests is that an effect size is produced in addition to a p or q value .", "This allows us to sort the results of multiple tests by the magnitude of the difference between groups , not only by q values , as the two are not necessarily correlated .", "In the case of hierarchically organized groups ( e . g . , bacterial clades , or KEGG pathways ) , this lack of correlation can arise from differences in the number of hypotheses considered at different levels in the hierarchy .", "For example , at the genus level , there may be 1 , 000 tests performed , requiring a high level of significance to pass multiple testing correction , whereas at the phylum level , only 10 tests may be performed , requiring a less stringent threshold for significance .", "Paired-end reads 100 bp in length were trimmed from both ends to yield the largest contiguous segment where all per-base QVs were >= 25 .", "Reads < 50 bp in length after this step were discarded .", "Quality-filtered reads were then aligned to the human reference genome ( hg19 ) using bowtie2 in—very-sensitive-local mode , keeping only those reads that failed to align .", "Human-filtered reads were then sorted into complete pairs and singletons ( whose mates were removed by filtering ) for downstream analyses .", "The P . copri DSM 18205-reference genome ( assembly GCA_000157935 . 1 ) was first concatenated into a pseudo-contig in order of increasing contig number .", "Filtered Illumina reads from P . copri positive NORA and healthy ( including HMP subjects , Supplementary file 1A ) subjects were aligned to the reference using bowtie2 in—very-sensitive-local mode .", "Paired-end reads aligning to non-overlapping 1 kb windows across the length of the genome were counted and normalized to FPKM ( fragments per kilobase per million reads ) .", "The interquartile range ( 25th to 75th percentile ) , mean , and median FPKM for each window was calculated and displayed as a boxplot with R . Filtered paired-end reads from P . copri positive subjects were first assembled according to the HMP Whole-Metagenome Assembly SOP ( Pop , 2011 ) using SOAPdenovo ( Luo et al . , 2012 ) .", "Briefly , paired-end and singleton reads were used concurrently with the parameters -K 25 -R -M 3 -d 1 .", "The resulting contigs >300 bp in length were then aligned to the P . copri reference genome with BLASTN at an e value cutoff of 1e-5 .", "A stringent cutoff requiring at least one hit of 97% identity across 300 bp was used to infer that a contig originated from a strain of P . copri ( Figure 3—figure supplement 1D ) .", "ORFs were then called on the resulting contigs using MetaGeneMark ( Zhu et al . , 2010 ) .", "The resulting ORFs were then clustered using USEARCH at an identity threshold of 97% to yield a final set of P . copri genes ( Figure 3—figure supplement 1D ) .", "Samples were excluded from further analyses if they had less than 7 million reads aligning to P . copri ( Figure 3—figure supplement 1C ) .", "This resulted in a catalog of 20 , 387 putative P . copri ORFs with 9 , 274 +/− 1 , 640 ( mean , SD ) present in each subject .", "Further filtering of partially assembled ( i . e . , containing gaps , lacking stop codons ) , short ( i . e . , less than 300 bp ) , and low-coverage ( i . e . , present in fewer than five subjects ) ORFs yielded a final set of 3 , 291 high-confidence P . copri ORFs .", "Filtered reads were aligned to the P . copri pangenome catalog using bowtie2 in–very-fast mode .", "ORFs were said to be present in a sample if at least 97% of their length , minus one read length ( i . e . , 100 bp ) to account for edge alignment artifacts , was covered at an identity of 97% or greater ( Figure 3—figure supplement 1A ) .", "The presence or absence of ORFs in each sample was determined as above , and Fisher’s exact test was used on 2 × 2 contingency tables for each ORF .", "Resulting p were adjusted for multiple hypothesis testing by converting to false discovery rate ( FDR ) q values using the Benjamini-Hochberg procedure .", "ORFs with q<0 . 25 were considered statistically significant .", "Effect size was calculated using the below equation . Effect Size = Absent in NORATotal Absent−Present in NORATotal Present In western cohorts , such as the Human Microbiome Project and our own , the prevalence of P . copri is approximately 19% , that is P ( Prevotella ) = 0 . 19 .", "The approximate incidence of RA is thought to be 1% , that is P ( NORA ) = 0 . 01 .", "In our cohort , we found that 75% of new-onset RA ( NORA ) subjects had 5% or more Prevotella OTU4 , which we determined to be P . copri , that is P ( Prevotella|NORA ) = 0 . 75 .", "We therefore applied Bayes’ theorem as given below . P ( NORA|Prevotella ) =P ( Prevotella|NORA ) P ( NORA ) P ( Prevotella ) The solution to this equation gives a 3 . 95% probability of NORA status if P . copri is present in the gut , compared to a 1% probability of NORA ( i . e . , the incidence of RA ) given no prior information .", "Long reads were obtained for several high-Prevotella abundance subjects ( 028B , 030B , 061B , 089B ) on the 454 GS FLX Titanium platform .", "These reads were assembled with Newbler v2 . 6 to obtain metagenomic assemblies ( Table 2 ) .", "The resulting contigs were subsequently filtered by alignment to the P . copri DSM 18205 reference genome , keeping those with at least one hit of 97% across 300 bp , to obtain draft patient-derived P . copri genomes .", "If each gene ( boxes in Figure 3B , rows 61 boxes in length ) is considered independently and can be in one of two states ( i . e . , present or absent ) , the probability of an exact match between any two individuals is 2−61 , or 2−60 with one mismatch .", "Qualitatively , it can be seen that any intra- or inter-individual comparison is highly statistically significant .", "Further , if we concede that genes within an island are not truly independent , and there are six such islands which are considered identical with 1–2 mismatches allowed , the probability of such a match is 2−6 , or 0 . 015625 , less than a 0 . 05 threshold for significance .", "Filtered paired-end reads were aligned separately to all genomes in KEGG with USEARCH 6 . 0 ( Edgar , 2010 ) using parameters—usearch_local—maxaccepts 2—maxrejects 8–evalue 0 . 1–id 0 . 80 .", "The results from each read in a pair ( and singletons ) were combined and processed with HUMAnN 0 . 96 ( Abubucker et al . , 2012 ) with default parameters .", "Output tables containing per-sample abundance estimates of KEGG modules were then processed with LEfSe ( Segata et al . , 2011 ) using an alpha cutoff of 0 . 001 and an effect size cutoff of 2 . 0 .", "Genomic DNA was isolated from the peripheral blood of RA patients and controls using QIAamp Blood Mini Kit ( Qiagen GmbH , Halden , Germany ) according to the manufacturer’s instructions .", "HLA-DRB1 alleles were determined by Sequence-Based Typing ( SBT ) and by Single Specific Primer-Polymerase Chain Reaction ( SSP-PCR ) methodologies ( Fred H Allen Laboratory of Immunogenetics , NY , USA; Weatherall Institute for Molecular Medicine , Oxford , UK ) ( Supplementary file 1E ) .", "Alleles considered to have the shared-epitope conferring higher risk for RA included: HLA-DRB1*01:01 , 01:02 , 04:01 , 04:04 , 04:05 , 04:08 , 10:01 , 13:03 , and 14:02 , corresponding to S2 and S3P RA risk classification ( du Montcel et al . , 2005 ) .", "Subjects with at least one copy of these alleles have >1 . 95 times the relative risk of disease compared to the least at-risk genotype studied .", "C57BL/6 mice ( Jackson Laboratories ) were treated with ampicillin , neomycin , metronidazole ( all 1 g/l ) for 7 days prior to gavage .", "P . copri ( CB7 , DSMZ ) or B . thetaiotamicron ( gift from E Martens ) was grown to log phase under anaerobic conditions in PYG liquid media ( Anaerobe Systems , CA , USA ) and 107 CFU were used to inoculate mice .", "Feces were collected at 1 and 2 weeks post-gavage to confirm colonization .", "Fecal DNA was extracted with mechanical bead beating with 0 . 1 mm zirconia silica beads ( Biospecs Inc . ) in 2% SDS followed by phenol chloroform extraction .", "Confirmation of colonization was achieved with P . copri genome specific primers ( F: CCGGACTCCTGCCCCTGCAA , R: GTTGCGCCAGGCACTGCGAT ) ; Prevotella 16S primers ( F: CACRGTAAACGATGGATGCC , R: GGTCGGGTTGCAGACC ) , B . thetaiotamicron SusC ( F: CACAACAGCCATAGCGTTCCA , R: ATCGCAAAAATAAGATGGGCAAA ) ( Benjida et al JBC 2011 ) , and Universal 16S Primers ( F: ACTCCTACGGGAGGCAGCAGT , R: ATTACCGCGGCTGCTGGC ) .", "QPCR was performed with a Roche Lightcycler ( Roche USA , South San Francisco , CA , USA ) and the following cycling conditions: 9°C for 5 m , 40 cycles of 95°C for 10 s , and 60°C for 30 s , 72°C for 30 s .", "Genomic DNA from P . copri was used to generate a standard curve to quantitate ng of P . copri present per mg of total feces .", "Mice were given 2% dextran sulfate sodium ( DSS ) in drinking water ad libitum for 7 days .", "Body weight was evaluated every 1–2 days over 14 days .", "Colonic mucosal damage 0 to 3 cm proximal to the anal verge was evaluated by direct visualization using the Coloview ( Karl Storz Veterinary Endoscopy , Tuttlingen , Germany ) .", "Endoscopic scoring was performed as previously described: assessment of colon thickening ( 0–3 points ) , fibrinization ( 0–3 points ) , granularity ( 0–3 points ) , morphology of the vascular pattern ( 0–3 points ) , and stool consistency ( normal to unshaped; 0–3 points ) ( Becker et al . , 2006 ) .", "Lamina propria mononuclear cells were isolated from colonic tissue as previously described ( Diehl et al . , 2013 ) .", "Cells were stimulated with phorbol myristate acetate and ionomycin with brefeldin for 4 hr and prepared as per manufacturer’s instruction with Cytoperm/Cytofix ( BD Biosciences ) for intracellular cytokine evaluation of IL-17A ( eBiosciences 17B7 ) and IFNγ ( eBiosciences XMG1 . 2 ) .", "For Foxp3 analysis , cells were fixed and permeabilized as per manufacturer’s instructions ( eBiosciences , Inc . , San Diego , CA , USA ) and stained intracellularly with anti-Foxp3 ( FJK-16s ) .", "Source files for the figures and figure supplements have been uploaded to github ( https://github . com/polyatail/scher_et_al_2013 ) and as Figure 1—source data 1 , Figure 1—source data 2 , Figure 2—source data 1 , Figure 2—source data 2 , Figure 3—source data 1 , Figure 3—source data 2 , Figure 4—source data 1 , Figure 5—source data 1 , and Figure 6—source data 1 .", "Any future updates will be made available on GitHub ." ] ]

[ "Rheumatoid arthritis ( RA ) is a prevalent systemic autoimmune disease , caused by a combination of genetic and environmental factors .", "Animal models suggest a role for intestinal bacteria in supporting the systemic immune response required for joint inflammation .", "Here we performed 16S sequencing on 114 stool samples from rheumatoid arthritis patients and controls , and shotgun sequencing on a subset of 44 such samples .", "We identified the presence of Prevotella copri as strongly correlated with disease in new-onset untreated rheumatoid arthritis ( NORA ) patients .", "Increases in Prevotella abundance correlated with a reduction in Bacteroides and a loss of reportedly beneficial microbes in NORA subjects .", "We also identified unique Prevotella genes that correlated with disease .", "Further , colonization of mice revealed the ability of P . copri to dominate the intestinal microbiota and resulted in an increased sensitivity to chemically induced colitis .", "This work identifies a potential role for P . copri in the pathogenesis of RA ." ]

[ "We share our bodies with a diverse set of microorganisms , known collectively as the human microbiome .", "Indeed , estimates suggest that our bodies contain 10 times as many microbial cells as human cells .", "Our stomach and intestines alone are home to many hundreds and possibly thousands of microbial species that break down indigestible compounds and help to prevent the growth of harmful bacteria .", "The immune system must therefore learn to tolerate these microorganisms , while retaining the ability to launch attacks against microorganisms that cause harm .", "Failure of this process may increase the risk of autoimmune diseases in which the body mistakenly attacks its own cells and tissues .", "Rheumatoid arthritis is a chronic autoimmune disease marked by inflammation of the joints .", "Although the causes of rheumatoid arthritis are unknown , mice with mutations that increase the risk of the disease remain healthy if they are kept under sterile conditions .", "However , if these mice are exposed to certain species of bacteria sometimes found in the gut , they begin to show signs of joint inflammation .", "Here , Scher et al . used genome sequencing to compare gut bacteria from patients with rheumatoid arthritis and healthy controls .", "A bacterial species called Prevotella copri was more abundant in patients suffering from untreated rheumatoid arthritis than in healthy individuals .", "Moreover , the presence of P . copri corresponded to a reduction in the abundance of other bacterial groups—including a number of beneficial microbes .", "In a mouse model of gut inflammation , animals colonized with P . copri had more severe disease than controls , consistent with a pro-inflammatory function of this organism .", "Current treatments for rheumatoid arthritis target symptoms .", "However , by highlighting the role played by gut bacteria , the work of Scher et al . suggests that novel treatment options focused on curbing the spread of P . copri in the gut could delay or prevent the onset of this disease ." ]

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

[ "neuroscience" ]

[ [ "Autism Spectrum Disorder ( ASD ) refers to a group of neurobiological disorders , which affect a growing proportion of the population .", "Patients with ASD suffer from a range of social and communication impairments , along with other characteristic behaviours and deficits .", "Behavioural treatment options are limited in their efficacy , and often do not generalize well beyond the specific training paradigm ( Otero et al . , 2015; Williams White et al . , 2007 ) .", "Numerous studies have documented widespread patterns of aberrant brain functional connectivity in patients with ASD , involving many cortical regions including frontal , parietal , and temporal lobes ( Müller et al . , 2011; Picci et al . , 2016; Di Martino et al . , 2014 ) .", "Specifically , these studies show that multiple cortical areas are significantly under-connected in patients with ASD compared to typically developing ( TD ) control subjects , although over-connectivity has also been reported ( Hahamy et al . , 2015; Belmonte et al . , 2004 ) .", "At the individual level , the degree of diminished connectivity as well as measures of cortical thickness were found to be correlated with symptom severity ( Gotts et al . , 2012; Wallace et al . , 2012; Di Martino et al . , 2009b; Assaf et al . , 2010 ) and this measure of connectivity was even predictive of future progression of autistic symptoms ( Plitt et al . , 2015 ) .", "Together , these findings suggest a causal link between connectivity and behavior , such that changing the connectivity might lead to a change in behavior .", "Traditional , explicit training paradigms , activate these aberrant networks ( Kana et al . , 2009; Di Martino et al . , 2009a ) , thus potentially reinforcing the sub-optimal connectivity .", "An implicit training paradigm , which allows the direct reinforcement of the desired networks , bypassing the atypical activation induced by explicit tasks , might be a better candidate for potential intervention in ASD .", "Real-time fMRI neurofeedback ( rt-fMRI-nf ) is an emerging technique with great potential for clinical applications ( Stoeckel et al . , 2014; Sulzer et al . , 2013; Weiskopf , 2012; Birbaumer et al . , 2013 ) .", "With this technique , network states can be monitored in real-time , and desired states can be reinforced through positive feedback .", "Covert neurofeedback is a variant of neurofeedback , in which participants are given no strategy with which to control the feedback , and might not even be cognizant that the feedback is related to brain activity .", "This tool is therefore extremely flexible , as it does not require the formulation of a specific strategy and is not limited by what we know about the networks or the ways in which they are typically activated .", "Instead , desired states are reinforced when they occur spontaneously , allowing for implicit training of networks , such as those found to be under-connected in ASD .", "We designed a covert neurofeedback experiment , to test whether it would be possible to change connectivity between these aberrantly connected network nodes , through direct reinforcement of spontaneously occurring network states .", "This decision is motivated by recent work showing that positive and negative reinforcement of brain activity patterns are sufficient for promoting small but widespread changes in network connectivity , even without any learning intention on the part of participants ( Ramot et al . , 2016 ) .", "Further evidence that covert neurofeedback can change networks , and that these changes can have behavioral effects , comes from other recent work .", "A number of studies have been successful at training complex patterns of activity within a given network using multi-voxel pattern analysis ( MVPA ) techniques ( deBettencourt et al . , 2015; Amano et al . , 2016; Shibata et al . , 2011 ) , with feedback related changes corresponding to robust behavioural changes after only a few sessions , and significant change being detectable after as little as one training session .", "Feedback induced behavioural changes have been shown to range from very local and low level , such as changes in perception of line orientation after V1 training ( Shibata et al . , 2011 ) , or inducing color association in V1 in a somewhat less local paradigm ( Amano et al . , 2016 ) , to changes in high level functions such as attention ( deBettencourt et al . , 2015 ) , and fear perception ( Koizumi et al . , 2016 ) .", "Such changes can even be bi-directional , both behaviourally and at the network level ( Cortese et al . , 2017 ) .", "This previous work sets up covert neurofeedback as a good candidate for a potential intervention in ASD , though whether specific , long-ranging connectivity changes can be induced through neurofeedback , which regions/networks are amenable to such reinforcement , how such training will affect wider brain networks , and how long these changes last , are all still open questions .", "In this study , we lay out a proof of principle of the plausibility of such training , showing robust , long-lasting feedback induced changes in these aberrant networks , coupled with preliminary results as to the behavioural correlates of these changes .", "17 ASD participants and 10 control participants were scanned over multiple sessions ( 123 sessions in total ) .", "These results reflect not only on the potential uses of such training in ASD , but also in other disorders with underlying aberrant connectivity at their core ." ], [ "We used previously collected resting state data on large groups of ASD and TD participants ( N = 56 ASD , 62 TD ) to identify two target brain regions that showed large under-connectivity in ASD compared with TD individuals , while also being physically distant from each other , and belonging to separate networks ( Figure 1 ) : target1 in superior temporal sulcus ( STS ) and target2 in somatosensory cortex , both of which have been consistently implicated in social processing ( Allison et al . , 2000; Frith and Frith , 2010; Adolphs , 2009; Damasio et al . , 2000 ) , and have previously been found to be under-connected and atypically activated in ASD ( Chen et al . , 2015; Gotts et al . , 2012; Müller et al . , 2001; Tuttle et al . , 2016; Khan et al . , 2015; Khan et al . , 2013 ) .", "This dataset is an expansion of previously reported data ( Gotts et al . , 2012 ) , which found very similar aberrant connectivity patterns , matching results from other studies using large datasets ( Cheng et al . , 2015 ) .", "As in the previously published subset of the dataset , we found that under-connectivity between these two networks ( STS and somatosensory ) in ASD was significant in both this large dataset ( p<4 . 3×10−5 ) and in the 17 participants recruited for the neurofeedback study ( p=0 . 002 ) , as well as significantly correlated to social symptom severity , as measured by the Social Responsiveness Scale ( SRS ) ( r = −0 . 35 p<0 . 009 without regressors , r = −0 . 31 , p<0 . 026 , using age and motion as regressors , r values represent Pearson’s correlation , p-values determined by permutation tests ) .", "The SRS is a parent filled questionnaire , which is designed to be a continuous , cardinal measure of social symptom severity in ASD , and has been shown to correlate with functional brain connectivity measures in multiple studies ( Anderson et al . , 2011; Di Martino et al . , 2009b ) .", "This result indicates that connectivity between these two networks is clinically relevant , i . e . the lower the connectivity , the more severe the social symptoms ( higher score on the SRS ) .", "The first goal of the training was therefore to increase the connectivity between target1 in STS , and target2 in somatosensory cortex .", "In order to ascertain that we would only be reinforcing connectivity between our two targets , rather than global changes that cause an overall increase in correlations across the entire brain in an undifferentiated manner , we selected a third control region ( in the inferior parietal lobule or IPL , part of the default mode network ) , which was chosen for being uncorrelated to the two target regions in our dataset of TD participants during resting state .", "IPL was significantly over-correlated to STS target1 in the ASD cohort participating in this study ( Figure 1C ) .", "This combination of under-connectivity between STS and somatosensory with over-connectivity to the default mode network , is in line with recent evidence of reduced within-network cohesion coupled with reduced between-network differentiation ( Hahamy et al . , 2015; Keown et al . , 2016 ) .", "The goal of the neurofeedback training was therefore to induce greater differentiation between these three regions of interest ( ROIs ) in participants with ASD , so as to bring connectivity levels between those three networks closer to those of TD individuals .", "This meant increasing connectivity between the two target regions , while simultaneously decoupling the two target-control pairs .", "To this end , we came up with the composite difference measure , combining the target-target and target-control correlations ( see Materials and methods ) .", "This measure was also significantly different between the ASD group and the TD group in both the previous large dataset ( p=0 . 001 ) , and in our cohort ( p<1×10−4 ) , Figure 1B–C ) .", "All p values were calculated through permutation tests , maintaining the original number of participants for each group .", "For the initial part of the study , 17 patients with ASD participated in four training sessions , over the course of 8 days ( two sessions of two consecutive days each , a week apart ) .", "Each session consisted of two rest scans , followed by four neurofeedback training scans , and finally two more rest scans ( Each scan was 9 min in duration . See Figure 2 ) .", "During the neurofeedback scans , participants started with a blank screen , and were instructed to attempt to reveal the picture hidden underneath ( see Figure 2—figure supplement 1 for an example ) .", "This was described to them as a puzzle task .", "No further instructions were given .", "Parents filled out behavioral questionnaires before training began , and two weeks after the last training session .", "An additional follow up study was then carried out , in which 15 of the 17 original participants returned for a final , slightly shorter training session .", "The interval between the original training and the follow up varied greatly between subjects , and ranged from 5 to 56 weeks .", "One of the barriers to carrying out connectivity-based rt-fMRI-nf has been the slow timescale of fMRI recordings , making online calculations of correlations very limited .", "We therefore developed a method that can approximate the correlations using only two time points: every two seconds , for each TR ( time to repetition ) , the signals from the three ROIs were analyzed in real-time ( see Materials and methods ) , and the trend in the signal compared to the previous TR was noted for each of the three ROIs ( increase/decrease ) .", "Positive feedback , in the form of revealing a part of the picture accompanied by an upbeat sound , was given whenever the network was deemed to have reached its desired state .", "As our goal was to increase correlation between the two target regions , and decrease correlations between the target and the control regions , feedback ( i . e . revealing a part of the picture ) was given whenever the signal trend in the two target ROIs was the same , and opposite from the trend in the control ROI ( Figure 2 , Materials and methods ) .", "This ‘two-point’ method was validated as being a good proxy for correlation analysis by comparing the results from this to standard Pearson’s correlation offline ( r = 0 . 61 , p<1×10−4 permutation test , Figure 2—figure supplement 2 ) .", "We carried out simulations using more time points ( 2–6 TRs ) to evaluate if longer time scales would lead to connectivity estimates for neurofeedback that better resemble actual correlations computed offline .", "We did not find any significant increase in the correlation of these extended measures to the whole series Pearson’s correlations , which were our ‘gold standard’ .", "We therefore chose to provide feedback on just two time points , to minimize the timescale of the feedback .", "At the end of each neurofeedback scan , participants were presented with their score , i . e . how many picture squares they had managed to reveal .", "They were then given a chance to attempt to beat their score on the next run , to win an additional bonus on top of the normal study compensation .", "The pictures were chosen to be neutral , depicting mostly scenes devoid of people and text , or abstract art/objects .", "Random pictures were chosen for each run , from a large set of such pictures .", "Participants completed 2–3 puzzles per scan on average , and there was no significant change in number of puzzles completed between day1 and day4 .", "Participants were blind to the purpose of the study , as well as to the mechanism of the neurofeedback , and even to the fact that it was neurofeedback .", "This was ascertained by exit questionnaires at the end of the last day of scanning , in which participants were interviewed regarding their thoughts on the study , their motivation , and their strategies during the training ( see Table 1 for responses ) .", "Responses as to the perceived nature of the ‘puzzle task’ varied widely , as did reported strategies , but none held any resemblance to the neurofeedback algorithm .", "Strategies mostly revolved around different ways of looking at the picture , as it was being revealed .", "Despite not knowing what they were supposed to do , most participants were highly motivated to solve the puzzles , with only 3 of the 11 participants for which responses are available reporting a motivation score of less than 5 ( on a scale of 1–10 , see Table 1 ) .", "An additional control group of 10 TD participants completed the same initial 4 day training regimen , following the same protocol as the above .", "This group received feedback on the same three nodes , but in a different network configuration: target1 in the STS remained the same , but somatosensory target2 and the IPL control region switched roles , so that feedback was given whenever STS ( target1 ) and IPL ( now target2 ) were co-modulated , and were opposite to somatosensory cortex ( now control ) , see Figure 2—figure supplement 3 .", "This provided feedback orthogonal to that given to the ASD group .", "Another key difference is that this feedback was antithetical to the normal connectivity patterns found in the typically developing brain , as STS and somatosensory are well correlated in the typically developing brain , whereas the IPL region used in this study was explicitly chosen to be as uncorrelated as possible with STS in TDs during rest ( Figure 1B–C ) .", "This control therefore served a dual purpose: in terms of the network that the ASD participants were being trained on , which rewarded increased connectivity between STS and somatosensory and decoupling of these from IPL , this was random feedback .", "That is to say , the feedback given to the TD participants was uncorrelated with the feedback they would have received had they been trained on the same network configuration as the ASD participants .", "This served as a control for any changes in connectivity in that direction being driven by something other than the feedback .", "At the same time , this control also examined whether it is possible to modulate any network , regardless of the native connectivity .", "To assess whether any learning took place over the course of these initial four training days , we examined the correlations between the two target regions ( which had been trained to increase connectivity ) , the two target-control pairs ( which were trained to decrease connectivity ) , as well as the composite difference measure .", "Figure 3 shows the results of this analysis for the ASD group .", "As can be seen , over the course of the four training days , correlations between the two target regions steadily increased ( with a significant difference between most days , p=4×10−4 between day1 and day4 , mean change in correlation = 0 . 11 , Figure 3A ) , while correlations between target1 and control decreased ( significant difference between day1 and all other days , p=8×10−4 between day1 and day4 mean change = −0 . 13 , Figure 3B ) .", "Though there is an overall decrease between target2 and control , this does not reach significance and the mean change is small ( Figure 3C ) .", "This is in line with the lack of differentiation between the ASD group and the TD group in target2-control correlations ( Figure 1C ) , suggesting neurotypical network connectivity is more resilient to change .", "Figure 3D shows the overall composite difference measure , taking into account all three correlation pairs , where there is a strong and consistent increase between day1 and day4 ( p<2×10−4 , mean change = 0 . 19 ) .", "15 of the 17 participants showed a positive change in this measure ( 14/17 had a positive change in target1-target2 correlations , as well as a negative change in target1-control correlations ) .", "The TD control group on the other hand , showed no significant changes between days in any of the three pairwise combinations , or in the composite difference measure .", "Only 4/10 participants in this group showed a change in the trained direction in the composite measure , within the range of chance , and the magnitude of change was minimal relative to the change seen in the ASD participants .", "Figure 4A shows these data for all the individual ASD participants , while Figure 4B shows the individual TD participants .", "The full results for all individual participants , for all days , are displayed in Figure 4—figure supplement 1 .", "To further ensure that the null result in the TD group was truly different from the significant result in the ASD group , and not simply due to lower statistical power because of the smaller sample size , we re-calculated the change in correlations for each of the possible subsets of 10 participants from our ASD group , and found that the significant difference between day1 and day4 was maintained for all subsets in the target-target correlations ( range of mean change 0 . 03–0 . 17 , p-value range 0 . 046 to 1 × 10−4 ) , for 83% of possible subsets in the target1-control correlations range of mean change −0 . 02 to −0 . 19 , 0 . 22 < p < , 1 × 10−4 and for all possible subsets in the composite correlation difference measure ( range of mean change 0 . 1–0 . 23 , p<0 . 007 for all subsets ) .", "To test for interaction between the TD and ASD groups , we carried out a two sample t-test ( without assuming equal variance to control for different sample size ) between the change in composite correlations in the ASD group vs . the change in composite correlations in the TD group .", "A significant difference ( p=9 . 2×10−4 ) was found between the learning seen in the TD and ASD groups in this analysis .", "This significant difference between the groups was maintained even when we chose the ASD subset of 10 participants with the smallest change ( p=0 . 013 ) .", "We next set out to test how long this learning would be maintained .", "To address this question , we called back the participants for a follow up study , in which they returned for another , shorter round of training .", "To get a good indication of the persistence of the training effect , participants were called back in a staggered manner , from as little as 5 weeks and up to 56 weeks from their original training .", "Our results indicate that the learning was mostly , though not fully , preserved , even after such an extended time period ( Figure 3A–D , follow up ) .", "In fact , although there was variation between subjects in the degree of retention , there was no correlation between the time that had elapsed and the rate of retention ( see Figure 3—figure supplement 1 ) .", "Since there were only two feedback runs in this follow up scan , we also compared them to just the first two feedback runs for the first four days , in order to account for any differences arising from the different number of runs .", "The results using just the first two runs for the first four days were not in any way different from the results using the full data ( see Figure 3—figure supplement 2 ) .", "So far we had only considered what happens in the regions that were trained .", "In order to get a more comprehensive picture of the effects of the training on the brain , we conducted a whole brain analysis , which looked for changes during the training period ( i . e . from day1 to day4 ) .", "We calculated three maps , one for target1 , one for target2 , and one for the control region , with each map showing the change from day1 to day4 in the correlation of each voxel in the brain to the corresponding region .", "We then carried out a t-test across all participants for each of these three analyses , and the resulting maps for the ASD group are displayed in Figure 5 .", "The changes were exactly as predicted by the training: the strongest positive change in correlation to target1 over the training period was in the somatosensory cortex ( with a peak at target2 ) , and the strongest negative change was in the control region .", "Changes to correlations with target2 were seen in the STS with a peak in target1 , and negative changes in correlation to control were seen in bilateral STS ( Figure 5A ) .", "Since we were training a network of three nodes , rather than a simple connection between two regions , we next calculated the composite change: for each voxel , the change between day1 and day4 in its correlation to target1 minus its correlation to control ( Figure 5B ) , and the same change in its correlation to target2 minus control ( Figure 5C ) .", "This analysis yielded similar but far stronger results .", "The maps of the composite correlations were corrected at a very conservative cluster threshold determined by random permutation testing , in accordance with recent statistical recommendations for analyses utilizing cluster size ( Eklund et al . , 2016 ) ( see Materials and methods ) .", "These results support a causative role for the feedback itself , as the specific relationship that was trained between the two targets and the control came up in completely independent , whole-brain analysis .", "That is , using target1 relative to the control seed , the largest change in the whole brain was found in target2 , even though that region was not pre-selected and the analyses did not constrain this to happen , and vice-versa using target2 and control .", "Note that we do not expect to find changes between day1 and day4 in either target1 or control in the target1-control map , as these regions did not change in relation to themselves .", "Rather , this analysis highlights all the other areas , outside of those two regions , which changed their correlation over the course of training in relation to target1 ( increasing ) and to control ( decreasing ) , finding the peak of this change in target2 .", "The same is true for the target2-control map , which shows an even greater effect focused on target1 , consistent with the ROI analysis results showing a greater decoupling of target1 from control than target2 from control .", "Note that Figure 5 shows results only for the ASD group , as no significant peaks were identified in any of the target or control regions for the TD control group , and no voxels survived the cluster correction threshold .", "The training-related changes we have demonstrated to this point were during the neurofeedback scans themselves .", "To be of any potential clinical value , these changes must also generalize beyond the training sessions , to the resting state scans , which reflect the baseline connectivity of the brain when not engaged in a specific task .", "In order to obtain as accurate an estimate of baseline as possible and to avoid any contamination by the task , only the two rest scans prior to the neurofeedback were used for this analysis .", "Changes were overall smaller than those seen during the training , but significant changes were found between day1 and day4 ( target1-target2 correlations mean change = 0 . 07 , p<0 . 038 , composite correlations measure mean change = 0 . 1 , p<2×10−4 ) , and between day1 and the follow up ( target1-target2 mean change = 0 . 09 , p<0 . 011 , composite correlations measure mean change = 0 . 11 , p<2×10−4 ) .", "Change in rest was significantly correlated to change during the neurofeedback scans ( r = 0 . 42 , p<0 . 04 , permutation test ) .", "Moreover , 14/15 participants who came in for the follow up showed an increase in the composite correlation measure ( Figure 6A ) .", "To assess whether the changes seen in the follow up could simply be a function of the elapsed time , we examined data from all participants in the previous study ( used to define the training regions ) which had at least two resting state scans from two different time points , and evaluated the change in connectivity seen between the sessions .", "19 participants had two such data points , and the average time between the sessions was 13 . 2 months .", "The change however was not significant for any of the pairwise correlations , or the composite correlation measure ( Figure 6A ) .", "We next looked at the composite measure for the 10 participants from our study who had also participated in the previous resting state experiment ( and in the follow up ) , and compared change from the previous experiment to the first rest sessions on day1 , before any training , and in this subset also the change from day1 of the training to day4 and to the follow up ( Figure 6B ) .", "While there was no significant change from the previous experiment to day1 ( mean time interval = 38 . 3 months , mean change = −0 . 04 ) , there was significant change to day4 ( mean change = 0 . 1 , p<0 . 019 ) and to the follow up ( mean change = 0 . 11 , p<0 . 003 ) .", "Taken together , these analyses provide strong evidence that the changes we observed were not a function of elapsed time but rather occurred as a direct result of our neurofeedback regime .", "Finally , we asked whether the changes we see as a result of the training are in any way correlated to behavior .", "To this end , we looked at changes in behavior as measured by the behavioral questionnaires filled out by the parents prior to training , and two weeks after the end of the initial training set .", "These behavioral results included two statistical outliers who were removed from the analysis ( definition of outliers was based on change scores greater than 3 standard deviation from that seen in an independent data set , see Materials and methods ) .", "We compared the change in these behavioral questionnaires to the change in correlations between the final resting state scans on the last day ( following neurofeedback ) , and the resting state scans on the first day .", "There were two measures of behavior: the first , the Social Responsiveness Scale ( SRS ) , has previously been found to correlate with functional connectivity in this network ( see section on target selection ) , and therefore the change in this rating was expected to correlate with the change in the network .", "The second , the Behavior Rating Inventory of Executive Function ( BRIEF ) , measures executive function rather than social abilities , and though patients with ASD show deficits on this measure ( Baron et al . , 2000 ) , it is expected to reflect prefrontal functioning , and we expected that changes on this measure would not correlate to changes in the social network being trained ( Anderson et al . , 2002; Anderson et al . , 2005; Mahone et al . , 2009 ) .", "Indeed , although there was no significant change in the mean SRS score across participants , with the mean score actually slightly ( though not significnatly ) increasing instead of decreasing as predicted ( mean SRS before training = 69 . 8 , mean SRS after training = 71 . 6 , p=0 . 11 ) , there was a significant correlation between changes in the resting state network and the change in SRS ( pre training minus post training , so that a positive change corresponds to a reduction in symptoms , r = 0 . 56 , p=0 . 016 , Figure 7 ) .", "There was no significant correlation between the baseline SRS score and the change in SRS seen during training ( r = 0 . 14 , p=0 . 29 ) , or the baseline rest and the change in rest ( r = −0 . 22 , p=0 . 2 ) .", "The partial correlation between change in rest vs . change in SRS , after controlling for baseline SRS and baseline rest , was higher ( r = 0 . 62 p=0 . 014 ) .", "No such correlation was found with the BRIEF ( change in SRS vs . change in resting state correlations: r = 0 . 56 , p=0 . 016; change in BRIEF vs . change in resting state correlations: r = 0 . 09 , p=0 . 39 ) .", "We further tested for the correlation between the change in SRS vs . change in the resting state correlations , after partialing out the contribution of the BRIEF .", "After removing the variance explained by the BRIEF , the correlation between the rest and SRS was even higher ( r = 0 . 6 , p=0 . 02 ) , indicating that change in resting state correlations was captured by the change in SRS scores , but not the BRIEF .", "Note that the SRS was chosen as our behavioral measure because it has consistently been shown to correlate with aberrations in network structure in ASD ( Gotts et al . , 2012; Wallace et al . , 2012; Di Martino et al . , 2009b; Anderson et al . , 2011 ) .", "Nevertheless , it was not designed to be used with such a short test-retest interval ( 3 weeks ) , and has only been validated for intervals of 6 months or more ( Hus et al . , 2013; Constantino et al . , 2003; Bölte et al . , 2008 ) .", "We can therefore make no claims regarding the absolute values of the change in scores .", "In the interest of completeness , we also ran the analysis with the outliers ( marked in red in Figure 7 ) .", "Without removing outliers , the correlation with SRS fell below significance , though the trend was still in the same direction ( r = 0 . 26 , r = 0 . 31 after controlling for baseline SRS and baseline rest , p=0 . 09 ) .", "A possible confound in using test-retest reliability on other data to calculate outliers ( as was done here ) is that our neurofeedback data could include large , training induced changes , which would not be present in other datasets , skewing the variance .", "Note however that the two outliers reported here had a large negative behavioral change , which is not the predicted direction of change due to neurofeedback based training .", "This is more likely due to the short assessment interval used here ( two weeks ) , which would give a lot of weight to negative incidents which occurred during this time .", "For instance , one of the participants whose data was determined to be an outlier , had moved to a new house during this time period ." ], [ "The study of rt-fMRI covert neurofeedback – feedback given on pre-specified brain activity , but without providing participants with further information regarding the nature of the task or an explicit strategy through which to control the feedback – is still in its infancy ( for a review see [Sitaram et al . , 2017] ) .", "These results not only help solidify the existing evidence that reward-mediated learning through covert neurofeedback is possible ( Shibata et al . , 2011; Ramot et al . , 2016; Amano et al . , 2016 ) , but also expand our knowledge in important ways .", "In this study , we have demonstrated that covert neurofeedback can be used to modulate correlations between distinct , physically distant networks ( Figure 3 ) , in the great majority of participants ( 15 of17 , Figure 4 , Figure 4—figure supplement 1 ) .", "We have further shown that this modulation is possible even in cases of aberrant network structure , in clinical populations , and is sustainable for extended periods of time ( some of our follow up sessions were a year after the original training , and we saw no evidence for an effect of time as a modulator of retention , Figure 3—figure supplement 1 ) .", "This kind of connectivity-based neurofeedback has previously been carried out in only a handful of studies ( Scharnowski et al . , 2014; Megumi et al . , 2015; Koush et al . , 2017 ) , though the network approach to neurofeedback is gaining traction and shows not only good preliminary findings but also has a sound theoretical basis ( Bassett and Khambhati , 2017 ) .", "It could be argued that the changes in functional connectivity that we found were not a result of the feedback , but rather of the multiple fMRI visits , or were somehow precipitated by the nature of the puzzle task .", "Though imperfect because of the different population , the control using the TD group , which received feedback largely orthogonal to the network trained in the ASD group , provided further evidence for the necessity of the feedback itself in inducing these changes .", "The TD group , which went through the exact same protocol as the ASD group but received feedback on a different network , did not demonstrate the changes in connectivity seen in the ASD group ( Figure 4 , Figure 4—figure supplement 1 ) .", "Moreover , the extraordinary specificity of the changes revealed by the whole brain analysis ( Figure 5 ) , peaking exactly at the small ROIs that were chosen for the training , would also seem to preclude an alternative explanation .", "The further significance of the whole-brain analysis is that this learning is spatially specific even when training disparate networks , meaning that it is possible to target specific regions of the brain .", "However , it is important to note in this regard that though the peaks were centered on the regions we were training , the changes spread to entire networks , as would be expected from the architecture of the brain , which is composed of large-scale networks of multiple brain regions , making it difficult to induce changes to just one region in isolation .", "In a larger context , the failure to induce change in the direction of training in the control subjects , suggests that while it is possible to train networks that are fundamentally connected , it is much more difficult to train networks that are uncorrelated or weakly correlated in the typically developing brain .", "This conclusion is also bolstered by the failure of the training to induce change in the ASD group between target2 and control , regions which did not differ in their connectivity from that of the TDs .", "It is also possible that some networks may be more difficult to train than others , as has previously been suggested ( Harmelech et al . , 2015 ) .", "Future studies will be needed to better understand the basic constraints in modifying these relationships .", "The prohibitive cost of rt-fMRI , or fMRI scans in general , limits the number of training sessions in these paradigms .", "Moreover , the under-connectivity between the target regions chosen here explains only some of the behavioral deficits , and is clearly not the sole underlying cause of autism .", "This study was therefore designed as a proof of principle that aberrant connectivity can be addressed through neurofeedback , rather than as a clinical intervention , and whatever behavioral effects we found were expected to be modest .", "Once such a causal relationship is established , future potential clinical applications might pursue more cost effective options , such as identifying EEG signatures that correspond to activity from these areas , as several groups have already begun to develop ( Zotev et al . , 2014; Meir-Hasson et al . , 2014 ) .", "From a clinical perspective , the most important result in this study is the successful transfer of the change in correlations to the resting state .", "By itself , change during training does not guarantee generalization of the learning , or in this case of the change in the network structure .", "Though the change in the baseline resting state ( before neurofeedback training each day ) was somewhat more modest than the change seen during training , it was reliable and consistent , and could not be explained simply by the passage of time ( Figure 6 ) .", "Note that the correlation seen with behavior ( Figure 7 ) is with the change from rest on day1 , to the resting state data collected immediately after the last training session on day4 .", "This resting state change was overall smaller than the change seen with the pre-training baseline rest on day4 shown in Figure 6 , but the day4 pre-training baseline did not show a correlation with behavior .", "The behavioral results , though they did not show an overall mean change , are preliminary in their scope and limited by the timescale on which they were measured , demonstrate that change in behaviorally relevant networks correlates with change in behavior , which is a crucial and entirely non-trivial point in terms of the potential clinical applications of neurofeedback .", "The lack of overall mean change together with the correlation with connectivity changes makes it unlikely that this result is driven by a placebo effect , and on debriefing after all data were collected , it was clear that the patients and their families did not conceive of this as an intervention , and had no expectation of behavioral change following the study .", "This finding also adds to the debate regarding the nature of the functional and structural hypoconnectivity found in ASD , whether it plays a causative role in ASD or is simply a downstream effect ( Vasa et al . , 2016 ) .", "A change in behavior following a change in connectivity suggests the former , bolstering the mechanistic approach to functional connectivity in ASD .", "However , it should be noted that underconnectivity is not the full story in ASD , and there is growing evidence for hyper cortico-thalamic connectivity , alongside the cortico-cortico hypoconnectivity ( Picci et al . , 2016 ) .", "It should also be noted that the under-connectivity seen in cortico-cortico correlations in ASD is dampened connectivity ( closer to 0 ) , and not anti-connectivity ( negative correlation ) .", "The nature of the feedback given here is that positive correlations between the two targets are rewarded , thus boosting connectivity that already exists at baseline ( as baseline connectivity is positive , just small ) .", "Future studies will have to replicate and expand on the behavioral findings , but this method of testing behavioral changes following connectivity changes could be a promising tool for assessing different models of autism as well as other neuropsychiatric disorders .", "The targets used in this paradigm were derived from a group analysis , and were not individually localized .", "As the variance within groups suggests , this is likely not the optimal method for ROI selection , especially if the focus is on behavioral change .", "These results are therefore probably an under estimation of what this tool can do , with individually tailored ROIs .", "It is not clear what the best method for individual ROI selection would be , or which localizer would best identify target regions , but it is another direction that should be pursued in future studies .", "Since the lack of an explicit strategy allows covert neurofeedback to be used to directly target all manner of abstract , behaviorally relevant networks , potential applications could be far ranging , encompassing many clinical disorders with underlying aberrant connectivity at their core .", "Moreover , this is a promising technique to be used in more basic science questions , as a tool to investigate questions of causality ." ], [ "19 Males aged 15–25 ( mean age = 20 . 93 ) who met the DSM-IV criteria for autistic disorder , an autism cut-off score for social symptoms on the Autism Diagnostic Review ( ADR ) and/or an ASD cutoff score from social +communication symptoms on the Autism Diagnostic Observation Schedule ( ADOS ) , all administered by a trained , research-reliable clinician , were recruited for this experiment .", "Additionaly , 11 age matched typically developing males were recruited for the control group .", "All participants had normal to corrected to normal vision .", "IQ scores were obtained for all participants , and all full-scale IQ scores were ≥85 as measured by the Wechsler Abbreviated Scale of Intelligence , the Wechsler Adult Intelligence Scale-III , or the Wechsler Intelligence Scale for Children-IV .", "Participant groups did not differ in terms of full-scale IQ .", "one ASD participant was removed due to discomfort in scanner on day1 , and another ASD participant was removed on day two due to anxiety .", "1 TD participant was removed after day1 for excessive motion .", "17 ASD and 10 TD participants completed all four days of neurofeedback training .", "15 ASD participants returned for the follow up experiment .", "The experiment was approved by the NIMH Institutional Review Board ( protocol 10_M-0027 ) .", "Written informed consent was obtained from all participants .", "Three Regions of Interest ( ROIs ) were selected for training: two targets and one control .", "The targets were chosen according to previous research as those with a large degree of reduced connectivity in Autism Spectrum Disorder compared with typically developing ( TD ) controls , based on between group analysis as explained in ( Gotts et al . , 2012 ) .", "For this analysis , we used an expansion of the dataset published in ( Gotts et al . , 2012 ) , N = 56 ASD , 62 TD .", "Of the 56 ASDs in this dataset , 11 participated in the neurofeedback study .", "Additional constraints placed on the choice of ROIs was for them to be physically distant from each other , and in different networks ( see ( Gotts et al . , 2012 ) for details ) .", "All ROIs were defined as spheres of 4 mm radius surrounding the focal points: Target1 - left Superior Temporal Sulcus ( Talairach coordinates: −49 , –29 , 0 ) , Target2 - left somatosensory cortex ( Talairach coordinates: −54 , 14 , 39 ) , and Control - right Inferior Parietal Lobe ( chosen to be as uncorrelated with these two targets as possible in the TD dataset , Talairach coordinates: 49 , –50 , 42 ) .", "Figure 1 shows the between group difference in the correlations between the ROIs .", "All scans were collected at the Functional Magnetic Resonance Imaging Core Facility on an 8 channel coil GE 3T ( GE Signa HDxT 3 . 0T ) magnet and receive-only head coil , with online slice time correction and motion correction .", "The scans included a 5 min structural scan ( MPRAGE ) for anatomical co-registration , which had the following parameters: TE = 2 . 7 , Flip Angle = 12 , Bandwidth = 244 . 141 , FOV = 30 ( 256 × 256 ) , Slice Thickness = 1 . 2 , axial slices .", "EPI was conducted with the following parameters: TR = 2 s , Voxel size 3 . 2*3 . 2*3 . 2 , Flip Angle: 60 , TE = 30 ms , Matrix = 72×72 , Total TRs = 270 , Slices: 37 .", "All scans used an accelerated acquisition ( GE's ASSET ) with a factor of 2 in order to prevent gradient overheating .", "The initial neurofeedback experiment consisted of 4 training sessions over 8 days .", "There were 2 consecutive training days , a 6 day delay , then a final set of 2 consecutive training days .", "Each training day had 2 initial rest scans , 4 neurofeedback sessions , and 2 final rest scans .", "All scans were 9 min long .", "Participants were instructed to maintain an eyes-open rest and look at the blank screen .", "Neuropsychological tests were administered at two timepoints: on the first training day before scanning , and two weeks following the last training day .", "Follow up scans were conducted 5–56 weeks after the final training day and consisted of a single , abbreviated neurofeedback session with two rest scans followed by two neurofeedback sessions .", "Regions of Interest ( ROIs ) were defined in Talairach space as described above .", "The standard Talairach brain was then co-registered to the structural scan collected that day , which was in turn co-registered to a short ( 10 TRs ) functional echo-planar imaging scan ( setup EPI ) collected for that purpose each day before the first resting state session , to bring the ROIs into the native space during neurofeedback processing .", "All coregistration was carried out with the AFNI ( Analysis of Functional Neuro-Images ) software package ( Cox , 1996 ) .", "During online processing of the data , 3D motion correction and slice time correction were carried out on all functional images .", "BOLD signal was extracted from each voxel in the ROIs and the mean signal was calculated for each ROI .", "Feedback decisions were determined by a difference measure , taking into account both the changes in the trend between the two target ROIs and the control ROI .", "This difference measure was calculated for each TR and for each of the three ROIs .", "Our rt-fMRI algorithm calculated the difference between the mean signal in the current TR minus the signal in the preceding TR , giving the signal trend in each ROI ( increasing or decreasing ) .", "If the trend in the two targets was the same , and opposite from the trend in the control ROI , then feedback was given , meaning both conditions had to be fulfilled for feedback to be given:ms ( Target1 ( TR=t ) ) −ms ( Target1 ( TR=t−1 ) ) ms ( Target2 ( TR=t ) ) −ms ( Target2 ( TR=t−1 ) ) >0&ms ( Target1 ( TR=t ) ) −ms ( Target1 ( TR=t−1 ) ) ms ( Control ( TR=t ) ) −ms ( Control ( TR=t−1 ) ) <0 ( ms = mean signal ) Each training session had four neurofeedback training scans .", "The scans started out with a uniformly grey screen .", "Participants were told that there is a picture hidden underneath , and were instructed to try to unveil the image during what was described as the puzzle task .", "Importantly , no further cognitive strategies or suggestions were given to the participant for the duration of the experiment .", "Participants were not informed that their performance on the puzzle task was determined by brain activation .", "Participants received two forms of positive reinforcement whenever the real-time algorithm determined that the network requirements had been met: a ‘puzzle piece’ , i . e . a square of the hidden picture , would become visible on the screen with a concomitant sound of positive valence .", "This feedback was chosen to maximize engagement with the paradigm during the scan by providing a complex and interesting visual stimulus in a game-like setting , and the auditory stimulus was paired to ensure that participants would be aware of positive feedback independent of their visual attendance .", "During rest scans participants were shown a uniformly grey screen .", "During Neurofeedback training participants would begin with a uniformly grey screen .", "Then the image would become visible in small rectangular blocks , described to the participants as ‘puzzle pieces . ’ There were 25 ‘puzzle pieces’ per board , which would be displayed piece by piece until a whole image was unveiled .", "After a full board was completed , the screen would become blank and a new image would begin to appear .", "The images were randomly selected from a set of 100 non-social images devoid of people or text , like a landscape or an abstract painting .", "At the end of each 9 min training round , participants viewed a scoreboard which told them how many individual pieces they had unveiled that round , as well as the top score that they had received that day .", "Participants were financially incentivized to beat their best score for that day .", "fMRI offline data preprocessing: Post-hoc signal preprocessing was conducted in AFNI .", "The first four EPI volumes from each run were removed to ensure remaining volumes were at magnetization steady state , and remaining large transients were removed through a squashing function ( AFNI's 3dDespike ) .", "Volumes were slice-time corrected and motion parameters were estimated with rigid body transformations .", "Volumes were coregistered to the anatomical scan .", "Volumes were smoothed with 6 mm blurring and normalized by the mean signal intensity of each voxel .", "The AFNI ANATICOR procedure was then applied to remove nuisance physiological and nonphysiological artifacts from the data ( Jo et al . , 2010 ) .", "The anatomical scan was segmented into tissue compartments with Freesurfer ( Fischl et al . , 2002 ) , Ventricle and white-matter masks were created and applied to the volume-registered EPI .", "Prior to smoothing , these masks gave pure nuisance times series for the ventricles and local estimates of the BOLD signal in white matter , averaged within a 15 mm radius .", "The measured respiration and heart rate signals were used to create Retroicor ( Glover et al . , 2000 ) and respiration volume per time ( RVT ) regressors ( Birn et al . , 2008 ) .", "All nuisance time series in every run ( average ventricle time series , average local white matter time series , 6 parameter estimates for head motion , and thirteen RVT and Retroicor regressors ) were detrended with fourth-order polynomials before least-squares model fit to each voxel time series .", "No other filtering of the data was done .", "All participant data was aligned by affine registration to AFNI’s TT-N27 template in standardized Talairach and Tournoux ( Talairach and Tournoux , 1988 ) space .", "Baseline neuropsychological tests were conducted before the initial training session , and post-experiment surveys were collected two weeks after the final neurofeedback session .", "Parents filled out the Social Behavior Scale ( SRS ) to identify common social behaviors in autism , as well as the Behavioral Rating Inventory of Executive Function ( BRIEF ) .", "The ‘informant’ report ( filled in by a parent ) was used as it has been shown to be more accurate ( McMahon and Solomon , 2015 ) .", "An independent dataset of ASD subjects who did not participate in this experiment but had SRS test-retest data was used to determine change reliability .", "Data points that were beyond three standard deviations from the mean as determined by this analysis were excluded as outliers .", "We developed a cognitive strategy questionnaire that was completed by 11 of the 17 participants .", "Following their final scan session on day4 of the training , each of these participants was asked what they thought the experiment was about .", "Participants were then asked what they were doing during the scans , and if they used a particular cognitive strategy .", "Finally , participants were asked to rate on a scale of 1–10 how hard they had been trying to solve the puzzles each day , how satisfied they felt when a new puzzle piece came up , and if there were differences between days .", "The first six participants did not complete this questionnaire , but were interviewed after the final scan and reported no knowledge of the objective of the task , and similar cognitive strategies to those later reported in the questionnaire .", "See Table 1 for the data from these questionnaires .", "All data were analyzed with in-house software written in Matlab , as well as the AFNI software package .", "Data on the cortical surface were visualized with SUMA ( SUrface MApping ) ( Saad et al . , 2004 ) .", "The composite difference measure was computed by subtracting the average correlation of the two target/control pairs , from the target/target correlation:corrTarget1 , Target2-12 ( corrTarget1 , Control+ corrTarget2 , Control ) All p-values for the changes in correlation between days were computed through permutation tests , randomly permuting the days for 5000 iterations .", "For each participant , for each neurofeedback scan on day1 and day4 , we first transformed the correlation values with Fisher’s z-transform to improve normality , then calculated a difference measure per voxel: corr ( voxel time series , avg . Target1 time series ) - corr ( voxel time series , avg . Control time series ) .", "The resulting maps held information regarding each voxel’s differential correlation to the Target1 vs . Control ROIs .", "We then averaged the maps for each participant across all four neurofeedback scans for each of the two days , and subtracted the average day1 map from the average day4 map .", "Each voxel in the resulting map now signified the change in correlation from day1 to day4 , in the differential correlation to the Target1 ROI vs . the Control ROI , where a positive value means that this voxel was differentially more correlated to Target1 than to Control on day4 compared with day1 .", "Normality of these data were ascertained using Lilliefor’s goodness of fit test .", "We then carried out a t-test across the 17 participants , to identify voxels with a consistent change across subjects .", "Maps were corrected using a permutation test to determine significant cluster size , with day1 and day4 randomly permuted for each participant across 5000 permutations ( as suggested by [Eklund et al . , 2016] ) .", "These permutations were carried out at p-value thresholds of 0 . 05 , 0 . 01 , 0 . 005 , 0 . 001 and 0 . 0005 , and a mask was created of voxels that survived any of these corrections .", "The mask was then applied to the map shown in Figure 5A , which was set at a p-value threshold of 0 . 05 .", "The same procedure was carried out for the Target2 minus Control differential correlation , and the resulting map is shown in Figure 5B .", "Data is available via the XNAT platform https://central . xnat . org/app/template/Index . vm ( dataset title 'Direct modulation of aberrant brain network connectivity through real-time neurofeedback' with ID number ASD_NF ) .", "As some of the participants in the experiment signed an older version of the consent form , which does not explicitly allow for data sharing , we are currently working on re-consenting all the participants with a new version .", "Hence for now , users will need to request access through the system .", "This can be done by creating a XNAT user account and pressing the request access link ." ] ]

[ "The existence of abnormal connectivity patterns between resting state networks in neuropsychiatric disorders , including Autism Spectrum Disorder ( ASD ) , has been well established .", "Traditional treatment methods in ASD are limited , and do not address the aberrant network structure .", "Using real-time fMRI neurofeedback , we directly trained three brain nodes in participants with ASD , in which the aberrant connectivity has been shown to correlate with symptom severity .", "Desired network connectivity patterns were reinforced in real-time , without participants’ awareness of the training taking place .", "This training regimen produced large , significant long-term changes in correlations at the network level , and whole brain analysis revealed that the greatest changes were focused on the areas being trained .", "These changes were not found in the control group .", "Moreover , changes in ASD resting state connectivity following the training were correlated to changes in behavior , suggesting that neurofeedback can be used to directly alter complex , clinically relevant network connectivity patterns ." ]

[ "Even when we are at rest , our brains are always active .", "For example , areas of the brain involved in vision remain active in complete darkness .", "Different brain regions that connect together to perform a given task often show coordinated activity at rest .", "Past studies have shown that these resting connections are different in people with conditions such as autism .", "Some brain regions are more weakly connected while others are more strongly connected in people with autism spectrum disorder compared to those without .", "Furthermore , people with more severe symptoms seem to have more abnormal connections .", "“Neurofeedback training” is a method of changing the resting connections between different brain regions .", "Scientists measure a brain signal – the connection between different brain regions – from a person in real time .", "They then provide positive feedback to the person if this signal improves .", "For example , if a connection that is too weak becomes stronger , the scientists might reinforce this by providing feedback on the success .", "Previous work has shown that neurofeedback training may even change people’s behaviour .", "However , it has not yet been explored as a method of treating the abnormal connections seen in people with autism when their brains are at rest .", "To address this , Ramot et al . used a technique known as “functional magnetic resonance imaging” ( or fMRI for short ) to measure brain activity in young men with autism .", "First , certain brain regions were identified as having abnormal resting connections with each other .", "The participants were then asked to look at a blank screen and to try to reveal a picture hidden underneath .", "Whenever the connections between the chosen brain regions improved , part of the picture was revealed on the screen , accompanied by an upbeat sound .", "The participants were unaware that it was their brain signals causing this positive feedback .", "This form of neurofeedback training successfully changed the abnormal brain connections in most of the participants with autism , making their connections more similar to those seen in the wider population .", "These effects lasted up to a year after training .", "Early results also suggest that these changes were related to improvements in symptoms , although further work is needed to see if doctors could reliably use this method as a therapy .", "These findings show that neurofeedback training could potentially help treat not only autism spectrum disorder , but a range of other disorders that involve abnormal brain connections , including depression and schizophrenia ." ]

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

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

[ [ "The spectra of genomic and transcriptomic alterations across cancer types are highly heterogeneous .", "A multitude of driver genes exist both within and across subtypes ( e . g . Armenia et al . , 2018; Nik-Zainal et al . , 2016 ) spanning a range of mutational types , from simple substitutions , to extensive genome-wide aneuploidies and complex rearrangements ( Ciriello et al . , 2013; Hoadley et al . , 2014; Kandoth et al . , 2013;Lawrence et al . , 2013b; Zack et al . , 2013 ) .", "This complex array of genetic alterations confounds efforts to assign function to driver genes and identify key driver mutations within patients .", "Despite this extensive molecular heterogeneity , tumors originating from a variety of tissues converge to several common hallmark cellular phenotypes ( Hanahan and Weinberg , 2011 ) .", "Many of these involve loss of features commonly associated with multicellularity , for example , uninhibited proliferation , tissue dedifferentiation , disruption of cell-cell adhesion and intercellular communication , suggesting the alteration to genes involved in the evolution of multicellular traits is central to tumor development ( Aktipis et al . , 2015; Aktipis and Nesse , 2013; Davies and Lineweaver , 2011; Vincent , 2012 ) .", "Through the addition of new genes and repurposing of existing genes , metazoan evolution has led to progressive formation of intricate and interconnected regulatory layers ( Arenas-Mena , 2017; Chen et al . , 2014; Schmitz et al . , 2016 ) , which suppress improper activation of replicative processes originating in single-celled organisms and ensure the complex phenotypes and cooperative growth required for multicellularity .", "Our investigation of gene expression data from a collection of solid tumors revealed extensive downregulation of genes specifying multicellular phenotypes and activation of genes conserved to unicellular organisms ( Trigos et al . , 2017 ) .", "This was accompanied by significant loss of coordinated expression of unicellular and multicellular processes within tumors , suggesting selection for the disruption of key regulators mediating communication between unicellular and multicellular genes .", "Evidence for such selection comes from the clustering of cancer genes at the evolutionary boundary of unicellularity and multicellularity ( Domazet-Loso and Tautz , 2010 ) and the accumulation of mutations in genes required for multicellular development during tumor progression ( Chen et al . , 2015 ) .", "Only a limited number of driver mutations are thought to be responsible for the transition from normal , healthy cell to a malignant state ( Martincorena et al . , 2017; Miller , 1980; Schinzel and Hahn , 2008; Stratton et al . , 2009 ) , suggesting individual mutations in highly connected genes in the regulatory network could bring about significant changes in cellular phenotypes .", "Under our model , the highest impact mutations would be those affecting proteins modulating the communication between the subnetworks that sustain multicellularity and the conserved core of fundamental cellular processes originated in single-celled organisms ( Trigos et al . , 2018 ) .", "This would enhance a more primitive phenotype and provide a strong selective advantage for individual cellular lineages .", "Therefore , the contribution of somatic mutations to the rewiring of transcriptional networks during tumor development can be contextualized and estimated by their effect on gene regulatory networks pieced together during evolution , aiding the identification of key driver mutations .", "Here , we elucidate how mutational heterogeneity across tumors results in common cellular hallmarks by accounting for their evolutionary ages and locations in the GRN .", "We found an overrepresentation of copy-number aberrations and point mutations in genes dating back to early metazoan ancestors .", "Point mutations disrupted key master regulators that evolved in early in the metazoan lineage , suggesting a primary role in the uncoupling of the subnetworks regulating multicellularity and the fundamental core of cellular processes dating back to single-celled organisms .", "CNAs were involved in a complementary mechanism of dysregulation , generally disrupting the downstream targets of each of these subnetworks .", "These results indicate that both point mutations and CNAs contribute to the dysregulation of multicellularity in cancer , but do so in different ways , impacting regions of transcriptional networks with distinct roles in the regulation of multicellularity .", "Finally , we show how our approach of integration of sequence conservation and transcriptome data with annotated regulatory associations provides a framework for identifying important driver mutations and prioritizing compounds for targeted therapy , at the level of both patient populations and individual tumors ." ], [ "We investigated the association between the evolutionary ages of genes and the frequency of copy-number aberrations ( CNAs ) and point mutations across tumor cohorts ( The Cancer Genome Atlas Network , 2017a; The Cancer Genome Atlas Network , 2017b ) .", "We collected CNA and point mutation data from 10256 and 9926 patients , respectively , from The Cancer Genome Atlas across 30 tumor types ( see Materials and methods ) .", "We selected a subset of genes that were consistently amplified or deleted in at least 10% of patients of each tumor cohort , and genes with either missense or loss-of-function ( LoF ) mutations in at least three patients and with a higher rate of occurrence than synonymous mutations ( Figure 1—figure supplement 1 ) ( see Materials and methods ) .", "Human genes were classified by their evolutionary age using phylostratigraphy ( Domazet-Loso and Tautz , 2010 ) , resulting in 16 phylogenetic groups ( phylostrata ) ( Figure 1—figure supplement 2 ) , ranging from genes found in unicellular ancestors ( Phylostrata 1–3 ) ( 6 , 719 UC genes ) , to genes found in early metazoans ( Phylostrata 4–9 ) ( 7 , 939 EM genes ) , and mammal-specific genes ( Phylostrata 10–16 ) ( 2 , 660 MM genes ) ( Figure 1—figure supplement 3 ) ( Trigos et al . , 2017 ) .", "We calculated the fraction of genes in each phylostratum with recurrent CNAs and point mutations , accounting for differences in CNA and mutation rates between tumor cohorts by ranking each phylostratum by the fraction of genes altered ( Figure 1A , B ) .", "We found an increasing trend of enrichment of CNAs starting from the earliest UC genes ( phylostratum 1 ) , but peaking in EM genes ( phylostrata 4–8 ) , with EM genes being the most enriched with both amplifications and deletions across tumors .", "A high proportion of tumor types ( 14/30 tumor types for amplifications and 27/30 tumor types for deletions ) had at least 3 EM phylostrata in the top five most recurrently altered phylostrata .", "In contrast , recurrent CNAs were consistently depleted from MM genes ( phylostratum 10 onwards ) , indicating a lack selection for CNAs in younger genes .", "The decreasing enrichment trend along the phylostrata was significant for amplifications in 20/30 tumor types , and for deletions in 27/30 tumor types ( Jonckheere-Terpstra tests Benjamini-Hochberg adjusted p<0 . 05 ) .", "Similar results were obtained using recurrent gains/losses as defined by Gistic2 , as the one used by Gistic2 ( see Materials and methods , Figure 1—figure supplement 4 ) , which employs a rigorous probabilistic model to identify recurrent CNAs .", "Among the recurrently amplified and deleted EM genes are well-known cancer genes .", "Examples include the EGFR oncogene , recurrently amplified in more than 10% of patients in 19/30 of the studied tumor types and having emerged together with bilaterians ( Phylostratum 6 ) , and the tumor suppressor TP53 which also dates back to early metazoan ancestors ( Phylostratum 5 ) and is found recurrently deleted in an average of 12 . 73% of patients across all tumor types studied .", "In contrast to the patterns obtained for genes with recurrent CNAs , genes not recurrently copy-numbered altered ( frequency <0 . 10 across patients ) were not enriched in EM genes , but were often enriched in MM genes ( Figure 1—figure supplements 5 and 6 ) .", "An exception is TNFRSF17 ( also known as BCMA , BCM ) , a mammal-specific gene involved in immune system processes that was amplified in >10% of BRCA , ACC , KIRP , THCA , PRAD and KIRC patients .", "TNFRSF17 has classically been associated with lymphomas ( Laâbi et al . , 1992 ) and has oncogenic properties ( Coquery and Erickson , 2012; Zhao et al . , 2008 ) .", "Overall , our results suggest that EM genes are specifically preferentially under selection for recurrent CNAs across patients .", "A similar enrichment of recurrent mutations in EM genes was identified for point mutations ( Supplementary file 1 ) .", "We found an increasing trend of enrichment of point mutations beginning at genes that date back to later unicellular ancestors ( phylostratum 2 ) , but peaking in early metazoan genes across tumors , with genes dating to the earliest metazoans ( phylostratum 4 ) being the most enriched ( Figure 1B , Figure 1—figure supplement 4C ) .", "In tumor types where ≥ 5 phylostrata had recurrent missense ( 23 overall ) or LoF mutations ( 14 overall ) , at least three of the five phylostrata most enriched for missense or LoF mutations were EM in 15/23 tumor types and 11/14 tumor types , respectively .", "In contrast , MM genes were consistently depleted of recurrent point mutations .", "The decreasing trend of enrichment associated with gene age was significant in the majority of tumor types for both missense ( 18/23 ) and LoF ( 12/14 ) mutations ( Jonckheere-Terpstra tests Benjamini-Hochberg adjusted p<0 . 05 ) .", "In contrast , this enrichment pattern was not observed for non-recurrently point-mutated genes ( Figure 1—figure supplements 7 and 8 ) .", "To determine the effect of somatic mutations on transcriptional states , we calculated the proportion of genes in each of our major phylostratigraphic groups where differential expression could be tied to mutation status at the per-patient level ( Figure 1C ) .", "EM genes with missense or LoF mutations were more likely to be differentially expressed when point mutated than were UC or MM genes ( one-sided Wilcoxon tests p=0 . 031 and p=3 . 78×10−6 , respectively ) .", "These results suggest that the strong selection for point mutations in EM genes across tumors could be linked to their dysregulation of expression providing an advantage to tumor development .", "We also investigated the association between gene age and signatures of selection for CNAs at the chromosome level ( Figure 1D , Figure 1—figure supplement 9 ) .", "For each patient and chromosome , we associated the evolutionary ages of the amplified or deleted genes with the fraction of chromosome affected by those CNAs , across tumor types ( Figure 1D ) .", "There was a strong and significant increasing trend along the phylostrata ( BH adjusted p<0 . 05 ) for amplifications in all tumor types and for deletions in >80% tumor types .", "These trends were not restricted to particular chromosomes ( Figure 1—figure supplement 9 ) .", "UC and EM genes were preferentially located in focally copy-numbered altered regions , suggesting stronger localized selection for the CNA of UC and EM genes .", "In contrast , MM genes were located in regions of broad copy-number changes , suggesting the CNA of MM genes are likely passenger events swept up in the large chromosomal rearrangements that occur during cancer development .", "Our results indicate a preferential recurrent alteration by both CNAs and point mutations of EM genes across tumor types ( Figure 1E , Figure 1—figure supplement 4D ) , suggesting that disruption of these genes by genetic changes likely provides an advantage in the development of multiple tumor types , whereas mutations in genes that evolved later in metazoan evolution , namely MM genes , are unlikely to be playing a significant role .", "The observed enrichment patterns across cancer types suggested alteration of EM genes provides a selective advantage to tumors .", "Known cancer drivers are mostly of EM origin ( Domazet-Loso and Tautz , 2010 ) and are highly interconnected in human molecular networks ( Cheng et al . , 2014 ) , suggesting EM genes hold regulatory roles with important pleiotropic effects in cancer ( Trigos et al . , 2018 ) .", "Since important innovations required for the regulation of transcriptional networks from unicellular ancestors evolved in early metazoan species , we investigated whether this could be evidenced in the current structure of the human gene regulatory network ( GRN ) .", "The GRN was obtained by subsetting the network from PathwayCommons ( Cerami et al . , 2011; Pathway Commons , 2017 ) to include only edges annotated with control-of-expression .", "Given the directed nature of the GRN , regulator genes can be distinguished from downstream target genes ( Figure 2A ) .", "As expected , many more genes act as targets ( 12 , 812 ) than as regulators ( 1 , 370 ) , indicating the presence of key regulatory hubs regulating a multitude of target genes .", "We found that over half ( 56 . 42% ) of regulators in the GRN were EM genes , whereas only 37 . 88% and 5 . 69% were UC and MM genes , indicating an enrichment of EM genes as regulators ( Fisher enrichment test p=6 . 48×10−6 ) ( Figure 2B ) .", "Focusing on the genes with key regulatory roles , we investigated regulators with at least 10 downstream targets ( out-degree >= 10 ) , which correspond to the upper quantile of the distribution of out-degree across all regulators ( Figure 2—figure supplement 1 ) .", "We found that 65 . 12% of these master regulators were EM genes , whereas only 28 . 49% and 6 . 40% were UC and MM genes , respectively , indicating that key master regulators with the largest pleiotropic effects in the network were mostly EM genes .", "This structure of the GRN substantially differed from general protein-protein interaction ( PPI ) networks ( e . g . ( Cerami et al . , 2011; Chatr-Aryamontri et al . , 2017; Li et al . , 2017 ) ) where UC genes are usually the most connected ( Figure 2—figure supplement 2 ) , suggesting specific evolutionary processes shaping the GRN resulted in key regulatory roles for EM genes .", "Additionally , EM genes were the most highly regulated downstream genes in the GRN , measured by the number of incoming edges ( in-degree ) , with EM genes having an average of 8 . 76 incoming edges , compared to only 6 . 59 and 4 . 54 in UC and MM downstream target genes ( Wilcoxon test p<2 . 2×10−16 in both cases ) ( Figure 2C , Figure 2—figure supplement 3 ) .", "The enrichment of EM genes as both regulators and highly regulated downstream targets in the GRN indicates that gene regulation in humans is predominantly under control of EM genes ( Figure 2D ) .", "Therefore , we hypothesized that the preference of somatic mutations in EM genes might stem from their key regulatory roles in the human GRN .", "To test this , we assessed whether selection for somatic mutations in EM genes was linked to their central regulatory roles ( Figure 2A ) .", "Given that broad CNAs involving large chromosome sections include a high number of genes with poor resolution of the genes under selection , we focused on recurrent CNAs that included less than 25% of the genes of a chromosome in at least 10% of patients of each tumor cohort .", "We found that a higher fraction of regulators were affected point mutations ( mean fraction altered = 0 . 20 ) than CNAs ( 0 . 12 ) in 80 . 77% of tumor types with at least three recurrent point mutations or CNAs ( Wilcoxon test p=2 . 95×10−5 ) ( Figure 2E ) , with LoF mutations driving most of the signal ( Figure 2—figure supplement 4 ) .", "In contrast , downstream target genes without a regulatory role were more likely to be affected by CNAs ( Wilcoxon test p=2 . 95×10−5 ) ( mean fraction altered = 0 . 88 for CNAs , 0 . 80 for point mutations ) ( Figure 2E ) .", "This dichotomy was even more pronounced in the top 5% most recurrently mutated genes , those with recurrent mutations in ≥7 tumor types .", "Over half ( 54% ) the regulators in this set are recurrently point mutated , but only 13% are recurrently CNA , a fourfold difference .", "In contrast , 87% of the most highly mutated targets are affected by CNAs but only 46% are affected by point mutations .", "Similar results were obtained when we used the significantly CNA and point mutated genes identified by MutSig2CV and Gistic algorithms ( Figure 2—figure supplement 5 ) .", "Therefore , recurrent point mutations are more likely to affect master regulators in the GRN , whereas recurrent CNAs are more likely to affect target genes .", "The complex regulatory interactions in the GRN result in many genes having a dual role , acting as both regulators and targets , with EM genes being both master regulators and under high degree of regulation ( Figure 2B–D ) .", "To account for this dual role , we calculated the ratio of the number of outgoing edges ( out-degree ) to the number of incoming edges ( in-degree ) , with greater ratios indicating a predominantly regulatory role ( Figure 2F , Supplementary file 2 ) , and calculated the median value across all genes with a dual role for each tumor type .", "We found that EM genes with point mutations held stronger regulatory role across tumors ( median ratio = 0 . 83 ) than UC and MM genes with point mutations ( median ratio = 0 . 51 and 0 . 31 , respectively ) .", "In contrast , EM genes with CNAs were skewed toward being highly regulated downstream targets ( median ratio = 0 . 38 ) , even compared to UC and MM genes with CNAs ( median ratio UC = 0 . 67 and MM = 0 . 60 ) .", "Similar results were obtained using MutSig2CV and Gistic to define recurrent point mutations and CNAs ( Figure 2—figure supplement 6 ) .", "Therefore , the observation of selection for point mutations in EM regulators and CNAs in EM target genes also holds for genes with dual regulatory and target roles in the GRN .", "Although there is a recurrent selection across tumor cohorts for the somatic mutation of EM genes , our results reveal that selection for point mutations and CNAs differentially disrupt the GRN .", "EM hub genes with key regulatory roles were preferentially disrupted by point mutations , indicating that few point mutations in key regulators are more likely to create large disruptions across the GRN .", "In contrast , downstream target genes were preferentially affected by recurrent CNAs , and are therefore more likely to have a localized effect .", "A main characteristic of cancer development is the loss of coordination in expression between UC and MC genes together with overexpression of UC genes and downregulation of MC genes ( Trigos et al . , 2017 ) , suggesting a compartmentalization of the GRN into UC and MC gene network regions interconnected by key regulatory links that get disrupted by mutations during cancer development ( Trigos et al . , 2018 ) ( Figure 3A ) .", "To distinguish these UC and EM network regions , we calculated the percentage of downstream UC and EM target genes for each regulator , and classified individual regulators as preferentially regulating UC targets ( >2/3 UC genes ) ( UC-t regulators ) , EM targets ( >2/3 EM genes ) ( EM-t regulators ) , MM targets ( >2/3 MM genes ) or being at the interface of UC and EM targets by regulating a mix of UC and EM downstream targets ( >1/10 UC and EM genes ) ( UC/EM-i regulators ) ( Figure 3B , main panel , Supplementary file 3 ) .", "We excluded regulators that primarily controlled mammalian genes from further analysis , as they only accounted for 2 . 04% of all regulators .", "Regulators not meeting any of the above criteria were also excluded ( 10 . 36% ) .", "UC-t regulators mostly dated back to UC ancestors ( 50 . 81% , Fisher test p=0 . 021 ) while both EM-t regulators ( 56 . 68% , Fisher test p=0 . 0011 ) and UC/EM-i regulators ( 61 . 73% , Fisher test p=0 . 00022 ) were mostly comprised of EM genes .", "We next investigated how point mutations and CNAs differentially affected UC-t , EM-t and UC/EM-i regulators .", "Given dysregulation of UC and MC gene expression and co-regulation has been found to be consistent across multiple tumors types ( Trigos et al . , 2017 ) and therefore likely to share similar drivers , we only considered the top 5% most recurrently mutated genes across tumor cohorts .", "The majority of regulators with recurrent point mutations were UC/EM-i ( 85 . 71% ) , whereas this percentage was only 32 . 81% and 32 . 33% for regulators affected by CNAs or not recurrently mutated ( Figure 3B density plot , Figure 3C , Figure 3—figure supplement 1 ) , indicating preference for the point mutation of UC/EM-i regulators , with no preference for those affected by CNA .", "These results indicate overrepresentation of highly recurrent point mutations across tumor cohorts in regulators at the UC/EM interface ( Figure 3—figure supplement 2 ) , which was not observed for CNA regulators or non-recurrently mutated regulators .", "To examine the functional downstream effects of point mutations in regulators , we calculated the percentage of downstream targets that were differentially expressed after point mutations in their regulator genes , and classified the magnitude of the downstream effect as being of low impact if less than 5% of the downstream genes were affected , and high impact otherwise .", "The majority of high impact mutations occurred in UC/EM-i regulators ( 75% ) , whereas only 43 . 13% of low impact mutations occurred in these regulators , indicating that high-impact mutations preferentially occur in in UC/EM-i regulators ( two-proportions Z-test p=0 . 026 ) ( Figure 3D ) .", "The actual ages of the UC/EM-i regulators were also associated with mutational impact .", "EM genes represented a higher proportion of UC/EM-i regulators with high impact mutations ( 67% ) than those with low impact mutations ( 50% ) , and as a whole , point-mutated early metazoan regulators were two-times more likely than other point-mutated regulators to have a high impact ( Figure 3—figure supplement 3 ) .", "Thus , point mutations creating the most substantial alterations to gene expression tend to be in regulator genes of early metazoan origin at the interface of UC and EM genes .", "Overall , our results suggest that the recurrence across tumor cohorts of point mutations in EM regulators is tied with transcriptional disturbances of the regulation between UC and EM genes in the GRN , making them potential gene drivers .", "Multiple known cancer genes were found among UC/EM-i regulators , including RB1 , PIK3CA , SMAD4 , NF1 TP53 , TERT and MDM4 .", "Functional enrichment analysis revealed that these regulators are involved in multiple signaling pathways , such as receptor tyrosine kinases , MAPK and PI3K-Akt signaling , but they are also involved in a diversity of other processes such as mitochondrial biogenesis and lipid metabolism ( Supplementary file 4 ) .", "Investigation of the pathways associated with UC/EM-i regulators revealed that their point mutation often had a significant effect on pathway expression levels ( Supplementary file 5 ) .", "Point mutation of well-studied genes such as p53 affected the expression of pathways such as MAPK signaling , p53 signaling , WNT , apoptosis , cell cycle across 18 tumor types; mutations in PTEN with changes in expression of p53 signaling in six tumor types , PIK3CA affecting the expression of multiple pathways such as ERBB signaling , MTOR , JAK-STAT , and VEGF signaling across 10 cancer types; and point mutations in SMAD4 in colon cancer affected WNT signaling .", "We also found other understudied effects .", "For example , mutation of EP300 was associated with differences in expression of cell cycle and JAK-STAT pathways in bladder , endometrial , cervical and endocervical cancers .", "Point mutation of PIK3R1 affected ERBB , MTOR and JAK-STAT signaling .", "Intriguingly , our analysis pointed to other other EM UC/EM-i regulator genes with mutational frequencies and percentage of differentially expressed downstream targets similar to known cancer genes .", "Many of these genes , including KEAP1 , HNF1A , NFE2L2 and LRRK2 , have implied roles in cancer but no strong mechanistic links to date ( Figure 5; Discussion ) .", "While somatic mutation of regulators could provide a major selective advantage via simultaneous dysregulation of a multitude of downstream target genes , where and when such mutations occur is largely based on stochastic events during tumor development .", "An alternate and complementary mechanism for disrupting conserved regions of the GRN without mutation of master regulators would be direct mutation of downstream target genes , as suggested by our finding that CNAs predominantly affected target genes of the GRN ( Figure 2E–F ) .", "To investigate the contribution of CNAs to the disruption of the regulatory links between UC and EM genes , we calculated the fraction of downstream targets with CNAs for each regulator in each individual patient .", "To exclude possible redundant mechanisms resulting from CNAs in both regulators and targets , we only included in the analysis samples where the regulator was copy-number normal ( CNN ) .", "We found that only a small fraction of downstream target genes of UC/EM-i regulators were CNA ( median = 0 . 11 ) , whereas a significantly larger fraction of downstream target genes of UC-t and EM-t regulators were affected ( median = 0 . 25 and 0 . 33 , respectively ) ( p=3 . 91×10−8 and p=8 . 42×10−27 comparing the fraction of CNA targets of UC/EM-i with that of UC-t and EM-t regulators , respectively ) ( Figure 4A ) .", "This indicates that CNAs preferentially affect target genes of UC-t and EM-t regulators , rather than directly disrupting the regulatory links at the interface of UC and EM regions of the GRN .", "We hypothesized the preferential of CNAs in targets genes of UC-t and EM-t regulators would be associated with the direct transcriptional modulation of genes by CNAs .", "To test this , we calculated the expression fold-change in tumor samples with respect to their paired normal samples , and used Wilcoxon tests to compare fold-change values in samples where the gene was CNA and those where the gene was CNN .", "We found a higher percentage of target genes than regulators were differentially expressed after amplifications across all tumor types , and for deletions in 64 . 64% of tumors types , indicating that CNAs more strongly influence the expression of target genes than regulator genes ( Figure 4—figure supplement 1 ) .", "Specifically , UC target genes showed the largest changes in expression after CNAs ( median values across tumor types: 7 . 80% upregulated after amplifications , 7 . 48% downregulated after deletions ) , followed by EM genes ( 4 . 45% and 4 . 79% , respectively ) , and lastly MM genes ( 2 . 46% and 1 . 62% ) ( Figure 4B , Figure 4—figure supplement 2 ) ( Jonckheere-Terpstra decreasing trend test: amplifications p-value: 0 . 0028 , deletions p-value: 0 . 0021 ) , indicating that UC and EM targets are more susceptible to changes in expression after CNA .", "However , the effect of CNAs on the expression of targets was dependent on their regulator .", "Targets of UC-t regulators were more likely to be differentially expressed after amplifications or deletions were the targets of UC/EM-i regulators ( Figure 4C ) .", "On the other hand , target genes of EM-t regulators mostly modulated their expression in response to deletions as opposed to amplifications ( one-sided Wilcoxon test p=0 . 043 ) ( Figure 4C ) , suggesting selection of CNAs in targets of EM-t regulators could be a mechanism for the direct down regulation of EM network regions , as an alternative to direct mutation of their regulators .", "In contrast , CNAs of the target genes of UC/EM-i regulators changed their expression much less often , no matter whether it was amplified or deleted ( two-sided Wilcoxon test p=0 . 95 ) , suggesting CNAs are playing a less prominent role in the dysregulation of UC and EM interface regions than do somatic point mutations .", "A model of transcriptional changes in UC-t and EM-t targets driven by CNAs as an alternative to direct mutation of the regulators themselves would predict the mutual exclusivity of concurrent CNAs of a regulator and its targets in the same tumor , as the co-occurrence of such mutations would be largely redundant .", "To test this hypothesis , we calculated the median fraction of targets with CNAs for each regulator across all patients in the tumor cohorts , and found that the fraction of targets with CNAs was significantly higher in patients where the regulator was CNN than when the regulator was CNA ( Wilcoxon test p-value=4 . 10×10−29 , Figure 4—figure supplement 3 ) .", "However , this trend was only observed for targets of UC-t and EM-t regulators ( one-sided Wilcoxon test p=1 . 36×10−7 and p=1 . 11×10−42 , respectively ) , but not for targets UC/EM-i regulators ( p=0 . 79 ) , where the trend seemed to be the opposite ( Figure 4D ) , indicating that preferential alteration of target genes by CNAs , as opposed to regulators , is a mechanism employed for the independent modulation of UC and EM network regions .", "We next investigated how the copy-number status of regulator ( CNA or CNN ) was associated with the fraction of CNA downstream target genes ( Figure 4E ) .", "We found that over two thirds of UC-t and EM-t regulators ( 71 . 70% and 71 . 30% , respectively ) had a higher fraction of copy-aberrant downstream genes when they were CNN than when they were CNA .", "These results were independent of the evolutionary ages of the regulators , since the percentages were similar for both UC and EM regulators ( Figure 4—figure supplement 4 ) .", "A potential explanation for this pattern is illustrated by MDM2 , where 33 . 33% of its downstream targets were CNA when MDM2 was CNN , but no target was CNA when MDM2 had changed in copy-number .", "Since MDM2 is an inhibitor of p53 , either amplification of MDM2 or deletion of p53 would have a similar effect , and therefore there is no selection for the simultaneous co-occurrence of both alterations in the same patient .", "In contrast to the strong trends observed for UC-t and EM-t regulators , this trend was only observed in 28 . 72% of UC/EM-i regulators .", "The preferential copy-number aberration of targets of CNN UC-t and EM-t regulators , but not in CNN UC/EM-i regulators , supports our proposed model of selection for the CNA of target genes of UC-t and EM-t regulators , but not for those of UC/EM-i regulators .", "Here we found that CNAs are a widespread mechanism of dysregulation of UC and EM target genes , directly modulating the expression of targets of UC-t and EM-t regulators .", "The effect of CNAs on the GRN is therefore distinct to the disruption by point mutations of the regulatory links between UC and EM genes , but are also important drivers of large transcriptional disturbances in tumors .", "UC/EM-i were found to be preferentially targeted by point mutations and are likely to be key points of vulnerability to cancer development given their regulatory role modulating UC and EM genes ( Trigos et al . , 2018 ) .", "Therefore , we investigated these regulators as potential drivers of tumourigenesis and their role in determining drug response .", "We compiled a set of known cancer drivers from the Cancer Census COSMIC database ( Forbes et al . , 2017 ) .", "These genes were enriched in EM genes ( Fisher test p=0 . 0015 , 57 . 36% EM genes , 39 . 58% UC genes and 3 . 06% MM genes ) .", "36 . 71% of the known cancer genes were regulators and were enriched in UC/EM-i regulators ( 46 . 88% , Fisher test p=0 . 0043 ) , but depleted in UC-t ( p=0 . 83 ) and EM-t regulators ( p=0 . 95 ) ( Figure 5A ) , supporting modulation of regulation between UC and EM genes is a common effect of cancer drivers .", "Furthermore , investigating drivers identified from multi-region sequencing data ( Caravagna et al . , 2018 ) in individual patients of non-small-cell lung cancers ( Jamal-Hanjani et al . , 2017 ) and breast cancers ( Yates et al . , 2015 ) revealed that 90 . 91% of lung cancer patients and 70 . 83% of breast cancer patients had at least one UC/EM-t regulator identified as a clonally mutated driver ( Figure 5—figure supplement 1 ) , supporting a fundamental role of the mutation of these regulators in cancer development .", "We further investigated the importance of these regulators to cancer development , we used the dependency scores from CRISPR-Cas9 essentiality screens of 364 solid-tumor tissue cell lines from the Avana CRISPR-Cas9 genome-scale knockout dataset made available by Project Achilles and the Cancer Dependency Map project ( Meyers et al . , 2017; Broad Institute , 2018b ) .", "A high probability of dependency indicates a gene is essential for proliferation of a given cell line ( Figure 5—figure supplement 2 ) ( for further details , see Meyers et al . , 2017 ) .", "We calculated the odds ratio ( OR ) of each regulator type having a large effect on cell line proliferation ( probability of dependency >= 0 . 95 ) , with OR greater than one indicating increased likelihood of a high dependency ( Figure 5B , Figure 5—figure supplement 3 ) .", "As expected , we found most cell lines were highly dependent on UC-t regulators ( OR >1 in 96 . 98% of cell lines ) , likely due to their role in regulating fundamental functions required for cell survival .", "However , we also found that 92 . 86% cancer cell lines were highly dependent on UC/EM-i regulators ( OR consistently greater than 1 ) , indicating these regulators are fundamental for cancer cell survival .", "In contrast , cancer cell lines were not nearly as dependent on EM-t regulators for their proliferation ( OR >1 in only 0 . 55% of cell lines ) , indicating dysregulation of EM processes might contribute to tumourigenesis , but not be sufficient by themselves .", "These results suggest that UC/EM-i regulators are indispensible for cancer cell line proliferation .", "We hypothesized the degree of dependency on UC/EM-i regulators would be tied to their mutational and copy-number status .", "For each regulator , we classified cell lines into those with or without a point mutation or amplification in the regulator based on data from the Cancer Cell Line Encyclopaedia ( CCLE ) ( https://portals . broadinstitute . org/ccle ) ( Broad Institute , 2013; Broad Institute , 2018a ) , and calculated the median dependency scores for mutated and non-mutated cell lines ( Supplementary file 6 ) .", "We only considered regulators whose difference in median dependency between mutated and non-mutated cell lines was at least 0 . 20 , and were significant by Wilcoxon tests ( p<0 . 05 ) .", "This revealed 40 regulators whose mutation was associated with cell-line dependency ( Figure 5C ) .", "Multiple well-known cancer genes were among these top hits .", "Amplification of the oncogenes ERBB2 , CDK6 , MDM2 affected dependency , as did point mutations in PIK3CA , KRAS , NRAS , VHL and BRAF , validating our approach to highlight genes with significance in cancer .", "Over half of these top hits correspond to UC/EM-i regulators ( 55 . 00% ) , indicating that mutations in UC/EM-i regulators are more likely to affect the dependency of a cancer cell line to a regulator .", "We found that in most cases cell lines were more dependent on a regulator when it was point mutated ( 80 . 00% ) or amplified ( 66 . 67% ) than when it was non-mutated , suggesting that mutation of these regulators are key to cancer cell line proliferation ( Figure 5C ) .", "We also investigated the implications of dependency to UC/EM-i regulators on drug sensitivity .", "Based on the half maximal inhibitory concentration values ( IC50 ) from drug sensitivity screens covering 250 drugs from the Genomics of Drug Sensitivity in Cancer database ( Genomics of Drug Sensitivity in Cancer Consortium , 2016; Yang et al . , 2013 ) , we calculated the Spearman correlation between dependency scores and the IC50 values ( Figure 5—figure supplement 4 ) , and identified 11 significant associations with UC/EM-i regulators , 38 with UC-t regulators and 23 with EM-t regulators ( correlation <−0 . 25 and adj . p<0 . 05 ) .", "These identified regulators were among the most highly correlated with the IC50 of the particular drug ( Figure 5—figure supplement 5 ) .", "As expected , we found strong correlation between dependency scores for UC/EM-i regulators and the IC50 of drugs targeting the regulator ( e . g . IGF1R and Linsitinib , BMS-536924 and BMS-754807 ) , as well as between MAPK1 and an inhibitor of related genes in the MAPK/ERK pathway ( ( 5Z ) −7-Oxozeaenol ) , validating our approach ( Figure 5D , Figure 5—figure supplement 6 ) .", "However , we also found unexpected strong correlations between the IC50 of particular drugs and the dependency scores of UC/EM-i regulators ( Figure 5D , Figure 5—figure supplement 6 ) .", "For example , the IC50 of XAV939 , an inhibitor of Wnt/β-catenin , was also strongly correlated with the dependency to ILK ( −0 . 30 ) , a regulator of integrin-mediated signal transduction involved in tumor growth and metastasis , supporting the use of Wnt/β-catenin inhibitors for cancers dependent on ILK , including colon , gastric and ovarian and breast cancers ( Hannigan et al . , 2005 ) .", "We also found strong correlation across cell lines between the dependency to PPRC1 and mTOR-inhibitors ( temsirolimus , used in the treatment of renal cancer ) , dual PI3K/mTOR-inhibitors ( dactolisib , in clinical trial for advanced solid tumors ( Wise-Draper et al . , 2017 ) ) , YK-4–279 ( showing pre-clinical efficacy for Ewing sarcoma ( Lamhamedi-Cherradi et al . , 2015 ) ) and the chemotherapy agent docetaxel , currently used in the treatment of breast , lung cancer , stomach cancer , head and neck and prostate cancer .", "Of the tumor types included in our study , the correlation with PPRC1 dependency was particularly strong ( <−0 . 25 ) in liver , lung and stomach cell lines for temsirolimus sensitivity , lung and stomach cell lines for docetaxel and dactolisib sensitivity and breast cell lines for YK-4–279 sensitivity , but were also held for a number of other solid tumor types ( Figure 5—figure supplement 7 ) , suggesting their use across multiple cancer types .", "With this , our novel approach has identified understudied potential vulnerabilities for cancer development and proposed drug repositioning possibilities ." ], [ "Detailed analyses of recurrent somatic mutations across tumor types revealed the prevalence of mutations related to both gene age and its position within the regulatory network .", "We provide evidence that point mutations and CNAs play complementary roles in the transcriptional dysregulation in cancer by affecting distinct regions of the underlying gene regulatory network , supporting the loss of communication between the core biological processes originating in ancient single-celled life and the regulatory controls acquired during metazoan evolution to control these processes .", "This would result in tumor convergence to similar transcriptional states of consistent activation of genes from unicellular ancestors and loss of cellular functions characteristic of multicellular organisms .", "Our results attribute key roles to genes at the interface of unicellular and multicellular regulation in tumourigenesis , with implications for conventional and experimental therapies .", "Common hallmarks shared by tumors of diverse genetic backgrounds suggest the consequences of mutations acquired during tumor development follow common principles , promoting the downregulation of genes and pathways associated with multicellularity and the activation of fundamental cellular processes evolved in early unicellular organisms ( Trigos et al . , 2017 ) .", "Here , we found genes central to the human gene regulatory network that arose in early metazoans were the most often recurrently affected by point mutations and CNAs across tumor types .", "Other studies have found that gatekeeper cancer drivers ( those that regulate cell cooperation and tissue integrity ) emerged at a similar evolutionary time , whereas caretaker genes ( those ensuring genome stability ) emerged at the onset of unicellular life ( Domazet-Loso and Tautz , 2010 ) .", "Our results suggest recurrent mutations mostly affect gatekeeper genes regulating fundamental aspects of multicellularity , whereas the disruption of caretaker activities by recurrent somatic mutations and CNAs is more limited .", "We found the impact of point mutations and copy-number aberrations was concentrated on specific regions of the gene regulatory network .", "Point mutations preferentially affected gene regulators at the interface of unicellular and early metazoan subnetworks , likely affecting the regulation of multicellular components over fundamental cellular processes .", "We found that these interface genes were involved in well-known cancer signaling networks ( e . g . TP53 , NF1 , PIK3CA ) , but found that other processes of emerging interest , such as metabolism and mithochondrial dysregulation are also associated with the rupturing of regulation of unicellular and multicellular components by point mutations .", "On the other hand , CNAs preferentially affected their downstream target genes , directly promoting the independent activation and inactivation of regions predominantly composed of unicellular and multicellular genes , as opposed to mixed regions , further supporting a loss of multicellularity and the tight association between gene expression level and gene age ( Trigos et al . , 2017 ) .", "Samples where UC/EM-i regulators were mutated tended not to have CNAs in the corresponding target genes and vice versa; in samples carrying CNAs for multiple target genes , unicellular and multicellular , the cognate UC/EM-i regulator were mostly unaltered .", "These complementary but distinct mechanisms of alteration to the same regulatory subnetworks by different classes of somatic mutations in different tumors provide mutually exclusive but highly convergent paths toward common hallmarks associated with the loss of multicellularity features in cancer .", "Our evolutionary network analysis approach also highlights potential early driver genes by elucidating the evolutionary regulatory context in which genes operate .", "Only a handful of driver mutations are thought to be responsible for the transition from a normal , healthy cell to a malignant state ( Martincorena et al . , 2017; Miller , 1980; Schinzel and Hahn , 2008; Stratton et al . , 2009 ) , but their identification amid large and diverse genetic alterations is challenging .", "Our results suggest the key positioning of early metazoan regulator genes at the interface of network regions from unicellular and multicellular ancestors makes cells susceptible to widespread dysregulation of transcriptional networks upon their disruption , as their alteration would uncouple the regulatory controls required for multicellularity ( Greaves , 2015 ) .", "This implicates these genes in key roles in the onset and progression of cancer and highlights them as potential gene drivers and drug targets .", "Our analysis of the effect on cell line dependency after knockout of these regulators revealed that their alteration is capable of modulating cell proliferation across 364 cell lines .", "Many of these interface regulators have not been significantly studied in the context of cancer , but drugs targeting these genes are currently in clinical trial for other diseases , opening the possibility for drug repurposing .", "LRRK2 encodes the dardarin protein , considered to be central to the aetiology of Parkinson disease ( Zimprich et al . , 2004 ) .", "Two inhibitors of dardarin , DNL201 and DNL151 , are currently undergoing testing in clinical trials as a means to slow down or regress neurodegenerative diseases ( Atashrazm and Dzamko , 2016 ) .", "We also identified that cell lines dependent on the UC/EM-i regulator PPRC1 , peroxisome proliferator-activated receptor gamma , co-activator-related 1 , were particularly susceptible to mTOR inhibitors , YK-4–279 and docetaxel .", "PPRC1 is an activator of mitochondrial biogenesis , a process regulated by mTOR ( Morita et al . , 2013; Ramanathan and Schreiber , 2009; Schieke et al . , 2006 ) , highlighting the use of mTOR inhibitors in cancers with aberrant mitochondrial activity .", "Furthermore , it suggests that YK-4–279 , a binding inhibitor of oncogenic fusion proteins in Ewing’s sarcoma and with encouraging pre-clinical efficiency in this cancer ( Lamhamedi-Cherradi et al . , 2015 ) , could also be broadly effective in general for tumors with aberrant mitochondrial activity .", "This study provides comprehensive evidence that both the frequency and types of mutations in genes in cancer are strongly influenced by a given gene’s evolutionary age and its regulatory functions .", "This furthers our understanding of how a limited number of genetic alterations could promote rapid tumor development through loss of multicellular features and provides an explanation as to how the widespread convergence to common hallmark phenotypes in solid cancer may occur .", "As we show , this approach can be used to prioritize genes as drivers and identify possible targeted therapies , creating a novel analytical framework that will become increasingly informative as the volume and resolution of cancer genomics data continue to increase ." ], [ "The evolutionary ages of genes were obtained from previously published work ( Trigos et al . , 2017 ) .", "Phylostratigraphy ( Domazet-Loso et al . , 2007 ) was used to map human genes onto a phylogenetic tree of 16 phylostrata , spanning genes found across all organisms ( Phylostratum 1 ) to those specific to humans ( Phylostratum 16 ) .", "Human genes with orthologs in primitive unicellular species were assigned to older phylostrata ( phylostrata 1–3 ) and referred to as unicellular ( UC ) genes , those with orthologs in early metazoan species ( phylostrata 4–9 ) are referred to as early metazoan ( EM ) genes , and those assigned to phylostrata 10–16 are considered to be mammal-specific ( MM ) genes .", "We included in our analysis all 30 solid cancer types available from TCGA GDAC for which there was CNA and point mutation data available .", "These were: Adrenocortical carcinoma ( ACC ) , Bladder urothelial carcinoma ( BLCA ) , Breast invasive carcinoma ( BRCA ) , Cervical and endocervical cancers ( CESC ) , Cholangiocarcinoma ( CHOL ) , Colon adenocarcinoma ( COAD ) , Esophageal carcinoma ( ESCA ) , Glioblastoma multiforme ( GBM ) , Head and Neck squamous cell carcinoma ( HNSC ) , Kidney Chromophobe ( KICH ) , Kidney renal clear cell carcinoma ( KIRC ) , Kidney renal papillary cell carcinoma ( KIRP ) , Brain Lower Grade Glioma ( LGG ) , Liver hepatocellular carcinoma ( LIHC ) , Lung adenocarcinoma ( LUAD ) , Lung squamous cell carcinoma ( LUSC ) , Ovarian serous cystadenocarcinoma ( OV ) , Pancreatic adenocarcinoma ( PAAD ) , Pheochromocytoma and Paraganglioma ( PCPG ) , Prostate adenocarcinoma ( PRAD ) , Rectum adenocarcinoma ( READ ) , Sarcoma ( SARC ) , Skin Cutaneous Melanoma ( SKCM ) , Stomach adenocarcinoma ( STAD ) , Testicular Germ Cell Tumors ( TGCT ) , Thyroid carcinoma ( THCA ) , Thymoma ( THYM ) , Uterine Corpus Endometrial Carcinoma ( UCEC ) , Uterine Carcinosarcoma ( UCS ) , Uveal Melanoma ( UVM ) ( The Cancer Genome Atlas Network , 2017b ) .", "We obtained somatic point mutation data from the Genomic Data Commons Data Portal ( https://portal . gdc . cancer . gov/ ) for all tumor types available .", "We selected the intersect of variants called by MuSE ( Fan et al . , 2016 ) , MuTect2 ( Cibulskis et al . , 2013 ) , VarScan2 ( Koboldt et al . , 2012 ) and SomaticSniper ( Larson et al . , 2012 ) by The Cancer Genome Atlas .", "We excluded variants found in ExAC , 1000 genomes , and only included variants from canonical transcripts not found in the last exon .", "We also excluded genes with large number of known false positives based on previous studies ( Lawrence et al . , 2013b ) , namely titin , mucins , ryanodine receptors , dyneins , PCLO , cub- and sushi-domain proteins , neurexins , contactins , PARK2 and olfactory receptors .", "Point mutations were classified as a synonymous mutation , missense mutation ( missense variant , inframe deletion or inframe insertion ) , or loss-of-function ( LoF ) mutation ( frameshift , stop-gain , splice acceptor variant and splice donor variant ) .", "Only missense mutations identified as being deleterious by SIFT and probably damaging by PolyPhen were included in our analyses .", "We also obtained a list of recurrently point-mutated genes derived using MutSig2CV ( Lawrence et al . , 2013b ) from Broad TCGA GDAC Firehose , using the firehose_get functionality ( version 0 . 4 . 13 ) .", "Genes with a q value less than 0 . 05 were considered significant .", "We obtained copy-number aberration ( CNA ) data from the Genomic Data Commons Data Portal ( https://portal . gdc . cancer . gov/ ) obtained with SNP arrays ( The Cancer Genome Atlas Network , 2017a ) .", "Only chromosome regions with at least 10 probes were considered .", "GenomicRanges ( Lawrence et al . , 2013a ) was used to assign chromosome regions to genes .", "Those with positive segment means were considered to be amplifications , and the rest deletions .", "A gene was considered amplified or deleted only in cases where genes were covered entirely by a region with a CNA .", "Amplifications were assigned the maximum segment mean and deletions the minimum , and only CNAs with a segment mean in the top 0 . 90 quantile were included in the analysis .", "We also obtained a list significantly CNAs genes derived using Gistic2 ( Mermel et al . , 2011 ) available from Broad TCGA GDAC Firehose , using the firehose_get functionality ( version 0 . 4 . 13 ) .", "Only genes with a confidence of 0 . 99 and that were in the CNA region were considered .", "We defined a gene as having recurrent point mutations if there were at least three patients across a particular tumor cohort with missense or LoF mutations in this gene .", "To account for the background mutation rate of the gene , only genes with a larger number of patients with missense or LoF mutations than with synonymous mutations were considered .", "We selected recurrent CNAs as those that were amplified or deleted genes in at least 10% of the patients .", "Note that these procedures were followed for each tumor type independently .", "We calculated the fraction of CNA or point mutated genes in each phylostratum for each tumor type as follows:FractionofgenesalteredPhyi=NRecurrentiNiwhere NRecurrenti corresponds to the number of genes with a recurrent genetic alteration in phylostratum i , and Ni the total number of genes in phylostratum i .", "To account for differences in ranges between tumor types , we ranked the phylostrata by the fraction of genes altered , ranging from 1 ( most altered ) to 16 ( least altered ) .", "To compare the trends of recurrent with non-recurrent alterations , we calculated the fraction of altered genes in each phylostratum that were non-recurrent . Fractionofnon−recurrentgenesalteredPhyi=NNon−recurrentiNNon−recurrenti+NRecurrentiwhere NNon-recurrenti corresponds to the number of genes with a non-recurrent genetic alteration in phylostratum i , and NRecurrenti the number of genes with recurrent alterations in phylostratum i .", "RNAseq gene expression data from the tumor samples and the corresponding normal samples were obtained from The Cancer Genome Atlas ( The Cancer Genome Atlas Network , 2015 ) .", "To determine how the mutational status of a gene affected its expression , we compared the expression of each gene with missense mutations in at least three patients or LoF mutations in at least three patients in the tumor type cohort against their levels in patients where the gene was unmodified .", "One-sided Wilcoxon tests were used to determine whether a gene was significantly over or underexpressed in patients where the gene was mutated , using a p-value cut-off of 0 . 05 .", "We subsequently calculated the ratio of the number of up- and downregulated genes in each phylostratum .", "We performed a similar procedure to calculate the effect of point mutations in regulators over the expression of their downstream targets , comparing the level of expression of each downstream target in tumor samples with and without the regulator being mutated using two-sided Wilcoxon tests ( p<0 . 05 ) .", "In this case , however , we pooled samples across tumor types to increase power due to the small number of point mutations that occur in regulators .", "Specifically , we only considered regulators that were point mutated in at least three samples across tumor types , and compared the expression of their downstream targets against those of samples of the tumor types where the regulator was not mutated .", "The magnitude of the downstream effect of mutating regulators was classified as low ( <5% down downstream targets affected ) , moderate ( 5–20% ) or high ( >20% ) impact .", "We defined the prevalence of EM genes as regulators as the ratio of the number of EM regulators over the number of UC regulators .", "The log10 values were calculated to normalize to zero .", "We defined the fraction of copy-number aberrant chromosome for each patient and chromosome as the ratio of the number of genes affected by amplifications or deletions and the total number of genes in the chromosome .", "To associate this chromosomal context with evolutionary ages of genes , we determined the presence or absence of genes of a specific phylostratum in the copy-number aberrant chromosome regions of each patient .", "The information was aggregated across patients by averaging the fraction of chromosome altered for each chromosome and phylostratum .", "Genes in shorter CNA regions ( smaller fraction ) were considered to be under stronger , focal selection , whereas CNAs in genes found in longer CNA regions ( larger fraction ) were considered to result from broad amplifications or deletions .", "We defined focally amplified or deleted genes as those in which their chromosomal context was in the upper quantile ( 0 . 25 ) of the distribution of the mean fraction of copy-number aberrant chromosome across patients for each chromosome .", "We obtained a human gene regulatory network ( GRN ) from PathwayCommons ( version 9 ) ( Cerami et al . , 2011; Pathway Commons , 2017 ) by selecting pairs of genes connected by an edge of the type ‘control-expression-of’ , resulting in a directed network with 95 , 651 edges .", "We defined regulator genes as those with at least one downstream target .", "We defined ‘out-degree’ as the number of outgoing edges of a regulator , representing the extent of its downstream regulatory network .", "In contrast , the ‘in-degree’ of a gene was defined as the number of incoming edges and it is proportional to how highly regulated the gene is .", "Greater out-degree/in-degree ratios indicate bias towards a higher number of outgoing edges ( i . e . regulatory role ) , whereas smaller ratios indicate bias towards a higher number of incoming edges ( i . e . target role ) .", "We also obtained protein-protein ( PPI ) networks of humans from PathwayCommons ( Cerami et al . , 2011 ) version 9 , BioGRID ( Chatr-Aryamontri et al . , 2017 ) version 3 . 4 . 152 and the InWeb_IM network ( Li et al . , 2017 ) .", "Only nodes and edges corresponding to genes and links between two genes were considered .", "Since these networks are undirected , we only calculated the degree of a gene as the total number of edges associated with the gene .", "Regulators were classified as UC-t , EM-t , MM-t or UC/EM-i regulators based on the ages of their target genes , independently of the evolutionary age of the regulator .", "First , we calculated the percentage of downstream UC , EM and MM target genes for each regulator .", "A regulator was classified as UC-t if more than 2/3 of its targets were UC genes , as EM-t if more than 2/3 of its targets were EM genes , or MM-t if more than 2/3 if its targets were MM .", "Regulators that did not meet the above criteria , but at least 1/10 of their target genes were UC genes or EM genes , and less than 1/10 were MM genes , were classified as UC/EM-i regulators .", "Functional enrichment of UC/EM-i regulators was performed using the R package of gProfiler ( Reimand et al . , 2007 ) ( version 0 . 6 . 4 ) .", "Electronic annotations and gene sets with more than 1000 genes were excluded from the enrichment analysis .", "We compared the effect of point mutations on the level of the expression of pathways they are involved in by calculating the ssGSEA scores ( Barbie et al . , 2009 ) using GSVA ( version 1 . 20 . 0 ) ( Hänzelmann et al . , 2013 ) of KEGG pathways available in the Molecular Signatures Database from the Broad Institute ( v6 . 2 ) ( Liberzon et al . , 2011; Subramanian et al . , 2005 ) and performing Wilcoxon test with Benjamini-Hochberg correction .", "Pathways that did not include an UC/EM-i regulator or referred to generic disease pathways were excluded from the analysis .", "Only pathways with an adjusted p-value less than 0 . 05 were considered significant .", "To determine whether the copy-number status of a regulator was associated with the fraction of downstream CNA targets , we calculated the fraction of downstream CNA targets for each regulator in each patient .", "We compared the fraction of CNA downstream target genes when regulators were CNA and CNN for each patient , and used Wilcoxon tests to determine if there were significant differences in these fractions between patients where the regulator was CNA or CNN .", "We only considered regulators that were CNA and CNN in at least three patients across all tumor cohorts , and had at least two targets .", "p-Values were corrected for multiple testing using Benjamini-Hochberg correction .", "A summary fraction of CNA targets was obtained for each regulator by calculating the median across patients , and we calculated the difference in percentage of targets with CNAs by subtracting the fraction of targets when the regulator was CNA minus the fraction when the regulator was CNN .", "RNAseq gene expression data from the tumor samples and the corresponding normal samples were obtained from The Cancer Genome Atlas .", "We evaluated the effect of CNAs on gene expression by calculating the expression fold-change between matched tumor and normal samples for each gene , and comparing the fold-changes of samples where the gene was CNA and where it was CNN using one-sided Wilcoxon tests .", "Only genes that were amplified or deleted in at least three samples were considered .", "Benjamini-Hochberg correction was used for correction for multiple testing .", "Note that due to the poor overlap between patient gene expression data for tumor and matched normal samples and CNA values , data presented is only of a subset of the 30 tumor types .", "We next calculated the percentage of significantly over or underexpressed amplified or deleted UC , EM and MM target genes , respectively .", "We calculated the percentage of differentially expressed CNA targets as the ratio between the number of differentially expressed CNA targets of each regulator and the total number of CNA target genes multiplied by 100 .", "We subsequently calculated the median percentage of differentially expressed CNA targets for each regulator class in each tumor type , and Wilcoxon tests were calculated to determine significant differences .", "Scores of the probability of dependency to genes across 364 solid-tumor tissue cell lines were obtained from the Avana CRISPR-Cas9 genome-scale knockout dataset generated by Project Achilles and the Cancer Dependency Map project ( 18Q1 version ) ( https://portals . broadinstitute . org/achilles; Trigos et al . , 2018 ) .", "We excluded all cell lines from haematopoietic and lymphoid tissues .", "A cell line was considered to be dependent on a regulator if its probability of dependency was greater than 0 . 95 .", "The enrichment of a regulator class among the regulators to which cancer cell lines were dependent was determined using the odds ratio .", "Mutation and CNA information of cell lines was obtained from the Cancer Cell Line Encyclopaedia ( CCLE ) ( https://portals . broadinstitute . org/ccle; Broad Institute , 2013; Broad Institute , 2018a ) .", "Significant differences in the dependency scores of cell lines with mutated and non-mutated regulators , or amplified or copy-number normal regulators were obtained using Wilcoxon tests ( p<0 . 05 ) .", "We only considered regulators that are either amplified or point mutated in at least three cell lines .", "IC50 values of cancer cell lines after treatment with 250 cancer drugs were obtained from the Genomics of Drug Sensitivity in Cancer database ( version 17 , release", "6 ) ( Yang et al . , 2013 ) ( https://www . cancerrxgene . org/; Genomics of Drug Sensitivity in Cancer Consortium , 2016 ) .", "We calculated the Spearman correlation of IC50 values with dependency scores from the Avana CRISPR-Cas9 databases .", "We only considered negative correlations < −0 . 25 and with a adjusted p-values after Benjamini and Hochberg correction <0 . 05 , since we were interested in cell lines with high dependency to a regulator and that showed greater drug sensitivity at lower concentrations .", "R code to perform all analyses and generate all figures is available at https://github . com/cancer-evolution/Evolutionary-analysis-of-somatic-mutations-in-cancer ( Trigo , 2019; copy archived at https://github . com/elifesciences-publications/Evolutionary-analysis-of-somatic-mutations-in-cancer ) ." ] ]

[ "Extensive transcriptional alterations are observed in cancer , many of which activate core biological processes established in unicellular organisms or suppress differentiation pathways formed in metazoans .", "Through rigorous , integrative analysis of genomics data from a range of solid tumors , we show many transcriptional changes in tumors are tied to mutations disrupting regulatory interactions between unicellular and multicellular genes within human gene regulatory networks ( GRNs ) .", "Recurrent point mutations were enriched in regulator genes linking unicellular and multicellular subnetworks , while copy-number alterations affected downstream target genes in distinctly unicellular and multicellular regions of the GRN .", "Our results depict drivers of tumourigenesis as genes that created key regulatory links during the evolution of early multicellular life , whose dysfunction creates widespread dysregulation of primitive elements of the GRN .", "Several genes we identified as important in this process were associated with drug response , demonstrating the potential clinical value of our approach ." ]

[ "Cancers arise when harmful changes happen in the genetic information of certain cells .", "These ‘mutations’ are different from person to person , but overall , they disrupt healthy cells in similar ways .", "In particular , cancer cells tend to lose features that help cells work together in the body .", "Researchers have suggested that cancers may emerge when cells stop being able to cooperate with each other as part of an organism .", "Our bodies still rely on old genes that were present in our earliest , single-cell ancestors .", "However , we also have newer genes that evolved when the organisms in our lineage started to have more than one cell .", "A complex network of signals exists to ensure that both sets of genes work together smoothly , and previous studies have suggested that cancers may appear when this delicate balance is disrupted .", "To address this question , Trigos et al . have now used a computational approach to analyse different types of tumours from over 9 , 000 patients .", "This showed that , in cancer , many mutations disrupt the genes that coordinate old and new genes .", "These mutations were usually small , punctual changes in the genetic sequence .", "However , large modifications , such as an entire gene being deleted or repeated , took place more often in the old or the new genes themselves .", "Therefore , different classes of mutations have specific roles when disrupting how old and new genes work in cancer .", "While certain genes highlighted during this analysis were already known to be associated with cancer , others were not – including genes present during the evolution of the earliest animals on Earth .", "Looking more closely into how these genes can cause disease may help us better understand and fight cancer ." ]

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

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

[ [ "Early mammalian development progresses through a series of landmark events that are regulated by transcriptional and epigenetic mechanisms .", "Accordingly , establishment of the pluripotent inner cell mass ( ICM ) in E3 . 5 blastocysts is linked with global DNA hypomethylation ( Figure 1A ) ( Hackett et al . , 2013a; Smith et al . , 2012; Wang et al . , 2014 ) .", "Subsequent development of the ICM leads to the formation of primed postimplantation epiblast cells by E5 . 5–6 . 25 , which are poised to initiate lineage-specification .", "This developmental transition is accompanied by epigenetic programming , including genome-wide de novo DNA methylation , and potentially accumulation of histone H3 lysine 9 dimethylation ( H3K9me2 ) and redistribution of histone H3 lysine 27 trimethylation ( H3K27me3 ) ( Figure 1A ) ( Borgel et al . , 2010; Leitch et al . , 2013; Marks et al . , 2012 ) .", "Methyltransferase enzymes , G9a ( encoded by Ehmt2 ) and enhancer of zeste homolog 2 ( EZH2 ) , respectively , are responsible for the establishment of these chromatin modifications ( O'Carroll et al . , 2001; Tachibana et al . , 2002; 2005 ) .", "However , the precise contribution of epigenetic programming for setting and regulating the transcriptional circuitry in early development remains to be fully elucidated ( Li et al . , 1992; O'Carroll et al . , 2001; Okano et al . , 1999; Tachibana et al . , 2002 ) . 10 . 7554/eLife . 09571 . 003Figure 1 . G9a-dependent programming occurs at implantation .", "( A ) Schematic of early mouse development and their in vitro equivalents .", "Genome-wide DNA demethylation after fertilisation leads to an epigenetic basal state with low 5meC in ICM of blastocysts .", "Shortly after implantation , the epiblast cells undergo epigenetic programming , which includes de novo DNA methylation .", "By E6 . 25 , the epiblast is primed for somatic development , while being competent for germline specification .", "Gastrulation follows at E6 . 75 .", "Naïve ESCs , primed EpiLCs and EpiSCs represent different stages of in vivo development .", "ESCs grown in 2i/LIF medium resemble the ICM , while EpiLCs , induced from ESCs after 48 h in response to FGF2 and Activin A , are equivalent to epiblast .", "Their prolonged culture results in EpiSCs , which are reminiscent of the anterior primitive streak ( Kojima et al . , 2014 ) .", "( B–D )", "Whole-mount IF staining for H3K9me2 ( B ) , G9a ( C ) and GLP ( D ) in E3 . 5 and E5 . 5 embryos .", "Dotted line shows the ICM , and the EPI .", "It is noteworthy that a single confocal plane is shown to maintain original IF intensity .", "For anti-G9a staining of E5 . 5 embryo , visceral endoderm was removed to reduce the background signal ( scale bar = 20 μm ) .", "( E–G )", "Box plots showing IF signal quantification for H3K9me2 ( E ) , G9a ( F ) and GLP ( G ) .", "Data shows IF intensity normaliseormalized to DAPI for individual ICM or epiblast cells .", "At least 3 embryos and 20 cells were quantified for each time point .", "( *p<0 . 05 in Wilcoxon rank sum test ) .", "DAPI: 4' , 6-diamidino-2-phenylindole; EPI: epiblast; EpiLCs: epiblast-like cells; ESCs: embryonic stem cells; FGF2: fibroblast growth factor 2; GLP: G9a-like protein; ICM: inner cell mass; IF: immunofluorescence; 2i/LIF: two-inhibitor/leukemia inhibitory factor . DOI: http://dx . doi . org/10 . 7554/eLife . 09571 . 003 The involvement of specific enhancer elements in these events is of particular interest as they undergo rapid epigenetic setting , for example , by becoming activated or poised with histone H3 lysine 27 acetylation ( H3K27ac ) and H3K27me3 modifications , respectively ( Creyghton et al . , 2010; Heintzman et al . , 2009; Nord et al . , 2013; Rada-Iglesias et al . , 2011; Zentner et al . , 2011 ) .", "Similarly , the transition from naïve embryonic stem cells ( ESCs ) to epiblast-like cells ( EpiLCs ) and epiblast stem cells ( EpiSCs ) is accompanied by rapid changes in enhancer usage ( Buecker et al . , 2014; Factor et al . , 2014 ) .", "These in vitro states are equivalent to the ICM , E6 . 25 epiblast and the primitive streak , respectively ( Figure 1A ) ( Boroviak et al . , 2014; Hayashi et al . , 2011; Kojima et al . , 2014 ) .", "Similar events likely occur in postimplantation epiblast in vivo when cell cycle shortening is linked with extensive transcriptional and epigenetic alterations , in preparation for key cell fate decisions , including specification of somatic and germline fates ( Borgel et al . , 2010; Snow and Bennett , 1978 ) .", "The modifications of enhancers in this context may be of crucial importance for ensuring appropriate response to the ongoing developmental cues .", "In this study , we have focused on the contribution of G9a-mediated H3K9me2 and EZH2-dependent H3K27me3 to early mouse development .", "We found that during the formation of postimplantation epiblast , there is a dramatic increase in H3K9me2 levels and a concomitant H3K27me3 redistribution .", "These events are necessary for repression of a distinct set of genes , including regulators of the germline , cell cycle , apoptosis , and development .", "The rapid acquisition of H3K9me2 extends to key enhancer elements , thereby reinforcing their repression .", "We propose that such epigenetic programming of epiblast primes a specific gene regulatory network , which is a necessary prerequisite for embryogenesis ." ], [ "First , we investigated the dynamics of epigenetic programming of repressive H3K9me2 during early mouse development .", "Immunofluorescence ( IF ) analysis of E3 . 5 and E5 . 5 embryos revealed significant enrichment of H3K9me2 in the epiblast of postimplantation embryos ( Figure 1B , E ) .", "Accumulation of this modification , which is dependent on G9a and its binding partner G9a-like protein ( GLP ) , coincides with increased levels of the enzymes ( Figure 1C , D , F , G ) .", "To address the function of H3K9me2 , we examined the consequences of Ehmt2 deletion ( Ehmt2−/− ) .", "Loss of G9a by E6 . 5 resulted in reduced levels of H3K9me2 modification , and an increase in apoptotic and non-proliferative cells as judged by IF staining for cleaved Caspase 3 and Ki67 , respectively ( Figure 2—figure supplement 1A , C , D ) .", "These changes led to a developmental delay of mutant embryos by E7 . 5 ( Figure 2A , B ) , consistent with previous reports ( Tachibana et al . , 2002; Yamamizu et al . , 2012 ) . 10 . 7554/eLife . 09571 . 004Figure 2 . G9a represses germline and proliferation-related genes in the postimplantation epiblast .", "( A , B ) bright-field images of Ehmt2+/+and Ehmt2−/− embryos at E7 . 5 ( A ) ( scale bar = 0 . 1 mm ) .", "At least nine embryos of each type were staged ( B ) ( *Chi2 test p-value= <0 . 05 ) .", "( C ) Scatter plot showing transcript expression levels in Ehmt2+/+or +/− and Ehmt2−/− E6 . 25 epiblast .", "Blue points are differentially expressed genes ( Log2RPKM>1 , p-value<0 . 05 , Log2 ( FC ) >1 . 4 ) .", "Shown is the geometric average from four biological replicates .", "( D ) Heatmap showing expression of selected genes from enriched GO categories .", "( E , F )", "Single-cell RT-qPCR validation of RNA-seq performed on individual epiblast cells isolated from E6 . 25 Ehmt2+/+ or Ehmt2−/− embryos ( minimum 2 embryos and 14 cells ) .", "Dot plots show levels of Ehmt2 , pluripotency ( Nanog , Pou5f1 ) , germline ( Asz1 , Rhox5 , Sohlh2 ) and proliferation regulators ( Cdkn1a , Cd3e , Foxj1 ) .", "Expression is normalised to Arbp and relative to average in Ehmt2−/− and for Ehmt2 relative to Ehmt2+/+ .", "Statistical significance was calculated using Wilcoxon rank sum test for Pou5f1 and Nanog , where majority of WT and KO cells show detectable expression .", "For remaining genes a Chi2 test was used .", "( *p-value<0 . 05 ) .", "Also see Figure 2—source data 1–4 and Figure 2—figure supplement 1–3 .", "LHF: late head fold; EHF: early head fold; LB: late allantoic bud; OB: no allantoic bud; LS: late streak; PS: pre-streak .", "RT-qPCR: real-time quantitative polymerase chain reaction; RNA-seq: RNA sequencing; WT: wild-type: KO: knockout; GO: gene ontology; FC: fold change . DOI: http://dx . doi . org/10 . 7554/eLife . 09571 . 00410 . 7554/eLife . 09571 . 005Figure 2—source data 1 . List of differentially expressed genes in E6 . 25 Ehmt2−/− epiblast from RNA-seq analysis data is based on four individual Ehmt2−/− and control ( Ehmt2+/+ or Ehmt2+/− ) epiblasts . Differentially expressed genes were identified using a minimum Log2 ( FC ) >1 . 4 and maximum Fisher combined test of p-value<0 . 05 .", "In the upregulated sample , expression was 1<log ( RPKM ) .", "RNA-seq: RNA sequencing; RPKM: Reads Per Kilobase of transcript per Million mapped reads; FC: fold change . DOI: http://dx . doi . org/10 . 7554/eLife . 09571 . 00510 . 7554/eLife . 09571 . 006Figure 2—source data 2 . List of enriched GO terms in genes upregulated in E6 . 25 Ehmt2−/− epiblast GO term enrichment for biological processes was calculated using DAVID software with minimum five genes in a category and EASE p-value<0 . 05 . EASE: Expression Analysis Systematic Explorer; GO: gene ontology . DOI: http://dx . doi . org/10 . 7554/eLife . 09571 . 00610 . 7554/eLife . 09571 . 007Figure 2—source data 3 . List of differentially expressed genes in E6 . 25 Ezh2−/−epiblast from RNA-seq analysis data is based on 3 individual Ezh2−/− epiblasts and 3 individual control ( Ezh2+/+ or Ezh2+/− ) epiblasts . Differentially expressed genes were identified using a minimum Log2 ( FC ) >1 . 4 and maximum Fisher combined test of p-value<0 . 05 .", "In the upregulated sample , expression was 1<log ( RPKM ) .", "RNA-seq: RNA sequencing; RPKM: Reads Per Kilobase of transcript per Million mapped reads; FC: fold change . DOI: http://dx . doi . org/10 . 7554/eLife . 09571 . 00710 . 7554/eLife . 09571 . 008Figure 2—source data 4 . List of enriched GO terms in genes upregulated in E6 . 25 Ezh2−/− epiblast GO term enrichment for biological processes was calculated using DAVID software with minimum five genes in a category and EASE p-value<0 . 05 . EASE: Expression Analysis Systematic Explorer; GO: gene ontology . DOI: http://dx . doi . org/10 . 7554/eLife . 09571 . 00810 . 7554/eLife . 09571 . 009Figure 2—figure supplement 1 . Epigenetic programming by G9a in postimplantation epiblast .", "( A ) Cryosection IF of E6 . 25 Ehmt2+/+ and Ehmt2−/− embryos stained with antibodies raised against G9a and H3K9me2 .", "Scale bar = 20 μm .", "( B ) Top 10 enriched GO terms in genes upregulated in Ehmt2−/− E6 . 25 embryos .", "( C , D )", "Whole-mount IF of E6 . 5 Ehmt2+/+ and Ehmt2−/− embryos ( C ) stained with antibodies raised against cleaved Caspase 3 and Ki67 , markers of apoptosis and proliferation , respectively .", "White arrows show Ki67 negative cells .", "Scale bar = 20 μm .", "Shown is maximum projection from 5 z-stacks .", "( D ) Bar plot showing the number of apoptotic cells ( cleaved Caspase 3 positive ) and non-proliferative cells ( Ki67 negative and Caspase 3 negative ) dependent on the genotype .", "Data are represented as mean ( ± SEM ) from five control and five KO embryos .", "( *Student’s t-test p-value< 0 . 05 ) .", "Also see Figure 2—source data 1 and 2 .", "GO: gene ontology; H3K9me2: histone H3 lysine 9 dimethylation; IF: immunofluorescence; SEM: standard error of the mean; KO , knockout . DOI: http://dx . doi . org/10 . 7554/eLife . 09571 . 00910 . 7554/eLife . 09571 . 010Figure 2—figure supplement 2 . Loss of G9a does not affect the exit from pluripotency and germline specification .", "( A , B )", "Whole-mount IF of E6 . 5 GGOF Ehmt2+/+ and Ehmt2−/− embryos stained with antibodies raised against Nanog ( A ) , GFP and G9a ( B ) .", "Shown is a sum of an equal number of z-stacks .", "Scale bar 20 = μm .", "( C , D )", "Whole-mount IF of E8 . 5 Ehmt2+/+ and Ehmt2−/− embryos stained with antibodies raised against GFP and AP2γ ( D ) .", "GFP is expressed under GGOF reporter , which marks nascent PGCs .", "The different size of the Ehmt2+/+ and Ehmt2−/− embryos are noteworthy .", "Higher magnification images reveal that E8 . 5 PGCs show upregulation of a germline marker gene AP2γ ( D ) irrespective of the genotype .", "Scale bar 10 = μm .", "GGOF: ΔPE-Pou5f1-EGFP; GFP: green fluorescent protein; IF: immunofluorescence; PGCs: primordial germ cells . DOI: http://dx . doi . org/10 . 7554/eLife . 09571 . 01010 . 7554/eLife . 09571 . 011Figure 2—figure supplement 3 . EZH2 represses multiple developmental regulators in vivo .", "( A ) Scatter plot showing transcript expression levels in Ezh2−/− versus Ezh2cntr E6 . 25 epiblast .", "Red points are differentially expressed genes ( Log2RPKM>1 , p-value<0 . 05 , Log2 ( FC ) >1 . 4 ) .", "Shown is geometric average from three biological replicates .", "( B ) RT-qPCR validation of RNA-seq at selected target genes .", "Data are represented as mean ( ± SEM ) from three independent biological replicates ( *Student’s t-test p-value< 0 . 05 ) .", "( C ) Top 10 enriched GO terms in genes upregulated in Ezh2−/− E6 . 25 embryos .", "Also see Figure 2—source data 3 and 4 .", "GO: gene ontology; RT-qPCR: Real-time quantitative polymerase chain reaction; RNA-seq: RNA sequencing; SEM: standard error of the mean; FC: fold change . DOI: http://dx . doi . org/10 . 7554/eLife . 09571 . 011 To gain further insight into the underlying causes of the phenotype , we performed RNA sequencing ( RNA-seq ) on individual Ehmt2−/− epiblasts at E6 . 25 .", "This revealed misregulation of 180 genes , of which 147 ( ~82% ) are upregulated ( Figure 2C , Figure 2—source data 1 ) ( Log2[fold change[FC]]>1 . 4 , p-value<0 . 05 ) .", "Among the upregulated genes , ~27% of them were located on the X chromosome ( p-value=1 . 6 × 10-15 ) , notably in clusters of Xlr , Rhox and Mage-a genes .", "Consistent with the known functions of these clusters , we found significant enrichment of gene ontology ( GO ) terms linked to hematopoiesis , sexual reproduction , and regulation cell proliferation ( Figure 2D , Figure 2—figure supplement 1B , Figure 2—source data 2 ) .", "To validate these findings , we analysed individual E6 . 25 epiblast cells by single cell real-time quantitative polymerase chain reaction ( RT-qPCR ) .", "A significant proportion of Ehmt2−/−cells showed upregulation of the cyclin-dependent kinase inhibitor Cdnkn1a , and late germline markers Asz1 and Rhox5 but not Pou5f1 ( coding for OCT4 ) or Nanog ( Figure 2E , F , Figure 2—figure supplement 2A , B ) .", "This indicates that , contrary to a previous report ( Yamamizu et al . , 2012 ) , the phenotypic effects cannot be attributed to a delayed exit from naïve pluripotency .", "Furthermore , loss of G9a did not abrogate the establishment of a population of primordial germ cells ( PGCs ) , as judged by the expression of AP2γ and OCT4 , key germline regulators ( Figure 2—figure supplement 2C , D ) .", "These observations show that G9a promotes growth of the embryo by repressing apoptotic and late germline genes , but it does not affect the exit from naïve pluripotency and establishment of the PGC lineage .", "Next , we examined the consequences of loss of Ezh2 and thus of the H3K27me3 modification , which likely undergoes significant redistribution during epiblast development ( Marks et al . , 2012 ) .", "For this reason , we performed RNA-seq on individual E6 . 25 epiblasts lacking EZH2 ( Ezh2−/− ) .", "We found upregulation of 165 , and downregulation of 24 transcripts ( Log2 ( FC ) >1 . 4 , p-value<0 . 05 ) ( Figure 2—figure supplement 3A , B , Figure 2—source data 3 ) , among which were homeobox and gastrulation-related genes , including Hoxd13 and Lefty2 , but pluripotency regulators such as Nanog and Pou5f1 were not affected ( Figure 2—figure supplement 3B , C , Figure 2—source data 4 ) .", "Importantly , we only found five significantly upregulated genes that were shared between Ehmt2−/− and Ezh2−/− embryos .", "Thus , G9a and EZH2 appear to stabilise silencing of distinct sets of germline , proliferation and developmental regulators , but neither of them has an effect on the pluripotency transcription programme in postimplantation embryos .", "To understand the roles of H3K9me2 and H3K27me3 modifications during the transition from naïve pluripotency in the ICM of blastocysts to a primed pluripotent state in postimplantation embryos , we investigated the genome-wide distribution of these modifications .", "For this purpose , we optimised a low cell number chromatin immunoprecipitation with sequencing ( lcChIP-seq ) protocol to analyse ~25 , 000 pregastrulation E6 . 25 epiblast cells in two biological replicates ( Figure 3—figure supplement 1A–C ) ( Ng , et al . , 2013 ) .", "We intersected this information with our RNA-seq data and with the published whole genome bisulfite sequencing ( WGBSeq ) datasets ( Seisenberger et al . , 2012 ) .", "This enabled us to generate a comprehensive overview of the epigenetic and transcriptional state of primed pluripotent epiblast cells in vivo .", "The enrichment of H3K9me2 and H3K27me3 modifications in E6 . 25 epiblast is associated with low and high CpG content , respectively ( Figure 3—figure supplement 2A ) .", "This is also the case in ESCs cultured in conventional media with serum ( sESC ) ( Lienert et al . , 2011; Wen et al . , 2009 ) .", "By contrast , naïve ESCs grown in 2i/LIF ( 2i/LIF ESCs ) show spreading of H3K27me3 outside the CpG dense loci ( Marks et al . , 2012 ) .", "Thus , there is redistribution of H3K27me3 in E6 . 25 epiblast , relative to both naïve ESCs and possibly ICM in vivo .", "The association of H3K9me2 and H3K27me3 modifications on promoters is mutually exclusive , since only 0 . 3% of them are enriched for both marks ( Figure 3A , anticorrelation with Chi2 p-value=0 . 0024 ) .", "These differences are in line with H3K9me2 and H3K27me3 being linked to high and low 5-methylcytosine ( 5meC ) levels , respectively ( Figure 3—figure supplement 2B ) .", "Nonetheless , despite marking distinct chromatin regions , both H3K9me2 and H3K27me3 are linked to transcriptional repression ( Figure 3B ) .", "Notably , this gene repression is correlated with histone modification enrichment at promoters as well as in gene bodies .", "The H3K9me2 modification in gene bodies could impede transcriptional elongation , splicing , or activity of regulatory elements ( Allo et al . , 2009 ) .", "Our evidence suggests that H3K9me2 and H3K27me3 modifications in vivo are linked to distinct repressive chromatin states .", "We confirmed this by means of self-organizing maps , which cluster promoters and gene bodies based on similarity of their cumulative epigenetic signature with respect to transcriptional activity ( Figure 3C ) ( Wehrens and Buydens , 2007 ) . 10 . 7554/eLife . 09571 . 012Figure 3 . In vivo lcChip-seq from E6 . 25 epiblast reveals distinct epigenetic state of primed pluripotent cells .", "( A ) Density contour plot showing the relationship between H3K9me2 and H3K27me3 enrichment .", "Shown are all promoters associated with genes repressed in the epiblast .", "( B ) Density contour plots showing correlation between H3K9me2 ( blue ) and H3K27me3 ( red ) at promoters ( left panels ) and gene bodies ( right panels ) , with transcriptional activity in epiblast .", "( C ) Unbiased clustering of promoters ( left panels ) and gene bodies ( right panels ) based on their cumulative epigenetic and transcriptional signature in epiblast .", "Analysis was performed using self-organizing maps .", "Each circle on the map represents a set of regions with very similar modification and expression profiles; neighbouring circles on the map are also similar .", "Black line separates H3K9me2-enriched regions .", "Scale is in relative enrichment calculated by centring input-normalised RPKM ( for ChIP ) , expression and DNA methylation and dividing by standard deviation .", "( D ) Schematic of early mouse development showing numbers of genes becoming activated ( red ) and repressed ( green ) in E6 . 25 when compared with E3 . 5 ICM ( Log2 ( RPKM ) <4 , p-value<0 . 05 , Log2 ( FC ) >1 ) .", "( E ) Pie charts showing proportion of genes repressed or activated in E6 . 25 with enrichment of H3K9me2 or H3K27me3 at promoter and/or gene body .", "Histone modification enrichment was classified using k-means .", "( F ) Bar plots show fold enrichment of selected GO terms in genes repressed and activated in E6 . 25 epiblast compared with E3 . 5 ICM .", "* p-value<0 . 05 using Fisher test .", "( G ) GO term enrichment analysis of genes repressed in E6 . 25 epiblast when compared with E3 . 5 ICM in relation to their histone methylation status .", "X axis shows the GO term enrichment bias between H3K9me2- and H3K27me3-marked genes ( GO term fold enrichment in H3K9me2 marked genes vs . that in H3K27me3 enriched ones ) .", "Y axis is minimum Fisher p-value in the H3K9me2- or H3K27me3-marked genes .", "Complexity of enriched GO terms was reduced by removing terms with highly overlapping gene sets .", "Also see Figure 3—figure supplement 1–3 .", "lcChIP-seq: low cell number chromatin immunoprecipitation with sequencing; H3K9me2: histone H3 lysine 9 dimethylation; H3K27me3: histone H3 lysine 27 trimethylation; RPKM: Reads Per Kilobase of transcript per Million mapped reads; FC: fold change; ICM: inner cell mass; GO: gene ontology . DOI: http://dx . doi . org/10 . 7554/eLife . 09571 . 01210 . 7554/eLife . 09571 . 013Figure 3—figure supplement 1 . LcChIP-seq on E6 . 25 epiblast .", "( A ) Validation of lcChIP .", "bar plots comparing enrichment of H3K27me3 and H3K9me2 in EpiSCs detected using large scale ( 5–10 × 106 cells ) xChIP or lcChIP .", "Data are represented as mean ( ± SEM ) fold enrichment relative to Gapdh from at least two independent biological replicates .", "( B ) Validation of lcChIP-seq .", "Heatmap of unbiased Spearman’s rho correlation of H3K27me3 levels at promoter elements showing that all EpiSC ChIP-seq samples cluster together independently of the technique used .", "( C ) Genome browser tracks showing H3K27me3 ( red ) and H3K9me2 ( blue ) enrichment in two biological replicates of E6 . 25 epiblast lcChIP-seq .", "Hoxc cluster and Pcsk5 are control regions for enrichment of H3K27me3 and H3K9me2 , respectively .", "Otx2 is a highly expressed gene in the epiblast .", "Data is shown as a sliding window ( 1 kb and 300 bp for H3K9me2 and H3K27me3 , respectively ) of enrichment over input: Log2 ( RPM ChIP/RPM input ) .", "lcChIP-seq: low cell number chromatin immunoprecipitation with sequencing; H3K9me2: histone H3 lysine 9 dimethylation; H3K27me3: histone H3 lysine 27 trimethylation; EpiSCs: epiblast stem cells; xChIP: fixed ChIP; SEM: standard error of the mean , RPM: reads per million mapped . DOI: http://dx . doi . org/10 . 7554/eLife . 09571 . 01310 . 7554/eLife . 09571 . 014Figure 3—figure supplement 2 . H3K9me2 and H3K27me3 correlate with distinct CpG and DNA methylation states .", "( A ) Density contour plots showing H3K9me2 and H3K27me3 enrichment in E6 . 25 epiblast at promoters of HCP , ICP , and LCP .", "( B ) Density contour plots showing correlation between levels of H3K9me2 ( blue ) or H3K27me3 ( red ) and DNA methylation at promoters in the epiblast .", "H3K9me2: histone H3 lysine 9 dimethylation; H3K27me3: histone H3 lysine 27 trimethylation; HCP: high CpG density; ICP: intermediate CpG density; LCP: low CpG density . DOI: http://dx . doi . org/10 . 7554/eLife . 09571 . 01410 . 7554/eLife . 09571 . 015Figure 3—figure supplement 3 . Accumulation of H3K9me2 or H3K27me3 at promoters of genes becoming repressed depends on CpG content .", "( A ) Density contour plots showing H3K9me2 and H3K27me3 enrichment in E6 . 25 epiblast at promoters of HCP , ICP , and LCP .", "Shown are only promoters associated with genes becoming repressed in E6 . 25 epiblast when compared with E3 . 5 ICM ( Log2 ( RPKM ) <4 , p-value<0 . 05 , Log2 ( FC ) >1 ) .", "( B , C )", "Top 10 enriched GO terms in genes repressed in E6 . 25 epiblast when compared with E3 . 5 ICM and associated with HCP ( B ) and ICP ( C ) promoters .", "LCP promoter-associated genes did not show significant GO term enrichment .", "GO: gene ontology; H3K9me2: histone H3 lysine 9 dimethylation; H3K27me3: histone H3 lysine 27 trimethylation; HCP: high CpG density; ICP: intermediate CpG density; LCP: low CpG density; ICM: inner cell mass; FC: fold change; RPKM: Reads Per Kilobase of transcript per Million mapped reads . DOI: http://dx . doi . org/10 . 7554/eLife . 09571 . 015 To gain insight into the epigenetic regulation of developmental progression from naïve to primed pluripotent cells in vivo , we integrated our dataset from E6 . 25 epiblasts with RNA-seq of E3 . 5 ICM ( ERP005749 ) ( Boroviak et al . , 2014 ) .", "First , we identified genes that become robustly activated or repressed in E6 . 25 epiblasts relative to ICM ( Figure 3D ) ( Log2 ( RPKM ) <4 , p-value<0 . 05 , Log2 ( FC ) >1 ) .", "These genes generally corresponded to the expected developmental progression .", "For example , the transcripts that become silenced by the postimplantation stage ( E6 . 25 ) are enriched for GO terms such as ‘blastocyst formation’ and ‘STAT ( Signal Transducer and Activator of Transcription ) signalling regulation’ ( Figure 3F ) .", "These repressed genes are generally enriched for H3K9me2 or H3K27me3 , especially when they have high or intermediate CpG density , respectively ( Figure 3E , Figure 3—figure supplement 3A ) .", "Moreover , consistent with global DNA hypomethylation of the ICM , there is transient expression of 5meC-sensitive germline genes at E3 . 5 that are subsequently repressed by E6 . 25 .", "On the other hand , genes activated in E6 . 25 epiblast include ‘neural tube’ and ‘polarized epithelium’ genes ( Figure 3F ) .", "Finally , both activated and repressed genes are enriched for regulators of transcription , embryonic development and metabolic processes ( Figure 3F ) .", "This analysis reveals that the transcriptional changes between the naïve state associated with the ICM , and the primed state of postimplantation epiblast in vivo reflect the dramatic alterations in signalling , morphology , and metabolism occurring during development .", "Next , we focused on genes that are repressed upon implantation and accumulate H3K9me2 or H3K27me3 .", "GO term analysis revealed preferential acquisition of H3K27me3 on genes associated with transcriptional regulation , embryonic organ development , blastocyst formation , and metabolism ( Figure 3G ) .", "On the other hand , meiotic genes and those involved in immune responses were more likely targeted by H3K9me2 .", "This analysis reveals that different functional pathways are inactivated via establishment of distinct epigenetic states .", "Despite being derived from postimplantation embryos , EpiSCs have reduced competence for the germline fate ( Hayashi and Surani , 2009 ) .", "To investigate this , we directly compared the epigenetic states of E6 . 25 epiblast and EpiSCs .", "Global and metagene analysis revealed little differences in the distribution of H3K9me2 and H3K27me3 between E6 . 25 epiblast and EpiSCs ( Figure 4A , D , Figure 4—figure supplement 1A ) .", "Similarly , over 97% of genes marked by H3K9me2 showed enrichment in both cell types ( Figure 4B , C ) .", "On the other hand , H3K27me3 underwent a significant rearrangement in EpiSCs when compared with in vivo epiblast cells , with genes regulating meiosis and bone development preferentially losing H3K27me3 at promoters ( Figure 4E , F , H ) .", "We reasoned that this might be associated with DNA hypermethylation of such elements since H3K27me3 mark is anti-correlated with 5meC ( Figure 3—figure supplement 2B ) .", "Indeed , our whole genome bisulfite sequencing ( WGBSeq ) of EpiSCs revealed a ~10% increase in global DNA methylation levels ( Figure 4G ) .", "More specifically , promoters that had lost H3K27me3 acquired significantly more 5meC in EpiSCs ( Figure 4—figure supplement 1B ) .", "This observation indicates that EpiSC derivation leads to an aberrant epigenetic state , especially at germline promoters .", "Such stable silencing of these genes might contribute to a reduced competence of EpiSC for PGC fate . 10 . 7554/eLife . 09571 . 016Figure 4 . EpiSC show aberrant H3K27me3 distribution and DNA hypermethylation at germline genes .", "( A , D )", "Distribution of H3K9me2 ( A ) and H3K27me3 ( D ) by metagene analysis in epiblast and EpiSCs .", "Genes were classified based on promoter CpG density .", "( Scale = base pairs . ) ( B , E ) Scatter plots showing H3K9me2 ( C ) and H3K27me3 ( D ) enrichment at gene bodies and promoters , respectively , in EpiSC versus E6 . 25 epiblast .", "Regions were classified as marked in both samples ( purple ) , epiblast ( pink ) or EpiSC only ( blue ) .", "Classification was performed using k-means and EdgeR ( pval<0 . 05 , Log2 ( FC ) >2 ) .", "( C , F )", "Venn diagrams showing overlap of H3K9me2 ( E ) or H3K27me3 ( F ) enrichment between EpiSC and E6 . 25 epiblast .", "( G ) Box plot showing global DNA methylation levels from WGBSeq in E6 . 5 epiblast and EpiSC .", "***p-value<0 . 001 from Wilcoxon rank sum test .", "( H ) Bar plot showing top 10 enriched GO terms in genes uniquely marked by H3K27me3 in the E6 . 25 epiblast .", "Also see Figure 4—figure supplement 1 .", "HCP: high CpG density; ICP: intermediate CpG density; LCP: low CpG density; TSS: transcriptional start site; TTS: transcriptional termination site; EpiSCs: epiblast stem cells; H3K27me3: histone H3 lysine 27 trimethylation; H3K9me2: histone H3 lysine 9 dimethylation; FC: fold change; GO: gene ontology; WGBSeq: whole genome bisulfite sequencing . DOI: http://dx . doi . org/10 . 7554/eLife . 09571 . 01610 . 7554/eLife . 09571 . 017Figure 4—figure supplement 1 . H3K9me2 , H3K27me3 and DNA methylation dynamics between E6 . 25 epiblast and EpiSC .", "( A ) Bar plots showing distribution of H3K9me2 ( top ) and H3K27me3 ( bottom ) genome-wide .", "1 kB tiles were calculated for all chromosomes with a 500 bp offset , and each tile was intersected with annotated genomic regions .", "For each tile , enrichment was calculated .", "Shown are top and bottom 20% of enriched tiles .", "( B ) Box plots showing gain of DNA methylation in EpiSC compared with E6 . 25 epiblast at promoters .", "Promoters were classified based on differential enrichment for H3K27me3 in EpiSC and E6 . 25 epiblast .", "Significance was calculated using unpaired Wilcoxon rank sum test with continuity correction .", "*p-value<0 . 01 .", "H3K27me3: histone H3 lysine 27 trimethylation; H3K9me2: histone H3 lysine 9 dimethylation; EpiSC: epiblast stem cell . DOI: http://dx . doi . org/10 . 7554/eLife . 09571 . 017 Next , we integrated our results on the transcriptional and epigenetic state of epiblast cells from embryos in vivo to identify genes under direct epigenetic regulation by G9a and EZH2 .", "We found that >97% of genes enriched with either H3K9me2 or H3K27me3 modifications do not exhibit differential expression in the Ehmt2−/− and Ezh2−/− embryos ( Figure 5A ) .", "This is consistent with only a minority of Polycomb Repressive Complex 2 ( PRC2 ) targets being dependent on H3K27me3 for their repression in naïve ESCs ( Riising et al . , 2014 ) .", "Thus , the epigenetic programming of epiblast cells by G9a and EZH2 appears to directly regulate silencing at only a limited set of genes , possibly owing to redundant epigenetic mechanisms .", "Nevertheless , we found that ~63% of genes upregulated in Ezh2−/− and ~36% in Ehmt2−/− show enrichment for H3K27me3 and H3K9me2 , respectively ( Figure 5A , Figure 5—figure supplement 1A , Figure 5—source data 1 , 2 ) .", "In the case of Ehmt2−/− , a larger proportion ( ~53% ) of genes seem to be regulated by H3K9me2 deposition at the gene bodies ( Figure 5A ) .", "Among direct G9a targets in the epiblast are Asz1 , Casp14 and Cdkn1a , but not Otx2 , or polycomb targets such as Hoxc10 ( Figure 5B ) .", "These overlaps are significantly higher than the 10% previously observed when analysing Suz12 knockout ( KO ) sESC , which self-renew without PRC2 and accumulate secondary transcriptional alterations ( Pasini et al . , 2007 ) .", "Thus , by focusing on in vivo epiblast just prior to the onset of an overt phenotype , we were able to identify direct primary targets of both G9a and EZH2 . 10 . 7554/eLife . 09571 . 018Figure 5 . H3K9me2 and H3K27me3 are directly involved in repression of genes and transposable elements .", "( A ) Venn diagrams showing overlap between H3K27me3 enrichment at promoters ( left panel ) or H3K9me2 at gene bodies ( right panel ) ; genes upregulated in Ezh2−/− and Ehmt2−/− epiblasts are shown .", "The overlaps are statistically significant ( p-value< 0 . 01 using Chi2 test ) .", "( B ) Genome browser tracks showing H3K9me2 and H3K27me3 enrichment at genes that are: derepressed in Ehmt2−/− ( Asz1 , Casp14 , Cdkn1a ) , active epiblast markers ( Otx2 ) , and PRC2 targets ( Hoxc10 ) .", "Data is shown as a sliding window ( 1kb and 300bp for H3K9me2 and H3K27me3 , respectively ) of enrichment over input: Log2 ( RPM ChIP/RPM Input ) .", "( C ) lcChIP-qPCR validation for H3K9me2 and H3K27me3 at the promoter of Asz1 and gene body of Cdkn1a during EpiLC and EpiSC induction .", "Signal was scaled relative to average enrichment on negative ( Gapdh: H3K9me2 and H3K27me3 ) and positive control regions ( Pcsk5: H3K9me2 , Hoxc10: H3K27me3 ) .", "Data are represented as mean ( ± SEM ) from three independent biological replicates ( *Student’s t-test p-value<0 . 05 relative to 2i/LIF ESC sample ) .", "( D ) Scatter plot showing correlation between the number of unique repeat loci in each subfamily with the number of loci upregulated in Ehmt2−/− E6 . 25 epiblast .", "Red points are subfamilies with significant H3K9me2 enrichment and increased proportion of upregulated loci .", "( E ) Single cell RT-qPCR validation of repeat upregulation in individual E6 . 25 Ehmt2+/+ and Ehmt2−/−epiblast cells .", "Statistical significance was calculated using Wilcoxon rank sum test for IAP where the majority of WT and KO cells show detectable expression .", "For remaining repeats , a Chi2 test was used .", "( *p-value<0 . 05 ) .", "( F ) LcChIP-qPCR measuring H3K9me2 levels at selected repeat elements in Ehmt2F/− CreER+ve d2 EpiLCs treated with EtOH or TAM .", "Data are mean ( ± SD ) from two independent biological replicates .", "( *Student’s t-test p-value<0 . 05 of EtOH compared with TAM treated sample ) .", "Also see Figure 5—figure supplement 1 , 2 and Figure 5—source data 1 , 2 .", "H3K9me2: histone H3 lysine 9 dimethylation; H3K27me3: histone H3 lysine 27 trimethylation; EZH2: Enhancer of zeste homolog 2; ChIP: chromatin immunoprecipitation; EpiLCs: epiblast-like cells; EpiSCs: epiblast stem cells; SEM: standard error of the mean; TAM: tamoxifen; EtOH: ethanol; IAP: intracisternal A particle . DOI: http://dx . doi . org/10 . 7554/eLife . 09571 . 01810 . 7554/eLife . 09571 . 019Figure 5—source data 1 . List of promoters enriched for H3K27me3 in lcChIP-seq from E6 . 25 epiblast data is based on two biological replicates of lcChIP-seq . Enrichment was called by k-means clustering . DOI: http://dx . doi . org/10 . 7554/eLife . 09571 . 01910 . 7554/eLife . 09571 . 020Figure 5—source data 2 . List of gene bodies enriched for H3K9me2 in lcChIP-seq from E6 . 25 epiblast Data is based on two biological replicates of lcChIP-seq . Enrichment was called by k-means clustering . DOI: http://dx . doi . org/10 . 7554/eLife . 09571 . 02010 . 7554/eLife . 09571 . 021Figure 5—figure supplement 1 . H3K9me2 accumulates at G9a-regulated promoters in the epiblast .", "( A ) Venn diagrams showing overlap between H3K9me2 enrichment at promoters and genes upregulated in Ehmt2−/− epiblasts .", "The overlaps are statistically significant ( p-value<0 . 01 using Chi2 test ) .", "( B ) Heatmap showing H3K9me2 and H3K27me3 enrichment at promoters of genes upregulated in Ehmt2−/− .", "Data shown is from two biological replicates of lcChIP-qPCR from 2i/LIF ESCs and day 1–3 EpiLCs .", "Scale is in relative enrichment normaliseormalized to a positive control region of Pcsk5 ( for H3K9me2 ) and Hoxc10 ( for H3K27me3 ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 09571 . 02110 . 7554/eLife . 09571 . 022Figure 5—figure supplement 2 . H3K9me2 accumulates at G9a-regulated repeat elements in the epiblast .", "( A ) Box plots showing fractions of unique loci significantly marked by H3K9me2 or H3K27me3 within classes of repeat elements .", "Shown is data from lcChIP-seq of E6 . 25 epiblast .", "( B ) Scatter plot showing correlation between the number of unique repeat loci in each subfamily with the number of loci upregulated in Ezh2−/− E6 . 25 epiblast .", "Red points are subfamilies with significant H3K27me3 enrichment and increased proportion of upregulated loci .", "( C , D )", "Tables with subfamilies of repeat elements showing significantly increased proportion of unique loci marked by H3K9me2 ( C ) or H3K27me3 ( D ) and upregulated in Ehmt2−/− ( C ) or Ezh2−/− ( D ) E6 . 25 epiblast .", "( E ) Density contour plots showing correlation between H3K9me2 and H3K27me3 enrichment at loci within two subclasses regulated by G9a .", "( F ) Scatter plots FC in expression levels of unique loci in Ehmt2−/− versus Ehmt2+/+ E6 . 25 epiblast .", "Red triangles show loci that are significantly upregulated . DOI: http://dx . doi . org/10 . 7554/eLife . 09571 . 022 Due to very limited material , we confirmed our genome-wide analysis of epiblast by using a more tractable in vitro model of priming cells for gastrulation .", "The in vivo developmental progression is represented in vitro by 2i/LIF ESCs , which are equivalent to ICM , followed by primed EpiLCs and EpiSCs , which represent postimplantation epiblast and the primitive streak , respectively ( Figure 1A ) ( Hayashi et al . , 2011; Kojima et al . , 2014 ) .", "The lcChIP-qPCR analysis showed rapid increase in H3K9me2 levels in primed cells at direct targets of G9a , including Asz1 promoter and Cdkn1a gene body ( Figure 5C ) , as well as promoters of other germline and proliferation regulators ( Figure 5—figure supplement 1B ) .", "Thus , our genome-wide dataset together with the analysis of single loci reveal that G9a represses regulators of germline and cell proliferation , and a subset of them accumulate H3K9me2 mark shortly after implantation .", "We next investigated the role of G9a in regulating transposable elements ( TEs ) as previous studies have shown that loss of G9a in sESC leads to aberrant expression of murine endogenous retroviruses with leucine tRNA primer ( MERV-L ) ( Maksakova et al . , 2013 ) .", "To this end , we combined our lcChIP-seq and RNA-seq from E6 . 25 epiblast and mapped only unique reads to all mouse repeat loci with sufficient coverage .", "We found that ~15% ( 4 . 8 × 105/3 . 3 × 106 ) and ~18% ( 4 . 8 × 105/2 . 6 × 106 ) of repeats show significant H3K9me2 and H3K27me3 enrichment , respectively ( Figure 5—figure supplement 2A ) .", "On the other hand , only ~0 . 11% ( 3636/3 . 3 × 106 ) and ~0 . 06% ( 1646/2 . 6 × 106 ) of all repeats were significantly upregulated in Ehmt2−/− and Ezh2−/− epiblast , respectively .", "By intersecting fractions of upregulated and epigenetically marked loci in each subfamily , we identified repeats that are enriched for and regulated by H3K9me2 or H3K27me3 ( Figure 5D , Figure 5—figure supplement 2B–F ) .", "Direct targets for EZH2 are limited and exhibited no subfamily trend .", "However , 13/15 subfamilies repressed by G9a are notable as they correspond to endogenous retroviral elements ( ERV ) , especially ERV-L and ERV-LMaLR ( Figure 5—figure supplement 2C ) .", "Consistent with the RNA-seq analysis , single cell RT-qPCR from individual E6 . 25 Ehmt2+/+ or Ehmt2−/− epiblast cells showed upregulation of mouse transposon D ( MTD ) , mouse transposable element b ( MTEb ) and Lx8 but not of IAP repeat elements ( Figure 5E ) .", "To confirm G9a-dependent deposition of H3K9me2 at these transposons , we used an in vitro model .", "We generated two Ehmt2F/− ESC lines expressing a tamoxifen ( TAM ) -inducible Cre recombinase ( CreER ) , which were cultured in 2i/LIF medium .", "Following TAM treatment , day 2 EpiLCs were used for lcChIP-qPCR , which showed significant depletion of H3K9me2 upon loss of G9a at MTEb and Lx8 loci , but not IAP ( Figure 5F ) .", "Thus , G9a deposits H3K9me2 at some repeat elements in postimplantation epiblast , where it is necessary to repress TEs that predominantly belong to the ERVs class .", "This implies a potentially important role for G9a in maintaining genomic integrity .", "H3K9me2 modification , unlike H3K27me3 , accumulates rapidly in the postimplantation epiblast ( Figure 1B ) , preferentially in the intergenic regions where many cis regulatory elements reside ( Figure 4—figure supplement 1A ) .", "Visual inspection of our lcChIP-seq analysis revealed that multiple putative enhancers show H3K9me2 enrichment in the epiblast , for example , in close proximity to Esrrb and Prdm1 ( Figure 6A ) , but the role of this modification at such elements is thus far unknown . 10 . 7554/eLife . 09571 . 023Figure 6 . H3K9me2 marks enhancers undergoing inactivation during exit from naïve pluripotency .", "( A ) Genome browser tracks showing p300 , H3K27ac , and H3K4me1 at inactive Esrrb and Prdm1 putative enhancers ( black boxes ) in day 2 EpiLCs ( GSE56138 ) ( Buecker et al . , 2014 ) .", "Bottom two tracks show H3K27me3 ( red ) and H3K9me2 ( blue ) enrichment in the E6 . 25 epiblast .", "Green and grey tracks show read density , while blue and red tracks show Log2 enrichment of ChIP sample over the input sample .", "( B ) Heatmaps showing H3K9me2 and H3K27ac enrichment at enhancers active in ESCs grown in 2i/LIF .", "All ESC enhancers in 2i/LIF enriched for p300 , H3K27ac and H3K4me1 were clustered using kohonen package based on H3K9me2 and H3K27ac .", "Individual classes were ranked based on H3K9me2 levels in EpiLCs .", "H3K27ac tracks were extracted from ( GSE56138 , GSE57409 ) ( Buecker et al . , 2014; Factor et al . , 2014 ) .", "( C ) LcChIP-qPCR measuring H3K9me2 levels at putative enhancer elements in Ehmt2F/−CreER+ve d2 EpiLCs treated with EtOH or TAM .", "H3K9me2- ( blue ) , H3K27me3- ( red ) marked , as well as active ( green ) regulatory elements are shown .", "Data are mean ( ± SD ) from two independent biological replicates .", "( *Student’s t-test p-value<0 . 05 of EtOH compared with TAM treated sample ) .", "Also see Figure 6—figure supplement 1 , 2 .", "H3K27ac: histone H3 lysine 27 acetylation; EpiLCs: epiblast-like cells; H3K27me3: histone H3 lysine 27 trimethylation; ChIP: chromatin immunoprecipitation; H3K9me2: histone H3 lysine 9 dimethylation; lcChIP-seq: low cell number chromatin immunoprecipitation with sequencing; TAM: tamoxifen; EtOH: ethanol; SD: standard deviation; ESCs: Embryonic stem cells . DOI: http://dx . doi . org/10 . 7554/eLife . 09571 . 02310 . 7554/eLife . 09571 . 024Figure 6—figure supplement 1 . Genome-wide accumulation of H3K9me2 extends to multiple enhancer elements .", "( A ) Bar plot showing H3K9me2 levels in ESC , d2 EpiLC and EpiSC measured by quantifying total DNA in anti-H3K9me2 ChIP relative to the input sample .", "Shown is mean ( ± SD ) from at least two independent biological replicates .", "( B ) Enrichment of H3K9me2 in 2i/LIF ESCs and EpiLCs visualized with a metagene analysis .", "Genes were classified based expression level .", "Data is based on nChIP-seq scaled to absolute H3K9me2 levels ( A ) .", "( C ) Heatmap showing H3K9me2 and H3K27ac enrichment at all enhancers active in ESC , EpiLC and EpiSC .", "Individual enhancer elements were clustered and then sorted based on H3K27ac enrichment in EpiSC .", "( D ) Genome browser tracks showing H3K9me2 ( blue ) and H3K27ac ( green ) putative enhancers in 2i/LIF ESC and d2 EpiLCs .", "H3K27ac tracks are from published datasets ( Buecker et al . , 2014 ) .", "Green tracks show read density , while blue tracks show Log2 enrichment of ChIP over the input sample .", "( E ) Box plots showing H3K9me2 levels at enhancers in 2i/LIF ESC , EpiLCs and EpiSCs .", "Significance was calculated using unpaired Wilcoxon rank sum test with continuity correction .", "Effect size: *r≤0 . 10; **0 . 10<r≤0 . 15; ***r>0 . 15 . DOI: http://dx . doi . org/10 . 7554/eLife . 09571 . 02410 . 7554/eLife . 09571 . 025Figure 6—figure supplement 2 . H3K9me2 marks a distinct set of enhancers .", "( A ) Unbiased dynamics of ESC enhancers upon exit from naïve pluripotency .", "Classification was performed using self-organizing maps .", "Some enhancers become inactivated via acquisition of H3K27me3 or H3K9me2 .", "Each of these modes is associated with distinct DNA methylation levels .", "( B ) Box plots showing DNA methylation levels at enhancers in EpiLCs and EpiSCs .", "Significance was calculated using unpaired Wilcoxon rank sum test with continuity correction .", "Effect size: *r≤0 . 10; **0 . 10<r≤0 . 15; ***r>0 . 15 .", "( C ) Chromatin profiles over enhancers in EpiLC and EpiSC .", "Active ESC enhancers ( p300 , H3K4me1 and H3K27ac enriched ) were classified using k-means clustering based on H3K9me2 and H3K27me3 enrichment in EpiLCs . DOI: http://dx . doi . org/10 . 7554/eLife . 09571 . 025 To examine likely functions of H3K9me2 at enhancers , we turned to the in vitro model of ESC priming towards EpiLCs and EpiSCs .", "We generated high quality native ChIP-seq datasets for H3K27me3 and H3K9me2 from 2i/LIF ESCs , EpiLCs and EpiSCs .", "Enhancers in these pluripotent cells have recently been identified based on enrichment of p300 , H3K4me1 and H3K27ac ( GSE56138 , GSE57409 ) ( Buecker et al . , 2014; Factor et al . , 2014 ) .", "Consistent with programming of epiblast in vivo by H3K9me2 modification ( Figure 1B ) , we found genome-wide accumulation of H3K9me2 in primed pluripotent cells ( Figure 6—figure supplement 1A ) .", "As H3K9me2 ChIP-seq measures only the relative enrichment , we have scaled it to indicate absolute quantities of histone modification .", "Our analysis revealed increased enrichment of H3K9me2 at genes upon exit from naïve pluripotency ( Figure 6—figure supplement 1B ) .", "This effect extends to many enhancers but significant H3K9me2 enrichment is observed at elements , which show DNA hypermethylation and cluster separately from poised H3K27me3-enriched elements ( Figure 6—figure supplement 1D , E , Figure 6—figure supplement 2A , B ) .", "As with H3K27me3 modification , H3K9me2 is not a distinct enhancer mark , but rather is associated with larger domains within which regulatory elements reside ( Figure 6—figure supplement 2C ) .", "Interestingly , of all the enhancers that are active in 2i/LIF ESCs ( p300 , H3K4me1 and H3K27ac enrichment ) , ~12% ( 3884 ) gain H3K9me2 after 2 days of induction towards EpiLCs ( Figure 6B ) , while only 3% ( 1117 ) become enriched for H3K27me3 .", "Such elements are also enriched for H3K9me2 in E6 . 25 epiblast ( Figure 6B ) .", "To validate these findings , we performed lcChIP-qPCR from ethanol or TAM treated Ehmt2F/− EpiLCs expressing CreER .", "We confirmed that previously identified enhancer elements gain H3K9me2 in a G9a-dependent manner but active or H3K27me3 poised elements do not ( Figure 6C ) .", "Thus , acquisition of H3K9me2 occurs at distal regulatory elements , perhaps to direct or reinforce their inactivation .", "Notably , H3K9me2-marked enhancers retain partial enrichment of active H3K27ac mark in EpiLCs , but exhibit near-complete H3K27ac loss in EpiSCs ( Figure 7A , Figure 6—figure supplement 1C ) .", "Consistently , enhancer classification based on their epigenetic state has shown that ~57% ( 2212 ) of H3K9me2-enriched enhancers in EpiLCs still retain significant H3K27ac ( Figure 7—figure supplement 1 ) , which we further confirmed using lcChIP-qPCR ( Figure 7B ) .", "To exclude possible effects of cell population heterogeneity , we performed sequential ChIP-qPCR , which showed dual H3K9me2 and H3K27ac enrichment ( Figure 7C ) .", "Such transient dual marking might indicate their continued responsiveness to signalling cues .", "In addition , H3K9me2-enriched elements showed no change in the active H3K27ac modification in EpiLCs lacking G9a ( Figure 7D ) .", "Taken together , our findings show that the repressive H3K9me2 and activating H3K27ac modifications transiently co-occur at enhancers during epiblast priming . 10 . 7554/eLife . 09571 . 026Figure 7 . H3K9me2 spreading to enhancers results in transient coenrichment with H3K27ac .", "( A ) Density contour plots showing correlation between H3K27ac enrichments at enhancers in 2i/LIF ESCs versus EpiLC ( left panels ) or 2i/LIF ESCs versus EpiSC ( right panels ) .", "Green panels show all regulatory elements active in 2i/LIF ESCs ( p300 , H3K4me2 and H3K27ac enrichment ) , while blue panels show only the subset that becomes enriched with H3K9me2 in EpiLCs .", "( B ) lcChIP-qPCR results measuring levels of H3K9me2 , H3K27me3 and H3K27ac at putative Prdm1 and Esrrb enhancers .", "Samples were collected during EpiLC and EpiSC induction .", "Signal was scaled relative to average enrichment on negative ( Pcsk5: H3K27ac , Gapdh: H3K9me2 and H3K27me3 ) and positive control regions ( Pcsk5: H3K9me2 , Gpr20: H3K27ac , Hoxc10: H3K27me3 ) .", "Data are represented as mean ( ± SEM ) from three independent biological replicates ( *Student’s t-test p-value<0 . 05 relative to 2i/LIF ESC sample ) .", "( C ) Sequential ChIP-qPCR performed Ehmt2F− CreER+ve d2 EpiLCs treated with EtOH or TAM .", "Upper panel shows samples precipitated first with anti-H3K9me2 antibody and later H3K27ac .", "Bottom panel shows results from an inverse experiment .", "Samples were scaled to a positive ( Gpr20 ) control region .", "Data are mean ( ± SD ) from two independent biological replicates .", "( *Student’s t-test p-value<0 . 05 ) ( D ) LcChIP-qPCR measuring enrichment of H3K27ac at selected enhancers in EtOH- or TAM-treated Ehmt2F/− CreER+ve d2 EpiLCs .", "H3K9me2-marked ( blue ) and control H3K27me3-poised or active regions are presented .", "Data are mean ( ± SD ) from two independent biological replicates .", "( *Student’s t-test p-value<0 . 05 ) .", "Also see Figure 7—figure supplement 1 .", "H3K27ac: histone H3 lysine 27 acetylation; 2i/LIF: two-inhibitor/leukemia inhibitory factor; EpiLCs: epiblast-like cells; EpiSCs: epiblast stem cells; lcChIP-seq: low cell number chromatin immunoprecipitation with sequencing; H3K9me2: histone H3 lysine 9 dimethylation; H3K27me3: histone H3 lysine 27 trimethylation; SEM: standard error of the mean; ESCs: Embryonic stem cells; TAM: tamoxifen; EtOH: ethanol; SD: standard deviation . DOI: http://dx . doi . org/10 . 7554/eLife . 09571 . 02610 . 7554/eLife . 09571 . 027Figure 7—figure supplement 1 . H3K9me2-marked enhancers retain some H3K27ac in EpiLCs . Box plots showing H3K27ac levels at enhancers in 2i/LIF ESC , EpiLCs and EpiSCs .", "Significance was calculated using unpaired Wilcoxon rank sum test with continuity correction .", "Effect size: *r≤0 . 10; **0 . 10<r≤0 . 15; ***r> 0 . 15 . DOI: http://dx . doi . org/10 . 7554/eLife . 09571 . 027 Enrichment of H3K9me2 generally coincides with transcriptional repression in epiblast cells that prompted us to incorporate our RNA-seq and published data from microarray experiments ( GSE30056 ) ( Hayashi et al . , 2011 ) .", "We found that enhancers marked by H3K9me2 are in close proximity to repressed genes , both in the epiblast in vivo and EpiLCs in vitro ( Figure 8A , B ) .", "Consistent with the Ehmt2−/− embryonic phenotype affecting growth and development , these genes are enriched for the regulators of apoptosis ( Figure 8—figure supplement 1A ) .", "To further address potential functions of these enhancer elements , we performed de novo motif analysis .", "H3K9me2-marked enhancers showed significant enrichment for regulators of early development ( T and SOX2 ) and apoptosis ( p53 and p63 ) ( Figure 8—figure supplement 1B ) .", "In line with the PRC2 function , H3K27me3 poised enhancers showed enrichment for motifs of skeletal development regulators SOX9 and HOXD8 ( Figure 8—figure supplement 1C ) .", "Such non-overlapping motif signatures further validate that H3K27me3 and H3K9me2 accumulate at distinct enhancers that regulate early development . 10 . 7554/eLife . 09571 . 028Figure 8 . G9a promotes transcriptional inactivation of enhancers .", "( A , B )", "Box plots showing transcript levels of genes in epiblast ( A ) and EpiLCs ( B ) , which lie in proximity of H3K9me2- or H3K27me3-marked enhancers .", "All comparisons are statistically significant ( p<0 . 05 ) .", "The effect size relative to active set is shown in the graphs: *r≤0 . 10; **0 . 10<r≤0 . 15; ***r>0 . 15 calculated using Wilcoxon rank sum test .", "For analysis of EpiLCs , a published microarray experiment was used ( GSE30056 ) ( Hayashi et al . , 2011 ) .", "( C , D )", "RT-qPCR for eRNAs at selected H3K9me2-marked ( blue ) and control ( red: H3K27me3 enriched , green: active ) enhancers shown as line plots ( D ) and summarized in a heatmap ( C ) .", "Data are represented as mean ( ± SEM ) from three independent biological replicates .", "Samples are normalised to ESC 2i/LIF sample with the exception of Otx2 , which is normalised to d3 EpiLCs .", "( *Student’s t-test p-value<0 . 05 ) ( E ) FC of eRNA expression in Ehmt2F/− CreER+ve d2 EpiLCs treated with TAM relative to EtOH control .", "Transcripts are originating from H3K9me2- ( blue ) , H3K27me3-marked ( red ) or active ( green ) loci .", "Data are presented as mean ( ± SEM ) from three independent biological replicates .", "( *Student’s t-test p-value <0 . 05 ) ( F ) FC of eRNA expression in individual Ehmt2−/− E6 . 25 epiblasts normalised to Ehmt2+/+ littermates .", "Lines show geometric means .", "( *p<0 . 05 Wilcoxon rank sum test ) .", "Also see Figure 8—figure supplement 1 .", "EpiLCs: epiblast-like cells; H3K9me2: histone H3 lysine 9 dimethylation; H3K27me3: histone H3 lysine 27 trimethylation; RT-qPCR: real-time quantitative polymerase chain reaction; eRNA: enhancer RNA; ESCs: Embryonic stem cells; TAM: tamoxifen; EtOH: ethanol . DOI: http://dx . doi . org/10 . 7554/eLife . 09571 . 02810 . 7554/eLife . 09571 . 029Figure 8—figure supplement 1 . H3K9me2 enriched enhancers are preferentially linked to the p53 pathway .", "( A ) Selected enriched GO terms in H3K9me2-marked enhancers .", "( B , C )", "Bar plots showing top 20 enriched motifs in H3K9me2- ( B ) or H3K27me3- ( C ) marked enhancers .", "Source data file legendsDOI: http://dx . doi . org/10 . 7554/eLife . 09571 . 029 Transcriptional analysis of genes most proximate to specific enhancers is subject to errors , since these elements can regulate distant promoters ( Sanyal et al . , 2012 ) .", "Therefore , we determined the correlation between H3K9me2 enrichment and enhancer activity by analysing the expression levels of enhancer RNAs ( eRNA ) , which is a hallmark of active enhancers ( Kim et al . , 2010 ) .", "We performed RT-qPCR for candidate robustly expressed eRNAs originating from regions enriched for H3K9me2 or H3K27me3 .", "Upon EpiLC induction , such elements underwent robust transcriptional inactivation ( Figure 8C , D ) .", "This is not a global effect because enhancers for genes , such as Otx2 that are linked with primed pluripotency , show increased eRNA expression in EpiLCs ( Figure 8D ) .", "Thus , upon blastocyst implantation , H3K9me2 domains extend to active enhancers targeted for initiation of silencing , which accounts for the co-enrichment with H3K27ac .", "To determine the functional relevance of H3K9me2 on enhancers , we used two Ehmt2F/− ESC lines expressing CreER .", "Following TAM treatment , day 2 EpiLCs showed increased eRNA expression at 4 of 8 previously identified H3K9me2-marked enhancers , confirming that , in principle , this histone modification promotes repression of enhancer activity ( Figure 8E ) .", "This was specific to H3K9me2 , since the H3K27me3-enriched and active regulatory elements were unaffected ( Figure 8E ) .", "Finally , we sought to validate our findings in vivo since multiple enhancer elements showed increased levels of H3K9me2 in the post-implantation epiblast .", "To this end , we isolated RNA from individual Ehmt2+/+ and Ehmt2−/− E6 . 25 epiblasts .", "After complementary DNA ( cDNA ) preamplification , we measured eRNA expression in these samples .", "We were able to detect 7/8 eRNAs associated with H3K9me2 marked enhancers , and 4 of these showed significant increase in expression in Ehmt2−/− embryos ( Figure 8F ) .", "Of all four control eRNAs detected in vivo , none showed altered expression .", "Taken together , these results reveal that G9a contributes to transcriptional and epigenetic repression of a subset of enhancers .", "These elements are typically active in naïve pluripotent cells but become repressed during priming of the epiblast ." ], [ "We present here evidence that global H3K9me2 is established by a wave of G9a activity during early postimplantation development and contributes to establishing a crucial chromatin signature at promoters and gene bodies .", "Once acquired , H3K9me2 represses a specific subset of genes , including key regulators of proliferation and germline development .", "The enrichment of H3K9me2 additionally extends into domains that contain multiple enhancer elements , leading to their developmentally-linked inactivation .", "This uncovers an important role for G9a in setting the regulatory circuitry in the epiblast that enables the subsequent developmental programme to unfold .", "A wave of heterochromatization occurs during early postimplantation development that contributes to the establishment of a specific ‘primed’ epigenetic state in epiblast cells ( Borgel et al . , 2010; Gilbert et al . , 2010 ) .", "While both G9a and PRC2 are involved in this epigenetic programming process , we found limited overlap between H3K9me2 and H3K27me3 targets or , indeed , between genes that were upregulated in Ezh2−/−and Ehmt2−/− mutant embryos .", "This indicates that they likely have independent functions , as observed in sESC , which is unrelated to the previously reported G9a-dependent recruitment of PRC2 complex ( Lienert et al . , 2011; Mozzetta et al . , 2013 ) .", "Thus , prior to gastrulation , H3K9me2 and H3K27me3 generate distinct repressive chromatin states , linked to DNA hyper- and hypomethylation , respectively .", "These modifications therefore act as complementary systems to target specific regulatory pathways , ensuring that they become repressed during the exit from naïve pluripotency .", "Following PGC specification , however , there is germline-specific resetting of the epigenome , including the erasure of H3K9me2 and DNA methylation ( Hajkova et al . , 2002; Seki et al . , 2005; 2007 ) , which allows for the expression of germline-specific genes that have been silenced by these epigenetic modifications .", "We have characterised the epigenetic landscape of the in vivo primed pluripotent cells relative to its in vitro model represented by EpiSCs .", "It is noteworthy that the two cell types show highly similar distribution of H3K9me2 , but EpiSCs globally gain DNA methylation and lose H3K27me3 from germline-related genes , which might contribute to the reduced competence of EpiSCs towards PGCs .", "Indeed , stable promoter DNA methylation has been previously reported for two germline genes , Stella and Rex1 ( Bao et al . , 2009; Hayashi and Surani , 2009 ) .", "Thus we show that in vitro derivation and self-renewal promotes aberrant accumulation of epigenetic modifications , as is the case with sESC and hematopoietic stem cells ( Ludwig et al . , 2014; Weidner et al . , 2013 ) .", "Our study on a transient and highly dynamic state of the pregastrulation epiblast reveals an unexpected level of epigenetic regulation .", "It is established that enhancer elements acquire H3K4me1 and H3K27ac coincident with their developmental activation at this point , while others become poised and enriched for H3K27me3 ( Buecker et al . , 2014; Factor et al . , 2014 ) .", "We additionally reveal that G9a is involved in the rapid switching of epigenetic states at regulatory elements by depositing H3K9me2 ( Figure 9 ) .", "The accumulation of H3K9me2 at enhancers is mostly linked with transcriptional repression , although they transiently retain significant levels of H3K27ac .", "Thus , we show uncoupling of histone acetylation from the transcriptional state , which indicates that loss of an active epigenetic signature is often secondary to enhancer inactivation .", "Such contrasting epigenetic marks at enhancers might confer responsiveness and plasticity of cells to signalling cues .", "This coincides with the rapidly unfolding developmental programme in postimplantation epiblast , resulting in potential for diverse cell fate decisions .", "In line with this role , the loss of H3K9me2 , as seen at an oestrogen-induced enhancer in breast cancer cells , promotes reactivation of the apoptosis regulator bcl2 ( Perillo et al . , 2008 ) . 10 . 7554/eLife . 09571 . 030Figure 9 . Proposed model for G9a-mediated enhancer inactivation . Distal regulatory elements active in ICM are typically associated with H3K4me1 and H3K27ac enrichment ( green ) as well as eRNA expression .", "Such elements activate promoters ( purple ) to increase gene transcription .", "Following implantation , the majority of enhancers undergo inactivation .", "In many cases , this process is aided by spreading of G9a-dependent H3K9me2 enrichment domains .", "Despite retaining H3K27ac and H3K4me1 enrichment , these enhancers typically loose eRNA expression . DOI: http://dx . doi . org/10 . 7554/eLife . 09571 . 030 The regulation of enhancer elements likely contributes to the phenotype of G9a null embryos , but is unrelated to increased expression of pluripotency genes , contrary to a previous suggestion ( Yamamizu et al . , 2012 ) .", "Decreased proliferation and increased apoptosis is more likely due to de-repression of negative regulators of cell cycle , including a potent cyclin-dependent kinase inhibitor , Cdkn1a ( also called p21 ) .", "Furthermore , G9a preferentially represses three clusters of X-linked genes: Rhox , Xlr and Mage-a .", "Two of these are imprinted , suggesting a role for H3K9me2 in this process , which merits further investigation ( Maclean et al . , 2011; Raefski and O'Neill , 2005 ) .", "Pluripotency is a transient state during mammalian development that is established at the blastocyst stage , and undergoes changes after implantation and development of the epiblast .", "Importantly , G9a-dependent programming entails spreading of H3K9me2 modification to enhancer elements where it regulates their activity; this contributes to rapid and dynamic changes at a critical period of epiblast development prior to gastrulation ." ] ]

[ "Early mouse development is accompanied by dynamic changes in chromatin modifications , including G9a-mediated histone H3 lysine 9 dimethylation ( H3K9me2 ) , which is essential for embryonic development .", "Here we show that genome-wide accumulation of H3K9me2 is crucial for postimplantation development , and coincides with redistribution of enhancer of zeste homolog 2 ( EZH2 ) -dependent histone H3 lysine 27 trimethylation ( H3K27me3 ) .", "Loss of G9a or EZH2 results in upregulation of distinct gene sets involved in cell cycle regulation , germline development and embryogenesis .", "Notably , the H3K9me2 modification extends to active enhancer elements where it promotes developmentally-linked gene silencing and directly marks promoters and gene bodies .", "This epigenetic mechanism is important for priming gene regulatory networks for critical cell fate decisions in rapidly proliferating postimplantation epiblast cells ." ]

[ "The genome contains full instructions for the development of the whole organism .", "The genes within the genome encode for all the proteins , but specific genes are selected to be active at the appropriate time .", "For this reason , there are mechanisms that can turn the genes on and off as and when required .", "One such mechanism is called methylation , in which a chemical group called a methyl tag is added to either the DNA or to histone proteins .", "DNA wraps around histone proteins to form a structure called chromatin .", "When histones are tagged with a methyl group , they become closely packed , and the resulting compaction of the chromatin around a gene inactivates that gene .", "For gene activation , the methyl tag is replaced by another tag called acetyl , which allows the chromatin to de-compact and become more accessible .", "Soon after fertilisation , most of the methyl tags an embryo inherits from its parents are removed and a group of stem cells is established .", "An opposite process takes place once the embryo “nests” in the uterus .", "Two enzymes called G9a and EZH2 add methyl tags to specific residues on specific histones to regulate the expression of genes at the time when cells begin to decide what tissue to become .", "Zylicz et al . have now investigated how these enzymes contribute to the changes in chromatin packing that occur during early mouse development that is essential for the progression of normal development .", "In mouse embryos that lacked the G9a enzyme , Zylicz et al . found that specific genes were inappropriately and prematurely activated early in development , including those generally involved in regulating ‘reproduction’ and 'cell death' .", "Similarly , embryos that lacked the EZH2 enzyme experienced the early and inappropriate activation of a different set of developmentally important genes .", "In addition , switches called enhancers control gene expression , and Zylicz et al . found that these regulators are also turned off by histone modifications made by the G9a enzyme .", "Thus , these findings provide important insights on how genes are regulated during a critical period of mouse development , when cells prepare to acquire their specific identities .", "This should lead to further work on the role of G9a in the hours after fertilisation; that is before the ‘programming’ events described here ." ]

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

[ "chromosomes and gene expression", "genetics and genomics" ]

[ [ "Aicardi–Goutières syndrome ( AGS ) is a severe inflammatory encephalopathy characterized by neurological dysfunction , psychomotor retardation , seizures , unexplained fevers , joint stiffness , basal ganglia calcification , and chilblain skin lesions ( Rice et al . , 2007b ) .", "Despite its rarity , AGS has captured the attention of the scientific community due to its clinical , molecular , and genetic overlap with another , common , systemic autoimmune disorder , systemic lupus erythematosus ( SLE ) ( Rice et al . , 2007a , 2007b; Ravenscroft et al . , 2011 ) ( Gunther et al . , 2015 ) .", "Both diseases are characterized , and to a large extent driven , by excessive expression of interferon alpha , a potent anti-viral cytokine associated with heightened innate immune response and inflammation ( Crow , 2011 ) .", "AGS is a monogenic disorder caused by mutations in any one of the six AGS genes .", "The AGS genes encode for the 3′ to 5′ single-stranded DNA exonuclease TREX1 ( AGS1 ) ( Crow et al . , 2006a ) , the RNA:DNA hybrid-specific ribonuclease H2 subunits ( RNase H2A , B and C , corresponding to AGS4 , 2 and 3 , respectively ) ( Crow et al . , 2006b ) , the 3′ to 5′ exonuclease and dNTP hydrolase SAMHD1 ( AGS5 ) ( Rice et al . , 2009; Goldstone et al . , 2011 ) , and the RNA adenosine deaminase ADAR1 ( AGS6 ) ( Rice et al . , 2012 ) .", "Recently , gain-of-function mutations in the cytosolic double-stranded RNA receptor gene IFIH1 have also been shown to be associated with AGS ( Rice et al . , 2014 ) .", "Interestingly , mutations in TREX1 and the RNASEH2 genes have also been implicated in SLE , further illustrating the genetic link between AGS and SLE ( Lee-Kirsch et al . , 2007; Gunther et al . , 2015 ) .", "Given the role of AGS enzymes in DNA and RNA metabolism , the innate immune response in AGS is thought to be triggered by the accumulation of incompletely metabolized endogenous nucleic acid elements ( Crow and Rehwinkel , 2009 ) .", "However , the identity and source of such immunogenic nucleic acid elements remain undefined .", "While genetically well defined , the potential contribution of epigenetic deregulation to AGS has not been addressed .", "Characterizing putative epigenetic deregulation in AGS is all the more justified given that DNA hypomethylation , either spontaneous or caused by drug exposure , triggers increased B cell autoreactivity and the development of drug-induced and idiopathic human lupus ( Richardson , 1986; Quddus et al . , 1993; Yung et al . , 1995; Jeffries et al . , 2011; Absher et al . , 2013; Zhang et al . , 2013 ) .", "Furthermore , loss of DNA methylation , albeit modest and restricted to specific autoimmunity-related genes , was reported in SLE patients ( Richardson et al . , 1990; Ballestar et al . , 2006; Javierre et al . , 2010; Jeffries et al . , 2011; Absher et al . , 2013 ) , raising the question of whether the same epigenetic deregulation is shared in AGS .", "To uncover the mechanism driving inflammatory responses in AGS , we sought to identify transcriptional , genetic , and epigenetic perturbations shared among AGS subtypes .", "For this , we extensively profiled a series of primary fibroblast cells from AGS patients with mutations in AGS1 , AGS2 , and AGS4 and AGS5 using high-throughput sequencing technologies .", "Transcriptional profiling via RNA-seq showed that AGS fibroblasts are characterized by an activated antiviral , immune response with a characteristic interferon signature .", "Genome-wide profiling of RNA:DNA hybrid formation revealed that AGS cells accumulate excessive loads of RNA:DNA hybrids .", "Whole-genome DNA methylation profiling further indicated that AGS genomes experience global loss of DNA methylation .", "Our data therefore provide the first evidence that TREX1 , RNASEH2 , and SAMHD1 mutations share common molecular abnormalities including genome-wide DNA hypomethylation and accumulation of RNA:DNA hybrid species ." ], [ "We first performed RNA-seq to ascertain transcriptional signatures of AGS using primary fibroblasts from four patients with mutations in AGS1 , AGS2 , AGS4 , or AGS5 , and an age-matched healthy control ( see Supplementary file for detailed genotype information ) .", "We identified a total of 98 and 209 genes that were significantly up- and down-regulated , respectively , in at least one AGS sample ( Figure 1A; Figure 1—figure supplement 1A; Supplementary file 2 ) .", "AGS down-regulated genes significantly associated with ontologies linked to cell adhesion and the extracellular matrix ( Supplementary file 2 ) .", "These ontologies may be relevant to the skin lesions often observed in AGS and SLE patients ( Rice et al . , 2007b ) .", "Genes involved in inflammation , immune responses , chemokine signaling pathways , and sensing of viral nucleic acids were up-regulated in AGS patient cells .", "Genes for the major pro-inflammatory cytokine IL1β ( Zhao et al . , 2013b ) and the CXCL5 and CXCL6 chemokines were up-regulated in several AGS patients ( Figure 1A , B ) .", "Additional immune signaling genes ( IL-33 , CXCL3 , CXCL1 , IL-8 , CCL11 ) were significantly up-regulated in at least one AGS sample ( Supplementary file 2 ) and gene ontology analysis of AGS deregulated genes identified cytokine and chemokine signaling pathways as one of weakly enriched terms ( Figure 1—figure supplement 1B ) .", "These observations are indicative of a heightened immunomodulatory and chemotactic response previously identified in multiple autoimmune conditions , including SLE ( Tuller et al . , 2013 ) .", "Genes involved in anti-viral responses ( RSAD2 , OASL , IGF2BP1 , BST2 ) , in particular interferon-inducible genes , were also up-regulated in multiple AGS samples ( Figure 1A , B ) .", "Additional interferon-inducible genes ( IFI6 , IF44L , ISG15 ) described previously as representing an interferon signature in SLE ( Baechler et al . , 2003 ) and AGS ( Rice et al . , 2013 ) showed up-regulation in at least one AGS sample ( Supplementary file 2 ) .", "AGS fibroblasts thus exhibit an activated immune , inflammatory , and anti-viral state , underscoring the systemic nature of the disease and indicating that fibroblasts are an appropriate cell type to study AGS . 10 . 7554/eLife . 08007 . 003Figure 1 . AGS fibroblasts exhibit activated immune antiviral response .", "( A ) RNA-seq heatmap showing log2 expression level of genes up-regulated in at least two Aicardi–Goutières syndrome ( AGS ) samples .", "Two biological replicates are shown for each sample .", "Immune-related genes are marked with green dots .", "( B ) Gene expression changes between AGS samples and control were measured for five immune-related genes by real time reverse-transcription PCR ( RT-qPCR ) .", "Y-axis represents fold change in gene expression , normalized to GAPDH gene , relative to the control sample .", "SDHA is another housekeeping gene used as control .", "Each value is the average of at least two technical replicates .", "Error bars represent SEM . DOI: http://dx . doi . org/10 . 7554/eLife . 08007 . 00310 . 7554/eLife . 08007 . 004Figure 1—figure supplement 1 . RNA-seq of AGS and control fibroblasts .", "( A ) Heatmap showing log2 expression level of genes down-regulated in at least two AGS samples .", "( B ) Pathways analysis of differentially regulated genes . DOI: http://dx . doi . org/10 . 7554/eLife . 08007 . 004 While it is likely that dysfunction in AGS enzymes results in an accumulation of incompletely metabolized immunogenic nucleic acid species ( Crow and Rehwinkel , 2009 ) , the identity and source of these molecules remain unclear .", "RNase H2 generally degrades RNA:DNA hybrids ( Cerritelli and Crouch , 2009 ) and is specifically responsible for removing single ribonucleotides that are misincorporated during DNA replication ( Reijns et al . , 2012 ) .", "We therefore determined whether ribonucleotide accumulation is a common feature of AGS .", "For this , we treated genomic DNA from AGS and control primary fibroblasts with purified human RNase H2 and measured the presence of resulting nicks by DNA polymerase I-dependent nick translation in the presence of [α-32P] dCTP .", "Genomic DNA from wild-type and RNase H2A-deficient ( rnh201Δ ) yeast cells that readily incorporate ribonucleotides ( Nick McElhinny et al . , 2010 ) was used as a control .", "Relative to wild-type , rnh201Δ yeast cells showed a fourfold to fivefold increase in ribonucleotide accumulation ( Figure 2A , B ) , consistent with prior results ( Nick McElhinny et al . , 2010 ) .", "Cells from two patients mutated in RNASEH2B ( AGS2 ) showed a twofold to threefold increase in labeling ( Figure 2A , C ) .", "Cells from two patients mutated in the catalytic RNase H2A subunit ( AGS4 ) showed a pronounced 10–25-fold increase in ribonucleotides ( Figure 2A , C ) .", "Thus , as observed in yeast ( Nick McElhinny et al . , 2010 ) and mouse ( Reijns et al . , 2012 ) models , a reduction of human RNase H2 activity leads to elevated levels of ribonucleotides in genomic DNA .", "By contrast , no significant increase in ribonucleotide loads could be detected in patients mutated in TREX1 ( AGS1 ) or SAMHD1 ( AGS5 ) ( Figure 2A , B ) .", "Thus , while aberrant accumulation of ribonucleotides may contribute to the severity of the disease in RNase H2-defective AGS patients , it is unlikely to represent a common form of disease-causing nucleic acids in AGS . 10 . 7554/eLife . 08007 . 005Figure 2 . Incorporation of ribonucleotides in genomic DNA is only observed in RNase H2-deficient AGS fibroblasts .", "( A ) Genomic DNA was treated with RNase H2 to reveal the presence of ribonucleotides as single-strand nicks .", "An untreated control was used to reveal background single-stranded breaks in genomic DNA preparations .", "The intensity of labeling after gel electrophoresis was measured .", "( B , C )", "Relative ribonucleotide loads are reported as fold-increase of radiolabel incorporation of RNase H2-treated over untreated sample .", "At least three independent replicates were performed and the error bars indicate SEM . DOI: http://dx . doi . org/10 . 7554/eLife . 08007 . 005 To identify other species of RNA:DNA hybrids that may be accumulating in AGS , we performed DRIP-seq , a technique originally developed to profile R-loop formation genome-wide ( Ginno et al . , 2012 ) .", "R-loops are long RNA:DNA hybrid structures that form co-transcriptionally upon re-annealing of the RNA transcript to the template DNA strand , forcing the non-template strand into a single-stranded state .", "R-loops are enriched at the 5′- and 3′-ends of human genes where they play roles in epigenetic control and transcription regulation ( Ginno et al . , 2012 , 2013 ) .", "DRIP-seq revealed that AGS and control fibroblasts share a total of 15 , 897 DRIP peaks representing approximately 141 megabases of genomic space ( Figure 3A , C , Figure 3—figure supplement 1A , B ) .", "Consistent with prior studies , 32% of these peaks mapped to promoters or gene ends , which represents a strong enrichment over expected genomic distribution and further indicates that gene ends correspond to R-loop formation hotspots ( Ginno et al . , 2013 ) .", "Our high-coverage DRIP-seq data also revealed that 56% of the R-loop signal mapped onto gene bodies , suggesting that RNA–DNA entanglements during transcription are prevalent ( Figure 3—figure supplement 1C ) .", "By contrast , only 15 . 6% of common R-loop peaks mapped to intergenic regions , consistent with a predominant genic origin for R-loop formation ( Figure 3—figure supplement 1C ) .", "Overall , common DRIP peaks showed strong overlap with GC skew ( Figure 3B ) , a key sequence determinant that favors co-transcriptional R-loop formation ( Ginno et al . , 2013 ) .", "The observation that AGS samples display R-loop formation over expected R-loop forming , GC-skewed , regions indicates that canonical R-loop formation is not significantly altered in AGS patients ( Figure 3—figure supplement 1A , B ) . 10 . 7554/eLife . 08007 . 006Figure 3 . AGS fibroblasts accumulate RNA:DNA hybrids .", "( A ) All genomic loci overlapping with a DRIP peak in at least one sample are stacked vertically; the position of each peak in a stack is constant horizontally across samples .", "Each patient subtype or control occupies a vertical bar , as labeled .", "Each bar corresponds to merged data sets from two independent samples .", "Common peaks ( i . e . , form in control and at least one AGS sample ) are represented in blue .", "Control-unique DRIP peaks are shown in pink; lack of DRIP signal over a given peak in any sample is shown as black .", "AGS-unique peaks are colored orange , yellow , green , and red in AGS1 , 2 , 4 , and 5 , respectively .", "Brackets on the right side demarcate common and AGS-specific peaks , respectively .", "( B , C )", "Graphs showing the % overlap between DRIP peaks and blocks of GC skew ( B ) ; and the total size of DRIP peaks in each category ( C ) .", "Color codes are as described for ( A ) .", "( D ) Enrichment or depletion of AGS-unique DRIP peaks over different genomic features is shown relative to common DRIP peaks .", "* indicates p < 0 . 002 and fold change >20% relative to common peaks .", "( E–G )", "Representative examples of AGS-specific DRIP peaks over an intergenic region ( E ) , a truncated long interspersed nuclear elements ( LINE ) element ( F ) and a truncated long terminal repeats ( LTR ) element ( G ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 08007 . 00610 . 7554/eLife . 08007 . 007Figure 3—figure supplement 1 . Canonical R-loop genomic patterns are not affected in AGS fibroblasts .", "( A ) Representative screenshot of the broad genomic landscape of R-loop formation over a 2-Mb region .", "DRIP-seq signal is represented by the accumulation of sequence reads along the genomic sequence .", "Each sample is color-coded as indicated .", "CpG islands are indicated together with the positions of genes along the region .", "( B ) A representative screenshot of R-loops formed at the 5′- and 3′-end of the DACT1 gene .", "( C ) Pie chart depicting the distribution of common DRIP peaks at different genomic regions .", "( D ) Percent length overlap of common and AGS-unique peaks over different genomic features .", "*p < 0 . 002 and fold change >20% relative to common peaks . DOI: http://dx . doi . org/10 . 7554/eLife . 08007 . 007 Despite the relative agreement between AGS and control R-loop profiles over a broad set of common peaks , a large excess of DRIP peaks ( 33 , 781 in total ) was observed specifically in AGS cells ( Figure 3A ) .", "These novel peaks of RNA:DNA hybrids were in many cases unique to each patient sub-type .", "While a small number of DRIP peaks were also unique to the control sample , AGS-specific DRIP peaks in each AGS subtype were both threefold to fourfold greater in numbers , and when combined , occupied a three to four times larger genomic space than control-specific peaks ( Figure 3C ) .", "On average , AGS-specific DRIP peaks occupied an additional 23 . 8 megabases of DNA sequence .", "This indicates that all AGS subtypes are burdened by higher loads of RNA:DNA hybrids in their genomes .", "Unlike common DRIP peaks , AGS-specific DRIP peaks were threefold to sevenfold less likely to overlap with GC skew ( Figure 3B ) , suggesting that they may originate according to a non-canonical mechanism .", "Additionally , AGS-specific DRIP peaks were depleted at transcription start sites ( TSSs ) and transcription termination sites ( TTSs ) ( Figure 3D ) , where R-loops are typically observed ( Ginno et al . , 2013 ) .", "In contrast , AGS2- and AGS4-specific peaks were significantly enriched over intergenic portions of the human genome ( Figure 3D , E ) .", "AGS1- and AGS5-specific DRIP peaks were instead enriched over gene body regions ( Figure 3D , Figure 3—figure supplement 1D ) .", "Interestingly , all AGS-specific DRIP peaks were significantly enriched in repeat classes corresponding to long interspersed nuclear elements ( LINE ) and long terminal repeats ( LTR ) retrotransposons ( Figure 3D , F , G , Figure 3—figure supplement 1D ) .", "Analysis of the overlap of AGS-specific DRIP peaks with human-specific , retrotransposition-competent , LINE-1 elements failed to reveal any significant trend ( data not shown ) .", "Therefore , whether the reported enrichment of AGS-specific DRIP peaks over LINE and LTR repeats carries biological significance or is simply a reflection of the increased repeat content of intergenic and intronic space remains to be determined .", "Altogether , our results indicate that AGS mutations in TREX1 , RNASEH2A , RNASEH2B , and SAMHD1 are associated with the accumulation of RNA:DNA hybrids over repeat-rich intergenic and gene body regions .", "The observation that RNA:DNA hybrids accumulate over intergenic regions is surprising given that they are normally maintained in a transcriptionally quiescent state owing to the deposition of silencing epigenetic marks such as DNA methylation ( Yoder et al . , 1997 ) .", "To determine if DNA methylation patterns were altered in AGS , we performed MethylC-seq ( Lister et al . , 2009 ) to profile DNA methylation at low coverage ( 3 . 21–7 . 82 X coverage ) across the same set of primary fibroblasts studied above ( Supplementary file 1 ) .", "All AGS cells displayed significant , global , DNA hypomethylation ( Figure 4A ) .", "AGS2 and 4 cells showed profound DNA hypomethylation , with a ∼20% reduction in genomic methylation levels overall .", "AGS1 and 5 showed a more moderate , but still highly significant , 5–10% reduction in DNA methylation .", "This reduction affected TSSs and TTSs ( Figure 4B , C; Figure 4—figure supplement 1A ) and spread along the lengths of entire chromosomes ( Figure 4D ) , significantly impacting nearly all genomic compartments , including genic , intergenic , and repeat regions ( Figure 4E ) .", "Analysis of repeat classes further revealed that LINE and LTR elements were significantly hypomethylated in all AGS subtypes ( Figure 4F ) .", "Pyrosequencing assays targeting human-specific LINE-1 elements also revealed a small but significant decrease in DNA methylation over 4 CpG sites carried at the LINE-1 5′-UTR promoter ( Figure 4—figure supplement 1B ) .", "SINE elements , satellite repeats , and other repeat types were significantly hypomethylated in AGS2 and 4 but not always in AGS1 and 5 ( Figure 4F ) .", "This profound reduction in DNA methylation was not caused by any detectable deregulation in the expression of genes encoding for the DNA methylation machinery or of components of a putative DNA de-methylation system ( Figure 4—figure supplement 1C ) . 10 . 7554/eLife . 08007 . 008Figure 4 . Genome-wide DNA hypomethylation in AGS fibroblasts .", "( A ) Circos plot depicting DNA methylation along human chromosomes .", "Each tick mark is colored according to the average percent methylation across a 2 Mb genomic region ( see color legend ) .", "( B , C )", "Metaplots of DNA methylation around transcription start site ( TSS ) ( B ) and transcription termination site ( TTS ) ( C ) .", "( D ) Percent methylation along chromosome 17 .", "( E , F )", "Violin plots depicting methylation levels at different genomic ( E ) and repeat ( F ) regions .", "p-values are relative to control .", "**p < 1e-9 , *p < 0 . 01 . DOI: http://dx . doi . org/10 . 7554/eLife . 08007 . 00810 . 7554/eLife . 08007 . 009Figure 4—figure supplement 1 . Methylation profiles of AGS and control cells .", "( A ) Percent methylation of lymphoblastoid cells around TSS , as determined by reduced representation bisulfite sequencing ( RRBS ) .", "AGS2 LCLs are significantly hypomethylated ( p < 2 . 2 × 10−16 ) .", "( B ) Percent methylation of control and AGS fibroblasts at four CpG sites , as surveyed by LINE-1 pyrosequencing .", "( C ) Gene expression levels ( measured by RNA-seq ) for a range of genes involved in the control of DNA methylation are shown for control and a variety of AGS fibroblasts . DOI: http://dx . doi . org/10 . 7554/eLife . 08007 . 009 To further characterize the DNA hypomethylation observed in AGS cells , we employed a Hidden Markov Model ( HMM ) to annotate highly and partially methylated domains ( HMDs and PMDs , respectively ) ( Schroeder et al . , 2011 ) .", "PMDs in control fibroblasts were generally shared by AGS fibroblasts but not by embryonic stem ( ES ) cells ( Figure 5A; Figure 5—figure supplement 1A ) , consistent with the notion that they represent fibroblast-specific PMDs .", "AGS2 and AGS4 fibroblasts displayed broader PMDs than the control cells , while the size of PMD regions in AGS1 and AGS5 cells was relatively unchanged ( Figure 5A; Figure 5—figure supplement 1A ) .", "Analysis of the chromatin features associated with PMDs showed that common PMDs resided within silenced genomic regions enriched for Lamin-B1 , a component of the nuclear lamina , and the silencing mark histone H3 Lysine 9 trimethylation ( H3K9me3 ) , as described previously ( Berman et al . , 2012 ) .", "Common PMDs were also strongly depleted for the H3K27me3 mark , a modification associated with facultative heterochromatin and silencing over developmental genes ( Figure 5B , Figure 5—figure supplement 1B ) .", "Interestingly , AGS-specific PMDs , while still associated with Lamin-B1 and to some extent with H3K9me3 , were enriched for H3K27me3 , particularly over regions corresponding normally to large H3K27me3 blocks ( Figure 5B , C ) .", "This suggests that the DNA hypomethylation observed in AGS is particularly associated with regions of silenced , condensed chromatin . 10 . 7554/eLife . 08007 . 010Figure 5 . AGS-specific PMDs are enriched over H3K27me3-marked genomic regions .", "( A ) Venn diagram displaying partially methylated domains ( PMDs ) in AGS4 and control fibroblasts compared to embryonic stem ( ES ) cells .", "( B ) Location analysis of PMDs over regions marked by H3K9me3 , Lamin-B1 , and H3K27me3 , shown as fold change relative to genome average ( represented by the dotted horizontal line ) .", "Stars indicate the p-value of the deviation from the genome average .", "( C ) Genome browser screenshot showing overlap of AGS-specific PMDs with H3K27me3 .", "( D ) Percent DRIP peaks ( common or AGS-unique ) in highly methylated domains ( HMDs ) and PMDs .", "The pie charts break down AGS2/4 PMDs to common and unique PMDs .", "( E ) Genome browser screenshot showing overlap between AGS2/4 PMDs with AGS2/4 DRIP-seq peaks .", "( F ) Percent DNA methylation of AGS-unique DRIP peaks measured in control and AGS fibroblasts ( as indicated at the bottom of the graph ) .", "*p < 7 . 18e-11 , ns = not significant . DOI: http://dx . doi . org/10 . 7554/eLife . 08007 . 01010 . 7554/eLife . 08007 . 011Figure 5—figure supplement 1 . HMDs and PMDs in AGS fibroblasts .", "( A ) Venn diagrams representing the overlap between PMDs identified in ES cells ( blue ) , control cells ( green ) , and AGS samples ( orange ) .", "The respective sizes of PMDs in each cell type are shown below .", "( B ) A representative genome browser screenshot showing that common fibroblast PMDs are depleted for H3K27me3 and enriched for Lamin-B1 . DOI: http://dx . doi . org/10 . 7554/eLife . 08007 . 011 We next determined if the regions that became hypomethylated also corresponded to regions that accumulated RNA:DNA hybrids in DRIP-seq analysis .", "In AGS1 and AGS5 fibroblasts , which showed only moderate DNA hypomethylation and limited additional PMDs , DRIP peaks , whether common or AGS-unique , mostly landed in HMD regions ( Figure 5D ) .", "This is consistent with AGS1- and AGS5-specific DRIP peaks being enriched over gene body regions ( Figure 3D ) since gene body regions are typically highly methylated ( Lister et al . , 2009 ) .", "In sharp contrast , nearly half of the AGS-unique DRIP peaks in AGS2 and AGS4 fibroblasts landed on PMD regions while only 10–15% of common DRIP peaks matched to PMDs in these two samples .", "Furthermore , two-thirds of these PMD-contained AGS-unique DRIP peaks matched to AGS-specific PMDs ( Figure 5D , E ) .", "Consistent with this , the DNA methylation levels measured over AGS1- , AGS2- , and AGS4- ( but not AGS5 ) unique DRIP peaks were significantly lower in their own respective samples compared to the DNA methylation levels of that same regions measured in control ( Figure 5F ) .", "Thus , our data show a remarkable agreement between regions that accumulate RNA:DNA hybrids in the AGS2 , AGS4 , and to a lesser extent AGS1 subtypes and regions that undergo DNA hypomethylation .", "To determine if the loss of DNA methylation observed in AGS patient cells could be directly due to AGS mutations , we focused on RNASEH2A since AGS4 fibroblasts displayed profound DNA hypomethylation ( Figure 4 ) .", "We employed a lentivirus-mediated CRISPR/Cas method to knockout exon 6 in RNASEH2A ( RNASEH2A KO ) in a HeLa cell line harboring a silent green fluorescent protein ( GFP ) reporter gene ( HeLa-GFP ) ( Poleshko et al . , 2010 ) .", "As a control , we treated the same cell line with a CRISPR vector containing a scramble guide sequence ( scramble ) .", "Effective gene knockout was validated by PCR genotyping ( not shown ) and by Western blots ( Figure 6A ) .", "To further validate the knockout , we measured the DNA damage response in RNASEH2A KO and scramble cells using an antibody against gamma H2AX .", "As expected from previous reports ( Reijns et al . , 2012; Gunther et al . , 2015 ) , we observed higher levels of gamma H2AX in RNASEH2A KO cells ( Figure 6B ) , indicative of an activated DNA damage signaling . 10 . 7554/eLife . 08007 . 012Figure 6 . CRISPR/Cas-mediated RNASEH2A knockout induces DNA hypomethylation .", "( A ) Western blot using an anti-RNase H2A antibody shows successful gene knockout in HeLa-GFP cells .", "Scramble cells were generated with a CRISPR vector carrying a scrambled guide RNA sequence .", "Tubulin was used as a loading control .", "( B ) Immunocytochemistry confirms elevated DNA damage response in RNASEH2A KO cells; DAPI ( blue ) , gamma H2AX antibody ( green ) .", "( C ) Bright field and green fluorescent protein ( GFP ) microscopy images of RNASEH2A KO and scramble control cells .", "Knockout of the RNASEH2A gene triggers the reactivation of the silent GFP reporter in HeLa-GFP cells .", "( D ) ( Top ) Western blot showing GFP expression in HeLa-GFP cells ( RNASEH2A KO and scramble ) ; ( bottom ) bar graph quantification of Western blot .", "( E ) Bisulfite methylation sequencing for two different LINE-1 loci in RNASEH2A KO and scramble control cells .", "Black and white circles represent methylated and unmethylated CpG sites , respectively .", "Missing bubbles indicate that a CpG site is absent from the sequence of that particular molecule .", "The coordinates for each fragment analyzed are indicated at top .", "( F ) Quantification of percent methylation at each CpG sites surveyed in panel E . p-value was calculated using a paired Wilcoxon test with the alternative hypothesis that RNASEH2A KO is less methylated than scramble . DOI: http://dx . doi . org/10 . 7554/eLife . 08007 . 012 Compared to scramble , RNASEH2A KO cells showed a clear increase in the number and brightness of GFP-positive cells ( Figure 6C ) .", "GFP expression in RNASEH2A KO cells was 14-fold higher than in scramble cells , as measured by Western blot ( Figure 6D ) , demonstrating reactivation of the GFP reporter gene .", "To survey DNA methylation levels in RNASEH2A KO and scramble cells , we focused on two 5′-UTR portions common to LINE-1 elements and performed DNA methylation sequencing after sodium bisulfite treatment .", "RNASEH2A KO cells showed significant DNA hypomethylation relative to scramble ( Figure 6E , F ) .", "This suggests a direct effect of RNASEH2A deficiency in triggering DNA hypomethylation and gene reactivation at least for the loci studied here ." ], [ "One of the key observations from this study is that primary fibroblasts from all four AGS subtypes studied here experienced global loss of DNA methylation .", "While additional samples encompassing other AGS mutations subtypes will need to be studied to confirm this trend , this observation nonetheless reinforces the similarities between AGS and SLE , although the DNA hypomethylation reported here is stronger than that previously reported in SLE .", "In AGS , DNA hypomethylation was found to affect every genomic compartment , including genic and intergenic regions , unique and repeated sequences ( Figure 4 ) .", "In AGS2 and AGS4 , where the effect was most marked , the DNA hypomethylation led to the formation of AGS-specific PMDs in addition to those normally observed in fibroblasts ( Lister et al . , 2009 ) .", "Similar to previously characterized PMDs in somatic tissues and cancer samples , common fibroblasts PMDs described here overlapped with inactive , late-replicating , heterochromatic regions associated with H3K9me3 and Lamin-B1 ( Hawkins et al . , 2010; Berman et al . , 2012 ) .", "Interestingly , AGS-specific PMDs , but not common PMDs , were also enriched over regions marked by H3K27me3 , a mark of facultative heterochromatin associated with developmental silencing ( Figure 5 ) .", "This suggests that silent , condensed genomic regions , may in fact be intrinsically prone to becoming PMDs , and that this tendency is made worse by AGS2 and AGS4 mutations .", "Our data further indicate that RNase H2 defects directly drive these epigenetic perturbations given that we could recapitulate DNA hypomethylation at a subset of LINE-1 sequences and trigger the reactivation of a silent GFP reporter by knocking out RNASEH2A ( Figure 6 ) .", "While the precise mechanism responsible for DNA hypomethylation in AGS remains to be elucidated , it is worth noting that defects in TREX1 , RNase H2 , and SAMHD1 all trigger the DNA damage response and are associated with increased genomic instability ( Yang et al . , 2007; Reijns et al . , 2012; Clifford et al . , 2014; Kretschmer et al . , 2015 ) .", "RNase H2 in fact associates with the DNA replication fork through the PCNA ( proliferating cell nuclear antigen ) clamp ( Bubeck et al . , 2011 ) and TREX1 was found to translocate to the nucleus upon replication stress where it might be involved in processing aberrant DNA replication intermediates ( Yang et al . , 2007 ) .", "It is therefore possible that the replication stress caused by AGS mutations indirectly results in defects in replication-coupled , DNMT1-mediated DNA methylation maintenance ( Figure 7 ) ( Law and Jacobsen , 2010 ) .", "In support of this notion , a recent report shows that the PCNA clamp unloads from the lagging strand of stalled DNA replication forks ( Yu et al . , 2014 ) .", "Given that the DNMT1 maintenance DNA methyltransferase binds to PCNA ( Leonhardt et al . , 1992 ) and requires PCNA binding for efficient activity ( Schermelleh et al . , 2007 ) , it is plausible that replication stress may lead to inefficient DNA methylation maintenance , particularly in silent , compact regions of the genome such as heterochromatic regions .", "An analogous mechanism was proposed to account for how defective DNA replication may trigger epigenetic instability by uncoupling DNA synthesis from histone recycling ( Sarkies et al . , 2010 ) .", "Interestingly , 5-aza-2′-deoxycytidine , a drug best known as a DNA-demethylating agent , in fact causes genome-wide DNA damage ( Karpf et al . , 1999; Palii et al . , 2008; Orta et al . , 2013 ) .", "Finally , pronounced DNA hypomethylation is also known to cause genomic instability ( Jackson-Grusby et al . , 2001; Chen et al . , 2007; Liao et al . , 2015 ) suggesting a possible positive feedback loop that may link and amplify defects in either pathways ( Figure 7 ) .", "It will be interesting to further investigate the connections between DNA replication stress , DNA hypomethylation , and epigenetic instability in the context of AGS or of other diseases associated with epigenetic aberrations such as cancers . 10 . 7554/eLife . 08007 . 013Figure 7 . Overall model by which AGS1-5 mutations may lead to DNA hypomethylation , RNA:DNA hybrid accumulation , and IFN-stimulated immune response . See ‘Discussion’ for details . DOI: http://dx . doi . org/10 . 7554/eLife . 08007 . 013 Understanding if and how DNA hypomethylation contributes to autoimmunity in AGS and SLE is more than ever a priority .", "One possibility is that DNA hypomethylation results in altered gene expression profiles .", "However , our RNA-seq characterization only identified a modest number of up- and down-regulated genes that were enriched for ontologies reflecting the biology of the disease itself , such as immunomodulatory genes ( Figure 1 ) .", "There is therefore no evidence for a global alteration of transcriptional profiles as a result of the DNA hypomethylation in AGS .", "This is consistent with the general absence of direct connections between DNA hypomethylation and gene reactivation ( Bestor et al . , 2015 ) , including in extremely hypomethylated mouse ES cells ( Li et al . , 1992; Tsumura et al . , 2006 ) .", "It remains possible , however , that DNA hypomethylation may enable the transcription of normally silent and methylated retrotransposable elements .", "DNA methylation is indeed the primary silencing force ensuring retrotransposon control in somatic cells ( Walsh et al . , 1998; Yoder and Bestor , 1998 ) .", "If correct , this could lead to the reactivation of endogenous retroviral-like particles , which if detected by innate immune sensors , could in turn induce the anti-viral IFNα cytokine , a hallmark of AGS and SLE ( Volkman and Stetson , 2014 ) .", "Here , we showed that the DNA hypomethylation observed in AGS samples affected LTR , LINE , and to a lower extent , SINE repeated DNA elements ( Figure 4 ) .", "However , methylation pyrosequencing focused on human-specific LINE-1s showed that the extent of DNA hypomethylation at these elements , while significant , was minimal and that they in fact remained mostly methylated ( Figure 4—figure supplement 1B ) .", "Likewise mining of our RNA-seq data sets or targeted real-time reverse-transcription PCR ( RT-qPCR ) assays aimed at the LINE-1 mRNA transcript failed to provide convincing evidence for LINE-1 transcriptional reactivation ( data not shown ) .", "Finally , Western blots aimed at detecting the LINE-1 Orf1p protein were negative for AGS fibroblasts even though the protein was easily detected in Ntera2 control cells ( data not shown ) .", "Altogether , while we can't rule out that LINE-1 , or other retroelements , undergo reactivation in AGS cells , particularly in other cell types or at earlier time points during development , the evidence suggests that the DNA hypomethylation observed in AGS primary fibroblasts is not accompanied by LINE-1 reactivation .", "The second major observation from this study is that all AGS subtypes tested here exhibit large accumulation of RNA:DNA hybrid species ( Figure 3 ) .", "RNA:DNA hybrids therefore represent candidate immunogenic nucleic acids in AGS .", "In contrast , ribonucleotide incorporation in genomic DNA was only observed in RNase H2-mutated samples ( Figure 2 ) .", "Our DRIP-seq profiling reveals that AGS2- and AGS4-specific RNA:DNA hybrids map to the intergenic space , away from traditional R-loop hotspots including genes , promoters , and terminators .", "AGS1- and AGS5-specific RNA:DNA hybrids were by contrast enriched over gene body regions ( Figure 3D ) .", "All AGS-unique RNA:DNA hybrid peaks showed a lower overlap with GC skew , an important sequence determinant of R-loop formation ( Figure 3B ) , suggesting that these hybrids may occur by a mechanism distinct from the well-accepted ‘thread back’ R-loop mechanism ( Aguilera and Garcia-Muse , 2012 ) .", "AGS-specific DRIP peaks were enriched in LINE-1 and LTR-containing sequences .", "This enrichment is intriguing in light of the proposed roles of SAMHD1 , RNase H2 , and TREX1 in the control of retroelement transposition ( Stetson et al . , 2008; Zhao et al . , 2013a; Volkman and Stetson , 2014 ) , although , as discussed above , we did not find convincing evidence for transposon reactivation here .", "Alternatively , it is possible that enrichment of AGS-specific RNA:DNA hybrids over LINE-1-rich regions simply reflects the fact that they map preferentially to intergenic and intronic regions .", "One possible clue as to the origin of AGS-specific DRIP peaks stems from the fact that a large fraction of such peaks in AGS2 and AGS4 samples overlap with AGS-specific PMDs in these two subtypes ( Figure 5 ) .", "Direct measurements in fact show that AGS-specific DRIP peaks are significantly hypomethylated in AGS1 , AGS2 , and AGS4 ( Figure 5 ) .", "Thus , the two main features observed in AGS samples , excessive RNA:DNA hybrid formation and DNA hypomethylation , often overlap , strongly supporting a link between both changes in cis .", "One possible explanation for this overlap is that regions experiencing DNA hypomethylation as a result of AGS mutations may undergo spurious transcription that lead to co-transcriptional R-loop formation ( Figure 7 ) .", "Another non-exclusive possibility is based on the proposition that PMD regions may result from DNA damage and inefficient maintenance DNA methylation ( see above ) .", "It was recently described that RNA transcripts may serve as a template for the repair of double-strand DNA breaks upon hybridization to DNA via Rad52 , a key member of the homologous recombination pathway .", "Interestingly , RNase H2 activity was shown to block the formation of these RNA:DNA hybrids ( Keskin et al . , 2014 ) .", "It is therefore possible that some of the DNA breaks caused by AGS mutations and replication stress are repaired according to a pathway prone to RNA:DNA hybrid formation ( Figure 7 ) .", "While speculative , this model may also account for the lack of overlap with GC skew since those hybrids are thought to be mediated by Rad52 and not to arise co-transcriptionally .", "Experiments aimed at testing this proposal will be important .", "One key unanswered question concerns the manner in which RNA:DNA hybrids trigger an immune response .", "The cGAS-STING pathway activates the interferon response upon sensing of not only cytosolic DNA ( Sun et al . , 2013 ) , but also RNA:DNA hybrids ( Mankan et al . , 2014 ) .", "Interestingly , several studies reported that multiple dsDNA break repair proteins can sense damaged DNA and induce a STING-mediated type I IFN response ( Zhang et al . , 2011; Kondo et al . , 2013; Hartlova et al . , 2015 ) , thereby highlighting previously underappreciated links between the DNA damage response and innate immune signaling .", "Besides STING , it is worth noting that the endosomal receptor TLR9 has also been identified as an RNA:DNA hybrid sensor ( Rigby et al . , 2014 ) in addition to its canonical role in sensing unmethylated CpG-rich DNA ( Hemmi et al . , 2000 ) .", "It is therefore possible that AGS mutations-induced DNA hypomethylation also contributes to the increased interferon response in AGS ( Figure 7 ) .", "Interestingly , treatment of colon adenocarcinoma with 5-aza-2′-deoxycytidine , which causes DNA damage and DNA demethylation , triggers a prominent interferon response ( Karpf et al . , 1999 ) .", "Future work will be necessary to dissect the pathways by which unmethylated DNA and RNA:DNA hybrids may be sensed to trigger the innate immune response characteristic of AGS , and by extension , SLE patients ." ], [ "Detection of incorporated ribonucleotides was performed as previously described ( Hiller et al . , 2012 ) .", "Briefly , 200 ng of human primary fibroblasts or yeast genomic DNA ( wild-type and RNASEH2-deficient ) was treated with 20 nM of purified recombinant human RNase H2 ( Loomis et al . , 2014 ) or water in RNase H2 reaction buffer ( 50 mM Tris-HCl pH 8 , 60 mM KCl , 10 mM MgCl2 , 0 . 01% BSA ( Bovine Serum Albumin ) , 0 . 01% Triton ) at 37°C for 1 hr . 20 μM of unlabeled dATP , dGTP and dTTP , plus 3 . 7 × 105 Bq [α-32P]-dCTP ( PerkinElmer , Santa Clara , CA ) and 5 U of Escherichia coli DNA polymerase I ( New England Biolabs , Inc . , Ipswich , MA ) were added and the reaction was incubated at 16°C for 30 min and was run on a 1% TAE ( Tris-acetate-EDTA ) agarose gel .", "Visualization was performed using a Storm PhosphorImager , and bands were quantified using ImageQuant ( GE Healthcare , United Kingdom ) .", "Relative ribonucleotide loads were calculated by dividing the radiolabel incorporation in the RNase H2-treated sample over the untreated sample .", "Experiments were performed at least in triplicate .", "DRIP-seq was performed on primary fibroblasts as previously described ( Ginno et al . , 2012 ) .", "Sequencing reads were trimmed with FastqMcf ( Aronesty , 2011 ) before mapping to the hg19 reference genome using BWA 0 . 6 . 1 ( Li and Durbin , 2009 ) .", "Peak calling was first performed by MACS 1 . 4 . 2 ( Zhang et al . , 2008 ) using input library as control .", "DRIP peaks were further assigned onto restriction fragments using BEDtools ( Quinlan and Hall , 2010 ) .", "For control and each AGS subtype , DRIP peaks from two independent samples were merged into one sample for downstream analysis .", "All overlap analysis was performed using BEDTools ( Quinlan and Hall , 2010 ) .", "GC skew annotation was according to low stringency SkewR peaks as previously described ( Ginno et al . , 2013 ) .", "The enrichment or depletion of AGS-unique DRIP peaks over different genomic regions was measured as fold-change of percent base-pair overlap of AGS-unique DRIP peaks relative to common DRIP peaks .", "The statistical significance of enrichment or depletion of AGS-unique DRIP peaks over a specific genomic feature was measured as follows: for each AGS-unique peak , 500 shuffled peaks of equal lengths were extracted from the common peak set and their overlap with various genomic features was recorded as % length overlap .", "The significance of the difference in overlap between observed ( AGS unique ) and expected ( shuffled ) was calculated using empirical p-values according to the Monte Carlo method .", "2 μg of genomic DNA was sheared by sonication down to 100–500 bp in size .", "Sequencing libraries were constructed as described before ( Schroeder et al . , 2011 ) .", "We then performed bisulfite treatment using EZ DNA methylation-direct kit ( Zymo Research , Irvine , CA ) following manufacturer's instructions .", "50 ng of bisulfite-treated DNA was amplified for 15 cycles using Pfu Cx Turbo Hotstart DNA polymerase ( Agilent Technologies , Santa Clara , CA ) , and the quality of the library was checked on a 2100 Agilent Bioanalyzer prior to sequencing on an Illumina HiSeq 2000 .", "Sequencing reads were trimmed as described in DRIP-seq .", "Mapping was performed using Bismark 0 . 7 . 7 ( Krueger and Andrews , 2011 ) , and percent methylation was called using custom Perl script , combind_strand_meth . pl .", "C to T conversion rate was determined as the ratio of converted C in CHG and CHH context relative to the total number of CHG and CHH .", "CpG coverage was calculated as the average number of reads over CpG sites in the genome .", "Both C to T conversion rate and CpG coverage are reported in Supplementary file 1 .", "To ensure good coverage and eliminate PCR bias , CpG sites with coverage below 4× or more than 99 . 9th percentile were discarded .", "Circos plot was generated using Circos 0 . 62–1 ( Krzywinski et al . , 2009 ) .", "TSS and TTS metaplots were generated using custom Perl script , wig_to_metaplot_lowmem . pl .", "Methylation levels at different genomic regions were calculated BEDtools ( Quinlan and Hall , 2010 ) .", "Wilcoxon paired test was performed using R with the alternative hypothesis that the AGS samples are less methylated than the control .", "PMDs and HMDs were called using StochHMM , a HMM-based software ( Lott and Korf , 2014 ) as previously described ( Schroeder et al . , 2013 ) with modifications: PMDs were trained using random 25-kb regions with 25–55% methylation and HMDs were trained using random 25-kb regions with 60–100% methylation .", "H3K27me3 and H3K9me3 data sets were obtained from ENCODE normal human dermal fibroblasts ( NHDF-Ad ) .", "The lamin B1 data set was obtained from Gene Expression Omnibus ( GSE49341 ) .", "Reduced representation bisulfite sequencing ( RRBS ) library was prepared according to the Myers lab protocol ( Varley et al . , 2013 ) prior to sequencing on an Illumina HiSeq 2000 .", "Sequencing reads were trimmed with Trim Galore 0 . 3 . 3 ( Krueger , 2014 ) and mapped to hg19 genome using Bismark 0 . 7 . 7 ( Krueger and Andrews , 2011 ) .", "TSS metaplot was generated using wig_to_metaplot_lowmem . pl .", "RNA-seq libraries were constructed using the Illumina TruSeq kit prior to sequencing on an Illumina HiSeq 2000 .", "Raw reads were trimmed as before and mapped to the hg19 genome using TopHat 2 . 0 . 5 ( Trapnell et al . , 2009 ) .", "Read counts for each gene were calculated using HTSeq .", "Pearson's correlation between each pair of biological replicates was calculated by comparing every gene's log2 normalized read count ( Supplementary file 4 ) .", "Differential gene expression was identified using DESeq ( Anders and Huber , 2010 ) using fold change > 2 and FDR ( false discovery rate ) < 0 . 1 .", "Pathway analysis was performed using DAVID ( Huang da et al . , 2009 ) .", "DRIP-seq , MethylC-seq , RRBS , and RNA-seq data are available at the Gene Expression Omnibus ( GEO ) database , under the accession number GSE57353 .", "Custom Perl scripts are available at https://github . com/ywlim/Perl .", "Total RNA was extracted from 80% confluent primary fibroblasts using TRI reagent ( Life Technologies , Grand Island , NY ) and Direct-zol RNA miniprep kit ( Zymo Research ) .", "RNA was reverse transcribed to first strand cDNA using iScript Reverse Transcription Supermix for RT-qPCR ( Bio-Rad , Hercules , CA ) .", "cDNA was cleaned up using DNA clean and concentrator ( Zymo Research ) and resuspended in 10 μl water .", "For RT-qPCR , 5 μl of 1:50 dilution of the cDNA was used per well in a 20 μl reaction , along with 2 μl of 10 μM primer sets and 10 μl of SsoAdvanced Universal SYBR Green Supermix ( Bio-Rad ) .", "Reactions were run in duplicate at least on a CFX96 Touch Real-Time PCR Detection System ( Bio-Rad ) with the following protocol: 95°C ( 2 min ) , 40 cycles of 95°C ( 10 s ) and 60°C ( 15 s ) .", "Quantification was calculated using the CFX Manager software ( Bio-Rad ) .", "Primer sequences are listed on Supplementary file 3 .", "RNASEH2A guide RNA sequence was designed using E-CRISP ( http://www . e-crisp . org/ ) and the corresponding oligonucleotides ( top: CACCGTTGGATACTGATTATGGCTC; bottom: AAACGAGCCATAATCAGTATCCAAC ) and scramble oligonucleotides ( top: CACCGGCACTACCAGAGCTAACTCA; bottom: AAACTGAGTTAGCTCTGGTAGTGCC ) were purchased from Life Technologies .", "The oligonucleotides were annealed and ligated into lentiCRISPR v2 ( Addgene plasmid 52961 ) at the BsmBI restriction site .", "The resulting lentiCRISPR-RNASEH2A and lentiCRISPR-scramble plasmids were transformed into DH10B cells and purified using Qiagen Plasmid Midi Kit .", "To produce lentivirus carrying the CRISPR plasmids , 5 μg lentiCRISPR-RNASEH2A or lentiCRISPR-scramble , 4 μg psPAX2 , and 1 . 5 μg of pMD2 . G were transfected into HEK293T cells on a 10-cm plate using Turbofect ( Life Technologies ) following manufacturer's protocol .", "Viral supernatant was collected 48 hr later , filter sterilized with a 0 . 45-μm filter ( Millipore , Billerica MA ) , and 1 ml of it was used to infect HeLa-GFP cells on a 6-well plate .", "1 μg/ml puromycin was added 24 hr later .", "22 days later , HeLa-GFP-RNASEH2A KO and HeLa-GFP-scramble cells were imaged using GFP microscopy .", "Whole-cell protein was extracted and Western blot was performed using antibodies against RNase H2A ( 1:1000 , ProSci ) or GFP ( 1:500 , UC Davis/NIH NeuroMab Facility , Davis , CA ) , and tubulin ( 1:1000 , Sigma–Aldrich , St . Louis , MO ) .", "Immunocytochemistry was performed on the same cells using antibody against γH2AX ( 1:150 , Abcam , United Kingdom ) .", "600 ng of genomic DNA was bisulfite converted using EZ DNA methylation-direct kit ( Zymo Research ) and eluted in 30 μl elution buffer .", "1 μl of it was then PCR amplified using two sets of LINE-1 primers ( Supplementary file 3 ) ( 95°C for 4 min , 20 cycles of 95°C , 55°C and 72°C for 30 s each , 72°C for 4 min ) .", "PCR products were gel purified and cloned into pDRIVE cloning vector using Qiagen PCR cloning kit ( Qiagen , Valencia , CA ) and the resulting vectors were transformed into DH10B cells .", "Miniprep was performed using QIAprep Spin Miniprep Kit ( Qiagen ) and 16 clones were sequenced for each LINE-1 locus .", "Percent methylation at each CG site was the ratio of unconverted ( CG ) vs the sum of unconverted and converted ( TG ) molecules ." ] ]

[ "Aicardi–Goutières syndrome ( AGS ) is a severe childhood inflammatory disorder that shows clinical and genetic overlap with systemic lupus erythematosus ( SLE ) .", "AGS is thought to arise from the accumulation of incompletely metabolized endogenous nucleic acid species owing to mutations in nucleic acid-degrading enzymes TREX1 ( AGS1 ) , RNase H2 ( AGS2 , 3 and 4 ) , and SAMHD1 ( AGS5 ) .", "However , the identity and source of such immunogenic nucleic acid species remain undefined .", "Using genome-wide approaches , we show that fibroblasts from AGS patients with AGS1-5 mutations are burdened by excessive loads of RNA:DNA hybrids .", "Using MethylC-seq , we show that AGS fibroblasts display pronounced and global loss of DNA methylation and demonstrate that AGS-specific RNA:DNA hybrids often occur within DNA hypomethylated regions .", "Altogether , our data suggest that RNA:DNA hybrids may represent a common immunogenic form of nucleic acids in AGS and provide the first evidence of epigenetic perturbations in AGS , furthering the links between AGS and SLE ." ]

[ "The immune system protects the body from attack by bacteria , viruses , and other microbes .", "A key feature of this system is the ability to discriminate between the body's own cells and potential foreign invaders .", "Occasionally , this process can go wrong and the immune system starts attacking its own tissues , which can lead to arthritis , diabetes , lupus , and other ‘autoimmune’ diseases .", "Aicardi–Goutières syndrome ( AGS ) is an autoimmune disease that leads to severe mental and physical symptoms .", "Recent research has revealed that the disease is caused by mutations in genes that make enzymes called nucleases .", "In healthy people , these enzymes destroy DNA molecules and other nucleic acids .", "In AGS patients , the failure of the nucleases to act is thought to lead to the accumulation of unwanted DNA and RNA molecules .", "These molecules , in turn , are thought to be mistakenly identified by the immune system as ‘foreign’ and to cause an autoimmune response .", "However , it is not clear how this works .", "Here , Lim et al . studied skin cells called fibroblasts from patients with Aicardi–Goutières syndrome .", "The experiments found that the patients' cells had excessive numbers of RNA molecules binding to sections of matching DNA .", "These unusual DNA–RNA ‘hybrids’ accumulated in regions of the genome that do not contain many genes , perhaps as a result of breaks in the DNA .", "It is possible that they may mimic nucleic acids from viruses and could trigger an autoimmune response .", "In healthy individuals , small ‘methyl’ groups are often attached to DNA in a process known as DNA methylation .", "This serves to maintain the stability of the genome and controls the activity of genes .", "Unexpectedly , Lim et al . found that the DNA in AGS patients had far fewer methyl groups , especially in areas where the DNA–RNA hybrids had accumulated .", "This may lead to genome destabilization , alterations in gene activity , and may mean that the DNA in these regions may be mistaken for foreign DNA by the immune system .", "Altogether , Lim et al . 's findings suggest that Aicardi–Goutières syndrome may be caused by immune responses triggered by the accumulation of RNA–DNA hybrids and lower levels of DNA methylation .", "These findings may aid the development of new therapies to treat Aicardi–Goutières syndrome , lupus , and other similar diseases ." ]

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

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

[ [ "Studies of organismal diversity have traditionally focused on plants and animals , and more recently on prokaryotes and viruses , while much less attention has been given to microbial eukaryotes ( Pawlowski et al . , 2012; del Campo et al . , 2014 ) ( mostly unicellular protists , plus small multicellular organisms between 0 . 5 µm and 1 mm in size ) .", "On the other hand , genetics together with molecular , cell , and developmental biology have used a limited number of model organisms to reveal over the past 40 years key principles underpinning the organization of living matter ( Alberts , 2014 ) .", "However , recent holistic meta-omics surveys of biodiversity across the full spectrum of life ( Bork et al . , 2015 ) are revealing nowadays a massive amount of unknown taxa and genes in microbial eukaryotes ( de Vargas et al . , 2015 ) implying that much more eukaryotic diversity and functions need to be explored .", "Phylogenomics ( Burki et al . , 2016 ) , geological ( Knoll , 2014; Falkowski and Knoll , 2007 ) , and ecological ( de Vargas et al . , 2015; Mahe et al . , 2016 ) records also show how intense symbiogenesis and diversification in protists have shaped the evolution of eukaryotes together with that of biogeochemical cycles .", "In order to better understand how such processes have led to the complexification of life and the Earth system , it is necessary to bridge contemporary diversity studies to environmental eukaryotic cell biology in an ecological context .", "As a first step , we need new automated high-content 3D-imaging systems that can cope with the broad size , abundance , and complexity ranges characterizing environmental microbial eukaryotes .", "Automated imaging techniques to tackle the diversity of uncultivated aquatic organisms include in-flow systems that couple high-throughput low-resolution imaging with feature extraction from single micro-organisms ( Sieracki et al . , 1998; Sosik and Olson , 2007 ) , and widefield microscope-based methods applied to phytoplankton recognition and quantification ( Embleton et al . , 2003; Rodenacker et al . , 2006; Schulze et al . , 2013 ) .", "These approaches can characterize eukaryotic organisms from low contrast bright field images acquired at a single focal plane , sometimes in association with auto-fluorescence measurements of photosynthetic pigments ( Schulze et al . , 2013; Hense et al . , 2008 ) .", "The identification and classification of eukaryotes is then based on semi-automated machine learning approaches for a few to tens of taxonomic classes ( Benfield et al . , 2007 ) with a peak performance at 70–90% accuracy , which is comparable to what trained annotators can achieve ( MacLeod et al . , 2010; Culverhouse , 2007 ) .", "None of these tools provide images of sufficient quality and resolution to assess the structural complexity of eukaryotic cells whose diversity peaks in the 5 to 50 μm size-range ( de Vargas et al . , 2015 ) .", "To improve both information content and the automated capture of morphological complexity of microbial eukaryotes over a broad size-range , we developed a 3D multichannel imaging workflow denoted e-HCFM ( environmental High Content Fluorescence Microscopy ) ( Figure 1a ) .", "Here , we introduce this new method , which adapts recent protocols developed in model cell biology ( Pepperkok and Ellenberg , 2006; Eliceiri et al . , 2012 ) to the large diversity of scattered objects that characterizes environmental samples ( organisms , debris , aggregates , abiotic objects , etc . ) .", "We then apply e-HCFM to 72 plankton samples collected during the Tara Oceans expeditions ( Karsenti et al . , 2011 ) ( Figure 1—source data 1 ) , containing communities of planktonic organisms in the 5–20 µm size range , which are typically analyzed automatically by flow cytometry with a very low morpho-taxonomic resolution ( Marie et al . , 2014; Zubkov et al . , 2007 ) .", "We show that e-HCFM allows its users", "( i ) to obtain accurate 3D images of micro-eukaryotes over a broad taxonomic range;", "( ii ) to enrich significantly the information content of each imaged organism;", "( iii ) to segregate automatically the high diversity of planktonic micro-particles , and", "( iv ) to archive annotated digital images of perishable samples .", "These advances allow linking identification and quantification of uncultured eukaryotes with ecological and functional traits ." ], [ "The main advantage of e-HCFM is the use of 3D fluorescence microscopy in an automated and quantitative manner for environmental microbiology .", "The relative slowness of the CSLM acquisition can be overcome by widefield microscopy with deconvolution or spinning-disk scanning microscopy , which can both benefit from the high sensitivity and dynamical range of recent cameras .", "Overall , e-HCFM can be applied to samples from any aquatic environment and be adapted for other habitats .", "For the sake of taxonomic comprehensiveness we have used a limited set of fluorescent markers highlighting the broad features of eukaryotic cells .", "However , many other subcellular structures or expressed genes ( FISH ) can potentially be visualized and quantified by e-HCFM , to provide information about their variation and evolution across the diversity of understudied microbial eukaryotic clades ( del Campo et al . , 2014; de Vargas et al . , 2015 ) , thus filling the gap between cell biology , evolution , and ecosystem structure and function .", "A key future challenge will be to combine automated , 3D multicolor fluorescent imaging of complex natural assemblages of eukaryotic cells with online detection of cells of interest and their isolation for single-cell –omics ( Kolisko et al . , 2014 ) .", "This will allow bridging the deluge of novel environmental eukaryotic genetic data to the complex cellular structure , shape , and behaviour of eukaryotic biota , while scaling up the information , through global meta-omics datasets , to the level of the ecosystems ." ], [ "The environmental samples used in this study were collected during the Tara Oceans expeditions ( Pesant et al . , 2015; Tara Oceans Consortium , Coordinators and Tara Oceans Expedition , Participants , 2017 ) .", "At each station , an identical sampling protocol was used to collect and preserve organisms in the 5 to 20 µm size range from surface ( 5 m depth ) and/or deep chlorophyll maximum ( DCM ) depths .", "A dedicated net with a 5 µm mesh size was gently towed for 10 min to avoid net saturation and preserve plankton morphology .", "A flowmeter at net’s mouth allowed quantification of the volume of filtered marine water .", "Concentrated plankton sample in the cod-end was then carefully recovered and poured through a 20 µm sieve to remove larger organisms .", "The filtrate was resuspended with 0 . 2 µm-filtered seawater up to 3 L , providing enough material for the various Tara Oceans morpho-genetic protocols ( Pesant et al . , 2015 ) .", "For e-HCFM , 45 ml were poured into a 50 ml tube pre-aliquoted with 5 ml of 10% monomeric formaldehyde ( 1% final concentration ) buffered at pH7 . 5 ( prepared from paraformaldehyde powder ) and 500 μl of 25% EM grade glutaraldehyde ( 0 . 25% final concentration ) ( Marie et al . , 1999 ) .", "After a gentle homogenization , the samples were kept at 4°C .", "This combination of formaldehyde and glutaraldehyde balances fixative penetration and cell stiffening ( Kiernan , 2000 ) .", "Live imaging was based on both cultures of marine protist from the Roscoff Culture Collection ( RCC ) and specimen isolated from the environment ( Villefranche-sur-mer , France ) .", "The labeling/mounting protocol was optimized to minimize operational steps and cell manipulations .", "After a gentle resuspension of the cells in the sample tube , an aliquot was loaded into a 8-well Lab-Tek II chambered cover glass ( Nunc 155382; Thermo Fisher Scientific , MA , USA ) , with bottom pre-coated with Poly-L-lysine ( Sigma-Aldricht P4707; Merck , Germany ) .", "This optical multi-well plate allows loading about 1 ml of sample per cm² .", "In particular , two Tara Oceans samples were placed in separated wells of the same plate .", "Four other wells were dedicated to fluorescent beads as internal calibration standards of fluorescent signals ( see below ) .", "A gentle centrifugation ( 1 , 000 rpm ) of the plate on a swinging bucket rotor allowed settling down of planktonic particles onto the sticky poly-L-lysine layer .", "This concentrated the objects on the well bottom while decreasing their orientation variability in relation to the optical axis ( Figure 1—figure supplement 3a ) .", "The volume of sample in each well was empirically adjusted by visual inspection of the wells for avoiding cell saturation ( Figure 1—figure supplement 3c ) .", "The sample was then washed for 5 min with 1 ml of artificial seawater ( AS media recipe ( Berges et al . , 2001 ) without vitamins and trace metals ) to remove the fixative .", "All washing steps were performed using pipette tips capped by a 1 µm mesh piece to avoid cell losses .", "AS washing maintains medium buffering capacity and an ionic composition close to natural seawater ( without any organic material ) , thus preventing modifications of cell shape and dissolution of mineral covering .", "The cells were first stained with 0 . 1 mg . ml−1 of a Poly-L-lysine conjugated with Alexa Fluor 546 ( see below ) in 500 µl of AS for 15 min , and then washed with 1 ml of AS for 5 min .", "Further fluorescent labeling was performed , for at least 30 min , with 500 µl of an AS solution containing 10 µM of Hoechst33342 ( Invitrogen H21492; Thermo Fisher Scientific , MA , USA ) and 1 . 5 µM of DiOC6 ( 3 ) ( Invitrogen D273; Thermo Fisher Scientific , MA , USA ) .", "These dyes do not require washing , reducing again sample manipulations .", "We developed a fluorescent labeling compound able to delineate the fine structures of all protist surfaces independently of their biochemistry .", "The poly-L-lysine ( PLL ) conjugated with Alexa Fluor 546 ( see below ) was found to be an effective probe for this task .", "α-poly-L-lysine is a cationic polymer under neutral pH conditions , but the length of the lysine aliphatic side chain confers a slight amphiphilic behavior to this polymer .", "Electrostatic binding occurs thus with both mineral ( e . g . silica , calcium carbonate , strontium sulfate ) and organic ( e . g . DNA , proteins , or polysaccharides ) materials .", "Furthermore , the glutaraldehyde used to fix the plankton sample contributes to crosslink PLL to cellular proteins .", "Examples of the labeling efficiency of Alexa-PLL are shown in Figures 1 and 2 and Figure 1—figure supplements 1 , 2 and 5 and Figure 2—figure supplement 1 .", "Note that a broad choice of dye are available for protein conjugation ( e . g . the Alexa Fluor family ) , allowing selection of spectrally relevant fluorochromes , a convenient feature for combination with other dyes in multi-channel methods .", "High throughput imaging leads to a trade-off between time constraints , the amount of region of interest , and the precision of information that can be extracted for each of them .", "In this study , we optimized settings for an increased throughput rather than spatial resolution .", "Microscopy was conducted using a commercially available inverted SP8 laser scanning confocal microscope ( Leica Microsystem , Germany ) equipped with a compact supply unit which integrates a LIAchroic scan head and several laser lines ( 405 nm , 488 nm , 552 nm , 638 nm ) .", "A two-step sequential acquisition was designed to collect the signal from five channels .", "The first step aims at recording DiOC6 ( 3 ) signal ( Ex488 nm/Em505-520 nm ) simultaneously with the chlorophyll autofluorescence signal ( Ex638 nm/Em680-720 nm ) , and the transmitted light channel .", "The second step is then dedicated to acquisition of the Hoechst signal ( Ex405 nm/Em415-475 nm ) and the AF546 signal ( Ex552 nm/Em570-590 nm ) .", "AF546 is poorly excited by 488 nm and 638 nm beam lines and its emission was not detected between 505 and 520 nm , and between 680 and 720 nm .", "The chlorophyll fluorescence was recorded in the spectral range 680–720 nm where AF546 and Hoechst do not emit with 488 nm and 638 nm illumination .", "Chlorophyll is also excited at 405 nm but it does not emit in both the hoechst ( 415–475 nm ) and AF456 ( 570–590 nm ) channels .", "The hoechst fluorescence spectrum is broad and may slightly bleed through the AF546 channel .", "However the brightness of AF546 signal imposed low sensitivity settings which reduced the impact of other dim signals that might bleed through this channel .", "The potential signal from Hoechst was finally not subtracted because the AF546 fluorescence was mostly used to delineate cell edges .", "The laser power and exposure settings were tuned to reach a tradeoff between the dimmest and brightest object of interest whereas the limited dynamic range of our photomultiplier tube detector ( PMT ) cannot avoid some signal saturation ( the variability of environmental samples cannot be anticipated ) .", "Signals were not averaged .", "The automation was piloted through the HCS A module of the LASAF software ( Leica Microsystem ) .", "We used the water immersion lens HC PL APO 40x/1 , 10 mot CORR CS2 .", "The scanning was bidirectional with a speed set at 600 Hz .", "The pinhole was adjusted to 1 Airy unit for all channels .", "Each square field of view ( fov ) is 385 . 62 µm wide and 21 . 80 µm thick .", "Field of views were organized in a rectangular mosaic with 10% overlapped in X and Y axes .", "The spatial sampling frequency was 0 . 188*0 . 188*1 . 090 µm voxel size ( 2048*2048*per frame ) .", "The Z-stack steps match the full width half maximum as a measure of the optical slice thickness at our lower signal emission wavelength ( 415 nm , refractive index 1 . 33 , numerical aperture 1 . 1 , 1 Airy unit ) while the pixel size matches the objective XY resolution .", "This is larger than the Niquist sampling rate ( 75*75*300 nm , with λemission at 415 nm ) to maximize throughput .", "A software autofocus procedure estimated the interface plane separating the sample from the coverslip .", "Based on this estimate , we initiated the Z-stack at a −1 µm offset .", "At a high-level , image analysis proceeds in three steps: ( 1 ) identification of objects , ( 2 ) computation of per-object features , and ( 3 ) classification of objects .", "In total , it takes circa 40 CPU hours to process one acquisition ( which consists of two wells , 768 data fovs , and 48 control fovs ) .", "However , after the background estimation step , the process is embarrassingly parallel as each fov can be processed in parallel ( a computationally inexpensive post-processing of the data relabels all objects so that they are numbered sequentially within each sample ) .", "Our software is designed to take advantage of a multi-core machine computer cluster by automatically splitting the computation across available CPUs .", "Image analyses of one microscopy run are performed in 20 min with sufficient resources .", "All organisms from a single sample were considered ( data points from multiple technical replicates of the same sample , when available , were concatenated ) .", "Counts were normalized by the volume of seawater represented by the imaged sample ( s ) .", "Correlations were evaluated using Spearman correlation ( implemented by Python's scipy . stats module ) .", "Samples which do not contain the relevant taxonomic group were excluded from each taxonomic-specific analysis .", "The software code is made available under an open source license ( https://git . embl . de/coelho/eHCFM ) .", "All raw images and preprocessed thumbnails are publicly available at EBI-BioStudies ( accession ID: S-BSST51 , https://www . ebi . ac . uk/biostudies/studies/S-BSST51 ) .", "Images can also be explored through the Image Data Resource platform ( Williams et al . , 2017; https://doi . org/10 . 17867/10000108 ) .", "The preprocessed thumbnails will be released for online expert annotations at EcoTaxa ( http://ecotaxa . sb-roscoff . fr ) , and taxonomically and ecologically informed thumbnails will be available for public exploration .", "The inventory of experiment metadata , samples and their associated contextual data from the Tara Oceans expedition are available at PANGAEA ( Tara Oceans Consortium , Coordinators and Tara Oceans Expedition , Participants , 2017; https://doi . org/10 . 1594/PANGAEA . 881193 ) .", "The eHCFM dataset presented herein constitutes an additional Tara Oceans global resource available for the wider community , and complementing other eco-morpho-genetic datasets generated in the project toward a holistic understanding of the world plankton ( Pesant et al . , 2015 ) ." ] ]

[ "We present a 3D-fluorescence imaging and classification tool for high throughput analysis of microbial eukaryotes in environmental samples .", "It entails high-content feature extraction that permits accurate automated taxonomic classification and quantitative data about organism ultrastructures and interactions .", "Using plankton samples from the Tara Oceans expeditions , we validate its applicability to taxonomic profiling and ecosystem analyses , and discuss its potential for future integration of eukaryotic cell biology into evolutionary and ecological studies ." ]

[ "Our planet’s ecosystems – from its oceans to its forests – are teeming with microbes .", "DNA analysis of environmental samples shows that many of these microbes belong to a group known as protists .", "This group consists of single-celled organisms that are close relatives of fungi , plants and animals .", "Though protists are a widespread and diverse group , scientists know little about them .", "One reason for this is the lack of high-throughput ways to recognize and count protists in environmental samples .", "Colin , Coelho et al . set out to tackle this blind spot in ecology and cell biology by developing an automated imaging system .", "The system needed to image many kinds of protist cells in enough detail to see the features inside .", "The end-result was a 3D-imaging technique called e-HCFM – which is short for “environmental high content fluorescence microscopy” .", "Colin , Coelho et al . went on to use the technique on 72 samples collected on an expedition across the world’s oceans .", "This allowed them to automatically image , recognize and classify over 330 , 000 organisms .", "This approach and new dataset will benefit researchers working in many fields , from cell biology to ecology , computational biology and beyond .", "In the future , this imaging method might integrate with techniques that can analyze the DNA in individual cells .", "This would allow scientists to link protists’ visible features to their genetic information , in a way that will scale from single cells up to entire ecosystems ." ]

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

[ "biochemistry and chemical biology" ]

[ [ "The CRISPR system provides adaptive immunity against viruses and other Mobile Genetic Elements ( MGE ) in bacteria and archaea ( reviewed in Wright et al . , 2016; Koonin and Makarova , 2017 ) .", "Type III CRISPR systems are widespread in archaea and found in many bacteria ( Makarova et al . , 2011 ) , including the human pathogen Mycobacterium tuberculosis ( Grüschow et al . , 2019 ) .", "Type III effectors utilise a Cas7 backbone subunit to bind CRISPR RNA ( crRNA ) ( Rouillon et al . , 2013 ) .", "This allows detection of the RNA encoded by invading MGE via base-pairing to the crRNA .", "Binding of this ‘target RNA’ results in subtle conformational changes in the effector complex , activating the catalytic Cas10 subunit which uses its HD-nuclease domain to degrade DNA ( Elmore et al . , 2016; Estrella et al . , 2016; Kazlauskiene et al . , 2016; Jung et al . , 2015; Han et al . , 2017b ) and its cyclase domain to synthesise cyclic oligoadenylate ( cOA ) molecules by polymerisation of ATP ( Kazlauskiene et al . , 2017; Niewoehner et al . , 2017; Rouillon et al . , 2018 ) .", "These cyclic molecules , which range from 3 to 6 AMP monomers in size ( cA3 to cA6 ) , act as second messengers in the cell , signalling viral infection and activating cellular defences .", "cA4 and cA6 bind specifically to two subtypes of CRISPR Associated Rossman Fold ( CARF ) domain ( Makarova et al . , 2014 ) .", "These domains are fused to a range of effector domains that carry out defensive duties .", "The best characterised is the Csx1/Csm6 family that utilises a C-terminal HEPN ( Higher Eukaryotes and Prokaryotes , Nucleotide binding ) domain to cleave RNA non-specifically ( Niewoehner and Jinek , 2016; Sheppard et al . , 2016 ) .", "The Csx1/Csm6 ancillary ribonucleases are crucial for CRISPR-based immunity in vivo in several different organisms , emphasizing the importance of cOA signalling for type III CRISPR defence ( Jiang et al . , 2016; Grüschow et al . , 2019; Foster et al . , 2019; Deng et al . , 2013 ) .", "A wide variety of alternative CARF-domain proteins associated with type III CRISPR loci have been identified but not yet described ( Shmakov et al . , 2018; Shah et al . , 2019 ) , and recent work has characterized the cA4-activated DNA nickase Can1 , which is present in Thermus thermophilus ( McMahon et al . , 2020 ) .", "Thus , type III CRISPR systems are capable of directing a multi-faceted antiviral defence on detection of foreign RNA in the cell .", "However , activation of this anti-viral state is known to generate collateral damage to host nucleic acids ( Rostøl and Marraffini , 2019 ) that could lead to dormancy or cell death .", "While this could be an acceptable outcome for an infected cell , if a viral infection can be cleared then the cOA-signalling pathway needs a mechanism to remove the cOA molecules and return the cell to a basal state .", "Some archaea encode a dedicated ring nuclease ( Athukoralage et al . , 2018 ) , recently named as the Crn1 family ( CRISPR-associated ring nuclease 1 ) ( Athukoralage et al . , 2020a ) .", "Crn1 is a specialised CARF domain protein that splits the cA4 ring into two linear A2 products to switch off the antiviral response ( Athukoralage et al . , 2018 ) .", "In other systems , the CARF domains of Csx1/Csm6 family nucleases slowly degrade cOA , thus acting as bi-functional , self-limiting nucleases ( Athukoralage et al . , 2019; Jia et al . , 2019; Garcia-Doval et al . , 2020 ) .", "Many archaeal viruses and some bacteriophage also encode a specialised ring nuclease as an anti-CRISPR ( Acr ) .", "This enzyme , known as AcrIII-1 , binds cA4 by means of a distinct protein fold ( DUF1874 ) unrelated to the CARF domain , and degrades cA4 rapidly using conserved active site residues ( Athukoralage et al . , 2020a ) .", "These viral ring nucleases can abrogate type III CRISPR immunity by rapidly destroying the cA4 infection signal .", "This enzyme has been co-opted into some bacterial type III CRISPR systems , where it likely acts to degrade cA4 following clearance of phage infection .", "In this context , it has been named as CRISPR-associated ring nuclease 2 ( Crn2 ) ( Athukoralage et al . , 2020a; Samolygo et al . , 2020 ) .", "Ring nucleases thus appear to be an important constituent of virus:host conflict in the expanding arena of cyclic nucleotide signalling .", "Here , we focus on the Csx3 family of proteins , which is found associated with many type III CRISPR systems ( Shah et al . , 2019; Shmakov et al . , 2018 ) .", "Csx3 from Archaeoglobus fulgidus has been described as an RNA deadenylation enzyme and crystallised in complex with a pseudo-symmetric RNA tetraloop ( Yan et al . , 2015 ) .", "Subsequent structural analysis suggested that the Csx3 protein may be a distantly related member of the CARF family of proteins ( Topuzlu and Lawrence , 2016 ) .", "Spurred by these observations , we undertook a detailed study of the Csx3 protein .", "We demonstrate that Csx3 is , in fact , a cA4-specific ring nuclease , for which we propose the name Crn3 ( CRISPR-associated ring nuclease 3 ) .", "The enzyme has an unusual cooperative reaction mechanism involving the association of two dimers , bridged by the cA4 substrate , to complete the active site .", "This mechanism may allow the cell to respond appropriately to changing cA4 and Csx3 levels during viral infection and preserve type III CRISPR immunity ." ], [ "The Csx3 protein from A . fulgidus was originally crystallised in the absence and presence of a pseudo-symmetric RNA tetranucleotide , and shown to have RNA deadenylase activity in vitro ( Yan et al . , 2015 ) .", "Given what is now known about cA4 ring nucleases , we re-assessed the structure and potential function of the Csx3 protein .", "It has previously been suggested that the binding site for the RNA tetranucleotide could be compatible with binding of a symmetric cyclic oligonucleotide ( Topuzlu and Lawrence , 2016 ) , similar to those observed in other CARF family proteins .", "Examination of the genomic context of Csx3 in selected archaeal and bacterial species confirmed its close association with type III-B CRISPR systems and with CARF family proteins such as the ribonuclease Csx1 ( Figure 1A ) , implying a role in cyclic oligoadenylate signalling and providing further impetus to re-examine the function of Csx3 .", "Csx3 is most commonly observed in the euryarchaea and cyanobacteria ( Figure 1—figure supplement 1 ) .", "We therefore cloned , expressed and purified A . fulgidus Csx3 , allowing biochemical analysis .", "Initial studies showed that Csx3 binds cyclic tetra-adenylate with an apparent dissociation constant <0 . 1 µM .", "In contrast , an RNA oligonucleotide ( 49-9A ) with a 9A poly-adenylate 3’ tail bound 100-fold less tightly , with an apparent dissociation constant around 10 µM ( Figure 1B ) .", "The enzymatic specificity of Csx3 was tested against an RNA substrate oligonucleotide 49-9A .", "Csx3 cleaved this substrate in the presence of Mn2+ ions , removing nucleotides from the 3’ end ( Figure 2A ) , in keeping with previous observations ( Yan et al . , 2015 ) .", "However , Csx3 cleaved cA4 with a far higher rate ( Figure 2B ) .", "The single-turnover rate constants were determined as 0 . 0063 ± 0 . 0013 min−1 for RNA and 3 . 5 ± 0 . 16 min−1 for cA4 ( Figure 2C ) .", "The ~600 fold faster cleavage rate for cA4 compared to RNA strongly suggests that cA4 is the physiological substrate , and that RNA with a 3’ polyA tail represents a substrate analogue of the cyclic nucleotide , as observed previously for a Csx1/Csm6 family enzyme ( Han et al . , 2017a ) .", "Although the physiological growth temperature of A . fulgidus is around 70°C , we conducted these assays at 50°C to enable rate determination .", "The products of the cA4 cleavage reaction were identified by liquid-chromatography high-resolution mass spectrometry ( LC-HRMS ) as predominantly linear di-adenylate ( A2P ) , plus a small amount of A2P with a cyclic phosphate ( A2 >P ) ( Figure 2D ) .", "This suggests there are two active sites in the dimeric Csx3 structure , as seen for the ring nuclease Crn1 ( Athukoralage et al . , 2018 ) .", "Similar results were obtained for Csx3 from the mesophilic archaeon Methanosarcina mazei ( Figure 2—figure supplement 1 ) .", "To determine whether Csx3 displayed properties consistent with a function as a ring nuclease in vivo , we made use of the synthetic M . tuberculosis ( Mtb ) type III CRISPR system that we recently established in E . coli ( Grüschow et al . , 2019 ) .", "This system allows expression of the Mtb Csm complex , defined CRISPR RNA , and the processing enzyme Cas6 along with a cOA effector protein of choice .", "In this case , we used the previously characterised Csx1 ribonuclease from Thioalkalivibrio sulfidiphilus , which is activated by cA4 ( Grüschow et al . , 2019 ) .", "Csx1 provided significant immunity ( three logs ) against plasmid transformation when a targeting crRNA specific for the plasmid was provided ( Figure 3 ) .", "As a control , we added the phage ring nuclease AcrIII-1 from bacteriophage THSA-485A , which we previously showed could abolish immunity mediated by cA4 ( Athukoralage et al . , 2020a ) , and observed the expected loss of plasmid targeting , reflected in higher transformation efficiencies .", "When the phage ring nuclease gene was replaced by a gene encoding Csx3 from M . mazei ( chosen as the organism grows at close to 37°C , in common with E . coli ) we observed smaller but significant increases in plasmid transformation efficiency , with an effect that increased from 18 to 40 hr growth post-transformation .", "These observations were consistent with a partial deactivation of the Csx1 ribonuclease due to degradation of cA4 by the Csx3 enzyme .", "The effect is not as striking as when using a bona fide Acr protein , which is consistent with the prediction that Csx3 functions as part of the type III CRISPR defence in cells that express it – rather than an Acr .", "It should be noted that we expressed the Csx3 enzymes using a strong inducible promoter in these assays and we do not know what the relevant Csx3 concentrations are in virally-infected cells .", "Previously , the RNA cleavage activity of A . fulgidus Csx3 was shown to be dependent on the presence of manganese ions .", "The H60 residue was shown to be essential for RNA cleavage , as an H60A variant was inactive , while the H57A and H80A variants showed reduced activity ( Yan et al . , 2015 ) .", "As these residues are positioned on the opposite surface of the dimer from the RNA binding site ( Figure 4A ) , this led to the prediction that the RNA binding site and RNA cleavage site existed on opposing sides of the dimeric structure , which was plausible for an RNA substrate ( Yan et al . , 2015 ) .", "We tested the metal ion dependence of the cA4 cleavage activity and confirmed that the presence of Mn2+ or Co2+ ions was required for catalysis ( Figure 2—figure supplement 1 and Figure 4—figure supplement 1 ) .", "We recapitulated the H60A variant and found that , in agreement with the previous study , the variant lacked any detectable catalytic activity ( Figure 4B ) .", "Thus , it appears that H60 and the presumed metal binding site are essential for the ring nuclease activity of Csx3 despite being situated on the opposite face of the Csx3 dimer , over 20 Å away from the cA4 binding site .", "Examination of the multiple sequence alignment of Csx3 ( Figure 1—figure supplement 1 ) revealed the presence of a conserved aspartate residue , D69 , which is positioned adjacent ( ~4 Å ) to the bound RNA in the crystal structure ( Figure 4A ) .", "The importance of D69 was not explored in previous studies , so we mutated D69 to an alanine to test for a role in catalysis .", "The D69A variant was completely catalytically inactive as a ring nuclease , confirming the importance of D69 in the catalytic mechanism and suggesting that catalysis requires residues on both faces of the dimer ( henceforth denoted as the ‘D69 face’ and ‘H60 face’ ) .", "A possible explanation for this was that Csx3 has a shared active site that is formed when two dimers come together , forming a tetramer .", "A diagnostic test for this , as first proposed for the shared active site of aspartate transcarbamoylase ( Wente and Schachman , 1987 ) , is to mix inactive single variants and look for recovery of activity , as a fraction of intact active sites can be formed in the quaternary structure .", "Accordingly , we took the two inactive variants of Csx3 , H60A and D69A , and tested their ring nuclease activity when mixed together ( Figure 4B ) .", "Ring nuclease activity was recovered when the two inactive variants were combined .", "This result was strongly supportive of the hypothesis that the reaction mechanism involves two half-sites that combine to form a single active site that bridges two dimers of the enzyme .", "In parallel with the biochemical analysis , we crystallised the H60A variant of Csx3 and soaked the cA4 substrate into the crystals .", "The structure was solved using molecular replacement with data to 1 . 84 Å resolution ( Supplementary file 1 ) .", "The electron density clearly showed a molecule of cA4 bound between adjacent dimers of Csx3 ( Figure 5A ) .", "Notably , the Csx3 structure presented here crystallised in a different space group to those structures published previously ( Yan et al . , 2015 ) , which has allowed the arrangement of Csx3 dimers into a pseudo filament arrangement ( Figure 5—figure supplement 1 ) with a cA4 molecule sandwiched between the D69 and H60 faces of the protein .", "The rotation between adjacent dimers in the filament is 144 ° , coupled with a translation of 28 Å .", "The calculated buried surface between the complex arrangement of protein and ligand reflects the number of interactions formed .", "The interface of the two monomers forming the dimer is around 1020 Å2 .", "The interface area between the D69 face , which forms the majority of the interactions with cA4 , is around 940 Å2 ( 470 Å2 per monomer ) , and for the H60 face is around 340 Å2 ( 170 Å2 per monomer ) .", "Interestingly , although not annotated as a tetramer , there is a buried surface area of around 700 Å2 ( 350 Å2 per monomer ) between the two adjacent dimers .", "It is difficult to rationalise that the structure of Csx3 in complex with cA4 formed following overnight soaks of the compound with apo crystals , given the size of cA4 and rigidity of crystal packing .", "From comparison to the apo Csx3 structure , we hypothesize there are no significant conformational changes or large loop movements upon binding , but instead suggest subtle movements of active site and interacting residues to accommodate cA4 .", "There are surprisingly few residues that interact with cA4 in the active site of Csx3 , given the size of the ligand .", "The orientation of each monomer in the dimer means the interactions with cA4 are symmetrical .", "The majority of interactions are made by the D69 face .", "The cA4 hydrogen bonds with the main chain atoms of I22 , S44 , G45 , and I49 , and side chain atoms of S44 , R46 and R71 ( Figure 5—figure supplement 2 ) .", "Despite the number of main chain interactions , these residues are either absolutely ( G45 , I49 , and R71 ) or highly ( I22 , S44 and R46 ) conserved ( Figure 1—figure supplement 1 ) .", "The one interaction evident from the H60 face with cA4 is a strong ( 2 . 4–2 . 5 Å ) hydrogen bond between H80 and the 2’-OH of the ribose ring .", "H60 , H80 , and another histidine residue ( H57 ) nearby on the H60 face are all absolutely conserved in the Csx3 family ( Figure 5B , C; Figure 1—figure supplement 1 ) .", "The ribose of each AMP moiety of cA4 adopts a relaxed 2’-endo conformation , with the four adenine bases each filling a pocket .", "Given the Csx3 structure in complex with an RNA fragment ( Yan et al . , 2015 ) had two adenine , one uracil and one guanine bases in the same position as the four adenine bases described here , there is obviously some plasticity around these recognition sites .", "We anticipated that the comparison of the structure of Csx3 with cA4 with the apo structure published by Yan et al . , 2015 , in conjunction with activity assays on Csx3 variants , might provide clues as to the key residues in catalysis .", "Interestingly , R71 moves around 3 . 3 Å in order to form bidentate hydrogen bonds with an oxygen atom of a phosphate group , which we predict is adjacent to the phosphodiester bond that is cleaved ( Figure 5C and text below ) .", "This movement of R71 brings it within hydrogen bonding distance of D69 , which has been shown to be vital for activity ( Figure 5—figure supplement 3 ) .", "The position of H80 also differs between the two structures; the residue moves around 3 . 3 Å in order to hydrogen bond with the cA4 .", "However , there is the caveat that the packing arrangement differs in the apo Csx3 structure , where H80 does not interact with a ligand or the face of an adjacent dimer and thus has nothing to ‘anchor’ it in place .", "There are a number of histidine residues in or near the active site which could be involved in coordinating one or more Mn2+ ions ( Figure 5—figure supplement 4 ) .", "H60 and H57 are the most likely candidates; they are both absolutely conserved , and the position of the alanine residue in the H60A variant structure suggests this residue does not move significantly upon tetramer formation .", "The position of H57 is identical in the apo and cA4-bound structures .", "H60 and H57 are at the symmetry plane for the monomers constituting a dimer , meaning there are four histidine residues in close proximity .", "It is therefore feasible that one or both H60 and/or H57 residues coordinate one or more metal ions , and there is the possibility that just one metal ion bridges the two monomers .", "The structure of the Csx3:cA4 complex reveals further details of the catalytic mechanism employed by the enzyme .", "The other ring nucleases characterised to date are metal independent enzymes that are predicted to work by catalysing in-line nucleophilic attack by a 2’-hydroxyl group of the cA4 substrate on an adjacent phosphodiester bond .", "In this regard , the best candidate for cleavage is the P-O bond of the phosphate ( labelled * ) , as the angle formed between the 2’-OH , P and O moieties is 152° ( Figure 5C ) , which although lower to that seen for the ring nuclease AcrIII-1 ( Athukoralage et al . , 2020a ) , is still significantly higher than the angle ( 129° ) between the same atoms in both adjacent adenine moieties .", "The absolutely conserved residue R71 interacts with this phosphate group via a bidentate hydrogen bond and may participate in stabilisation of the transition state or oxyanion leaving group .", "The conserved catalytic residue D69 in turn makes polar contacts with R71 .", "These two residues may serve to position each other correctly ( and/or perturb their respective pKa’s ) to enhance catalysis .", "The conserved H80 residue forms a hydrogen bond with the cA4 .", "As it is one of only two residues from the H60 face that interacts with cA4 , H80 may play a ‘pinning’ role to ensure engagement of the H60 face in catalysis .", "Csx3 variants R71A and H80A both displayed highly reduced ring nuclease activity ( Figure 5—figure supplement 5 ) , consistent with important roles in cA4 binding and/or catalysis .", "The observations that Mn2+ is required for catalysis , and that the primary product of cA4 cleavage is A2P rather than A2>P ( Figure 2—figure supplement 1 and Figure 4—figure supplement 1 ) are suggestive ( but not diagnostic ) of a mechanism whereby a metal-activated hydroxyl ion initiates nucleophilic attack on the phosphodiester bond ( Yang , 2011 ) .", "Uncertainty arises from the observation that the metal independent ring nuclease AcrIII-1 also largely generates A2P , presumably due to the rapid hydrolysis of the cyclic phosphate following phosphodiester bond cleavage ( Athukoralage et al . , 2020a ) .", "The conserved histidine residues H57 and H60 are in appropriate positions to contribute to catalysis by coordination of the essential catalytic Mn2+ ion ( s ) .", "We see no evidence for the ion in our crystal structure , which may be due to the deletion of the H60 side chain .", "However , we note that the five Mn2+ ions modelled in the Csx3 structure by Yan et al . have ambiguous electron density , hampered by the lower resolution data ( Yan et al . , 2015 ) .", "In addition , the coordination distances for a Mn2+ ion with a nitrogen atom ( of the histidine residues ) are longer than expected ( Zheng et al . , 2017 ) .", "It remains possible that H80 participates in binding a second metal ion in addition to its observed interaction with cA4 .", "The crystal structure of the Csx3:cA4 complex neatly explains the observation of two distant active site regions , which come together in the complex .", "During the catalytic cycle , cA4 binding and dimer:dimer sandwiching would lead to rapid cA4 degradation that presumably in turn results in dissociation of the tetrameric active form .", "In support of this model , we observed that the inactive H60A variant of Csx3 had a milky , colloidal property on addition of cA4 , which we interpreted as being due to the formation of long Csx3 fibres bridged by multiple cA4 molecules ( Figure 5D ) .", "The wild-type protein did not show this behaviour , presumably because cA4 was rapidly cleaved .", "We confirmed the propensity of Csx3 to oligomerise upon cA4 addition by dynamic light scattering ( DLS ) measurements in buffer devoid of metal ions .", "Under these conditions , the addition of cA4 resulted in mixed oligomeric species with significantly increased particle size and molecular weight , whereas the molecular weight of particles formed in the absence of cA4 were consistent with homodimers of Csx3 ( Supplementary file 2 ) .", "The discovery that two dimers of Csx3 must associate to sandwich the cA4 substrate to effect cleavage opens the possibility of cooperative kinetic control in the Csx3 reaction cycle .", "To investigate this , we measured the initial reaction velocity of Csx3 ring nuclease activity under conditions of saturating cA4 at a range of enzyme concentrations .", "A plot of initial velocity against [E] revealed a sigmoidal relationship that could be fitted to the Hill equation with a Hill coefficient of 1 . 8 ( Figure 6 ) , consistent with obligate dimerization ( of Csx3 dimers ) in the reaction cycle .", "Practically , this cooperativity may be important to modulate the ring nuclease activity of Csx3 appropriately in the cell , particularly if the concentration of Csx3 changes in response to viral infection .", "The maximal multiple turnover rate constant observed under these conditions was 0 . 075 min−1 at 70°C , about 50-fold lower than the rate for the chemical step of catalysis measured under single turnover conditions at 50°C .", "Thus , the association of Csx3 dimers sandwiching cA4 , or the dissociation of the complex following product formation , limits the turnover number of the enzyme significantly ." ], [ "Here we re-examined the specificity of the Csx3 nuclease , which is found in association with type III CRISPR systems in bacteria and archaea .", "Csx3 was originally described as a manganese dependent RNA deadenylase ( Yan et al . , 2015 ) .", "The enzyme was co-crystallised with an RNA tetranucleotide , revealing a pseudo-symmetrical binding mode , and site directed mutagenesis revealed a crucial role for residues including H60 in catalysis – a residue that was situated on the opposite face of the protein from the RNA binding site ( Yan et al . , 2015 ) .", "This prompted the authors to make the reasonable speculation that RNA is bound on one side and wraps around the protein to be cleaved on the opposite side .", "Subsequently , Csx3 was identified as a divergent member of the CARF domain protein family ( Topuzlu and Lawrence , 2016 ) that bind cyclic oligoadenylates generated by the Cas10 subunit of type III CRISPR systems ( Niewoehner et al . , 2017; Kazlauskiene et al . , 2017 ) .", "Coupled with the lack of an obvious role for a RNA deadenylase in type III CRISPR defence , this led us to speculate on its function in vivo .", "While we confirmed the main experimental observations of the original study ( Yan et al . , 2015 ) , our analyses have revealed that Csx3 binds much more tightly to cA4 than to RNA , is considerably more active as a cA4-specific ring nuclease than an RNA deadenylase , and has properties consistent with a function as a ring nuclease in vivo .", "These data indicate that Csx3 functions as a cA4-specific ring nuclease in type III CRISPR systems .", "The default ‘Csx’ nomenclature was designed for proteins of unknown function lacking a clear association with a specific CRISPR subtype ( Haft et al . , 2005 ) .", "We therefore propose the family name Crn3 ( CRISPR-associated ring nuclease 3 ) for the Csx3 protein family .", "The Crn1 family of ring nucleases , based on the CARF domain fold , was originally identified in the Sulfolobales and related crenarchaea ( Athukoralage et al . , 2018 ) , where their function is thought to be to remove cA4 from the cell once a viral infection has been cleared .", "The anti-CRISPR ring nuclease AcrIII-1 appears in many archaeal virus and some bacteriophage genomes ( Athukoralage et al . , 2020a ) .", "Homologues of AcrIII-1 are also found associated with some bacterial type III CRISPR systems , and in this context have been named as CRISPR associated ring nuclease 2 ( Crn2 ) enzymes ( Athukoralage et al . , 2020a; Samolygo et al . , 2020 ) .", "Ring nuclease activity has also been identified associated with the CARF domains of some Csx1/Csm6 family ribonucleases , which are therefore bifunctional , self-limiting CRISPR ancillary defence enzymes ( Jia et al . , 2019; Athukoralage et al . , 2019; Garcia-Doval et al . , 2020 ) .", "Overall , the emerging picture is becoming clearer for the specific ring nucleases as they are identified in different archaeal and bacterial genomes ( Figure 7 ) .", "It appears to be an emerging paradigm that cells with a type III CRISPR defence require a mechanism to remove the cOA signal , either once viral infection is cleared , or when the system ‘fires’ inappropriately due to self-targeting ( Athukoralage et al . , 2020b ) .", "A . fulgidus Crn3/Csx3 is a much faster enzyme than Crn1 .", "The single turnover reaction rate for cA4 cleavage of 3 . 6 min−1 is closer to that of the AcrIII-1 family , which utilises a distinct active site architecture , employing a conserved histidine residue as a general acid to stabilise the oxyanion leaving group ( Athukoralage et al . , 2020a ) .", "This raises an important question: why does Crn3/Csx3 degrade cA4 so quickly when it plays a role in cellular defence rather than viral offense ?", "Clearly , removing a crucial signal of viral infection that mobilises cellular defences is not something that should be undertaken precipitously .", "A key observation is that Crn3/Csx3 functions via a highly unusual cooperative mechanism where two enzyme dimers associate to sandwich a cA4 substrate molecule .", "The active site is thus composed of two half-sites that are present on opposite faces of each dimeric moiety , with enzyme tetramers formed transiently to complete the catalytic cycle .", "There are many examples of allosteric control of enzyme quaternary structure and activity ( Selwood and Jaffe , 2012 ) and of active sites shared across subunit interfaces .", "However , the substrate-induced , non-allosteric , dimerization of Csx3 appears unprecedented in the literature – a partial exception being the example of the Arginine Finger of AAA+ ATPases ( Nagy et al . , 2016 ) , where interdomain interactions with ATP can influence quaternary structure ( Zhao et al . , 2016 ) .", "This results in cooperative kinetics where low concentrations of Crn3/Csx3 provide very low levels of ring nuclease activity that rapidly increases with increasing enzyme concentrations .", "Furthermore , the rate of cA4 turnover is limited by the formation of protein:cA4 complexes or dissociation of the products , rather than the catalytic step , which is significantly faster .", "Together , these factors may provide a means to control ring nuclease activity in an appropriate manner thus ensuring that cA4 activation of ancillary ribonucleases is allowed to proceed and provide immunity .", "Thus , alterations in the gene expression levels of Csx3 would have a large effect on the overall rate of catalysis .", "Finally , the observed specificity of Crn3/Csx3 for cA4 implicates the corresponding type III CRISPR systems as functioning via a cA4 second messenger .", "This conforms to the paradigm established from studies of the Crn1 and Crn2/AcrIII-1 family as well as analysis of the specificity of the Csx1/Csm6 ribonucleases , which are most often activated by cA4 ( Grüschow et al . , 2019 ) .", "Although this conclusion may be influenced by a sampling bias , it appears that cA4 is the default cyclic nucleotide employed to signal infection and activate defences in type III CRISPR systems .", "It is possible that the pressure applied by cA4-specific anti-CRISPRs has resulted in the utilization of cA3 and cA6 second messengers in some bacterial defence systems .", "Given the pace of discovery in this area , new cellular and viral enzymes implicated in cyclic nucleotide signalling are anticipated ." ], [ "For cloning , synthetic genes ( g-blocks ) encoding A . fulgidus or M . mazei Csx3 ( Figure 1—figure supplement 1 ) , codon optimised for expression in Escherichia coli , were purchased from Integrated DNA Technologies ( IDT ) , Coralville , USA , and cloned into the pEhisV5spacerTev vector between the NcoI and BamHI restriction sites ( Rouillon et al . , 2019 ) .", "Competent DH5α ( E . coli ) cells were transformed with the construct and sequence integrity was confirmed by sequencing ( Eurofins Genomics ) .", "The plasmid was transformed into E . coli C43 ( DE3 ) cells for protein expression .", "The following constructs were used in plasmid immunity assays: pCsm1-5_ΔCsm6 ( M . tuberculosis csm1-5 under T7 and lac promoter control ) and pCRISPR_TetR ( CRISPR array with tetracycline resistance gene targeting spacers and M . tuberculosis cas6 under T7 promoter control ) which have been described previously ( Grüschow et al . , 2019 ) ; pRAT-Duet constructs containing T . sulfidiphilus csx1 under arabinose-inducible promoter control with and without viral AcrIII-1 which have been previously described ( Athukoralage et al . , 2020b ) .", "M . mazei csx3 was cloned into the pRAT-Duet_tsuCsx1 vector using the NdeI and XhoI restriction sites .", "Constructs were verified by sequencing .", "For expression of A . fulgidus or M . mazei Csx3 , the standard protocol described in detail previously was followed ( Rouillon et al . , 2019 ) .", "Briefly , a culture was grown in 2L Luria-Broth at 37°C to an OD600 of 0 . 8 AU with shaking at 180 rpm .", "Protein expression was induced with 0 . 4 mM isopropyl β-D-1-thiogalactopyranoside and cells were grown at 25°C overnight before harvesting by centrifugation .", "For protein purification the cell pellet was resuspended in lysis buffer containing 50 mM Tris-HCl 7 . 5 , 0 . 5 M NaCl , 10 mM imidazole and 10% glycerol supplemented with EDTA-free protease inhibitor tablets ( Roche; 1 tablet per 100 ml buffer ) and lysozyme ( 1 mg/ml ) .", "Cells were lysed by sonication and the lysate was ultracentrifuged at 40 , 000 rpm ( 70 Ti rotor ) at 4°C for 35 min .", "The lysate was loaded onto a 5 ml HisTrap FF Crude column ( GE Healthcare ) equilibrated with wash buffer containing 50 mM Tris-HCl pH 7 . 5 , 0 . 5 M NaCl , 30 mM imidazole and 10% glycerol .", "Unbound protein was eluted with 20 column volumes ( CV ) of wash buffer prior to elution of His-tagged protein using a step gradient of imidazole ( holding at 10% for 3 CV , and 50% for 3 CV ) of elution buffer containing 50 mM Tris-HCl pH 7 . 5 , 0 . 5 M NaCl , 0 . 5 M imidazole and 10% glycerol .", "The His-tag was removed by incubating concentrated protein overnight with Tobacco Etch Virus ( TEV ) protease ( 1 mg per 10 mg protein ) while dialysing in buffer containing 50 mM Tris-HCl pH 7 . 5 , 0 . 5 M NaCl , 30 mM imidazole and 10% glycerol at room temperature .", "Cleaved protein was passed through a 5 ml HisTrapFF column and further purified by size-exclusion chromatography ( S200 16/60 column; GE Healthcare ) in buffer containing 20 mM Tris-HCl pH 7 . 5 , 0 . 125 M NaCl .", "After SDS-PAGE , pure protein was pooled , concentrated and stored at −80°C .", "H60A , D69A , R71 and H80 variants of A . fulgidus Csx3 ( Figure 1—figure supplement 1 ) were generated using the QuikChange Site-Directed Mutagenesis kit as per manufacturer’s instructions ( Agilent Technologies ) and purified as for the wild-type protein .", "For RNA cleavage , Csx3 ( 8 μM protein dimer ) was incubated with 50 nM radiolabelled RNA oligonucleotide 49-9A in buffer containing 20 mM Tris-HCl pH 7 . 5 , 150 mM NaCl , 1 mM DTT , three units SUPERase•In Inhibitor and supplemented with 2 mM MnCl2 at 50°C .", "A control reaction incubating RNA in buffer without protein was also carried out .", "Reactions were quenched by adding EDTA ( 17 mM final concentration ) and incubated with 20 μg Proteinase K ( Invitrogen ) for 30 min at 37°C .", "Subsequently , the reactions were deproteinised by phenol-chloroform extraction and 8 μl reaction product was extracted into 5 μl 100% formamide xylene-cyanol dye .", "All experiments were carried out in triplicate and RNA cleavage was visualised by phosphor imaging following denaturing polyacrylamide gel electrophoresis ( PAGE ) as previously described ( Rouillon et al . , 2019 ) .", "Oligo 49-9A: 5’-AGGGUACAGUUUGGGUAUUAGCCGUUCUGGUCCUUAUACGAAAAAAAAA .", "Cyclic oligoadenylate ( cOA ) was generated using S . solfataricus Csm complex as detailed in Rouillon et al . , 2019 .", "Single turnover kinetics experiments for A . fulgidus or M . mazei Csx3 and variants ( 8 μM protein dimer ) were performed by incubating protein with radiolabelled SsoCsm and cOA ( ~200 nM cA4 ) in buffer containing 20 mM Tris-HCl pH 7 . 5 , 150 mM NaCl , 2 mM MnCl2 , 1 mM DTT and three units SUPERase•In Inhibitor at 50°C .", "At desired time points , 10 μl aliquot was removed from the reaction and quenched by adding to phenol-chloroform and vortexing .", "Products were further isolated by chloroform extraction for thin layer chromatography ( TLC ) .", "A control reaction incubating cOA in buffer without protein to the endpoint of each experiment was also carried out .", "All experiments were carried out in triplicate and cA4 degradation was visualised by phosphor imaging .", "For experiments examining metal dependence , Csx3 was incubated with 1 mM EDTA in buffer as above before adding cOA and supplementing with 3 mM MgCl2 , CaCl2 or CoCl2 .", "Pilot experiments showed that Csx3 was specific for cA4 and did not degrade cA6 .", "Multiple turnover kinetics of Csx3 was carried out by varying enzyme concentration ( 240 , 320 , 640 , 800 , 960 , 1120 , 1280 , 1920 , 2560 , 3840 , 5120 nM dimer ) and incubating with a mix of unlabelled and radiolabelled cA4 ( total concentration 128 . 5 μM ) in buffer containing 20 mM Tris-HCl pH 7 . 5 , 150 mM NaCl , 2 mM MnCl2 , 1 mM DTT and three units SUPERase•In Inhibitor at 70°C .", "Reaction products were visualised by phosphor imaging following TLC .", "All experiments were done in triplicate .", "Initial rates were calculated and adjusted for labelled:unlabelled cA4 concentration before fitting to the Hill equation on Kaleidagraph ( Synergy Software ) .", "For activity rescue of Csx3 variants , D69A ( 4 μM dimer ) variant and H60A variant ( 4 μM dimer ) were mixed together and incubated with cA4 ( ~200 nM ) in a reaction supplemented with 2 mM MnCl2 at 70°C .", "Reactions were quenched at 10 , 60 and 600 s by adding phenol-chloroform and vortexing .", "All experiments were done in triplicate .", "TLC was carried out as previously described ( Han et al . , 2017b ) , by spotting 1 μl of radiolabelled product 1 cm from the bottom of a 20 × 20 cm silica gel TLC plate ( Supelco Sigma-Aldrich ) .", "TLC was carried out at 35°C in a humidified chamber with running buffer composed of 30% H2O , 70% ethanol and 0 . 2 M ammonium bicarbonate , pH 9 . 2 .", "Sample migration was visualised by phosphor imaging the TLC plate .", "For kinetic analysis , cA4 cleavage was quantified using the Bio-Formats plugin ( Linkert et al . , 2010 ) of ImageJ as distributed in the Fiji package ( Schindelin et al . , 2012 ) .", "The data were fitted to a single exponential curve ( y = m1 + m2* ( 1 - exp ( -m3*x ) ) ) using Kaleidagraph , as described previously ( Sternberg et al . , 2012 ) .", "Csx3 and variants ( 0 . 01 , 0 . 1 , 0 . 5 , 1 , 10 and 20 μM dimer ) were incubated with 20 nM radiolabelled cA4 in binding buffer containing 20 mM Tris-HCl pH 7 . 5 , 150 mM NaCl and 1 mM EDTA supplemented with UltraPure BSA ( Thermofisher ) for 10 min at 25°C .", "To examine RNA binding by Csx3 , 50 nM 5’-end radiolabelled 3’ poly-adenylate tailed RNA oligonucleotide 49-9A was incubated with Csx3 as above .", "Subsequently , a reaction volume equivalent of 20% glycerol was added to each reaction and native ( 15% acrylamide , 1X TBE ) gel electrophoresis at 30°C and 250V was performed .", "Gels were phosphor imaged overnight at −80°C .", "All experiments were done in triplicate .", "Crystallisation conditions were tested with JCSG and PACT 96 well commercial screens ( Jena Biosciences ) with Csx3 H60A at a concentration of 13 . 5 mg/ml .", "Following optimisation , crystals were obtained from 25% ( v/v ) Jeffamine M-600 and 100 mM HEPES pH 7 . 5 using hanging drops in a 24 well plate .", "3 μl drops in a 2:1 or 1:1 protein:mother liquor ratio were added to a silanized cover slip over 400 μl mother liquor and sealed with high-vacuum grease ( DOW Corning , USA ) and left to grow at room temperature .", "Prior to addition of the cA4 ligand , crystals were harvested into a fresh 2 μl drop of mother liquor and 1 μl of 16 mM cA4 solution was added and left to soak for 12 hr .", "Crystals were harvested and cryoprotected with the addition of 2 μl 50% ( v/v ) Jeffamine M-600 in 0 . 5 μl increments , mounted on loops and vitrified in liquid nitrogen .", "Data on Csx3 H60A crystals soaked with cA4 were collected at Diamond Light Source ( DLS ) on beamline I04 at a wavelength of 0 . 9795 Å to 1 . 84 Å resolution .", "Diffraction images were automatically processed through the Xia2 pipeline ( Winter , 2010 ) using DIALS ( Gildea et al . , 2014 ) and AIMLESS ( Evans , 2006 ) .", "After checking the likely cell content by the Matthews’ coefficient , MOLREP ( Vagin and Teplyakov , 1997 ) was used to solve the structure using molecular replacement with the Csx3 structure ( PDB 3WZI ) ( Yan et al . , 2015 ) as the search model with ligand and water molecules removed .", "REFMAC5 ( Murshudov et al . , 2011 ) and Coot ( Emsley and Cowtan , 2004 ) were used for automated and manual refinement respectively , which included addition of the ligand and water molecules .", "cA4 was drawn using Chemdraw ( Perkin Elmer ) and restraints generated in JLigand ( Lebedev et al . , 2012 ) .", "The model was corrected and validated using tools in PDB-redo ( Joosten et al . , 2014 ) and Molprobity ( Chen et al . , 2010 ) .", "The Molprobity score is 1 . 04; centile 100 .", "Ramachandran statistics are 97 . 34% allowed , 0% disallowed .", "Data processing and refinement statistics are shown in Supplementary file 1 .", "The coordinates and data have been deposited in the Protein Data Bank with accession code 6YUD .", "Dynamic light scattering experiments were carried out in a 20 µl quartz cuvette using a Zetasizer Nano S90 instrument ( Malvern ) .", "80 µM AfCsx3 was either measured alone or when mixed with an equimolar concentration of cA4 in phosphate buffered saline , pH 7 . 5 at 25°C .", "Three technical replicates were carried out and by default triplicate measurements were made to produce an average for each technical replicate .", "For visual inspection of the effect of adding cA4 to AfCsx3 and variants , 80 µM AfCsx3 was added to equimolar cA4 in PBS supplemented with 2 mM MnCl2 in a 100 µl reaction volume and heated to either 25°C or 70°C for 10 min before photographing using a 12-megapixel ƒ/1 . 8-aperture camera .", "Samples were generated by incubating Csx3 ( 10 μM dimer ) with 100 μM synthetic cA4 ( BIOLOG Life Science Institute , Bremen ) in buffer containing 20 mM Tris-HCl pH 7 . 5 , 150 mM NaCl , 1 mM DTT and 2 mM MnCl2 for 10 min at 50°C .", "Reactions were quenched by adding EDTA to a final concentration of 25 mM and acidified with trifluoroacetic acid .", "The acidified sample was bound to a C18 cartridge ( Harvard Apparatus ) , and salts and buffer were washed away with 0 . 1% trifluoroacetic acid , 2% acetonitrile .", "Adenylates were eluted from the cartridge with 20 mM ammonium bicarbonate , 50% acetonitrile leaving most protein bound to the resin .", "Liquid chromatography-high resolution mass spectrometry ( LC-HRMS ) analysis was performed on a Thermo Scientific Velos Pro instrument equipped with HESI source and Dionex UltiMate 3000 chromatography system as previously described ( Grüschow et al . , 2019 ) .", "Data were analysed using Xcalibur ( Thermo Scientific ) .", "These assays were performed largely as described previously ( Athukoralage et al . , 2020a ) .", "Cells containing Mycobacterium tuberculosis ( Mtb ) Csm1-5 , Cas6 and a CRISPR array targeting the tetracycline resistance gene of pRAT-Duet were transformed with the target plasmid containing genes encoding the cA4-activated ancillary nuclease T . sulfidiphilus ( Tsu ) Csx1 and a ring nuclease ( M . mazei Csx3 or the anti-CRISPR ring nuclease AcrIII-1 from Thermoanaerobacterium phage THSA-485 ) .", "Transformants were allowed to recover on selective LB plates in the presence of 0 . 2% lactose and 0 . 02% arabinose ( the former for induction of the Cas genes and the ring nuclease , the latter induces the Csx1 plus ring nuclease ) .", "The experiment was run with increasing arabinose concentrations ( 0 , 0 . 002 , 0 . 02 , 0 . 2 % w/v ) ; there was no difference between 0% and 0 . 002% , and a slight difference between 0 . 02% and 0 . 2% arabinose .", "We did not test directly for toxicity of the Csx3 enzyme in E . coli , but judge this to be unlikely due to its specificity for cA4 degradation .", "Four technical replicates on two biological replicates were carried out , N = 8 .", "Unpaired Welch t test was performed using GraphPad Prism version 8 . 3 . 1 , GraphPad Software , La Jolla California USA , www . graphpad . com ." ] ]

[ "Type III CRISPR systems detect foreign RNA and activate the cyclase domain of the Cas10 subunit , generating cyclic oligoadenylate ( cOA ) molecules that act as a second messenger to signal infection , activating nucleases that degrade the nucleic acid of both invader and host .", "This can lead to dormancy or cell death; to avoid this , cells need a way to remove cOA from the cell once a viral infection has been defeated .", "Enzymes specialised for this task are known as ring nucleases , but are limited in their distribution .", "Here , we demonstrate that the widespread CRISPR associated protein Csx3 , previously described as an RNA deadenylase , is a ring nuclease that rapidly degrades cyclic tetra-adenylate ( cA4 ) .", "The enzyme has an unusual cooperative reaction mechanism involving an active site that spans the interface between two dimers , sandwiching the cA4 substrate .", "We propose the name Crn3 ( CRISPR associated ring nuclease 3 ) for the Csx3 family ." ]

[ "Bacteria protect themselves from infections using a system called CRISPR-Cas , which helps the cells to detect and destroy invading threats .", "The type III CRISPR-Cas system , in particular , is one of the most widespread and efficient at killing viruses .", "When a bacterium is infected , the CRISPR-Cas system takes a fragment of the genetic material of the virus , and copies it into a molecule .", "These molecular ‘police mugshots’ are then loaded into a complex of Cas proteins that patrol the cell , looking for a match and destroying any virus that can be identified .", "Some Cas proteins also produce alarm signals , called cyclic oligoadenylates ( cOAs ) , which can trigger additional defences .", "However , this process can damage the genetic material of the bacterium , harming or even killing the cell .", "Enzymes known as ring nucleases can promptly degrade cOAs and turn off this defence system before it causes harm .", "However , ring nucleases have only been found in a few species to date; how most bacteria deal with cOA toxicity has remained unknown .", "Here , Athukoralage et al . set out to determine whether a widespread enzyme known as Csx3 , which is often associated with type III CRISPR-Cas systems , could be an alternative off switch for cOA triggered defences .", "Initial ‘test tube’ experiments with purified Csx3 proteins confirmed that the enzyme could indeed break down cOAs .", "A careful dissection of Csx3’s molecular structure , using biochemical and biophysical techniques , revealed that it worked by ‘sandwiching’ a cOA molecule between two co-operating portions of the enzyme .", "As a final test , Csx3 was introduced into strains of bacteria genetically engineered to have a fully functional Type III CRISPR-Cas system .", "In these cells , Csx3 successfully turned off the Type III immune response .", "These results reveal a new way that bacteria avoid the toxic side effects of their own immune defences .", "Ultimately , this could pave the way for the development of anti-bacterial drugs that work by blocking Csx3 or similar proteins ." ]

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

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

[ [ "Aurora B , a component of the chromosomal passenger complex ( CPC ) , is an essential kinase that is highly enriched at different intracellular locations from which it regulates cell division: it localizes initially at the inner centromere and subsequently at the anaphase spindle midzone ( Carmena et al . , 2012 ) .", "Accumulating evidence indicates that Aurora B is capable of phosphorylating substrates that are located at a significant distance from its major binding sites .", "In anaphase , a long-range phosphorylation gradient is established around the spindle midzone ( Fuller et al . , 2008; Tan and Kapoor , 2011 ) , but extending well beyond major sites of kinase localization ( Figure 1A ) .", "This phosphorylation gradient controls the stability and length of the central spindle ( Ferreira et al . , 2013; Uehara et al . , 2013 ) , chromosome decondensation and nuclear envelope reassembly ( Afonso et al . , 2014 ) .", "Similar distance-dependent phosphorylation is observed prior to anaphase onset , but at this stage Aurora B localizes to chromatin with highest concentration at the inner centromere , where CPC binding sites are enriched .", "During metaphase the primary targets for Aurora B , such as the microtubule-binding protein Hec1/Ndc80 , are located hundreds of nanometers away at the outer kinetochore .", "As in anaphase , phosphorylation is lower on substrates positioned farther from Aurora B binding sites , indicating existence of a gradient of Aurora B activity ( Keating et al . , 2009; Liu et al . , 2009; Welburn et al . , 2010; DeLuca et al . , 2011; Suzuki et al . , 2014 ) .", "Interestingly , changes of position of as little as 30–50 nm are associated with different levels of phosphorylation of both endogenous and exogenous Aurora B substrates at kinetochores ( Welburn et al . , 2010; Suzuki et al . , 2014 ) , indicating that the spatial regulation of Aurora B activity is very precise . 10 . 7554/eLife . 10644 . 003Figure 1 . Spatial phosphorylation patterns in mitotic HeLa cells .", "( A ) A HeLa cell expressing the chromatin-targeted Aurora B sensor and Aurora B-mCherry was imaged in anaphase , 10 min after addition of an Mps1 inhibitor , reversin , to increase occurrence of lagging chromosomes .", "The FRET ratio image shows the YFP/CFP emission ratio , color-coded as indicated .", "Scale bar is 5 µm .", "The plot shows normalized sensor phosphorylation ( left axis ) calculated from the FRET ratio data ( see Materials and methods ) and Aurora B localization signal ( right axis ) along the white lines which were drawn along the spindle axis in images on the left .", "( B ) HeLa cells expressing CENP-B-FKBP , mCherry-INbox-FRB and miRNAs to deplete endogenous FKBP and INCENP , and the chromatin-targeted Aurora B sensor .", "Cells were treated with the kinesin-5 inhibitor STLC to generate monopolar spindles , then imaged live during rapamycin addition to induce INbox and Aurora B recruitment to centromeres .", "Images show INbox recruitment ( bottom panels ) and the YFP/CFP emission ratio ( top panels ) for one cell .", "Graph shows the FRET emission ratio averaged over chromatin in multiple cells ( n≥10 ) treated at 3 min ( arrow ) with or without rapamycin .", "FRET ratio = 1 . 3 ( horizontal dotted line ) represents maximal Aurora B activity in cells with no INCENP depletion .", "The experiment was repeated three times with similar results . DOI: http://dx . doi . org/10 . 7554/eLife . 10644 . 00310 . 7554/eLife . 10644 . 004Figure 1—figure supplement 1 . Phosphorylation of the chromatin-targeted Aurora B sensor after INbox recruitment to centromeres .", "( A ) Schematic of the experiment in which the Aurora B-INbox complex labeled with mCherry ( mCH ) is recruited to centromeres by addition of rapamycin; see Materials and methods for details .", "( B ) Cells were treated as in Figure 1B , but arrested with nocodozole instead of STLC; scale bar is 5 µm .", "Graph on the right shows FRET emission ratio averaged over chromatin in n≥11 cells , N = 3 independent experiments .", "FRET ratio = 1 . 24 ( horizontal dotted line ) represents maximal Aurora B activity in cells with no INCENP depletion . DOI: http://dx . doi . org/10 . 7554/eLife . 10644 . 004 One model to explain such well-controlled long-range spatial activity is by a specialized pool of Aurora B localized in close proximity to its targets ( Krenn and Musacchio , 2015 ) .", "At the outer kinetochore , for example , the observed gradient of substrate phosphorylation could correspond to the outermost region of the localization gradient of chromatin-bound kinase ( Liu et al . , 2009 ) , or reflect the ability of Aurora B to reach these substrates by an elongated INCENP tether , the CPC component that directly binds Aurora B and is important for its mitotic functions ( Samejima et al . , 2015 ) .", "In this view , the less abundant but proximally located Aurora B pool plays a more physiologically important role than the distant centromeric pool .", "Support for the kinetochore pool model comes from experiments in budding yeast , which show that the centromere localized pool of Aurora B ( Ipl1 ) can be removed without major consequences for mitotic progression ( Campbell and Desai , 2013 ) .", "In several other systems , however , disrupting CPC targeting to centromeres leads to strong mitotic defects ( Vader et al . , 2006; Tsukahara et al . , 2010; Wang et al . , 2010; 2012; Yamagishi et al . , 2010 ) , suggesting that the centromere-localized pool is essential for normal cell division .", "An alternative model to explain how Aurora B activity is controlled at distances away from its most abundant localization sites is that this pattern depends on a biochemical crosstalk between the bound Aurora B and its cytosolic pool , which recent quantitative measurements estimate as ~25% of total Aurora B ( Mahen et al . , 2014 ) .", "Cytosolic gradients of another mitotic regulator , RanGTP , play important roles in regulating spindle assembly ( O’Connell and Khodjakov , 2007; Kalab and Heald , 2008 ) , and a similar mechanism could contribute to long-range Aurora B activity .", "In this reaction-diffusion model , activation of Aurora B takes place at sites with high kinase concentration , such as the inner centromere or anaphase spindle midzone ( Lampson and Cheeseman , 2011 ) .", "These sites exchange quickly with a cytosolic pool ( Fernández-Miranda et al . , 2010 ) , so they could serve as a source of active kinase , which has been proposed to spread to distant targets via diffusion ( Fuller et al . , 2008; Wang et al . , 2011 ) .", "However , it is not clear whether a gradient based only on the diffusion of soluble activated kinase from the inner centromere could account for changes in Aurora B substrate phosphorylation within the length scale of the kinetochore ( Krenn and Musacchio , 2015 ) .", "In contrast , bistable reaction-diffusion systems can in principle exhibit complex spatial patterns and support sharp boundaries of system components ( Kapral and Showalter , 1995; Lobanova and Ataullakhanov , 2003; Liehr , 2013 ) .", "Bistable homogeneous systems ( i . e . , with mixing ) can switch between the alternative states , characterized by high and low activity , with no intermediate states .", "Furthermore , unlike in regular trigger systems , in bistable systems the high and low states can co-exist , leading to hysteresis , when the output of the system depends on its prior history ( Martinov et al . , 2000; Angeli et al . , 2004; Tsyganov et al . , 2012; Noori , 2013 ) .", "Published results indicate that Aurora B kinase could in principle engage in complex non-linear behaviors .", "Most importantly , Aurora B can activate itself via phosphorylation of its activation loop and of a conserved TSS motif in the C-terminus of INCENP ( Bishop and Schumacher , 2002; Honda et al . , 2003; Yasui et al . , 2004; Sessa et al . , 2005; Kelly et al . , 2007; Xu et al . , 2010 ) .", "Conversely , phosphatase can inactivate the kinase by dephosphorylating sites on Aurora B and INCENP ( Sessa et al . , 2005; Kelly et al . , 2007; Rosasco-Nitcher et al . , 2008 ) , which could potentially help to shape the spatial gradient of Aurora B activity .", "Whether these reactions can lead to bistability in a coupled Aurora B-phosphatase system has not been investigated .", "Here , we examine the mechanisms that control Aurora B activity using cellular and simplified in vitro systems and mathematical modeling .", "First , we designed a novel molecular system to control Aurora B localization in cells , to directly test the importance of the centromeric pool of Aurora B in long-range activity .", "Second , we used purified components to reconstitute a simplified coupled Aurora B kinase-phosphatase system in vitro and showed that it exhibits bistability and hysteresis in the physiological range of Aurora B concentration .", "Because the complex , non-linear dynamics of reaction-diffusion systems and their spatial behavior are not intuitive , we constructed quantitative models to assist analysis of homogeneous biochemical reactions and formation of phosphorylation patterns in cells .", "We then developed experimental methods to analyze bistability and hysteresis of Aurora B-dependent phosphorylation in live mitotic cells , linking our biochemical findings with Aurora B regulation in cells .", "With these multiple approaches we provide strong evidence for a model in which spatiotemporal regulation of Aurora B is governed by a bistable reaction-diffusion mechanism ." ], [ "Because experiments in budding yeast have raised questions about whether concentrating Aurora B at centromeres is necessary for its mitotic function ( Campbell and Desai , 2013 ) , we designed an experiment to measure phosphorylation in live human cells while manipulating Aurora B localization with temporal control .", "We made a cell line that inducibly knocks down endogenous INCENP , while expressing an INbox construct that can bind and activate Aurora B ( Sessa et al . , 2005 ) but does not interact with other CPC components .", "The Aurora B–INbox complex is sufficient for enzymatic activity but does not localize to any particular intracellular structure because it does not form the full CPC .", "To control localization , we used rapamycin-based dimerization ( Putyrski and Schultz , 2012 ) , with FRB fused to INbox and FKBP fused to the centromere protein CENP-B ( Figure 1—figure supplement 1A ) .", "FKBP and FRB are domains that dimerize in the presence of rapamycin .", "This system allows us to measure immediate effects in live cells within minutes of concentrating Aurora B at centromeres .", "To monitor changes in Aurora B kinase activity at a distance from sites of localization at centromeres , we used a FRET-based biosensor targeted to chromatin by fusion to histone H2B ( Fuller et al . , 2008 ) .", "When endogenous INCENP is replaced with INbox , which is freely diffusing in the cytosol , phosphorylation is uniformly low , indicating that the cytosolic kinase pool on its own is incapable of maintaining high kinase activity along chromosome arms .", "Addition of rapamycin led to INbox recruitment to centromeres within minutes , accompanied by sensor phosphorylation; importantly the signal was visible all over the chromatin ( Figure 1B ) .", "For these experiments cells were arrested in mitosis with a kinesin-5 inhibitor , so that chromosomes were positioned radially around a monopolar spindle with centromeres oriented toward the center ( Mayer et al . , 1999 ) .", "With this arrangement of chromosomes , a transient phosphorylation gradient was evident extending from centromeres , similar to previous experiments in which Aurora B activity was manipulated by global inhibition ( Wang et al . , 2011 ) .", "Similar results were observed for cells arrested with nocodazole ( Figure 1—figure supplement 1B ) .", "Thus , concentrating Aurora B at centromeres of a mammalian cell is necessary and sufficient to regulate kinase activity at distal cellular locations , warranting further investigation of the kinetic mechanisms of Aurora B autoactivation .", "Highly concentrated centromeric kinase may become a source of active kinase for establishing spatial patterns if Aurora B can robustly activate itself in trans , i . e . intermolecularly ( Sessa et al . , 2005; Kelly et al . , 2007; Lampson and Cheeseman , 2011 ) .", "To determine the kinetic constants for Aurora B autoactivation , we measured phosphorylation in vitro in real time using purified recombinant Aurora B kinase with an INbox fragment , which is sufficient for kinase autoactivation ( Sessa et al . , 2005; Rosasco-Nitcher et al . , 2008 ) ( see Materials and methods and Figure 2—figure supplement 1 ) .", "With purified kinase , the INCENP TSS motif , an established autophosphorylation site associated with kinase activation ( Bishop and Schumacher , 2002; Honda et al . , 2003; Sessa et al . , 2005 ) , was phosphorylated , as determined by immunoblotting with a phospho-specific antibody ( Salimian et al . , 2011; Figure 2—figure supplement 1D ) .", "This phosphorylated kinase was highly active , as shown using a chemosensor composed of a peptide containing an Aurora kinase substrate consensus site conjugated to a sulfonamido-oxine ( Sox ) fluorescent probe ( Figure 2—figure supplement 2 ) ( Gonzáles-Vera et al . , 2009 ) .", "Phosphorylation-induced increase in fluorescence of the chemosensor was followed in real time with a spectrofluorimeter , and the Michaelis–Menten , Lineweaver–Burk and Hanes–Woolf plots were analyzed ( see Materials and methods ) , giving KM = 320 μM and kcat = 19 s-1 , similar to a previous report for Aurora A kinase ( Gonzáles-Vera et al . , 2009 ) .", "To examine activity of Aurora B in the dephosphorylated state , we incubated the kinase with λ phage phosphatase , which has previously been reported to dephosphorylate INCENP ( Rosasco-Nitcher et al . , 2008 ) , and observed loss of INCENP phosphorylation ( Figure 2—figure supplement 1D ) .", "Phosphonoacetic acid was then added to inhibit the phosphatase ( Reiter et al . , 2002 ) and chemosensor phosphorylation was measured .", "The dephosphorylated Aurora B kinase was two orders of magnitude less active than the phosphorylated Aurora B , consistent with previous studies ( Eyers et al . , 2005; Sessa et al . , 2005 ) , so we refer to this kinase state as partially active .", "Next , we sought to determine the kinetic parameters of Aurora B autoactivation .", "At 10–30 nM of partially active kinase , chemosensor phosphorylation was barely detected .", "This finding is consistent with our results using INbox replacement in cells with no rapamycin , since this low concentration range was reported for cytosolic Aurora B ( Mahen et al . , 2014 ) .", "At 0 . 16–1 . 5 µM kinase , chemosensor phosphorylation increased nonlinearly with time , indicating autoactivation ( Figure 2A , Figure 2—figure supplement 2G ) .", "Previous studies have reported that this autoactivation takes place in trans ( Sessa et al . , 2005; Rosasco-Nitcher et al . , 2008 ) ( Figure 2B ) , predicting that the coefficient for this increase vs . kinase concentration is close to 2 when plotted on a logarithmic scale .", "The measured slope in our experiments with low kinase concentrations was 1 . 23 ± 0 . 02 ( Figure 2C ) , implying that the partially active Aurora B can activate itself in cis , i . e . intramolecularly ( Figure 2B ) . 10 . 7554/eLife . 10644 . 005Figure 2 . Aurora B kinase autoactivation in vitro .", "( A ) Phosphorylation of 20 µM chemosensor by the indicated concentrations of partially active Aurora B kinase .", "Data are averages of N = 2 experiments for each kinase concentration; error bars are SEMs .", "Black lines are theoretical fittings with the reaction scheme in panel E . ( B ) Molecular scheme for Aurora B autoactivation in trans or in cis .", "A and A* denote partially active ( dephosphorylated ) and active kinase; S and P indicate substrate and product ( unphosphorylated and phosphorylated chemosensors , respectively ) .", "( C ) Coefficient k for the quadratic phase of chemosensor phosphorylation by partially active Aurora B kinase vs . kinase concentration ( A ) plotted on a log-log scale .", "Line is linear fit .", "( D ) Diagram of the experimental procedure to evaluate Aurora B autoactivation at high kinase concentration ( 4 µM ) .", "Experimental graph on the right shows changes in concentration of active Aurora B , calculated as described in Materials and methods .", "Data points are mean ± SEM for N≥4 experiments .", "Solid line is theoretical fitting with the reaction scheme in panel E . Dashed line is theoretical fit using the analytical solution for A*", "( t ) for the reaction scheme with only in cis activation of Aurora B . ( E ) Molecular scheme for the Aurora B kinase two component autoactivation in the presence of chemosensor and the corresponding reactions , see system equation 2 in Materials and methods .", "All other symbols are listed in Tables 1 and 2 . DOI: http://dx . doi . org/10 . 7554/eLife . 10644 . 00510 . 7554/eLife . 10644 . 006Figure 2—figure supplement 1 . Bicistronic construct of Aurora B-INbox and its dephosphorylation .", "( A ) Schematic of a bicistronic DNA construct for the Aurora B-INbox complex ( top ) and the expected protein product .", "( B ) Elution profile from size-exclusion chromatography and SDS gel ( below ) show that Delta60N Aurora B and INbox co-purify .", "Predicted molecular weights for Delta60N Aurora B and INbox are 36 and 7 kDa , respectively .", "( C ) Dephosphorylation of purified Aurora B-INbox complex by Lambda protein phosphatase ( 25 nM ) added at time 0; phosphatase was inhibited by 10 mM phosphonoacetic acid .", "A phospho-specific antibody to INCENP ( Salimian et al . , 2011 ) was used for western blots; dilutions of purified Aurora B-INbox complex with no phosphatase were used to confirm linearity of the detection procedure .", "Fluorescent signals were quantified as described in Materials and methods .", "( D ) Western blot using the phospho-specific antibody to INCENP was done for 8 µM Aurora B before and after treatment with 0 . 2 µM phosphatase for 90 min at 30°C . DOI: http://dx . doi . org/10 . 7554/eLife . 10644 . 00610 . 7554/eLife . 10644 . 007Figure 2—figure supplement 2 . Aurora B activity towards chemosensor .", "( A ) Molecular scheme for the reaction of chemosensor phosphorylation by Aurora B kinase; see Table 1 and 2 and legend to Figure 2 for details .", "( B ) Example trace for phosphorylation of commercial Omnia sensor .", "Recording is interrupted when Aurora B kinase is added; shaded area shows time interval with a roughly linear slope , from which the initial rate was calculated .", "( C ) Standard curves for chemosensor substrate and product florescence .", "Lines are linear fits .", "( D ) Initial rate of chemosensor phosphorylation by Aurora B kinase as a function of chemosensor concentration .", "Solid lines are Michaelis-Menten fittings .", "Here and in panels ( E ) and ( F ) , green color depicts data for the commercial Omnia chemosensor , and orange is our synthesized chemosensor .", "Graphs represent data from 2 independent experiments; error bars are SEM .", "( E ) Lineweaver–Burk plot of the inverse phosphorylation rate as a function of the inverse chemosensor concentration .", "Solid lines are linear fits .", "( F ) Hanes–Woolf plot of the ratio of substrate to the reaction rate as a function of substrate concentration .", "Solid lines are linear fits .", "( G ) Enlargement of the initial stage for chemosensor phosphorylation curves from Figure 2A .", "Dashed lines are best fits with quadratic functions , yielding coefficient k plotted in Figure 2C .", "( H ) Phosphorylation of 20 µM chemosensor by 0 . 5 µM partially active Aurora B kinase .", "Orange line – experimental data from Figure 2A , solid black line – calculated concentration of the chemosensor product phosphorylated by Aurora B activated in cis , dashed black line - by Aurora B activated in trans .", "( I ) Time course for the fraction of active Aurora B kinase; initial concentration of partially active Aurora 0 . 5 µM .", "Solid line – fraction of Aurora B phosphorylated in cis , dashed line – autoactivation in trans . DOI: http://dx . doi . org/10 . 7554/eLife . 10644 . 00710 . 7554/eLife . 10644 . 008Figure 2—figure supplement 3 . Results for Aurora B two sites phosphorylation model .", "( A ) Results of the unconstrained fitting for chemosensor phosphorylation curves; experimental data same as in Figure 2A .", "( B ) Same experimental data as in panel ( A ) but fitted using limiting case model parameters for the rapid conversion of kinase A# .", "( C ) Changes in concentrations of three Aurora B forms during autoactivation at 4 µM total Aurora B concentration using unconstrained fitted parameters .", "( D ) Same calculation as in panel ( C ) but using limiting case parameter values .", "( E ) Fitting of the experimental data from Figure 2D .", "Solid line - unconstrained fitting , dashed line – limiting case values .", "( F ) Fitting of the experimental data from Figure 4C .", "Solid lines - unconstrained fitting with two site model . DOI: http://dx . doi . org/10 . 7554/eLife . 10644 . 008 To reveal the in trans component , we carried out experiments using high concentration of partially active Aurora B , mimicking its clustering at cellular binding sites .", "At high kinase concentration the chemosensor becomes depleted quickly , so we modified our assay to uncouple the Aurora B autophosphorylation reaction from the activity measurement with the chemosensor ( Figure 2D ) .", "With 4 µM kinase , kinase activity increased strongly with time , and the best-fit curve based only on in cis autoactivation provided a poor fit ( Figure 2D ) , confirming the presence of the in trans component .", "With a computational model combining both reactions ( Figure 2E ) , we generated a global fit to experimental curves in Figure 2A , D and determined molecular constants for the two-component autoactivation mechanism for Aurora B kinase ( Table 2 , Materials and methods ) .", "This model demonstrates that kinase autoactivation in cis dominates over the trans-activation during initial activation at low kinase concentration ( Figure 2—figure supplement 2 , panels H and I ) .", "Our findings above imply that if Aurora B kinase , phosphatase and ATP are mixed together , two reactions should take place simultaneously: Aurora B autoactivation and its inactivation by phosphatase .", "We constructed a quantitative model for such a coupled kinase-phosphatase system ( Figure 3A ) , which takes into account the determined molecular constants for two-component Aurora B autoactivation and a Michaelis–Menten mechanism for a phosphatase with variable enzymatic constants .", "Solving the differential equations describing this system in silico ( see Materials and methods ) reveals that at high kinase concentration three steady-state solutions could coexist ( Figure 3B ) .", "Figure 3C shows region of bistability in the parametric plane of Aurora B kinase-phosphatase concentrations .", "Bistability arises when Aurora B kinase concentration exceeds 4 μM , and further increasing Aurora B concentration broadens the range of permissible phosphatase concentrations .", "In this region , a homogeneous mixture of kinase and phosphatase can exist in one of two stable states with different kinase activity , high or low , depending on initial conditions ( Figure 3C ) .", "This prediction is important because , as we will show later , bistable behavior is essential for accurate regulation of Aurora B kinase activity away from sites of high kinase concentration . 10 . 7554/eLife . 10644 . 009Figure 3 . Theoretical analysis of the coupled Aurora B kinase-phosphatase system .", "( A ) Molecular scheme for the coupled system and the corresponding reactions .", "For reactions 1 and 2 see Figure 2E; see Table 1 and 2 for more details .", "( B ) Steady-state solutions for concentration of active Aurora B kinase as a function of phosphatase concentration ( Equation 4 in Materials and methods ) .", "For 8 µM Aurora B , three steady states can co-exist: two stable states with high and low activities and one unstable state ( dashed line ) , corresponding to the region of bistability .", "( C ) Bistability region in the parametric plane of phosphatase and total ( phosphorylated and not ) Aurora B kinase concentrations .", "In this region the model has two coexisting stable steady-state solutions , while enzymatic concentrations outside this region lead to only one steady state .", "Colored lines correspond to the solutions shown in panel B for active kinase .", "( D ) Theoretical predictions for the changes in concentration of active Aurora B kinase , plotted as a fraction of total kinase concentration , for two different initial conditions .", "The initial concentration slightly higher than the threshold ( horizontal line ) has a steady-state solution with a larger fraction of active kinase ( high state ) .", "The fraction of active Aurora B kinase declines when its initial concentration is below the threshold ( low state ) .", "Calculations were done for 8 µM total Aurora B kinase and 0 . 47 µM phosphatase .", "( E ) Simulation of perturbations to reaction with 0 . 47 µM phosphatase and 8 µM total Aurora B kinase .", "Active kinase is added 3 times as indicated ( vertical arrows ) .", "The system returns to the steady state with low Aurora B kinase activity until the threshold is exceeded .", "( F ) Hysteresis loop in the kinase-phosphatase system with 8 µM kinase .", "Phosphatase concentration was initially low , so almost all Aurora B kinase was active .", "As the phosphatase concentration was gradually increased up to 0 . 8 µM , the steady-state concentration of active Aurora B kinase decreased ( top line with downward arrow ) .", "Different solutions were obtained when phosphatase concentration was decreased gradually back to 0 µM ( lower line with two upward arrows ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 10644 . 00910 . 7554/eLife . 10644 . 010Figure 3—figure supplement 1 . Aurora B hysteresis dependency on phosphatase .", "( A ) Kinetics of the fraction of active Aurora B kinase in simulations to study hysteresis .", "Horizontal dashed line shows the steady-state level for active Aurora B; results are for total Aurora B concentration 8 µM and phosphatase 0 . 45 µM .", "( B ) Hysteresis loop was calculated for four different Michaelis constants KMP for phosphatase ( PPase ) .", "Hysteresis is observed for all tested parameters but it requires slightly lower PPase concentration for smaller KMP .", "( C ) Hysteresis loops for three different PPase catalytic rate constants kcatP plotted vs . normalized PPase concentration ( PPase concentration divided by kcatP ) ; three curves overlap completely , illustrating that hysteresis in this coupled system does not depend on the catalytic rate of phosphatase . DOI: http://dx . doi . org/10 . 7554/eLife . 10644 . 010 As expected for the bistable regime , increasing concentration of active Aurora B above a threshold causes this biochemical system to switch between two states with no intermediate steady-states ( Figure 3D , E ) .", "The model also predicts hysteresis in the region of bistability .", "Hysteresis becomes evident at intermediate levels of phosphatase concentration ( e . g . 0 . 4 μM in Figure 3F ) , when almost the entire kinase pool can be either phosphorylated ( high kinase activity state ) or dephosphorylated ( low kinase activity state ) depending on the prior state of this reaction mixture .", "Importantly , we find that these non-linear regimes are determined mostly by the parameters of the two component Aurora B autoactivation mechanism , but not by the enzymatic constants of the protein phosphatase ( Figure 3—figure supplement 1; see Materials and methods ) .", "Thus , Aurora B kinase , coupled with an inactivating phosphatase , is predicted to exhibit robust hysteresis and bistability .", "Using the reconstituted in vitro system , we next designed an experiment to test the prediction of our theoretical model that at high Aurora B concentration the same mixture of kinase and phosphatase will result in different degrees of Aurora B activity depending on the initial conditions .", "We combined Aurora B kinase ( 8 μM ) , ATP ( 4 mM ) and variable concentrations of λ phage phosphatase , such that both Aurora B activation and inactivation could take place simultaneously .", "Importantly , these reactions were carried out for two different initial conditions: using either the active kinase or active kinase pretreated with phosphatase ( Figure 4A ) .", "All other reactants in these two mixtures were adjusted to achieve the same final concentrations for all components , including the phosphatase .", "The progress of these reactions was followed by taking samples at the indicated times; phosphatase inhibitor was then added to stop kinase dephosphorylation and kinase activity was measured via the initial rate of chemosensor phosphorylation .", "As expected , at low phosphatase concentration ( 0 . 25 µM ) the partially active kinase gradually activated itself , reaching a steady-state with high activity , while the active kinase slightly lost its activity ( Figure 4B , top graph ) .", "At high phosphatase concentration ( 0 . 5 µM , bottom graph ) the active kinase was overpowered by the phosphatase and became gradually inactivated , reaching a level close to the fully dephosphorylated kinase .", "Importantly , the model accurately predicted the behavior for these two reactions and when intermediate phosphatase concentration was used ( lines in Figure 4B ) .", "At 0 . 45 µM phosphatase , the kinase that was initially active robustly retained its high state ( red data points , Figure 4B , middle graph ) , while the activity remained low for the kinase that was initially in the low state ( blue data points , Figure 4B middle graph ) .", "These outcomes demonstrate bistability because in both of these enzyme mixtures the final concentrations of all components were identical and the reactions were allowed to proceed long enough to reach the steady-states ( 120 min ) .", "Similar experiments were carried out for additional phosphatase concentrations , and the steady-state levels of active Aurora B were obtained by averaging measurements for ≥60 min incubation times .", "These data , plotted as a function of phosphatase concentration in Figure 4C , define the bistable region for the homogeneous system in vitro , in quantitative agreement with model predictions .", "In this range of concentrations , the coupled kinase-phosphatase system exhibits hysteresis , with different activity levels observed depending on the initial conditions . 10 . 7554/eLife . 10644 . 011Figure 4 . Reconstitution of the coupled Aurora B kinase-phosphatase system in vitro .", "( A ) Diagram of the experimental procedure to study bistability and hysteresis .", "Active kinase was preincubated with phosphatase ( PPase ) in the absence of ATP to generate partially active kinase , and ATP was added at time = 0 ( 'initially low' experiment ) .", "In a parallel experiment , the same reagents were used but active kinase , phosphatase and ATP were mixed together at time = 0 ( 'initially high' experiment ) .", "Samples were taken to analyze kinase activity until the corresponding steady states were reached .", "( B ) Experimental results ( dots ) for changes in kinase activity vs . incubation time for 8 µM kinase and 0 . 25 , 0 . 45 or 0 . 5 µM phosphatase , as indicated .", "Each point shows mean ± SEM ( N≥2 ) for experiments with active ( red ) or partially active ( blue ) Aurora B kinase .", "( C ) Fraction of active kinase at steady state as a function of phosphatase concentration .", "Points are mean ± SEM for N≥4 independent experiments .", "These data are in close agreement with the model built using our experimentally determined kinetic constants ( solid lines in panels B and C ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 10644 . 011 If the biochemically simplified coupled kinase-phosphatase system in our in vitro experiment represents the behavior in the more complex in vivo setting , we predict that complex non-linear regimes should also be observed in mitotic cells , where Aurora B kinase is highly concentrated at the sites of its localization and various cellular phosphatases , such as PP1 ( Trinkle-Mulcahy et al . , 2003 ) , may inactivate it by dephosphorylation .", "First , we tested whether the endogenous Aurora B–phosphatase system can exhibit bistability , using a FRET-based phosphorylation sensor targeted to centromeres by fusion to CENP-B ( Fuller et al . , 2008 ) .", "If the system is bistable , this sensor should reveal that Aurora B exists in either a high or low activity state and that these states can co-exist under the same experimental conditions .", "We manipulated Aurora B activity by incubating mitotic cells with varying concentrations of its specific inhibitor , ZM447439 .", "Cells were imaged live , and the average FRET ratio was calculated for each individual cell , representing the overall phosphorylation state of that cell .", "Analysis of a population of cells expressing the centromere-targeted sensor shows that the distribution of phosphorylation states is clearly bimodal , with distinct high and low FRET states ( Figure 5A ) .", "In the absence of inhibitor , all cells are in the high phosphorylation ( low FRET ratio ) state , as expected .", "As the inhibitor concentration increases , the distribution shifts so that a greater fraction of cells is in the low phosphorylation state , but intermediate phosphorylation states are rare .", "Importantly , for some intermediate concentrations of Aurora B inhibitor , both peaks are observed in the same cell population , likely because individual cells differ slightly in their parameters , for example in membrane permeability to kinase inhibitor .", "Similar results were obtained for the sensor targeted to chromatin by fusion with histone H2B ( Fuller et al . , 2008 ) .", "Here , cells were blocked in mitosis with either monastrol or nocodazole ( Figure 5B ) , indicating that these results do not depend on which sensor is used or how cells are arrested . 10 . 7554/eLife . 10644 . 012Figure 5 . Bistability and hysteresis of Aurora B kinase in dividing cells .", "( A ) Cells expressing the centromere-targeted Aurora B sensor were arrested in mitosis with the proteasome inhibitor MG132 and incubated with various concentrations of the Aurora B inhibitor ZM447439 , then imaged live .", "Images show representative cells at 0 . 75 µM of Aurora B inhibitor: top images CFP emission , bottom images YFP/CFP emission ratio .", "Histograms below show fraction of cells with the indicated FRET ratio ( average YFP/CFP over a single cell ) , N > 50 cells for each Aurora B inhibitor concentration .", "( B ) Data from ( A ) are plotted together with similar experiments for cells expressing the chromatin-targeted sensor , arrested in mitosis with either nocodazole or monastrol and incubated with different ZM447439 concentrations .", "Each data point represents the FRET ratio for one cell normalized as described in Materials and Methods .", "( C ) HeLa cells expressing the chromatin-targeted Aurora B sensor were arrested in mitosis with nocodazole and treated with either 0 or 1 . 5 µM ZM447439 for 100 min .", "Then , cells were imaged live and ZM447439 concentration was changed as indicated at t = 0 .", "Results of a single experiment are plotted with each line representing an individual cell; 'low' , 'intermediate' and 'high' correspond to ZM447439 concentrations 0 , 0 . 6 and 1 . 5 µM , respectively .", "( D ) Normalized steady-state sensor phosphorylation as a function of ZM447439 concentration .", "Each data point ( mean ± SEM ) is calculated from the average of final FRET ratios for cells imaged as in panel ( C ) ( see Materials and methods ) .", "When the final FRET ratio is at a minimum ( as in 0 µM inhibitor ) , the normalized sensor phosphorylation is maximal because phosphorylation decreases FRET in this biosensor .", "Data were averaged over two independent experiments , N>9 cells per condition in each experiment . DOI: http://dx . doi . org/10 . 7554/eLife . 10644 . 01210 . 7554/eLife . 10644 . 013Figure 5—figure supplement 1 . Aurora B and PP1γ localizations are not affected by Aurora B inhibition .", "( A ) Cells were incubated for 2 hr with nocodazole and the indicated concentrations of ZM447439 to mimic conditions of the in vivo bistability and hysteresis experiments ( Figure 5B , C ) .", "Cells were then fixed and stained for Aurora B and phospho-INCENP .", "Intensity of the phospho-INCENP , but not Aurora B , signal decreases in response to adding the inhibitor in an abrupt manner , consistent with bistability of the Aurora B kinase activity , but not its localization .", "( B ) Cells expressing PP1γ-GFP were treated as in ( A ) and stained for Aurora B . DOI: http://dx . doi . org/10 . 7554/eLife . 10644 . 013 To further test the bistable kinase-phosphatase system in vivo , we asked whether the prior history of Aurora B kinase activation affects the level of Aurora B activity in mitotic cells .", "We designed an experiment to manipulate Aurora B activity in a similar manner as in our in vitro experiments , in which hysteresis was observed .", "Cells were first incubated with low ( 0 µM ) or high ( 1 . 5 µM ) concentration of the Aurora B inhibitor to establish two different initial conditions of either high or low kinase activity , respectively .", "From these initial conditions , the inhibitor concentration was switched following one of four protocols: low to high ( 0 to 1 . 5 µm ) or high to low ( 1 . 5 to 0 µM ) , as experimental controls , and low to intermediate ( 0 to 0 . 6 µM ) or high to intermediate ( 1 . 5 to 0 . 6 µM ) to reach identical final conditions .", "If hysteresis is present , cells that end up at the same intermediate inhibitor concentration will show different phosphorylation levels depending on their past history , i . e . , whether they we preincubated with initially high or low inhibitor concentration .", "Cells were then imaged live to track changes in phosphorylation of the chromatin targeted FRET sensor .", "Switching from low to high inhibitor concentration led to kinase inhibition and sensor dephosphorylation , and conversely switching from high to low led to kinase activation and sensor phosphorylation , as expected .", "When the inhibitor concentration was switched to the intermediate level , however , kinase activity remained in the initial state in these mitotic cells: high if the initial condition was low inhibitor ( 0 to 0 . 6 µM ) and low activity if the initial condition was high inhibitor ( 1 . 5 to 0 . 6 µM ) ( Figure 5C , D ) .", "In addition , we found that cellular localizations of Aurora B and PP1γ phosphatase were not affected by treatment with this inhibitor ( Figure 5—figure supplement 1 ) , consistent with a previous report for Aurora B localization ( Ditchfield et al . , 2003 ) .", "Together , these results strongly indicate that bistability and hysteresis of Aurora B phosphorylation in mitotic cells are driven by the intrinsic properties of Aurora B kinase coupled with the inactivating phosphatase", "( s ) .", "To gain insight into the physiological significance of the bistability of the Aurora B kinase-phosphatase system , we built a spatial model of Aurora B kinase activity in cells .", "This model simplifies or leaves out many mitotic features while focusing on molecular processes that are essential for Aurora B kinase activity in cells .", "Specifically , we used deconvolved intensity profiles for Aurora B localization at the centromere and along chromosome arms to define the spatial distribution of Aurora B binding sites on chromatin ( Figure 6—figure supplement 1 ) .", "Peak Aurora B concentration at the centromere is estimated at 10 µM , dropping down to 1 . 5 µM along chromosome arms and 1–2 µM in the kinetochore area ( Figure 6A ) .", "In the model soluble kinase molecules bind and unbind these sites dynamically to achieve the steady-state fractions of the bound and diffusing soluble kinase pools of 75% and 25% , respectively ( Mahen et al . , 2014 ) .", "Soluble kinase in the model behaves identically to our in vitro experiments , activating itself via the two component mechanism with the kinetic constants listed in Table 2 .", "The activity of chromatin-bound Aurora B is not known , but the elongated flexible structure of the INCENP subunit is thought to permit some mobility for the tethered Aurora B kinase ( Krenn and Musacchio , 2015; Samejima et al . , 2015 ) .", "We therefore assume that chromatin bound Aurora B kinase can interact with soluble Aurora B molecules freely ( with the same kinetic constants as in Table 2 , but phosphorylation in trans between the chromatin-bound molecules is limited ( see Materials and methods ) .", "Both bound and soluble Aurora B molecules can be inactivated by a phosphatase , which for simplicity is assumed to be soluble and diffusive .", "This reaction-diffusion system was described with partial differential equations ( Equation 6 in Materials and methods ) and solved numerically . 10 . 7554/eLife . 10644 . 014Figure 6 . Spatial model of Aurora B activity in the cell .", "( A ) Schematics of the essential features of our spatial model , see Materials and methods for details .", "( B ) Parametric plane of phosphatase and total Aurora B kinase concentrations analogous to the plot in Figure 3C but calculated using the spatial model .", "Grey area shows region of bistability; KPM = 0 . 16 µM .", "( C ) Simulated ( right axis ) and experimental ( left axis ) results for the hysteresis experiment in cells ( data points reproduced from Figure 5D ) .", "Calculations are for KPM = 0 . 16 µM , phosphatase concentration 0 . 1 µM .", "( D , E )", "These plots are analogous to those in panels ( B , C ) , but they were calculated for kcis = 7 . 3 · 10–4 s-1 , all other model parameters were not changed .", "With this autoactivation constant , the model predicts no bistability in the physiological range of kinase concentrations ( D ) , and , the kinase activity vs . inhibitor concentration curve does not depend on the system’s history ( E ) , blue and red curves are slightly offset for clarity ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 10644 . 01410 . 7554/eLife . 10644 . 015Figure 6—figure supplement 1 . Theoretical model for spatial regulation of Aurora B kinase phosphorylation .", "( A ) Molecular and biochemical reactions in the spatial model of Aurora B kinase phosphorylation , see Table 1 , 2 and Materials and methods for details .", "( B ) Estimated Aurora B kinase concentration profile along the axis connecting sister kinetochores .", "Origin corresponds to centroid , i . e . , the midpoint between sister kinetochores .", "( C ) Estimated Aurora B kinase concentration profile along the chromosome arm .", "Origin corresponds to centroid , i . e . , the midpoint between sister kinetochores .", "( D ) GFP image of a HeLa cell expressing GFP-Aurora B kinase and arrested with monastrol .", "Scale bar 5 µm .", "( E ) GFP-Aurora B signal along chromosome arms averaged over N>16 chromosomes that were aligned at their centromere positions ( distance = 0 ) and normalized to maximal Aurora B signal at centromeres; points are mean ± SEM . DOI: http://dx . doi . org/10 . 7554/eLife . 10644 . 015 With this analytical framework we first tested if this model could reproduce the bistability and hysteresis observed for the overall state of Aurora B kinase in cell experiments .", "Consistent with our theoretical analysis of the homogeneous system , bistability was predicted for a range of phosphatase concentrations , overlapping with the physiological kinase concentrations ( Figure 6B ) .", "Aurora B inhibition was then simulated using the published relationship between ZM447439 concentration and Aurora B activity ( Ditchfield et al . , 2003 ) .", "The steady-state Aurora B activity was examined starting from two different initial conditions: when all cellular kinase had enzymatic activity of the fully active kinase or it was inactive .", "The inhibitor concentration was varied in 10-nM steps , and the spatial distribution of Aurora B kinase activity was calculated and averaged to represent the overall fraction of active kinase for each initial condition and inhibitor concentration ( Figure 6C , lines ) .", "As in the homogeneous system ( Figure 3—figure supplement 1 ) , the KPM value for phosphatase affected the exact shape and position of this theoretical hysteresis plot .", "The enzymatic constant for phosphatase acting on Aurora B kinase in cells is not known , but KPM = 0 . 16 µM provided an excellent match with experimental measurements in cells ( Figure 6C ) .", "Thus , Aurora B hysteresis in mitotic cells can be reproduced using the molecular and biochemical features which form the basis for our model and reasonable values of model parameters .", "To examine whether bistability of the coupled kinase-phosphatase system was essential for matching the experimental data , we modified our model by changing only one parameter kcis , which characterizes Aurora B kinase autophosphorylation in cis .", "Importantly , all other model features and the values of all other parameters were unchanged , such that Aurora B autoactivation and its inhibition by phosphatase were still present .", "With this modification , the bistable region could only be observed at much higher kinase and phosphatase concentrations , while bistability in the range of physiological Aurora B concentrations was lost ( Figure 6D ) .", "When this modified model was used to mimic the ZM447439 inhibition experiment , it predicted a reasonably good match to the gradual decrease in Aurora B activity in experiments with increasing inhibitor concentration .", "However , when calculations were done starting from the inactive kinase and the inhibitor was 'washed out' , the model prediction did not change , indicating a lack of hysteresis ( Figure 6E ) .", "Thus , bistability of the underlying biochemical pathways is required to explain hysteresis that we detected with the Aurora B phosphorylation sensor in cells .", "Next , we investigated model predictions for the regulation of Aurora B phosphorylation of substrates located remotely from centromeric sites of kinase enrichment .", "Previous experiments using cells arrested in mitosis found gradients of Aurora B phosphorylation spreading from centromeres , along chromosome arms , after Aurora B was inhibited with ZM447439 and then the inhibitor was washed out ( Wang et al . , 2011 ) ( Figure 7A ) .", "Analogous images were obtained in this work after inducible clustering of Aurora B at the centromere ( Figure 1B and Figure 1—figure supplement 1B ) , emphasizing that these large scale phosphorylation patterns are triggered by Aurora B localization and activation at the centromere .", "We modeled the kinase inhibitor washout experiment to determine the spatiotemporal distribution of activated Aurora B kinase , then calculated the resulting phosphorylation patterns for a chromatin-bound substrate ( see Materials and methods ) .", "Consistent with the in vivo experiment , the model exhibited spatially non-uniform large-scale distributions with phosphorylation high at the centromere and gradually decreasing along chromosome arms ( Figure 7A ) .", "In the model , and in cells , these gradients are transient , as Aurora B signal propagates from the centromere , eventually leading to uniformly high Aurora B phosphorylation of all chromatin bound substrates ( Figure 7B ) .", "This spreading appeared similar to a trigger wave , a hallmark feature of a bistable medium ( Kapral and Showalter , 1995 ) .", "Importantly , self-sustained trigger waves propagate at a constant speed , which discriminates them from other mechanisms of signal propagation in systems with diffusion .", "Indeed , the predicted plot for the timing of Aurora B activation as a function of distance from the centromere was linear , implying a constant rate of spreading ( Figure 7C ) .", "To test this model prediction we plotted the time of phosphorylation as a function of distance from centromeres for the chromatin bound FRET sensor , from experiments in which ZM447439 was washed out .", "This dependency was also linear , strongly suggesting that phosphorylation along chromatin propagates as a trigger wave ( Figure 7C ) .", "As expected , the model with no bistability in the concentration range of chromatin bound Aurora B predicted very different kinetics of phosphorylation spreading with a non-linear rate ( Figure 7A–C ) . 10 . 7554/eLife . 10644 . 016Figure 7 . Wave propagation of Aurora B activity .", "( A ) Color-coded plots showing spatial patterns of Aurora B phosphorylation .", "Top row: HeLa cell expressing the chromatin-targeted FRET sensor and arrested with monastrol is shown before ( t<0 ) and after Aurora B inhibitor washout .", "Time 0 min corresponds to FRET signal reaching half of its maximum level at the centromere .", "Lower FRET signal corresponds to higher sensor phosphorylation .", "Other two rows: color-coded substrate phosphorylation calculated in the models with and without bistability .", "Scale bar , 5 µm .", "( B ) Profiles of average substrate phosphorylation along chromosome arms in cells observed at different time after inhibitor washout and analogous model predictions .", "Signals were normalized to maximum level of substrate phosphorylation .", "( C ) Time of 50% sensor phosphorylation as a function of distance along chromosome arms .", "Closed symbols: experimental data with a linear fit .", "Open symbols correspond to model solutions with and without bistability . DOI: http://dx . doi . org/10 . 7554/eLife . 10644 . 016 Finally , we used our model to seek new insights into the spatial distribution of Aurora B kinase activity at kinetochores , where phosphorylation decreases from prometaphase to metaphase .", "Previous measurements in metaphase using Aurora B phosphorylation sensors targeted to different molecular locations at kinetochores revealed different phosphorylation levels at sites separated by only 10s of nm , indicating a sharp gradient of Aurora B activity ( Welburn et al . , 2010; Suzuki et al . , 2014 ) .", "With our model , we calculated the fraction of activated kinase as a function of distance from the centroid ( midway between the sister kinetochores ) for the unstretched centromere , corresponding to the microtubule-free kinetochores in prometaphase .", "Almost the entire centromere-bound pool of prometaphase Aurora B kinase is predicted to be active , with the fraction of active kinase decreasing slightly at the kinetochore ( bounded by CENP-A and Ndc80 ) ( Figure 8A ) .", "We then stretched this mechano-biochemical system to mimic the ~2-fold increase in distance between sister kinetochores seen in metaphase HeLa cells ( Wan et al . , 2009 ) .", "In stretched chromatin the distance between Aurora B binding sites increased correspondingly , as indicated with the white mesh in Figure 8 .", "As a result , the local concentration of chromatin-bound kinase decreased , and a region of bistability emerged at the kinetochore , hundreds of nm away from the centroid ( Figure 8—figure supplement 1 ) .", "As we have shown earlier , the bistable kinase-phosphatase system exhibits a highly nonlinear behavior .", "In the chromatin meshwork , these threshold-dependent reactions created a stable and steep gradient of Aurora B kinase activity .", "In contrast , in the model with no bistability , stretching induced a much more gradual gradient of Aurora B activity , reflecting a gradual decrease in density of centromere-bound Aurora B kinase ( Figure 8B , D ) . 10 . 7554/eLife . 10644 . 017Figure 8 . Predicted gradient of Aurora B kinase activity at kinetochores during prometaphase ( left ) and metaphase ( right ) .", "Color-coded plots of the profile of Aurora B kinase activity along the axis connecting the centromere centroid ( midway between the sister kinetochores ) and the outer kinetochore .", "Arrow for Ndc80 corresponds to the location of the N-terminus of Hec1 ( Wan et al . , 2009 ) .", "Density of the white mesh indicates concentration of Aurora B kinase; local Aurora B concentration is lower when mesh holes are larger .", "( A ) and ( B ) show model predictions for prometaphase kinetochores that are not under tension ( smaller centroid to Ndc80 distance ) .", "In metaphase ( C and D ) this distance increases due to forces generated by the end-on attached kinetochore microtubules .", "In the model without bistability ( B–D ) , the fraction of active Aurora B kinase simply reflects the total Aurora B kinase concentration . DOI: http://dx . doi . org/10 . 7554/eLife . 10644 . 01710 . 7554/eLife . 10644 . 018Figure 8—figure supplement 1 . Quantification of Aurora B activity gradient during mitosis .", "( A , B )", "Calculated concentration of active Aurora B as a function of total Aurora B concentration in the model with and without bistability .", "Red and blue curves correspond to the initially active and partially active Aurora B , correspondingly .", "In the model with bistability ( A ) , Aurora B kinase remains largely inactive ( 'low' state ) until total kinase concentration reaches ~1 . 2 µM .", "With higher kinase concentration , the active kinase increases roughly proportionally to the total concentration , but in the range of 1 . 2–1 . 5 µM both 'low' and 'high' activity states are possible ( bistable region ) .", "In the model with no bistability ( B ) , kinase activity increases roughly proportionally to total kinase concentration and the curves for different initial conditions overlap completely ( shown with slight offset for better visualization ) .", "( C–F ) .", "Graphs show the calculated fraction of total Aurora B kinase that is active ( black solid lines , right axes ) as a function of distance along the centromere-kinetochore axis .", "Shown in grey ( left axes ) is estimated total concentration of Aurora B kinase .", "In the model with bistability ( C , E ) , different concentration areas are colored as in panel ( A ) , indicating predicted activity states .", "Graphs for the less stretched centromere ( C , D ) correspond to distances observed at the microtubule-free kinetochores in prometaphase .", "Since total Aurora B ( grey areas ) is constant during prometaphase and metaphase , centromere stretching ( E , F ) reduces Aurora B concentration everywhere , and bistability becomes possible at the outer kinetochore ( E , yellow-colored area ) , where active Aurora B concentration drops below threshold .", "In the spatial region with bistability , a steep gradient of Aurora B activity can form . DOI: http://dx . doi . org/10 . 7554/eLife . 10644 . 018" ], [ "Our findings address the long-standing question of how Aurora B phosphorylates substrates at a distance from its major sites of localization .", "First , we use a rapamycin-induced targeting system to examine the immediate effects of concentrating Aurora B at centromeres .", "This approach represents an advance over previous manipulations of Aurora B localization , such as depleting CPC components , comparing different mutant forms of INCENP , or inhibiting mitotic kinases that control CPC localization ( Vader et al . , 2006; Tsukahara et al . , 2010; Wang et al . , 2010; 2011; Yamagishi et al . , 2010 ) .", "In the experiments reported here , Aurora B localization is controlled by rapamycin addition , while keeping other components of the system constant , so indirect effects from these manipulations are less likely .", "Importantly , in the absence of rapamycin , the cytosolic Aurora B/INbox protein complex shows little activity toward chromatin-localized targets .", "However , recruiting the same complex to centromeric binding sites leads to phosphorylation of the chromatin-localized probe within minutes , demonstrating that the highly-concentrated centromeric pool of Aurora B is essential for phosphorylation at other cellular locations ( Figure 1B ) .", "This result from mitotic cells suggests that specialized mechanisms enable long-range regulation of Aurora B kinase activity in mitosis .", "The theory of complex non-linear systems suggests a plausible molecular explanation for this phenomenon , since certain feedback-controlled reactions are known to lead to formation of a self-sustained source of active components and establishment of well-controlled spatial activity patterns ( Kapral and Showalter , 1995 ) .", "Testing such biochemical models requires knowledge of the underlying feedbacks and specific enzymatic constants and parameters values .", "In this work we build a quantitative foundation for such a mechanism using a reconstituted system with purified components .", "First , our work defines a quantitative biochemical mechanism for Aurora B autoactivation ( Table 2 ) .", "Previous experiments have suggested that Aurora B is activated in trans ( Bishop and Schumacher , 2002; Honda et al . , 2003; Sessa et al . , 2005; Kelly et al . , 2007 ) .", "We confirm these initial findings , but we also find that only the active , already phosphorylated Aurora B kinase can activate in trans .", "The analogous reaction by the unphosphorylated kinase is not as productive and is carried out in cis .", "This newly revealed cis component dominates at initial stages of Aurora B kinase activation , when the majority of kinase molecules are still unphosphorylated .", "The cis step may reflect autophosphorylation of the activation loop , as shown for Aurora A ( Dodson et al . , 2013 ) , while the TSS motif of INCENP is phosphorylated in trans .", "The significance of the in cis reaction is not fully understood , but we show that its rate has a large impact on the bistable region of the Aurora B-phosphatase system , as discussed below .", "Second , with a mathematical model we show that the kinetic constants we have defined for Aurora B kinase autoactivation can lead to non-linear behavior , bistability and hysteresis , when Aurora B is coupled with a phosphatase ( Figure 4 ) .", "Positive feedback in this system is provided by the two-component ( cis and trans ) autoactivation of Aurora B , while protein phosphatase inactivates the kinase by dephosphorylation .", "Importantly , we were able to observe this non-trivial behavior in vitro using purified Aurora B kinase and λ phosphatase , which to the best of our knowledge is the first reconstitution of this kind for any kinase-phosphatase pair .", "The experimental results in vitro are in quantitative agreement with model predictions ( Figure 4 ) , implying that we have reached a deep understanding of these phenomena .", "Importantly , our theoretical analyses predicted that bistability depends strongly on the Aurora B kinase autoactivation mechanism , while kinetic constants for the dephosphorylation reaction have much less impact ( Figure 3—figure supplement 1 ) .", "Thus , although our simplified reconstitution in vitro used the non-physiological λ phosphatase , the model suggested that these nonlinear regimes could exist in a physiological cell context , where Aurora B kinase is coupled with its native phosphatase partner ( s ) .", "Indeed , we were able to recreate bistability and hysteresis for Aurora B substrate phosphorylation in live mitotic cells .", "Our cellular experiments relied on sensors at different locations ( centromere or chromatin ) and used three methods to synchronize cells ( monastrol , nocodazole , or the proteasome inhibitor MG312 ) .", "Because these different experimental tools led to consistent results , our findings in cells likely reflect the same basic non-linear mechanisms that we recapitulated in vitro .", "We therefore explain the distinct phosphorylation states in mitotic cells as arising from the threshold-dependent autoactivation of the highly concentrated pool of centromere-localized Aurora B , propagated at long distance via the chromatin-bound and cytosolic pools of Aurora B . As in vitro , the high kinase activity state in cells can be sustained over a range of input signals , which in cells were generated using different concentrations of a specific Aurora B inhibitor .", "As the inhibitor concentration was increased , the system switched to the low kinase activity state ( Figure 5A , B ) , just as happened in vitro and in our model when the threshold was crossed .", "Moreover , these 'high or low' kinase activity states persisted in populations of mitotic cells under the same conditions , depending on the initial state ( Figure 5C , D ) .", "The consistency between our findings in vitro and in vivo indicates that we have captured key features of the Aurora B-phosphatase system that underlie cellular behaviors .", "This conclusion is also supported by our ability to describe the in vivo results for bistability and hysteresis using a spatial model of Aurora B kinase activity in mitotic cells .", "Since the cellular environment for Aurora B regulation is complex and many constants for the underlying biochemical and molecular reactions in cells are not known , it is currently not possible to provide a detailed quantitative description of Aurora B phosphorylation in cells .", "However , using reasonable assumptions and values for unknown model parameters , we were able to recapitulate our findings of hysteresis in live cells ( Figure 6 ) .", "Moreover , the model made a strong prediction for the propagation of self-sustained trigger wayves of kinase phosphorylation .", "Long-range propagation of Aurora B phosphorylation has been observed previously ( Wang et al . , 2011 ) , but it was thought to be caused by simple diffusion of activated Aurora B released from the sites of concentration .", "We quantified these waves and found that they propagate at constant speed ( Figure 7 ) , strongly implying that they are sustained by a more complex reaction-diffusion mechanism in a bistable system .", "Additionally , we demonstrate that a highly similar reaction-diffusion model , which also includes kinase autoactivation coupled with inactivating phosphatase but lacks bistability in the range of physiological Aurora B concentrations , fails to reproduce the trigger waves and other results in cells .", "We conclude that bistability of the coupled kinase-phosphatase system is an essential feature of Aurora B kinase regulation in cells .", "Different non-linear mechanisms operating in excitable media have been shown to play important roles in developmental biology and cell division ( Turing , 1952; Caudron et al . , 2005; Maini et al . , 2006; Karsenti , 2008; Chang and Ferrell , 2013 ) , the cardiovascular system and blood clotting ( Lobanova and Ataullakhanov , 2003; Sharma et al . , 2009 ) , kinase signaling ( Kholodenko , 2009 ) and intracellular patterning and size control ( Meyers et al . , 2006; Fischer-Friedrich et al . , 2010; Hachet et al . , 2011; Subramanian et al . , 2015 ) .", "Based on our findings , we propose that bistability of a coupled Aurora B-phosphatase system enables formation of an excitable medium , in which a source of localized active kinase can trigger complex spatial patterns , orchestrating Aurora B phosphorylation in a time and location-dependent manner .", "Specifically , our work offers a plausible physico-chemical mechanism to explain long-distance regulation of phosphorylation at the mitotic kinetochore in response to tension .", "We view the centromeric chromatin as a mechanical medium that is capable of sustaining biochemical reactions via a spatially non-uniform distribution of Aurora B kinase .", "Activity of this kinase in different chromatin areas depends on both the local concentration of chromatin-tethered Aurora B molecules and on the activity of the soluble Aurora B pool .", "Importantly , our work clearly shows that the level of Aurora B activity in each local area also depends on Aurora B activity in more distant locations of this mechano-biochemical medium .", "This is because in areas with higher kinase concentration , such as the inner centromere , kinase is strongly activated due to the two component autoactivation mechanism .", "This activity then propagates to more distant areas with lower kinase concentration via phosphorylation in trans by neighboring chromatin-tethered kinase molecules and via their cross-talk with diffusing soluble kinase .", "Since the concentration of chromatin-bound kinase decreases from the inner centromere to the outer kinetochore , this reaction-diffusion system can establish a gradient of kinase activity even if the underlying biochemical pathways are not bistable ( Figure 8 and Figure 8—figure supplement 1 ) .", "However , we demonstrate that when the bistable coupled kinase-phosphatase system is incorporated into this stretchable mechanical matrix , the resulting phosphorylation gradients can be much steeper .", "Moreover , the steep part of the gradient arises only upon kinetochore stretching and coincides with the outer kinetochore area , where the concentration range for Aurora B kinase causes its bistability .", "Thus , bistability affords versatile control of the position and steepness of the resulting gradient , which is thought to be essential for regulation of kinetochore-microtubule interactions ( Funabiki and Wynne , 2013; Krenn and Musacchio , 2015 ) .", "Bistability is also likely to play an important role in establishing a gradient of Aurora B activity around the spindle midzone in anaphase , though the mechanistic details may be different and need to be examined separately .", "In addition to the bistable system described here , other mechanisms may also regulate spatial patterns of Aurora B activity , such as changes in Aurora B enrichment at centromeres as chromosomes align ( Salimian et al . , 2011 ) and localized phosphatase activity at different cellular locations , such as kinetochores or centromeres or on chromatin ( Trinkle-Mulcahy et al . , 2003; 2006; Kitajima et al . , 2006; Riedel et al . , 2006; Tang et al . , 2006; Liu et al . , 2010; Foley et al . , 2011 ) .", "Localization of both PP1 and PP2A at kinetochores depends on microtubule attachment and tension , and changes in these local phosphatase activities may modulate the location of the bistable region of Aurora B activity or exert direct effects on substrates located in the immediate vicinity .", "These additional mechanisms are not mutually exclusive , and future experiments , building on our developed in vitro system and quantitative model , should examine how these mechanisms contribute to the establishment and maintenance of gradients at the appropriate spatial scales ." ], [ "PEFT was used to fit experimental results for kinase autoactivation , hysteresis and bistability .", "This program optimized the score function value - a sum of normalized squared differences between experimental and modeled data points .", "The software tool was developed in Mathematica software ( Wolfram Research ) similarly to the systems biology software in ( Zi , 2011 ) and it contained the following modules:", "1 ) experimental data parser ,", "2 ) ODEs solver ,", "3 ) score function calculator , and", "4 ) numerical optimizer ." ] ]

[ "Aurora B kinase , a key regulator of cell division , localizes to specific cellular locations , but the regulatory mechanisms responsible for phosphorylation of substrates located remotely from kinase enrichment sites are unclear .", "Here , we provide evidence that this activity at a distance depends on both sites of high kinase concentration and the bistability of a coupled kinase-phosphatase system .", "We reconstitute this bistable behavior and hysteresis using purified components to reveal co-existence of distinct high and low Aurora B activity states , sustained by a two-component kinase autoactivation mechanism .", "Furthermore , we demonstrate these non-linear regimes in live cells using a FRET-based phosphorylation sensor , and provide a mechanistic theoretical model for spatial regulation of Aurora B phosphorylation .", "We propose that bistability of an Aurora B-phosphatase system underlies formation of spatial phosphorylation patterns , which are generated and spread from sites of kinase autoactivation , thereby regulating cell division ." ]

[ "Cell division is a highly organized process that involves a series of major changes .", "First , the cell’s chromosomes are copied and arranged at the middle of the cell .", "Then , the pairs of copied chromosomes are separated and pulled towards opposite ends of the cell and , finally , the cell splits in two .", "These steps are mainly regulated by modifications to proteins , and enzymes called protein kinases play an important role because they add phosphate groups to , or phosphorylate , so-called 'substrate' proteins to change their activities .", "Other enzymes called phosphatases are also important because they remove the phosphate groups from the substrates to reverse the effects .", "The kinase Aurora B is required for several steps during cell division and has been widely studied .", "This kinase is enriched in specific locations within the cell , for example at the centromere regions of the chromosomes as they line up at the cell’s center .", "However , Aurora B phosphorylates substrates located at distant sites on the chromosome , with less phosphorylation at sites farther from the centromere .", "The level of phosphorylation also changes as chromosomes become aligned .", "Aurora B can activate itself and this ability was suspected to help this spatiotemporal regulation .", "However , it was not clear how the observed gradients of kinase activity might form .", "Zaytsev , Segura-Peña et al . set out to answer this question by first mixing in a test tube purified Aurora B and an inhibitory phosphatase .", "This revealed that this kinase-phosphatase system is 'bistable' , meaning that it has two stable states , low or high kinase activity , and that these states could switch in response to small changes in enzyme concentrations .", "Further experiments showed that this system has a kind of memory such that the level of activity ( low or high ) persists for a range of concentrations and depends on the system’s prior history .", "Zaytsev , Segura-Peña et al . then showed that both of these properties , the two stable states and the memory , exist in dividing human cells , and then went on to develop a mathematical model of how such bistability could set up gradients of Aurora B kinase activity .", "At the sites of highest concentration at the centromere , Aurora B can overcome inhibition by phosphatase and activates itself , as in the test tube .", "This activity spreads to more distant locations as active kinase molecules activate neighboring kinase molecules , establishing the areas with a high state of activity .", "As the local Aurora B concentration decreases further from the centromere , the phosphatase switches Aurora B into the low activity state , establishing a steep gradient of kinase activity in a region where its substrates that are important for chromosome segregation are located .", "Importantly , the shape and location of this gradient are predicted to depend on forces that stretch the lined chromosomes apart , offering a plausible mechanism to explain phosphorylation changes in response to tension .", "These theoretical insights and experimental approaches could be used to study other coupled kinase-phosphatase systems ." ]

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

[ "neuroscience" ]

[ [ "The regulated , Ca2+-triggered secretion of catecholamines from chromaffin cell LDCVs is an integral part of the physiological adaption to environmental stressors .", "Like the exocytosis of neuronal SVs , LDCV exocytosis is mediated by SNARE complex formation , in concert with Ca2+ sensors and essential regulatory proteins ( James and Martin , 2013; Neher , 2006; Ovsepian and Dolly , 2011; Pang and Sudhof , 2010 ) .", "Mammalian uncoordinated 13 ( Munc13 ) proteins are essential SV priming factors in neurons ( Augustin et al . , 1999; Richmond et al . , 1999; Rosenmund et al . , 2002 ) , and ultrastructural studies have shown that in synapses lacking Munc13s/Unc-13 , SVs also fail to physically dock to synaptic active zones ( Imig et al . , 2014; Siksou et al . , 2009; Weimer et al . , 2006 ) .", "At the molecular level , this morphological phenotype most likely corresponds to a role of Munc13s in mediating the formation of SNARE complexes at vesicular release sites ( Hammarlund et al . , 2007; Hammarlund et al . , 2008; Imig et al . , 2014; Ma et al . , 2011; 2013; Yang et al . , 2015 ) .", "The Munc13 family consists of five members , Munc13-1 ( Unc13a ) , Munc13-2 ( Unc13b ) , Munc13-3 ( Unc13c ) , the brain specific angiogenesis inhibitor I-associated protein 3 ( Baiap3 ) , and the non-neuronal isoform Munc13-4 ( Unc13d ) ( Koch et al . , 2000 ) .", "Genetic deletion of Unc13a and Unc13b completely eliminates SV exocytosis in hippocampal neurons ( Varoqueaux et al . , 2002 ) , and selectively reduces synaptic vs . extrasynaptic exocytosis of neuronal LDCVs ( van de Bospoort et al . , 2012 ) , which indicates that SV and LDCV exocytosis at active zones is mediated by similar molecular mechanisms .", "By contrast , studies in C . elegans and Drosophila have shown that Unc-13/dUnc-13 selectively regulate SV release , whereas the Ca2+-dependent activator proteins for secretion ( CAPS/Unc-31 ) specifically regulate LDCV release ( Hammarlund et al . , 2008; Renden et al . , 2001; Speese et al . , 2007; Zhou et al . , 2007 ) .", "In mammals , Munc13s and CAPSs appear to perform non-redundant functions critical for both SV and LDCV exocytosis in neurons ( Jockusch et al . , 2007; van de Bospoort et al . , 2012 ) , as well as for LDCV exocytosis in neuroendocrine cells ( Elhamdani et al . , 1999; Kabachinski et al . , 2014; Kang et al . , 2006; Kwan et al . , 2006; Liu et al . , 2010; Liu et al . , 2008; Speidel et al . , 2008 ) .", "Yet , to date , while CAPS-1 and CAPS-2 have been shown to be required for LDCV exocytosis in mammalian chromaffin cells ( Liu et al . , 2010; Liu et al . , 2008 ) , evidence that endogenous Munc13s are required for LDCV exocytosis is lacking .", "In fact , the role of Munc13-1 and ubMunc13-2 has only been examined in the context of overexpression studies , and other isoforms have not been investigated ( Ashery et al . , 2000; Bauer et al . , 2007; Liu et al . , 2010; Stevens et al . , 2005; Zikich et al . , 2008 ) .", "In the present study , we performed the first comprehensive analysis of all neuronal and neuroendocrine members of the Munc13 protein family in chromaffin cells , defining their respective roles in LDCV exocytosis .", "We identify the Ca2+-dependent step in the priming process at which Munc13-1 and ubMunc13-2 operate , and demonstrate that , although they are critical for LDCV priming and release , LDCV docking can occur without them ." ], [ "We first analyzed the expression of all Munc13 isoforms in the murine adrenal gland by western blotting ( Figure 1 ) .", "In perinatal adrenal glands , we detected Munc13-1 ( Figure 1A and Figure 1—figure supplement 1B ) , the ubiquitous isoform ubMunc13-2 ( Figure 1B and Figure 1—figure supplement 1B ) , and Baiap3 ( Figure 1D ) .", "Not detected were the brain-specific isoform of Munc13-2 ( bMunc13-2 ) , which is a splice variant expressed from the same gene as ubMunc13-2 ( Figure 1B ) , Munc13-3 ( Figure 1C ) , and the non-neuronal isoform Munc13-4 ( Figure 1E ) .", "To directly compare the expression levels of Munc13-1 , ubMunc13-2 , bMunc13-2 , and Munc13-3 , we used knock-in mice that express these proteins fused to enhanced yellow or green fluorescent protein ( EYFP/EGFP ) from the respective endogenous loci ( Cooper et al . , 2012; Kalla et al . , 2006 ) .", "We found that ubMunc13-2-EYFP is the only isoform readily detectable in the adrenal gland using an antibody to the GFP-derived tags ( Figure 1—figure supplement 1A ) . 10 . 7554/eLife . 10635 . 003Figure 1 . Expression of Munc13 isoforms in the mouse adrenal gland . KO mouse lines of the respective Munc13 isoform were used as control .", "The antibodies used to detect individual Munc13 isoforms and loading controls are indicated on the left .", "( A ) Munc13-1 ( * ) is barely detectable in perinatal adrenal gland .", "( B ) ubMunc13-2 , but not bMunc13-2 , is expressed .", "( C ) Munc13-3 was not detected .", "( D ) Baiap3 was detected , but not ( E ) Munc13-4 .", "Jx refers to mice homozygous for the Unc13dJinxmutation ( Crozat et al . , 2007 ) .", "( F ) Munc13-1 and Baiap3 are mainly located in the medulla ( Med ) , but ubMunc13-2 is present in cortex ( Cort ) as well .", "Please note that the difference in the position of ubMunc13-2 relative to the marker in panels ( B ) and ( F ) is due to how far the respective gels were run .", "Loading controls were valosin-containing protein ( VCP ) , glyceraldehyde-3-phosphate dehydrogenase ( GAPDH ) and CgA .", "Brain samples , and spleen tissue in the case of Munc13-4 , were used for comparison .", "Please note that the Baiap3KO ( Wojcik et al . , 2013 ) and Unc13aKO animals express truncated protein products , whereas the truncated product present in the Unc13bKO ( Cooper et al . , 2012 ) is not shown here .", "Based on previous analyses of the Unc13aKO and Unc13bKO mice ( Augustin et al . , 1999; Cooper et al . , 2012; Varoqueaux et al . , 2002 ) , the truncated Munc13-1 and Munc13-2 products are neither functional , nor do they have a dominant-negative effect .", "The truncated Baiap3 product was not detected in adrenal gland , and its effect in the brain , where it can be detected in young animals up to P21 , is currently unknown ( Wojcik et al . , 2013 ) .", "See also Figure 1—figure supplement 1 . DOI: http://dx . doi . org/10 . 7554/eLife . 10635 . 00310 . 7554/eLife . 10635 . 004Figure 1—figure supplement 1 . Comparison of Munc13-1 , Munc13-2 , and Munc13-3 expression .", "( A ) Adrenal gland and brain homogenates from Unc13a-EYFP , Unc13b-EYFP , and Unc13c-EGFP knock-in ( KI ) mice were probed using an antibody that recognizes the GFP-derived tags , which allowed us to compare expression levels of Munc13-1 , ubMunc13-2 , bMunc13-2 and Munc13-3 with the same antibody .", "The two bands detected in the Unc13b-EYFP KI brain sample represent ubMunc13-2 ( lower band ) and bMunc13-2 ( upper band ) .", "While all isoforms were detected in brain , ubMunc13-2-EYFP was the only isoform readily detectable in the adrenal gland .", "Valosin-containing protein ( VCP ) was used as a loading control .", "( B ) Adrenal gland and brain samples from Unc13aKOUnc13bKO ( DKO ) mice and WT controls , probed with antibodies to Munc13-1 , ubMunc13-2 and VCP as a loading control .", "Expression of Munc13-1 ( marked with * , to distinguish it from co-migrating bands of higher molecular weight ) is low in E18 adrenal glands , while ubMunc13-2 can be readily detected .", "The expression of both isoforms was abrogated in DKO mice .", "Please note that , like the single KO lines , the DKO animals express truncated Munc13-1 protein products and a truncated Munc13-2 product ( Cooper et al . , 2012 ) , the latter is not shown here . DOI: http://dx . doi . org/10 . 7554/eLife . 10635 . 004 To assess whether the isoforms detected in whole gland homogenates are present in the adrenal medulla , and/or the adrenal cortex , we dissected adult wild-type ( WT ) adrenal glands , and used an antibody to the LDCV marker Chromogranin A ( CgA ) to monitor effective separation of the medullary tissue , which consists mostly of chromaffin cells , from cortical tissue ( Figure 1F ) .", "The expression of Munc13-1 and Baiap3 in the adrenal gland is largely restricted to the medulla .", "Expression of ubMunc13-2 was detected in both adrenal medulla and cortex .", "Thus , a significant fraction of the ubMunc13-2 signal detected in whole gland homogenates ( Figure 1B ) appears to originate from the adrenal cortex , possibly due to innervation of the cortex by ubMunc13-2 positive synapses .", "Next , we analyzed cultured chromaffin cells from knockout ( KO ) mice deficient for the individual Munc13 isoforms ( Figure 2 ) .", "LDCV exocytosis was triggered using flash photolysis of caged Ca2+ , which causes a sharp global increase in intracellular [Ca2+] ( Neher , 2006 ) .", "Fusion of LDCVs with the plasma membrane was monitored by measurement of the membrane capacitance change ( △Cm ) .", "Fitting a sum of three exponentials to the exocytotic burst of each individual trace identifies the amplitudes and time constants of release , which are generally interpreted as two kinetically distinct vesicle pools , the fast burst as the Readily-Releasable Pool ( RRP ) , and the slow burst as the Slowly-Releasable Pool ( SRP ) ( Sorensen et al . , 2003a; Voets , 2000 ) .", "However , as will be discussed later , the slow burst component may in fact not be a releasable pool , but instead represent the conversion from a Non-Releasable Pool ( NRP ) , to the RRP ( Walter et al . , 2013 ) .", "The rate of sustained release was measured as a linear component after the exocytotic burst , and reflects the ongoing recruitment of LDCVs into the NRP/SRP and RRP .", "Deletion of Munc13-1 ( Unc13a ) , the major Munc13 isoform in SV exocytosis ( Augustin et al . , 1999; Varoqueaux et al . , 2002 ) , did not markedly alter LDCV exocytosis compared to WT littermate controls ( Figure 2A , D ) , nor did it affect the kinetics of the exocytotic burst ( Figure 2D ) . 10 . 7554/eLife . 10635 . 005Figure 2 . Flash photolysis induced LDCV exocytosis in chromaffin cells . For each KO line , the average intracellular [Ca2+] ± SEM and the average △Cm are shown in panels ( A–C , E ) .", "Single gene deletions of ( A ) Unc13a , ( B ) Unc13c , or ( C ) Baiap3 did not impair LDCV exocytosis .", "( D ) Summary of burst sizes , sustained release rates , and time constants .", "( E ) LDCV exocytosis is dramatically reduced in Unc13aKOUnc13bKO cells , Unc13aWTUnc13bKO cells , and Unc13aHetUnc13bKO cells .", "This reduction is primarily due to the absence of ubMunc13-2 .", "( F ) Fast burst , slow burst and the rate of sustained release are reduced in the absence of Munc13-1 and ubMunc13-2 , as well as in the absence of ubMunc13-2 alone ( ANOVA with post-hoc Tukey’s test ) .", "( G ) Compared to Unc13aWTUnc13bKO cells , the deletion of Munc13-1 causes significant reductions in the slow burst and the rate of sustained release ( Student’s t-test , two-tailed ) .", "( H ) Delay of the onset of exocytosis after the flash stimulus ( ANOVA with post-hoc Tukey’s test ) .", "( I ) Normalized traces show identical release kinetics of the exocytotic burst .", "( J ) Summary of the release components shown in panels ( E , F , G ) .", "Time constants are not significantly different ( ANOVA with post-hoc Tukey’s test ) .", "( *p < 0 . 05 , **p < 0 . 01 , ***p < 0 . 001 ) .", "See also Figure 2—figure supplement 1 . DOI: http://dx . doi . org/10 . 7554/eLife . 10635 . 00510 . 7554/eLife . 10635 . 006Figure 2—figure supplement 1 . Single gene deletion or overexpression of Baiap3 does not affect LDCV exocytosis in chromaffin cells .", "( A ) LDCV exocytosis was elicited by a series of depolarizing steps to analyze the Immediately-Releasable Pool ( IRP ) of vesicles localized in the vicinity of Ca2+ channels and the Readily-Releasable Pool ( RRP ) .", "Shown are the averaged [Ca2+] ± SEM , ΔCm , and whole-cell current traces .", "The inset in the whole-cell current panel shows an enlargement of the first 100 ms depolarization .", "( B ) The size of the IRP , estimated based on the release elicited by 6 short ( 10 ms ) depolarization pulses , was not altered .", "( C ) The total ΔCm caused by the depolarization train was also not altered in Baiap3KO cells .", "Note that the data from Baiap3KO and Baiap3WT were matched for equal ionic influx according to the ionic charge of the first 100 ms depolarization to eliminate effects of differential ionic influx .", "( D ) Overexpression ( OE ) of Baiap3 in WT chromaffin cells .", "EGFP expression in WT cells was used as control .", "Shown are the averaged [Ca2+] ± SEM and ΔCm traces .", "( E ) Burst sizes , rate of sustained release and the kinetics of release were not altered by the overexpression of Baiap3 ( Student’s t-test , two-tailed ) .", "( F ) Normalized traces of Baiap3WT and Baiap3KO cells overlapped with each other , indicating no alterations in release kinetics in Baiap3KO cells . DOI: http://dx . doi . org/10 . 7554/eLife . 10635 . 006 Although we did not detect Munc13-3 in the adrenal gland , we wanted to rule out possible physiological effects of protein expression below the detection limit of Western blot analysis ( Figure 1C ) , and included Unc13cKO mice in our analysis .", "However , as expected , LDCV exocytosis in Unc13cKO chromaffin cells was not perturbed ( Figure 2B , D ) .", "We then investigated the possible role of Baiap3 in LDCV exocytosis , as this isoform is prominently expressed in the adrenal medulla ( Figure 1D , F ) .", "Surprisingly , LDCV exocytosis in Baiap3KO cells was intact ( Figure 2C , D ) .", "Furthermore , Baiap3KO cells also did not show a release deficit when we stimulated the cells using a series of depolarization steps ( Figure 2—figure supplement 1A–C ) , nor did overexpression of Baiap3 in WT cells affect LDCV exocytosis ( Figure 2—figure supplement 1D–F ) .", "We then analyzed the role of ubMunc13-2 and Munc13-1 in chromaffin cell LDCV exocytosis .", "For this purpose , we used an Unc13a/b ( DKO ) mouse line .", "Heterozygous ( Het ) animals of this line express ~50% of WT levels of Munc13-1 and Munc13-2 , which does not affect neurotransmission ( Augustin et al . , 1999; Varoqueaux et al . , 2002 ) .", "Data were collected from genotype groups available for a given litter and were pooled for analysis .", "Because our breeding scheme did not produce littermate WT animals in sufficient numbers , and because deletion of Unc13a alone was without effect , data from Unc13aWTUnc13bHet and Unc13aHetUnc13bHet cells were pooled and used as control ( Figure 2E–J , Unc13aWT/HetUnc13bHet ) .", "Deletion of both Unc13a alleles together with a single Unc13b allele ( Unc13aKOUnc13bHet ) did not reduce LDCV release ( Figure 2E , F ) .", "By contrast , abrogation of ubMunc13-2 expression alone , irrespective of the Unc13a genotype , drastically diminished release ( Figure 2E , F ) .", "Furthermore , in the context of the Unc13bKO background , cells with Unc13aWT , Unc13aHet , and Unc13aKO genotypes showed a progressive reduction of LDCV release that depended on the number of Unc13a alleles present ( Figure 2F , G ) .", "The fast and slow burst components were reduced to 39% , 32% , and 27% , and to 54% , 52% , and 42% of control levels , respectively ( Figure 2F ) .", "The rate of sustained release was reduced even more dramatically , to 26% , 19% , and 12% of control levels ( Figure 2F ) .", "When one uses the Unc13aWTUnc13bKO genotype as a reference point ( Figure 2G ) , the deletion of Unc13a caused a reduction of the sustained release component to 48% .", "The rate of sustained release of Unc13aKOUnc13bKO cells was also significantly reduced when compared to Unc13aHetUnc13bKO cells .", "The deletion of both Unc13a and Unc13b significantly delayed the onset of vesicular exocytosis triggered by flash photolysis , compared to control and Unc13aKOUnc13bHet cells ( Figure 2H ) .", "Unc13aWTUnc13bKO cells also showed a mild increase in delay .", "However , this difference was significant only when compared to Unc13aKOUnc13bHet cells , but not compared to the other groups .", "Thus , ubMunc13-2 , the only isoform expressed from the Unc13b gene in mouse chromaffin cells , is the most critical isoform for LDCV release in this cell type .", "Moreover , in its absence it becomes apparent that endogenous Munc13-1 also regulates LDCV release in this cell type .", "We next assessed whether ubMunc13-2 affects LDCV release in response to Ca2+ entry through voltage-gated Ca2+ channels by stimulating the cells with a series of depolarization steps ( Figure 3 ) .", "The first six short depolarizations of the train release the Immediately-Releasable Pool ( IRP ) , that is , the subset of RRP vesicles located closest to Ca2+-channels ( Schonn et al . , 2010; Voets et al . , 1999 ) .", "We found a significant reduction in LDCV release; the size of the RRP in Unc13bKO cells was reduced to 53% of WT levels ( Figure 3C ) .", "This deficit is somewhat less pronounced than the reduction seen in the flash photolysis experiment ( reduction to 39% , Figure 2F ) , most likely because the depolarization protocol used to obtain the data shown in Figure 3 lasts several seconds and therefore causes some ongoing recovery of the RRP .", "By contrast , in flash photolysis experiments , the RRP is probed within ∼60 ms ( 3 times the time constant ) , which is much faster than the recovery of the RRP .", "Strikingly , in the depolarization experiment , impaired release in Unc13bKO cells was already evident in response to the first 10-ms depolarization ( Figure 3B ) , which implies that lack of ubMunc13-2 would even affect resting level catecholamine release driven by low frequency stimulation ( Zhou and Misler , 1995 ) . 10 . 7554/eLife . 10635 . 007Figure 3 . Absence of Munc13-2 results in a significant release deficit in response to depolarization .", "( A ) Shown are the averaged [Ca2+] ± SEM , △Cm , and whole-cell current traces of Unc13bWT and Unc13bKO cells .", "The inset in the whole-cell current panel shows an enlargement of the first 100 ms depolarization .", "( B ) △Cm elicited by individual depolarizations was significantly different between the two groups .", "RRP , Readily-releasable Pool; IRP , Immediately-Releasable Pool .", "( C ) The size of the RRP was measured as the △Cm after the train of depolarization pulses and was significantly reduced in Unc13bKO cells .", "( *p < 0 . 05 , **p < 0 . 01 , ***p < 0 . 001; Student’s t-test , two-tailed ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 10635 . 007 To understand how ubMunc13-2 affects the kinetics of single catecholamine release events , we performed single spike amperometry while infusing the cells with a solution with moderate ( ∼4 . 6 µM ) [Ca2+] ( Figure 4 ) .", "Unc13bKO cells showed a dramatic reduction in spike frequency ( Figure 4B , C ) , whereas basic spike parameters such as duration , half-width , maximum amplitude , charge , rise time , and decay time were unchanged ( Figure 4D–I ) .", "Amplitude , duration and charge of the spike foot signal , which is thought to reflect release during the initial formation of the fusion pore prior to full fusion , were also unchanged ( Figure 4J–L ) .", "However , we found that the number of spikes that did show these foot signals was slightly reduced in Unc13bKO cells ( Figure 4M ) , which may indicate that fusion pore dynamics are altered for some release events .", "However , overall , the LDCVs undergoing fusion in the absence of ubMunc13-2 do so without major alterations in fusion kinetics or vesicle content . 10 . 7554/eLife . 10635 . 008Figure 4 . Reduced number of fusion events of catecholamine-containing LDCVs in the absence of Munc13-2 . ( A ) Illustration of a single amperometric spike , corresponding to the release of catecholamines from a single LDCV , and the parameters analyzed .", "( B ) Representative amperometric recordings of a Unc13bWT and a Unc13bKO cell .", "( C ) Dramatic reduction in spike frequency in Unc13bKO chromaffin cells .", "( D ) Spike duration , ( E ) width at half amplitude ( t½ ) , ( F ) maximum spike amplitude , ( G ) amperometric charge , ( H ) rise time , and ( I ) decay time were unchanged in the Unc13bKO .", "The stability of fusion pores was also not altered , as shown by the unchanged ( J ) foot amplitude , ( K ) duration and ( L ) charge .", "( M ) The fraction of amperometric spikes with a detectable foot was reduced in Unc13bKO cells .", "( *p < 0 . 05 , ***p < 0 . 001; Student’s t-test , two-tailed ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 10635 . 008 As our experiments so far showed that , with the exception of Baiap3 , the contribution of the Munc13 isoforms to the regulation of LDCV release correlates with their level of expression in perinatal adrenal glands , we next wanted to compare the intrinsic properties of the different isoforms .", "To this end , we overexpressed Munc13-1 , ubMunc13-2 , Baiap3 , and its closest relative Munc13-4 using Semliki Forest Virus ( SFV ) in Unc13aKOUnc13bKO cells .", "Munc13-1 and ubMunc13-2 were expressed as EGFP fusion constructs whose functions are identical to those of the respective WT proteins ( Rosenmund et al . , 2002 ) , whereas Baiap3 and Munc13-4 were expressed as internal ribosome entry site ( IRES ) -EGFP constructs , to avoid possible confounding effects of a fusion tag .", "Unc13aKOUnc13bKO cells expressing only EGFP were used as control .", "For the purpose of comparison , the averaged traces obtained from the rescue experiments with the four isoforms were plotted in the same graph ( Figure 5A ) .", "The exocytotic burst was measured as the △Cm within the first 0 . 5 s after the flash stimulus , and the rate of sustained release was measured as the △Cm between 0 . 5 s and 4 s after the flash ( Figure 5B ) .", "Interestingly , Munc13-1 and ubMunc13-2 were both able to rescue the LDCV release deficit of Unc13aKOUnc13bKO cells ( Figure 5A , B ) .", "However , rescue with ubMunc13-2 resulted in an enormous enhancement of LDCV exocytosis to levels that by far exceeded the amount of exocytosis typical of WT cells , for both the exocytotic burst and the rate of sustained release ( Figure 5A , B ) .", "The direct comparison of Munc13-1 and ubMunc13-2 expressing cells with matching EGFP fluorescence intensity confirmed that the stronger enhancement of burst size and rate of sustained release in ubMunc13-2 expressing cells was not due to higher expression levels of ubMunc13-2 ( Figure 5—figure supplement 1 ) . 10 . 7554/eLife . 10635 . 009Figure 5 . LDCV exocytosis in Unc13aKOUnc13bKO chromaffin cells is rescued by overexpression ( OE ) of Munc13-1 , ubMunc13-2 and Munc13-4 , but not Baiap3 . Unc13aKOUnc13bKO cells were infected with SFV-Munc13-1-EGFP , SFV-ubMunc13-2-EGFP , SFV-Munc13-4-IRES-EGFP or SFV-Baiap3-IRES-EGFP using SFV-EGFP as control .", "( A ) Averaged [Ca2+] ± SEM and capacitance traces △Cm are shown in the same graph to compare the efficiency of rescue: ubMunc13-2 > Munc13-1 > Munc13-4 > Baiap3 .", "( B ) Burst sizes and rates of sustained release after analysis of individual traces .", "( *p < 0 . 05 , ***p < 0 . 001; Student’s t-test , two-tailed ) .", "See Figure 5—figure supplement 1 for a direct comparison of Munc13-1-EGFP- and ubMunc13-2-EGFP-expressing cell matched for fluorescence intensity , and Figure 5—figure supplement 2 for western blotting to confirm the expression of Munc13-4 and Baiap3 from the IRES-constructs . DOI: http://dx . doi . org/10 . 7554/eLife . 10635 . 00910 . 7554/eLife . 10635 . 010Figure 5—figure supplement 1 . Direct comparison of Munc13-1 and ubMunc13-2 over-expressing cells with matching EGFP fluorescence .", "( A ) Averaged [Ca2+] ± SEM and capacitance traces △Cm are shown in the same graph to compare the efficiency of rescue .", "( B ) Fluorescence intensity in arbitrary units ( a . u . ) of Unc13aKOUnc13bKO chromaffin cells overexpressing Munc13-1-EGFP and ubMunc13-2-EGFP .", "( C ) Expression of ubMunc13-2 resulted in a significantly larger enhancement of burst size and rate of sustained release than expression of Munc13-1 .", "( **p < 0 . 01 , p < 0 . 001; Student’s t-test , two-tailed ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 10635 . 01010 . 7554/eLife . 10635 . 011Figure 5—figure supplement 2 . Western blot analysis confirming expression of Munc13-4 and Baiap3 SFV constructs . Neuronal cultures were infected with SFV-Baiap3-IRES-EGFP and SFV-Munc13-4-IRES-EGFP , respectively .", "Tissue samples from Baiap3 and Unc13d ( Jx ) WT and KO mice were used as controls .", "Both Baiap3 and Munc13-4 were expressed .", "Please note that we had to use infected neurons for this analysis , because our chromaffin cell cultures do not provide enough material for western blotting . DOI: http://dx . doi . org/10 . 7554/eLife . 10635 . 011 Overexpression of Baiap3 failed to rescue the LDCV release deficit of the Unc13aKOUnc13bKO chromaffin cells ( Figure 5A , B ) .", "Yet , its closest relative , Munc13-4 , which regulates SNARE-mediated vesicular exocytosis in the hematopoietic system ( Feldmann et al . , 2003; Shirakawa et al . , 2004 ) , was able to rescue LDCV exocytosis in Unc13aKOUnc13bKO cells , albeit less efficiently than Munc13-1 or ubMunc13-2 ( Figure 5A , B ) .", "To exclude the possibility that these findings might be due to inefficient translation of Baiap3 and Munc13-4 , protein expression was confirmed with isoform-specific antibodies in SFV-infected neuronal cultures , which provide enough material for Western blot analysis ( Figure 5—figure supplement 2 ) .", "Thus , individual Munc13 isoforms appear to show inherent differences in their ability to promote LDCV release in chromaffin cells .", "We went on to investigate whether Unc13aKOUnc13bKO chromaffin cells show an LDCV docking defect analogous to the SV docking defect seen in Unc13aKOUnc13bKO synapses ( Siksou et al . , 2009 ) .", "SV docking deficits of Munc13/Unc-13 deficient synapses in mice and C . elegans were previously only detected when rapid cryo-fixation methods were employed instead of classical chemical fixation for ultrastructural analysis ( Siksou et al . , 2009; Weimer et al . , 2006 ) .", "Moreover , it has been shown that 3D electron tomography ( ET ) allows a more accurate assessment of SV docking at the active zone ( Imig et al . , 2014; Siksou et al . , 2009 ) .", "To study LDCV recruitment and docking in chromaffin cells , we therefore combined high-pressure freezing ( HPF ) and freeze-substitution of acute adrenal gland slices with classical 2D-EM ( Figure 6A–E ) and high-resolution 3D-ET analyses ( Figure 6F–N ) .", "Quantitative analysis of 2D-EM images of Unc13aHetUnc13bHet ( Figure 6A ) and Unc13aKOUnc13bKO chromaffin cells ( Figure 6B ) did not reveal any differences in LDCV distribution within 2 μm of the plasma membrane ( PM ) ( Figure 6C ) , in the number of membrane-proximal LDCVs within 40 nm of the PM ( Figure 6D ) , or in the total number of LDCVs ( Figure 6E ) .", "LDCV docking and recruitment into the vicinity of the PM were assessed using 3D-ET ( Figure 6F–N ) .", "From all LDCVs analyzed within 100 nm of the PM , the percentage of membrane-proximal LDCVs ( 0–40 nm ) ( Figure 6M ) and their distribution ( Figure 6L ) was unaltered between both groups , indicating that LDCV recruitment to the PM is intact in Munc13-deficient chromaffin cells .", "The number of docked LDCVs , defined as LDCVs in physical contact with the PM and assigned to the 0–4 nm bin in Figure 6L , and the number of docked LDCVs normalized to the number of membrane-proximal LDCVs ( Figure 6N ) were unchanged .", "Furthermore , the average LDCV diameter of docked or non-docked LDCVs , measured by 3D-ET , did not differ significantly between genotypes , although Unc13aKOUnc13bKO LDCVs tended to be smaller ( Figure 6—figure supplement 1 ) .", "Thus , in spite of the dramatic release deficit seen in Unc13aKOUnc13bKO chromaffin cells and the dramatic SV docking deficit seen in neurons of this genotype ( Siksou et al . , 2009 ) , we did not detect any changes in LDCV docking , nor a loss or accumulation of LDCVs in the vicinity of the PM .", "Thus , chromaffin cells can generate what appears to be a full-sized pool of morphologically docked LDCVs in the absence of Munc13-1 and Munc13-2 , which implies that the molecular requirements of morphological LDCV and SV docking are distinct .", "Additionally , this could either indicate that the mechanism of functional docking , that is , priming , differs between LDCV and SVs as well , or else , that the primed LDCVs ( i . e . , those that belong to the RRP ) are in the minority among the docked vesicles and therefore cannot be detected .", "To distinguish between these two possibilities , we estimated the total number of docked LDCVs per cell .", "To this end , we re-calculated the percentage of docked vesicles identified using 3D-ET , as the percentage of membrane proximal vesicles ( 0–40 nm ) identified in the 2D-EM analysis and converted LDCVs/μm PM to LDCVs/cell as described ( Parsons et al . , 1995 ) .", "This conversion was necessary due to the limited volume sizes analyzed by 3D-ET and the uneven distribution of LDCVs within the cells .", "The estimated size of the morphologically docked pool was ∼662 LDCVs per chromaffin cell in control cells .", "For Unc13aKOUnc13bKO cells we calculated ∼865 docked LDCVs per cell , which can be accounted for by two factors used in the calculation: Unc13aKOUnc13bKO cells are slightly larger , and their LDCVs are slightly smaller ( Figure 6—figure supplement 1 ) .", "Both values are lower than the previously reported ∼1607 for embryonic day ( E ) 18 murine chromaffin cells ( de Wit , 2010 ) , presumably reflecting improved discrimination between docked and undocked vesicles by 3D-ET .", "With a diameter of a docked LDCV of ∼170 nm ( Figure 6—figure supplement 1E ) and assuming a specific membrane capacitance of 1 μF/cm2 , this corresponds to a vesicular capacitance of 0 . 91 fF , in excellent agreement with recent electrophysiological measurements of 0 . 94 fF ( Pinheiro et al . , 2014 ) .", "Thereby , the size of the RRP , which is <40 fF at resting [Ca2+] ( Voets , 2000 ) , corresponds to <44 vesicles from the total of ∼662 in control cells , indicating that even with 3D-ET , the RRP will be very hard or even impossible to distinguish morphologically from other docked vesicles in adrenal chromaffin cells . 10 . 7554/eLife . 10635 . 012Figure 6 . Ultrastructural analysis of LDCV docking in adrenal chromaffin cells . 2D-EM of ( A ) Unc13aHetUnc13bHet ( CTRL ) and ( B ) Unc13aKOUnc13bKO ( DKO ) adrenal glands .", "( C ) Frequency distribution of LDCVs within 0–2 µm of the plasma membrane ( PM ) .", "( D ) Membrane-proximal LDCVs ( 0–40 nm of PM ) normalized to PM circumference .", "( E ) Total number of LDCVs normalized to cytoplasmic area .", "( F–K )", "Tomographically reconstructed subvolumes from 400 nm-thick sections through ( F–H ) Unc13aHetUnc13bHet and ( I–K ) Unc13aKOUnc13bKO cells in which docked LDCVs ( enlarged in panels G , J ) and undocked LDCVs ( enlarged in panels H and K , small gaps separating undocked LDCVs from the PM indicated with arrowheads ) can be distinguished .", "( L ) Frequency distribution of membrane-proximal LDCVs distributed within 0–40 nm of the PM .", "( M ) Number of membrane-proximal LDCVs expressed as a percentage of all LDCVs within 0–100 nm of the plasma membrane .", "( N ) Percentage of docked LDCVs with respect to all membrane-proximal LDCVs ( within 0–40 nm of PM ) .", "Scale bars represent 1 µm in ( A , B ) , 200 nm in ( F , I ) , and 50 nm in ( H , K ) .", "C: 5779 LDCVs in CTRL and 4120 LDCVs in DKO profiles .", "D , E: CTRL: N=2 , n=36; DKO: N=2; n=27 .", "L: 473 LDCVs in CTRL and 386 LDCVs in DKO tomographic subvolumes .", "M , N: CTRL: N=2 , n=24;DKO: N=2 , n=26 .", "Values indicate mean ± SEM .", "( Student’s t-test , two-tailed ) .", "See also Figure 6—figure supplement 1 . DOI: http://dx . doi . org/10 . 7554/eLife . 10635 . 01210 . 7554/eLife . 10635 . 013Figure 6—figure supplement 1 . Ultrastructural analysis of LDCV size in adrenal chromaffin cells . 2D-EM of ( A ) Unc13aHetUnc13bHet ( CTRL ) and ( B ) Unc13aKOUnc13bKO ( DKO ) adrenal glands .", "( C ) Frequency distribution of LDCV diameters measured by 3D-ET .", "( D ) Mean LDCV diameter in randomly imaged chromaffin cell areas .", "( E ) LDCV diameter of docked ( 0–4 nm of PM ) vesicles .", "Scale bar in B represents 5 µm .", "C: 846 LDCVs in CTRL and 831 LDCVs in DKO tomographic subvolumes .", "D: CTRL: N=2 , n=24; DKO: N=2 , n=26 .", "E: CTRL: N=2 , n=98; DKO: N=2 , n=140 .", "Values indicate mean ± SEM .", "( Student’s t-test , two-tailed; diameters of docked LDCVs were tested with Mann-Whitney U-test ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 10635 . 013 Thus far , our data indicate that although morphological docking of LDCVs does not require Munc13s , the priming of a functional RRP does .", "We therefore wanted to identify the Munc13-sensitive step in the LDCV priming process , and compare the intrinsic properties of Munc13-1 and ubMunc13-2 , the two most relevant isoforms in the adrenal medulla .", "In a recently published mathematical model for LDCV fusion , we showed that the fast and slow bursts of release originate from two serially arranged pools of vesicles , the RRP and the non-releasable NRP , respectively ( Walter et al . , 2013 ) ( Figure 7 ) .", "The NRP in turn is refilled from a larger depot pool .", "Thus , the model features two separate priming steps ( Liu et al . , 2010 ) , but only one fusion pathway ( Figure 7E ) .", "Since the deletion of Unc13a and Unc13b changed the fast and slow burst to nearly the same degree ( Figure 2F ) , within the framework of this model , Munc13s must act upstream of the NRP .", "Furthermore , since the sustained release rate is changed proportionally as well , Munc13s likely act to accelerate the forward priming rate , k1 ( Ashery et al . , 2000 ) .", "In most models , this rate constant k1 is Ca2+-dependent ( Voets , 2000; Walter et al . , 2013 ) , and confers overall Ca2+-dependence to the primed vesicle pool .", "We next investigated how the two relevant Munc13-isoforms in chromaffin cells ( ubMunc13-2 , Munc13-1 ) affect this priming step . 10 . 7554/eLife . 10635 . 014Figure 7 . Munc13-1 and ubMunc13-2 accelerate upstream vesicle priming ( 'priming step 1' ) with identical Ca2+ affinities , but distinct rates .", "( A ) Estimation of steady-state Ca2+ affinities of vesicle priming driven by Munc13-1 or ubMunc13-2 .", "Left two panels: binned and averaged secretory responses in Unc13aKOUnc13bKO cells overexpressing ( OE ) either Munc13-1 ( green ) or ubMunc13-2 ( blue ) .", "The release fraction at 30 ms ( traces normalized to their amplitude after 3 s ) after the stimulus was determined as the read-out for priming ( vertical broken lines ) .", "Right panel: the fraction of release plotted as a function of pre-flash [Ca2+] .", "Both genotypes are described by the same Hill function , suggesting a similar Ca2+-dependence of priming ( i . e . identical cooperativity and affinity ) .", "( B ) Fits of a secretion model ( see panel E ) to the capacitance responses observed experimentally in Unc13aKOUnc13bKO cells expressing Munc13-1 at intermediate and low pre-flash [Ca2+] .", "Top panel: measured Ca2+ values were used to drive the secretion model .", "The chemical equation shows a priming sensor ( PS ) , which is active in the Ca2+ bound state ( CanPS ) .", "The lower panel shows the experimental capacitance data ( solid lines ) together with simulations with the best fit parameters ( broken lines; see Table 1 ) .", "Insert: magnified view with horizontal/vertical scale bars: 200 ms/200 fF .", "( C ) Same as ( B ) , but for Unc13aKOUnc13bKO cells expressing ubMunc13-2 .", "The secretion model was fitted using identical KDs and n for the Ca2+ binding to the PS ( determined by the analysis shown in panel A ) .", "The best-fit values suggest a ∼2 . 5-fold slower on-rate ( activation rate ) , but a 4 . 5-fold higher maximal priming rate for ubMunc13-2 ( see Table 1 ) , resulting in a sigmoidal secretion response from low pre-flash [Ca2+] .", "( D ) Fitting our secretion model to the experimental data of several genotypes suggests that Munc13-1 and ubMunc13-2 both primarily act by increasing the forward priming rate ( k1 , see also panel E ) , while loss of ubMunc13-2 – the dominant endogenous isoform – has the opposite effect .", "The downstream priming step ( 'priming 2' , k2 ) changes in the opposite direction .", "( E ) Secretion model: Munc13-1 ( green ) and ubMunc13-2 ( blue ) regulate the Ca2+ binding rates ( kon ) to a PS , which controls the asymptotic forward priming rate k1 ( see text for details , Table 1 for fitted parameters , and Walter et al . , 2013 for model development ) .", "NRP: Non-Releasable Pool; RRP: Readily-Releasable Pool; F: Fused pool .", "See also Figure 7—figure supplement 1 . DOI: http://dx . doi . org/10 . 7554/eLife . 10635 . 01410 . 7554/eLife . 10635 . 015Figure 7—figure supplement 1 . Exocytotic burst size as a function of pre-flash [Ca2+] .", "Unc13aKOUnc13bKO cells were rescued by Munc13-1 or ubMunc13-2 overexpression ( OE ) .", "For ubMunc13-2 , data from 23 cells already analyzed for Figure 5 were included here as well .", "The average size of the exocytotic burst is plotted as the ΔCm 0 . 5 s after the flash stimulus ± SEM against average pre-flash [Ca2+] ± SEM .", "Cells were binned according to pre-flash [Ca2+] .", "For Munc13-1 , bins are 250–350 nM ( n = 17 ) , 350–450 nM ( n = 15 ) , 450–550 nM ( n = 11 ) , 550–800 nM ( n = 11 ) , 800–1200 nM ( n = 13 ) , 1200–1700 nM ( n = 7 ) .", "For ubMunc13-2 , bins are 250–350 nM ( n = 11 ) , 350–450 nM ( n = 20 ) , 450–540 nM ( n = 10 ) , 540–800 nM ( n = 12 ) , 800–1200 nM ( n = 10 ) and 1200–1700 nM ( n = 4 ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 10635 . 015 The Ca2+-dependence of LDCV-priming ( essentially k1 in Figure 7E ) can be assessed in an experiment by varying the pre-flash intracellular [Ca2+] , before an uncaging flash is used to probe the size of the primed vesicle pool ( Voets , 2000 ) .", "We expressed either Munc13-1 or ubMunc13-2 in Unc13aKOUnc13bKO cells and extended the range of pre-flash [Ca2+] values from the previously used 300–600 nM ( Figure 2 and Figure 5 ) to 250–1200 nM ( Figure 7A and Figure 7—figure supplement 1 ) .", "In order to compare the respective Ca2+-sensitivities rather than the absolute priming rates of Munc13-1 and ubMunc13-2 – and to overcome cell-to-cell variability – we normalized capacitance traces to their value after 3 s ( Figure 7A , left and middle panels ) .", "Using the fractional increase in capacitance after 30 ms as a read-out of the primed vesicle pool , we identified the characteristic Ca2+-dependence of priming .", "Strikingly , the Ca2+-dependence was almost identical for the two isoforms and could be fitted with a single Hill equation ( Figure 7A right-hand panel , Table 1 ) . 10 . 7554/eLife . 10635 . 016Table 1 . Parameters of the exocytosis model . DOI: http://dx . doi . org/10 . 7554/eLife . 10635 . 016ParameterControlUnc13bKOMunc13-1 OEubMunc13-2 OECommentVtot2350Total number of vesicles , best fitk1p ( Ca2+ ) ·k1maxp ( Ca2+ ) ( Ca2+ ) n ( Ca2+ ) n+ ( KD , cat ) nfraction of activated PSkon8 . 96*10-9 s-1 µM-n2 . 25*10-8 s-1 µM-n2 . 25*10-8 s-1 µM-n8 . 96*10-9 s-1 µM-nOn-rate calcium binding to PSk1Max1 . 99*10-2 s-16 . 89*10-3 s-17 . 44*10-2 s-13 . 42*10-1 s-1Maximal priming rate , best fitKDn0 . 407 µMExperiment , Hill plot Figure 7k-14 . 70*10-1 s-1Best fitn7 . 38Cooperativity PS , experiment , Hill plot Figure 7k2k20+gCa2+·k2cat ( Walter et al . , 2013 ) k-2k-20+gCa2+·k-2cat ( Walter et al . , 2013 ) g ( Ca2+ ) Ca2+Ca2++KD , cat ( Walter et al . , 2013 ) k202 . 37*10-2 s-12 . 95*10-2 s-11 . 29*10-2 s-15 . 80*10-3 s-1Best fitk2cat3 . 95*101 s-14 . 91*101 s-12 . 14*101 s-19 . 65*100 s-1Best fitk-202 . 10*10-2 s-1Best fitk-2cat=k2cat · k-20/k20 ( Walter et al . , 2013 ) KD , cat138 µMBest fitk34 . 4 s-1μM-1 ( Voets , 2000 ) k-356 s-1 ( Voets , 2000 ) k41450 s-1 ( Voets , 2000 ) Thus , Ca2+-dependent priming is supported with identical steady-state affinities in the presence of Munc13-1 or ubMunc13-2 .", "However , when applying Ca2+-uncaging flashes from a relatively low pre-flash [Ca2+] , the two isoforms induce quite different secretion kinetics ( Figure 7B , C ) .", "For ubMunc13-2 , secretion shows a clear sigmoid shape with acceleration after ∼0 . 5 s ( Figure 7C ) , which is absent when the pre-flash [Ca2+] is higher .", "This sigmoid shape of ubMunc13-2 driven secretion was noted before and was attributed to a slow association of ubMunc13-2 with Calmodulin and Ca2+ , resulting in a slow 'priming switch' ( Zikich et al . , 2008 ) .", "In contrast , Munc13-1 does not show this secondary acceleration ( Figure 7B ) , regardless of the pre-flash [Ca2+] .", "To understand the origin of this behavior , we modeled the Ca2+ association with the priming sensor ( PS ) explicitly – in our previous model ( Walter et al . , 2013 ) , this step had been assumed to be always in equilibrium .", "In accordance with the observed identical steady state Ca2+ dependencies ( Figure 7A ) , we used identical dissociation constant ( KD ) values for both isoforms , and varied only the on-rate , kon , ( the off-rate was changed simultaneously , koff = KD*kon , with constant KD ) .", "This led to a very satisfactory fit to both Munc13-1 and ubMunc13-2 data from both low and high pre-flash [Ca2+] ( Figure 7B , C ) .", "Also , this model made it possible to fit both the control trace , and the Unc13bKO trace ( Figure 7D ) .", "Importantly , the fit was performed simultaneously to all traces , so that we could ensure consistency between fits , simplify the interpretation of parameter changes , and ensure that all conditions could be reproduced by one version of our model ( for fitted parameters , refer to Table 1 ) .", "Our model ( Figure 7E ) assumes that the NRP vesicles can only fuse after maturing to the RRP state ( Walter et al . , 2013 ) .", "Earlier models assumed that the 'NRP pool' can fuse directly via an alternative pathway; in those cases , the corresponding pool was called ‘Slowly Releasable’ ( SRP ) ( Voets , 2000 ) .", "We note that our conclusion that Munc13-1/ubMunc13-2 exert their main effects upstream of both pools ( NRP/SRP and RRP ) , i . e . on k1 , is consistent with both ideas ( see also Ashery et al . , 2000 ) .", "Therefore , our observations here do not necessarily distinguish between the parallel pool model ( SRP and RRP are both releasable ) and the sequential pool model ( only the RRP is releasable ) ; but see ( Walter et al . , 2013 ) for data supporting the sequential pool model .", "The modeling resulted in two main conclusions ( Table 1 ) : First , as expected , the action of Munc13 ( either isoform ) is consistent with an increase in k1 – the forward rate of priming within the first priming step ( Figure 7E ) .", "This is seen both by the increase in the fitted k1 upon overexpression of either isoform , and by the decrease of k1 in the Unc13bKO cells .", "Second , ubMunc13-2 increases k1 4 . 5-fold more than Munc13-1 , but it does so after a longer delay .", "In the model , this delay is due to slower kinetics of Ca2+ binding to the priming sensor ( Zikich et al . , 2008 ) .", "Note that this step might coincide with the translocation of Munc13-1 or ubMunc13-2 to the membrane as a prerequisite for the priming action of the protein .", "Thus , the different delays might reflect differences in the membrane translocation step .", "As a minor note , we also noticed that k2 , the forward rate of downstream priming ( 'priming 2' , Figure 7E ) always changed in the opposite direction of k1 .", "The reason for this is unclear , but one explanation could be that the protein ( s ) driving downstream priming compete with Munc13 for association with the fusion machinery ." ], [ "Our data demonstrate that although Munc13s are critical for functional priming of LDCVs in chromaffin cells , morphological LDCV docking , even when assessed by 3D-ET at unprecedented resolution , is not impaired in the absence of Munc13s ( Figure 6 ) .", "Thus , in contrast to synapses , where most , if not all docked SVs are part of the RRP , the majority of docked LDCVs in chromaffin cells are not primed , and the functional RRP therefore cannot be distinguished from other docked LDCVs by current ultrastructural methods .", "Although we cannot completely exclude the possibility that Munc13-3 and Munc13-4 may be present at very low levels , that we were unable to detect ( Figure 1 ) , it seems unlikely that their presence could account for the full-sized pool of docked LDCVs in Unc13aKOUnc13bKO cells .", "This raises the question of how the non-primed LDCVs are docked .", "SV docking requires the SNARE proteins Stx-1 , SNAP-25 , and Syb-2 , as well as Munc13s and CAPSs , but not necessarily the Ca2+ sensor of fusion , Synaptotagmin-1 ( Syt-1 ) ( Imig et al . , 2014 ) .", "By contrast , current models imply that LDCV docking is mediated by Syt-1 , possibly via interaction with the Stx-1/SNAP-25 acceptor complex ( de Wit et al . , 2006; de Wit et al . , 2009; Parisotto et al . , 2012 ) .", "Additionally , Munc18-1 docks LDCVs via its interaction with the closed form of Stx-1 ( Gandasi and Barg , 2014; Gerber et al . , 2008; Gulyas-Kovacs et al . , 2007; Han et al . , 2011; Voets et al . , 2001 ) and is also involved in an additional tethering step ( Toonen et al . , 2006 ) .", "The vesicular SNAREs seem to be dispensable for docking in chromaffin cells ( Borisovska et al . , 2005; Gerber et al . , 2008 ) , although they have been implicated in PC12 cells ( Wu et al . , 2012 ) .", "Some of these discrepancies are most likely due to methodological and terminological differences as well as to limitations in assessing true membrane attachment .", "However , since we used the same experimental approach previously employed to detect SV docking deficits ( Imig et al . , 2014; Siksou et al . , 2009 ) , our data clearly show that the molecular requirements of SV and LDCV docking are distinct .", "More specifically , while the formation of the docked/primed RRP requires Munc13s in both cases , and thus appears to be mechanistically quite similar for SVs and LDCVs , the non-primed LDCVs in chromaffin cells appear to dock via a separate , Munc13-independent mechanism .", "Our findings are therefore consistent with the following model of LDCV docking and priming:", "( i ) LDCV docking mediated by Munc18-1/Stx-1 , this configuration would be the starting point for Munc13-mediated SNARE complex assembly , i . e . priming ( Ma et al . , 2011; Ma et al . , 2013 ) , and in parallel ,", "( ii ) LDCV docking mediated by a second configuration , that would not be expected to progress to SNARE complex assembly directly or as efficiently , and thus be consistent with the large unprimed , but docked LDCV pool .", "What this second configuration would look like in terms of molecular interactions is less clear .", "Docking via Syt-1/Stx-1/SNAP-25 complexes would be consistent with un-primed docking ( de Wit et al . , 2009 ) .", "This mode of docking would require the assumption that in chromaffin cells , Stx-1/SNAP-25 complexes can escape NSF/SNAP mediated disassembly .", "An additional or alternative mode of un-primed docking may involve the recruitment of vesicles based on the interaction of Syt-1 with phosphatidylinositol 4 , 5-bisphosphate ( PIP2 ) /Stx-1 clusters ( Honigmann et al . , 2013; Park et al . , 2015 ) , although further interactions may be required to achieve close membrane apposition .", "In the model suggested above , we explicitly included only molecular components for which docking deficits have been demonstrated in chromaffin cells .", "However , LDCV docking most likely involves additional factors .", "For instance , the docking of LDCVs to Munc18-1/Stx-1 complexes probably requires the interaction between Munc18 and the vesicle-associated small GTPases Rab3 and Rab27 ( Graham et al . , 2008; Tsuboi and Fukuda , 2006; van Weering et al . , 2007 ) , and additional docking/tethering factors may be involved in docking both primed and un-primed LDCVs to the membrane .", "Our analysis of how Munc13s prime LDCVs for fusion identifies the earliest phase of the priming process as the step most sensitive to Munc13s .", "We interpret our data according to a model that features Munc13 as a Ca2+-sensitive priming protein in a single pathway to LDCV fusion with two serially arranged vesicle states or pools ( NRP and RRP ) ( Figure 7E ) .", "This essentially allows us to describe what was previously interpreted as the release of two kinetically distinct LDCV pools ( SRP and RRP ) , as two sequential priming processes , priming 1 and priming 2 , resulting in only one releasable pool , the RRP ( Walter et al . , 2013 ) .", "According to previous data , the step most sensitive to Munc13s – priming 1 – is also the step affected by mutations designed to interfere with the initiation of N-terminal SNARE complex assembly ( Walter et al . , 2013; Walter et al . , 2010 ) .", "This is in line with a function of Munc13s in initiating SNARE-complex assembly ( Yang et al . , 2015 ) .", "The second priming step may involve a downstream , presumably more C-terminal phase of SNARE-complex assembly , although other options remain open .", "Thus , in the model ( Figure 7E ) , the formation of the NRP , i . e . the step most sensitive to Munc13s , most likely represents the initiation of N-terminal SNARE complex assembly .", "As our study demonstrates , Munc13 isoforms differ in their ability to facilitate LDCV priming ( Figure 5 ) .", "We detected three endogenously expressed isoforms in murine chromaffin cells , Munc13-1 , ubMunc13-2 , and Baiap3 ( Figure 1 ) .", "Baiap3 , somewhat surprisingly given its prominent expression and ability to translocate to membranes in a Ca2+-dependent manner ( Lecat et al . , 2015 ) , does not appear to be involved in LDCV priming in this cell type .", "However , Munc13-4 , which regulates SNARE- mediated vesicle exocytosis in the hematopoietic system ( Boswell et al . , 2012; Feldmann et al . , 2003; Shirakawa et al . , 2004 ) , and is the closest relative of Baiap3 ( Koch et al . , 2000 ) , can promote LDCV priming , albeit less efficiently than Munc13-1 and ubMunc13-2 .", "The two most relevant isoforms , Munc13-1 and ubMunc13-2 , promote LDCV priming with very similar steady-state Ca2+-affinities , but nonetheless confer unique release kinetics depending on the pre-stimulus [Ca2+] .", "Modeling of the secretion kinetics produced by overexpression of Munc13-1 and ubMunc13-2 in Unc13aKOUnc13bKO cells allowed us to isolate the intrinsic properties of each isoform ( Table 1 ) .", "Secretion driven solely by the dominant isoform ubMunc13-2 shows a characteristic sigmoid shape at low pre-stimulus [Ca2+] ( Figure 7C ) ( Zikich et al . , 2008 ) .", "In our secretion model , the best-fit parameters indicate 2 . 5-fold slower sensing of [Ca2+] for ubMunc13-2 , which can however accelerate priming dramatically when [Ca2+] increases .", "Thus , the fitted maximum priming rate ( k1 ) for ubMunc13-2 is 4 . 5-fold higher than for Munc13-1 .", "However , although Munc13-1 is unable to support the same maximum priming rate as ubMunc13-2 , it reacts faster to a change in the [Ca2+] concentration ( Figure 7B ) , which may reflect distinct conformational changes in response to [Ca2+] , and/or differences in a membrane translocation step .", "Thus , neuroendocrine cells can fundamentally modify the kinetics of secretion by expressing different Munc13 isoforms .", "Previous data from autaptic neurons showed that Munc13-1 causes short term depression , whereas ubMunc13-2 causes short-term facilitation ( Rosenmund et al . , 2002 ) , which parallels our findings in chromaffin cells from low basal [Ca2+] , raising the possibility that the functions of different Munc13 isoforms in priming LDCVs and SVs are conserved , even though their role in docking is not .", "Our model therefore provides a theoretical framework for how the molecular properties of priming factors may be linked to the kinetics of exocytosis .", "Although Munc13s have the strongest effect on the priming step 1 , i . e . the formation of the NRP vesicle state , they also influence priming step 2 , i . e . the formation of the RRP .", "Remarkably , overexpression and deletion of Munc13s change the rate constants k1 ( priming 1 ) and k2 ( priming 2 ) in opposite directions ( Figure 7D ) .", "One possible reason for this effect could be that the interaction of Munc13s with the SNARE fusion machinery may compete with that of another priming protein , which mainly catalyzes priming step 2 .", "A likely candidate for this second catalyst appears to be CAPS , as deletion of CAPS1 and CAPS2 leads to a significant reduction of the RRP , but has little effect on the NRP/SRP , placing Munc13 upstream of CAPS ( Liu et al . , 2010; Liu et al . , 2008 ) .", "Furthermore , in PC12 cells , strong stimulation bypasses the need for CAPS-1 in LDCV exocytosis , but not the need for ubMunc13-2 ( Kabachinski et al . , 2014 ) , and the ability of CAPS to promote membrane fusion is impaired by C-terminal mutations in Stx-1 ( Daily et al . , 2010 ) .", "We therefore propose an LDCV priming model , in which the Munc13-driven priming step 1 corresponds to the initiation of N-terminal SNARE-complex assembly , and the CAPS-driven priming step 2 represents a more C-terminal , and presumably more easily completed phase of zippering ( Gao et al . , 2012 ) .", "Assuming that priming step 2 is not catalyzed by CAPS alone but also influenced by Munc13 and possibly Syt-1 , such a model would also offer an explanation as to why in both SV and LDCV exocytosis , lack of CAPS can be compensated for by an increase in Ca2+ , whereas lack of Munc13 cannot ( Jockusch et al . , 2007; Kabachinski et al . , 2014 ) .", "In summary , our data show that mammalian neurons and neuroendocrine cells both require Munc13s to generate fusion-competent vesicles , although the molecular steps leading to LDCV docking prior to SNARE complex assembly appear to be unique .", "In LDCV priming , the step most sensitive to Munc13s is the initial phase ( priming step 1 ) , which most likely corresponds to the initiation of N-terminal SNARE-complex assembly .", "Individual Munc13 isoforms accelerate this step at distinct rates , thereby imparting distinct properties on the kinetics of LDCV release , which indicates that they may have specialized functions in the fine-tuning of catecholamine release in response to varying physiological stimuli ." ], [ "All experiments were performed in compliance with the regulations of the local Animal Care and Use Committee of Lower Saxony , Oldenburg , Germany .", "The generation and basic characterization of the KO lines of the Munc13 isoforms has been described previously ( Augustin et al . , 2001; Augustin et al . , 1999; Varoqueaux et al . , 2002; Wojcik et al . , 2013 ) .", "Unc13dKO ( Unc13dJinx ) mice ( Crozat et al . , 2007 ) were obtained from Jackson Laboratories .", "Adult and perinatal mice were killed by decapitation prior to the removal of adrenal glands and other tissues .", "Adrenal glands of perinatal animals were excised and stored at -80°C prior to use .", "Adrenal glands from around 20 perinatal animals were pooled for the preparation of homogenates .", "Homogenates of whole adrenal glands were prepared by homogenization in an ice-cold buffer ( 320 mM D-glucose , 20 mM HEPES , 2 mM EDTA , pH 7 . 4 , with 0 . 5 μg/ml leupeptin , 1 μg/ml aprotinin and 0 . 1 mM PMSF added freshly prior to homogenization ) , using a Potter S homogenizer .", "For the preparation of adrenal cortical and medullary homogenates , adrenal glands from WT animals were dissected in ice-cold buffer containing 19 mM NaH2PO4 and 81 mM Na2HPO4 and material from 5–6 animals was pooled .", "Spleen homogenates were prepared similarly and the DNA in the samples subsequently digested with 0 . 66 U/μl benzonase ( E1014 , Sigma-Aldrich ) in 3 . 86 mM MgCl2 for 10 min at 37°C prior to denaturation .", "Whole-brain homogenates were prepared using a Potter S homogenizer and centrifuged for 10 min at 1000 g at 4°C to remove the nuclear fraction .", "Homogenates were analyzed by western blotting with the following antibodies at the indicated dilutions: rabbit-anti-Munc13-1 ( 1:500 ) ( 126103 , Synaptic Systems ) , rabbit-anti-ubMunc13-2 ( 1:2000 ) , rabbit-anti-bMunc13-2 ( 1:1000 ) , rabbit-anti-Munc13-3 ( 1:500 ) ( Varoqueaux et al . , 2005 ) , rabbit-anti-Baiap3 ( 1:1000 ) ( Wojcik et al . , 2013 ) , goat-anti-Munc13-4 ( 1:250 ) ( NB100-41385; Novus Biologicals ) , rabbit-anti-Chromogranin A ( 1:8000 ) ( 259002; Synaptic Systems ) , mouse-anti-GFP ( 1:500 ) ( 11814460001; Roche ) , mouse-anti-valosin containing protein ( VCP ) ( 1:1000 ) ( 612182; BD Transduction Laboratories ) , and mouse-anti-GAPDH ( 1:25000 ) ( ab8245; Abcam ) .", "Secondary antibodies ( goat anti-rabbit IgG , 111035144; goat anti-mouse IgG , 115035146 , donkey anti-goat , 705-035-147 ) were obtained from Jackson ImmunoResearch .", "Chromaffin cell cultures were prepared as described in Sørensen et al . ( Sorensen et al . , 2003b ) .", "Cultures from Unc13aKO and Unc13aKOUnc13bKO mice were prepared on embryonic day ( E ) 18 , and from Unc13bKO , Unc13cKO , and Baiap3KO mice on postnatal day ( P ) 0 , in each case using littermates of the appropriate genotypes as controls .", "For overexpression of Baiap3 in WT cells , chromaffin cells from P0 WT C57Bl/6N mice were used .", "Briefly , adrenal glands were excised and placed into ice-cold Locke’s solution ( 154 mM NaCl , 5 . 6 mM KCl , 0 . 84 mM NaH2PO4 , 2 . 14 mM Na2HPO4 and 10 mM D-glucose , pH 7 . 0 ) .", "The glands were then transferred to 300 μl of a papain solution [20 U/ml papain ( Worthington Biochemical ) , 200 mg/L L-cysteine , 1 mM CaCl2 , 0 . 5 mM EDTA , in DMEM ( Gibco ) ] , which had been equilibrated for 15 min with 95% O2 and 5% CO2 , and incubated with gentle shaking for 45 min at 37°C .", "To terminate the papain digestion , 300 μl of inactivating solution [10% fetal bovine serum ( Gibco ) , 2 . 5 g/L trypsin inhibitor ( Gibco ) and 2 . 5 g/L albumin in DMEM ( Gibco ) ] were then added , followed by an incubation period of 5 min at 37°C .", "The mixture of solutions was then replaced by 160 μl of DMEM ( Linaris ) supplemented with 1% insulin-transferrin-selenium X ( Gibco ) and 200 U/L penicillin-streptomycin ( Gibco ) .", "The glands were triturated with a 200-μl pipette tip and the cell suspension was placed as 50 μl drops on coverslips in a 6-well plate .", "Following an incubation period of 30 min at 37°C at 8% CO2 to allow cells to settle , 2 ml of DMEM ( Linaris ) with the supplements described above were added per well and the cells were kept at 37°C and 8% CO2 .", "The cells were used for electrophysiological recordings on days in vitro 2–3 .", "Expression constructs based on the SFV plasmid ( pSFV1 ) for Munc13-1 and ubMunc13-2 , both subcloned in frame with a C-terminal EGFP , have been described previously ( Rosenmund et al . , 2002 ) .", "Munc13-4 and Baiap3 pSFV1 expression constructs were generated as IRES-EGFP constructs using the full-length cDNAs .", "Production of SFV particles was done according to published protocols ( Ashery et al . , 1999 ) .", "Briefly , pSFV1 constructs and pSFV-helper2 DNA were linearized with Spe I and transcribed into RNA using SP6 RNA polymerase .", "RNA from the pSFV1 constructs and the pSFV-helper2 construct , 10 μg each , were electroporated ( 500 V , 0 . 957 mF ) into baby hamster kidney 21 cells .", "Supernatant of cell cultures containing the virus was collected after 24 hr .", "In cases where the virus titer was low , the supernatant was concentrated approximately 25-fold using a filter unit with a nominal molecular weight limit of 100 kDa ( UFC910024 , AMICON ) .", "SFV particles encoding the respective Munc13 isoforms with EGFP or only EGFP as a control were added to chromaffin cell cultures , infected cells identified based on the EGFP fluorescence , and electrophysiological recordings performed 4–6 hr after addition of the virus .", "Whole cell patch-clamping was performed with Sylgard-coated 4–6 MΩ pipettes ( Science Products ) at a setup equipped with a Zeiss Axiovert 200 microscope ( Zeiss ) and a HEKA EPC-10 amplifier controlled by Patchmaster ( HEKA ) .", "Capacitance measurements were performed according to the Lindau-Neher technique using the 'sine+dc' mode in the Lockin Extension of Patchmaster .", "The frequency and peak-to-peak amplitude of the sine wave were 1042 Hz and 70 mV , respectively , and the holding potential was -70 mV .", "Recordings were sampled at 12 . 5 kHz and filtered at 2 . 9 kHz .", "Flash photolysis experiments were performed according to established protocols ( Voets , 2000; Walter et al . , 2010 ) .", "The extracellular solution contained 147 mM NaCl , 10 mM HEPES , 11 . 1 mM D-glucose , 2 . 8 mM KCl , 2 mM CaCl2 , 1 mM MgCl2 and 3 μM tetrodotoxin ( pH 7 . 2 , 300–310 mOsM ) .", "For flash experiments , the intracellular solution contained 109 mM L-glutamic acid , 35 mM HEPES , 5 mM nitrophenyl-EGTA ( Synaptic Systems ) , 5 . 65 mM CaCl2 , 2 mM Mg-ATP , 0 . 3 mM Na-GTP , 0 . 205 mM fura-4F ( Invitrogen ) , 0 . 3 mM furaptura ( Invitrogen ) and 1 mM ascorbic acid ( titrated to pH 7 . 2 with CsOH , osmolarity 290–295 mOsM ) .", "The flash stimulus was applied approximately 80 s after the whole-cell configuration was established using a xenon lamp ( Rapp OptoElectronics ) .", "Unless otherwise specified , only cells with pre-flash [Ca2+] in the range of 300–600 nM were used for analysis .", "In flash photolysis experiments requiring pre-flash [Ca2+] concentrations higher than 600 nM ( Figure 7 and Figure 7—figure supplement 1 ) , pulses of light at wavelengths of 340 and 380 nm were applied at varying frequencies to release Ca2+ from nitrophenyl-EGTA and the cell was kept at the target [Ca2+] for ∼20 s before the flash stimulus was given .", "The pre-flash [Ca2+] was taken as the averaged measured [Ca2+] during the 20 s period .", "In depolarization experiments , the same extracellular solution was used except that tetrodotoxin was omitted .", "The intracellular solution contained 111 mM L-glutamic acid , 35 . 5 mM HEPES , 17 mM NaCl , 1 mM MgCl2 , 2 mM Mg-ATP , 0 . 3 mM Na-GTP ( titrated to pH 7 . 2 with CsOH , osmolarity 290–295 mOsM ) , and fura-4F and furaptra at the same concentration used in flash experiments .", "In flash photolysis experiments , pool sizes ( fast and slow bursts ) and their time constants were obtained by fitting a sum of exponential functions to the capacitance traces ( Sorensen et al . , 2003b ) , using a custom macro ( Three-Exponential-Fit-Macro-Igor ) ( see Source code 1 ) with the software IgorPro ( WaveMetrics ) .", "The near-linear rate of release of the sustained component is measured as a linear component with the unit capacitance increase per second .", "The exocytotic delay was defined as the time point where the exponential fit meets the pre-flash capacitance .", "In flash experiments , exocytosis was stimulated by a sudden elevation of intracellular [Ca2+] using UV flash stimuli given by a xenon flash lamp ( Rapp OptoElectronics ) .", "[Ca2+] measurements were performed according to established protocols ( Voets , 2000; Walter et al . , 2010 ) .", "The ratiometric Ca2+ indicator dyes fura-4F and furaptra were alternately excited at 340 and 380 nm using a Polychrome V monochromator ( TILL Photonics ) , and the emitted light was detected with a photomultiplier .", "The area of fluorescence measurement was limited to the diameter of the cell .", "The 340/380 nm fluorescence ratio was independently calibrated at the same dye concentrations with a range of intracellular solutions with known [Ca2+] , buffered with Ca2+ buffers 1 , 2-bis ( o-aminophenoxy ) ethane-N , N , N’ , N’-tetraacetic acid ( BAPTA , Invitrogen ) and diethylenetriaminepentaacetic acid ( DPTA , Sigma-Aldrich ) .", "The [Ca2+] of the calibration solutions was calculated using KD of BAPTA = 0 . 222 μM and KD of DPTA = 80 μM .", "Amperometric recordings were performed using carbon fibers of 5 µm diameter ( Thornel P-650/42; Cytec , NJ , USA ) , insulated using the polyethylene method ( Bruns , 2004 ) .", "Vesicle fusion was triggered by infusing the cells through the patch pipette with a solution containing 4 . 6 µM free Ca2+ .", "The fibers were clamped at 700 mV and currents were hardware filtered at 3 kHz using an EPC-7 patch clamp amplifier ( HEKA ) .", "Currents were digitized at 25 kHz and filtered off-line using a Gaussian filter with a cut-off set at 1 kHz .", "Filtering , spike detection , and analysis were performed using a freely available , custom-written macro ( Mosharov and Sulzer , 2005 ) running under IgorPro ( Wavemetrics , Lake Oswego , OR ) .", "A spike detection threshold of 5 pA and a foot detection threshold of 2 pA were imposed .", "For each analyzed cell , the median of each parameter ( duration , halftime , amplitude , charge , rise time , decay time , foot amplitude , foot duration , foot charge ) was calculated from all spikes , and this value was used for averaging between cells ( giving the mean of cell medians ) .", "Adrenal glands from E18 animals were embedded in 3% low gelling agarose ( Sigma-Aldrich ) and adrenal gland slices were prepared according to published protocols ( Moser and Neher , 1997 ) .", "Slices were allowed to recover in bicarbonate-buffered saline ( 125 mM NaCl , 26 mM NaHCO3 , 2 . 5 mM KCl , 1 . 25 mM NaH2PO4 , 2 mM CaCl2 , 1 mM MgCl2 , 10 mM D-glucose and 0 . 2 mM ( + ) -tubocurarine ) at 37°C for 15 min and were subsequently kept in the same solution at RT before cryofixation .", "Slices were rapidly frozen in external cryoprotectant ( 20% BSA in bicarbonate-buffered saline ) using a HPM100 HPF device ( Leica ) .", "After freezing , samples were stored in liquid nitrogen until further processing .", "Freeze substitution was performed as previously published ( Rostaing et al . , 2006 ) .", "Briefly , samples were substituted in anhydrous acetone , fixed by 2% OsO4 in acetone for 7 h at -90°C prior to a temperature ramp ( 5°C/h ) to -20°C , an incubation for 16 hr at -20°C , and a final ramp ( 10°C/h ) to 4°C .", "Samples were washed in acetone and infiltrated with EPON resin at room temperature ( acetone/EPON 1:1 for 3 h , 90% EPON in acetone overnight , and pure EPON for 36", "h ) .", "Finally specimen carriers containing infiltrated samples were incubated for 24 hr at 60°C to polymerize .", "Aluminum sample carriers were trimmed off the EPON block with a specimen trimming device ( EM TRIM2 , Leica ) to expose the surface of the embedded tissue for ultramicrotomy .", "An Ultracut UCT ultramicrotome ( Leica ) was used to cut 500 nm-thick sections until the first tissue appeared .", "Ultrathin ( 50 nm ) and semithin ( 400 nm ) sections were then collected onto Formvar-filmed , carbon-coated copper slot or mesh grids for 2D and 3D ultrastructural analysis , respectively .", "Vitrified samples were subjected to rigorous quality control ( Mobius et al . , 2010 ) and samples exhibiting indications of ice-crystal damage were excluded from the analysis .", "Ultrathin sections were post-stained with 1% uranyl acetate in ddH2O for 30 min , washed several times in ddH2O , stained with 0 . 3% lead citrate for 2 min , washed , and dried with filter paper .", "For 3D-tomographic analysis , 400 nm-thick sections were briefly incubated in a solution of Protein A conjugated to 10 nm gold particles ( Cell Microscopy Center , Utrecht , The Netherlands ) to introduce fiducial markers .", "Electron micrographs ( 2048 × 2048 pixels ) were acquired with a sharp:eye CCD camera ( Tröndle , TRS ) at 5000 fold magnification using the multiple image acquisition and alignment feature of iTEM software ( version 5 . 1 , Olympus Soft Imaging Solutions ) .", "Assembled montages had dimensions of approximately 21 × 21 µm and typically contained 1–3 randomly chosen chromaffin cells .", "Only chromaffin cells with a clearly visible plasma membrane were included in the analysis .", "iTEM software was used to measure chromaffin cell plasma membrane circumference and the cytoplasmic area ( calculated by subtraction of the nuclear area from the total cell area ) .", "Additional parameters including the number of LDCVs and the shortest distance of each LDCV to the plasma membrane were quantified using ImageJ software .", "In regions where the plasma membrane did not appear as a clear cut , the shortest distance from the vesicle membrane to the middle of the membrane projection was measured .", "For this reason and due to the fact that LDCVs ( mean diameter of CTRL LDCVs ~160 nm; ~170 nm for docked CTRL LDCVs ) might have their centers outside of the imaged ultrathin ( 50 nm ) section , we did not quantify LDCV docking as defined by physical membrane-attachment in these 2D projection images , but rather calculated the number of membrane-proximal LDCVs ( within 0–40 nm of the plasma membrane ) .", "All vesicles with electron-dense cargo were included in the analysis .", "Secretory vesicles of both genotypes exhibited heterogeneity in size ( see Figure 6—figure supplement 1 ) and appearance ( e . g . compactness of the dense-core ) , possibly reflecting distinct LDCV types ( e . g . adrenaline vs . noradrenaline ) or different levels of maturity present in immature ( E18 ) chromaffin cells .", "Data are presented as LDCV density ( number of LDCVs per µm² area cytoplasm ) , the number of LDCVs within 40 nm of the plasma membrane normalized to the cell perimeter , and the mean frequency distribution of LDCVs from the plasma membrane in 40 nm bins .", "For high-resolution electron tomographic analysis of LDCV docking in adrenal chromaffin cells , we randomly selected regions between two neighboring cells that exhibited a high density of LDCVs in proximity of the plasma membrane in thick ( 400 nm ) sections .", "Single-axis tilt series were acquired from -60° to +60° with 1° increments and binned by the factor two at 10 , 000-fold magnification using an Orius SC1000 camera ( Gatan ) and the SerialEM software for automated tilt series acquisition ( Mastronarde , 2005 ) .", "Tomograms were reconstructed using the IMOD package ( Kremer et al . , 1996 ) , and exported as z-stacks for analysis with ImageJ ( National Institutes of Health ) .", "All analyses were performed blindly and manually .", "The smallest distance between the plasma membrane and the outer leaflet of each LDCV membrane was measured at its vesicular midline using the straight-line tool of ImageJ on individual virtual z-slices .", "Only vesicles observed in physical contact with the plasma membrane in tomographic volumes were considered ‘docked’ .", "In distribution analyses docked LDCVs were assigned to the 0–4 nm bin to account for the voxel dimensions of reconstructed tomograms ( isotropic voxel size = 2 . 86 nm ) .", "The number of membrane-attached ( 0–4 nm , ‘docked’ ) LDCVs identified in a tomographically reconstructed volume was normalized to the number of membrane-proximal ( 0–40 nm of plasma membrane ) LDCVs .", "The number of membrane-proximal ( 0–40 nm ) LDCVs was expressed as a percentage of all LDCVs within 100 nm of the plasma membrane and compared across genotypes to test for potential differences in the ability of recruiting LDCVs close to the plasma membrane .", "The frequency distribution displays the number of docked LDCVs ( 0–4 nm , first bin ) and subsequently the distances of LDCVs from the plasma membrane in 2 nm bins .", "Statistical analyses were performed using Student’s t-test .", "The mean LDCV diameter was calculated from the area of the LDCV measured at its midline including the vesicular phospholipid bilayer in electron tomograms by using the elliptical selection tool in ImageJ .", "All LDCVs in the randomly chosen field of view that contained their midline within the tomographic volume were analyzed ( Figure 6—figure supplement 1 ) .", "For illustrative purposes , figures depicting tomographic subvolumes represent an overlay of 3 consecutive slices produced by the slicer tool of the 3dmod software of the IMOD package to generate a ~8 . 6 nm thick subvolume .", "The size of the pool of docked LDCVs per cell can be calculated from the number of LDCVs per µm² PM area ( na ) ( Parsons et al . , 1995; Plattner et al . , 1997 ) .", "We measured the number of membrane-proximal LDCVs ( 0–40 nm of PM ) per µm PM length ( nl ) in ultrathin sections of 0 . 05 µm thickness .", "Our 3D-ET approach permitted us to accurately measure LDCV diameters ( dv in µm ) in chromaffin cells ( Figure 6—figure supplement 1 ) .", "The number of LDCVs per µm² PM area could then be calculated as na = nl/ ( dv + 0 . 05 ) ( Parsons et al . , 1995; Plattner et al . , 1997 ) .", "The average cell surface area ( ac in µm2 ) per genotype was estimated based on the average cell capacitance measured in cultured cells assuming 1 μF/cm2 .", "We chose this method , rather than using the cell circumference measured in ultrathin sections , because the chromaffin cells in adrenal slices are not round , but have rather complex shapes ( Figure 6—figure supplement 1 ) .", "Using either measurement , the Unc13aKOUnc13bKO cells were slightly larger , by 5% based on capacitance , and by 15% based on cell circumference measurements .", "Unc13aHetUnc13bHet control cells: nl: 0 . 984 ± 0 . 075 vesicles per µm length dv: 0 . 1627 ± 0 . 005 µm vesicle diameter na: 4 . 627 vesicles per µm² PM area ac: 421 . 23 µm² cell membrane area The estimated number of LDCVs within 40 nm of the PM in Unc13aHetUnc13bHet chromaffin cells is therefore ~1949 .", "Our 3D analysis of LDCV docking revealed that only 33 . 97% of LDCVs within 40 nm of the PM are physically attached to the PM , therefore we estimated the pool of docked vesicles to contain ~662 LDCVs in our acute adrenal slice preparation .", "Unc13aKOUnc13bKO cells: nl: 1 . 150 ± 0 . 083 vesicles per µm length dv: 0 . 1525 ± 0 . 003 µm vesicle diameter na: 5 . 679 vesicles per µm² PM area ac: 442 . 26 µm² cell membrane area The estimated number of LDCVs within 40 nm of the PM in Unc13aKOUnc13bKO chromaffin cells is therefore ~2511 .", "Our 3D analysis of LDCV docking revealed that only 34 . 43% of LDCVs within 40 nm of the PM are physically attached to the PM , therefore we estimated the pool of docked vesicles to contain ~865 LDCVs in our acute adrenal slice preparation .", "We simulated capacitance traces in Ca2+ uncaging experiments with an exocytosis model that was adapted from a previous study of ours ( Walter et al . , 2013 ) to explicitly describe the Ca2+-dependent vesicle priming reaction .", "Parameters ( Table 1 ) were either taken from the literature , directly from experiments ( Figure 7A ) , or determined by fitting the model to experimental capacitance traces .", "We assume that Munc13 proteins act on the Ca2+-dependent priming reaction , which ensures the refilling of vesicles from a large depot pool ( Voets , 2000 ) .", "We wanted to explicitly describe this reaction in terms of its thermodynamic steady state binding properties ( i . e . its dissociation constant KD and cooperativity n ) , and in terms of its Ca2+ binding kinetics .", "Let the chemical equation for Ca2+ binding to the priming sensor ( PS ) be: n * Ca2+ + PS ⇄koffkon ( CanPS ) Then its overall dissociation constant KD is defined as: KD=[Ca2+]n [PS][CanPS] In order to obtain the values of the KDand n experimentally , we pre-equilibrated chromaffin cells at different pre-flash [Ca2+] prior to uncaging .", "The secretion responses were averaged in bins and normalized to their respective values 3 s after the flash .", "Fitting the fraction of release 30 ms after the uncaging stimulus as a function of pre flash [Ca2+] with the following Hill equation ( non-linear curve fitting routine of Origin Pro 8 G , OriginLab Corporation ) allowed us to estimate KDand n: Fraction ( [Ca2+] ) =[Ca2+]nKD+[Ca2+]nFmax Where Fraction ( [Ca2+] ) is the relative release at 30 ms , and [Ca2+] is the pre-flash Ca2+ concentration .", "Fmax , n and KDare free parameters .", "The best fit is shown as solid line in Figure 7 and the values of n and KD can be found in Table 1 .", "We assume that priming is only increased when the proper number ( n ) of Ca2+ ions are bound to the PS .", "Therefore , the rate of priming is proportional to the fraction ( f ) of PS that has bound the correct number of Ca2+ ions divided by the total amount of PS: f ( Ca2+ ) =[CanPS][PStot] k1 ( Ca2+ ) =f ( Ca2+ ) k1Max Where k1Max is the asymptotic value of the priming rate for [Ca2+]≫KD .", "Since the total amount of PS is the sum of Ca2+-free and Ca2+-bound PS: f ( Ca2+ ) =[CanPS][CanPS]+[PS] At steady state , the following relationships hold: f ( Ca2+ ) SteadyState=[Ca2+]nKD+[Ca2+]n k1SteadyState ( Ca2+ ) =[Ca2+]nKD+[Ca2+]nk1Max In order to describe temporal changes in this fraction at non-equilibrium conditions , the Ca2+ concentrations ( [Ca2+] ) were interpolated from the experimental values and the concentration of [CanPS] was calculated at all time points by numerical integration using the 'ode15s' function in Matlab ( version R2013a , Mathworks ) of the kinetic equation: d[CanPS]dt=kon[PS][Ca2+]n−koff[CanPS] We assume that the amount of PS is not limiting ( i . e . that each vesicle contains a PS ) .", "Then , by investing the relationships [Vtot]=[CanPS]+[PS] and KD=koffkon we obtain d[CanPS]dt=kon ( [Vtot]−[CanPS] ) [Ca2+]n−KDkon[CanPS] Such that the fraction of activated PS can be calculated at time t: f ( Ca2+ , t ) =[CanPS] ( t ) [Vtot] This allows us to calculate k1 at all times t .", "k1 ( Ca2+ , t ) =f ( Ca2+ , t ) k1Max Our model consists of a sequence of mandatory steps for vesicle maturation and fusion ( Walter et al . , 2013 ) .", "Vesicles from the depot enter a non-releasable state ( NRP , Figure 7E ) from which they cannot fuse directly .", "Instead , these vesicles first need to mature into the readily releasable pool ( RRP ) , a transition that is governed by a Ca2+-dependent rate constant ( k2[Ca2+] ) .", "This Ca2+-dependence is modeled by a Ca2+-dependent catalyst as described in Walter et al . , 2013: k2 ( Ca2+ ) =k20+g ( Ca2+ ) k2catk-2 ( Ca2+ ) =k-20+g ( Ca2+ ) k-2catk−2cat=k−20k20k2cat As in our previous study , we assume that binding of one Ca2+ ion activates the catalyst and that the catalyst is in equilibrium with Ca2+ , which allows us to calculate g ( Ca2+ ) : g ( Ca2+ ) =[Ca2+]KD , cat+[Ca2+] Kinetic equations of the exocytosis model d[Depot]dt=−k1 ( Ca2+ ) [Depot]+k−1[NRP]d[NRP]dt=k1 ( Ca2+ ) [Depot]- ( k-1+k2 ( Ca2+ ) ) [NRP]+k-2 ( Ca2+ ) [RRP]d[RRP]dt=k2 ( Ca2+ ) [NRP]- ( k-2 ( Ca2+ ) +3k3[Ca2+] ) [RRP]+k-3[RRPCa]d[RRPCa]dt=3k3[Ca2+][RRP]- ( k-3+2k3[Ca2+] ) [RRPCa]+2k-3[RRPCa2]d[RRPCa2]dt=2k3[Ca2+][RRPCa]- ( 2k-3+k3[Ca2+] ) [RRPCa2]+3k-3[RRPCa3]d[RRPCa3]dt=k3[Ca2+][RRPCa2]- ( 3K-3+k4 ) [RRPCa3]d[F]dt=k4[RRPCa3] Modeling of exocytosis upon Ca2+ uncaging To find the steady state occupation of the system k1 ( Ca2+ ) , k2 ( Ca2+ ) and k−2 ( Ca2+ ) were calculated for the experimental pre-flash Ca2+ values , the first five kinetic equations were taken and set to zero , and mass conservation of vesicles was obeyed: 0=[Depot]0+[NRP]0+[RRP]0+[RRPCa]0+[RRPCa2]0+[RRPCa3]0−[Vtot] This system of 6 equations was solved using the function 'fsolve' of Matlab ( version R2013a , Mathworks ) .", "The steady state values were used as starting values for the numerical integration of the kinetic equations using the 'ode15s' function of Matlab ( version R2013a , Mathworks ) .", "For parameter optimization , model simulations were compared to experimental capacitance data .", "The parameters of the model were varied and the goodness of fit was determined in a cost function , which was the sum of squared deviations between data and model prediction .", "In order to avoid bias towards data with larger secretion , the cost function used relative deviations: deviations were normalized to the maximal value of either the experimental capacitance trace or the simulated one , whichever was smaller ( led to a larger cost ) : cost=weight ( i ) max ( yExperiment ) ∑i ( yExperiment ( i ) −yModel ( i ) ) 2for max ( yExperiment ) <max ( yModel ) cost=weight ( i ) max ( yModel ) ∑i ( yExperiment ( i ) −yModel ( i ) ) 2for max ( yExperiment ) ≥max ( yModel ) Three principal kinetic components have been described in capacitance traces: a fast component with a time constant of tens of milliseconds , a slower component with a time constant of hundreds of milliseconds and a sustained component with a time constant of several seconds ( Voets , 2000 ) .", "To account for the fact that more data points exist for slower components ( due to the constant sampling rate ) which would dominate the fit , deviations at shorter times after the uncaging flash were given larger weight: the weight was 100 for all datapoints upto 80 ms after the uncaging stimulus; the weight was 10 for all datapoints occurring later than 80 ms , but earlier than 1 . 2 s after the stimulus; the weight was 1 for all datapoints thereafter .", "To ensure consistency , all conditions depicted in Figure 7 were fitted simultaneously and the cost values from all data were summed .", "Only the parameters labeled with “best fit” in Table 1 were allowed to vary .", "Because the lowest bin of the capacitance traces of the DKO overexpressing Munc13-1 and ubMunc13-2 in Figure 7B and C contained relatively few cells , the total number of vesicles was also a free parameter under these conditions ( best fit values Vtot ( Munc13-1 low Ca2+ ) = 2120 fF , Vtot ( Munc13-1 low Ca2+ ) = 3340 fF ) .", "All parameters were optimized by minimizing the cost values using a Nelder-Mead Simplex algorithm implemented in the Matlab function 'fminsearch' ( version R2013a , Mathworks ) .", "Statistical analyses were performed using two-tailed Student’s t-test , ANOVA with post-hoc Tukey’s test , or Mann-Whitney U-test as specified in the figure legends ." ] ]

[ "It is currently unknown whether the molecular steps of large dense-core vesicle ( LDCV ) docking and priming are identical to the corresponding reactions in synaptic vesicle ( SV ) exocytosis .", "Munc13s are essential for SV docking and priming , and we systematically analyzed their role in LDCV exocytosis using chromaffin cells lacking individual isoforms .", "We show that particularly Munc13-2 plays a fundamental role in LDCV exocytosis , but in contrast to synapses lacking Munc13s , the corresponding chromaffin cells do not exhibit a vesicle docking defect .", "We further demonstrate that ubMunc13-2 and Munc13-1 confer Ca2+-dependent LDCV priming with similar affinities , but distinct kinetics .", "Using a mathematical model , we identify an early LDCV priming step that is strongly dependent upon Munc13s .", "Our data demonstrate that the molecular steps of SV and LDCV priming are very similar while SV and LDCV docking mechanisms are distinct ." ]

[ "Mammals have adrenal glands , which secrete the stress hormone adrenaline as well as other hormones into the bloodstream .", "These molecules are produced in chromaffin cells , where they are packaged into compartments called large dense-core vesicles ( LDCVs ) .", "To release the hormones into the bloodstream , the vesicles bind to and fuse with the membrane that surrounds the cell .", "This process – which is called exocytosis – is triggered by increases in the level of calcium ions inside the cells .", "Exocytosis also enables nerve cells to release chemical signals at junctions ( known as synapses ) with other nerve cells .", "These signals are packaged within another type of vesicle called 'synaptic' vesicles , which also release their contents by fusing with the cell membrane .", "However , it is not clear whether the two types of vesicle carry out exocytosis in the same way .", "Exocytosis requires that the vesicles physically attach to the membrane and undergo a process termed 'priming' , which enables them to fuse quickly with the membrane in response to an increase in calcium ion levels .", "In synaptic vesicles , both of these processes – physical membrane attachment and priming – appear to occur in a single step that requires a family of proteins called the Munc13 proteins .", "Here , Man et al . investigate whether the Munc13 proteins are also essential for LDCV exocytosis in the chromaffin cells of mice .", "The experiments reveal that in contrast to synaptic vesicles , the initial binding of LDCVs to membranes does not require Munc13 proteins .", "However , the loss of one member of the family called Munc13-2 dramatically reduces the fusion of LDCVs with the membrane of chromaffin cells .", "Further experiments reveal that different Munc13 proteins differ in their ability to drive the exocytosis of LDCVs .", "Man et al . use a mathematical model of LDCV exocytosis , which reveals that Munc13 plays an important role in the first part of the priming step .", "Together , these findings show that synaptic vesicles and LDCVs use different mechanisms to bind to membranes , but are primed for fusion in a similar way ." ]

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

[ "neuroscience" ]

[ [ "During development of the peripheral nervous system , specific neuronal connections are being established to ensure its proper function .", "Sensory neurons of the dorsal root ganglia ( DRG ) initially extend their axons toward their targets by responding to attractive and repulsive cues expressed in the extracellular environment along their trajectory ( Kolodkin and Tessier-Lavigne , 2011 ) .", "Once axons reach the targets , limiting amounts of neurotrophic factors control target innervation by regulating axonal growth , neuronal cell death , and pruning ( Huang and Reichardt , 2001 ) .", "Brain-derived neurotrophic factor ( BDNF ) acts as a survival factor for specific subsets of sensory neurons during development ( Kirstein and Fariñas , 2002 ) , and regulates mechanosensation ( Carroll et al . , 1998 ) .", "During early development of the mammary gland , BDNF is required to establish and maintain sensory innervation in both female and male embryos .", "In males only , at later stages of development , sequestering of BDNF by a truncated form of TrkB ( TrkB . T1 ) results in elimination of the axons innervating the gland .", "This elimination is not blocked in the Bax knockout ( KO ) , suggesting that it occurs through axonal pruning rather than cell death ( Liu et al . , 2012 ) .", "As a result , a sexually dimorphic pattern of mammary gland sensory innervations is formed .", "Whether other cues operate in concert with BDNF , both in females and males , is unknown .", "The Semaphorins belong to a large family of secreted and membrane-bound guidance cues .", "The 27 family members are divided into 8 subclasses according to structural homology ( Pasterkamp , 2012 ) .", "Initially characterized for their importance in axonal guidance during development , Semaphorins are now known to be involved in multiple developmental processes including axonal fasciculation , pruning , dendritic guidance , and modulation of the immune system ( Pasterkamp and Kolodkin , 2003; Yazdani and Terman , 2006; Tang et al . , 2007; Zhou et al . , 2008 ) .", "Two receptor families have been implicated in mediating Semaphorin family functions: Plexins and Neuropilins ( Fujisawa and Kitsukawa , 1998 ) .", "Most class 3 Semaphorins bind to Neuropilins , which associate with type A Plexin family members to transduce their signals through the plasma membrane .", "Other members of the Semaphorin family bind and signal directly through Plexin receptors ( Yoshida , 2012 ) .", "Moreover , previous in vitro and overexpression studies demonstrated a crosstalk between class 3 Semaphorins and neurotrophins ( Dontchev and Letourneau , 2002; Atwal et al . , 2003; Tang et al . , 2004; Ben-Zvi et al . , 2006; Tang et al . , 2007; Ben-Zvi et al . , 2008 ) .", "Here we show that opposing effects of neurotrophic ( BDNF ) and repulsive ( Semaphorins ) factors regulate the sensory innervation of the mammary gland , and that the repulsive factors may promote axonal pruning once trophic signaling is inhibited ." ], [ "The sexually dimorphic innervation of the mammary gland at late E13 mouse embryos is a result of differential expression of TrkB . T1 in mesenchymal cells surrounding the mammary epithelium .", "In male embryos , expression of TrkB . T1 results in sequestering of BDNF , which leads to pruning of the sensory fibers innervating the mammary gland ( Liu et al . , 2012 ) .", "In agreement , we have detected this expression pattern of TrkB . T1 using an antibody that recognizes the TrkB extracellular domain ( TrkBECD ) ( Figure 1B arrowheads ) .", "In addition , this staining revealed innervation of the gland by TrkB-positive axons in female embryos and reduced innervation in male embryos ( Figure 1A , B arrows ) .", "In order to find additional factors that regulate the innervation , we performed an in-situ hybridization screen to detect the mRNA that encodes the different Semaphorins ( Figure 1C–H and Figure 1—figure supplement 1 ) .", "Our screen detected clear expression of the secreted Semaphorins , Semaphorin3d ( Sema3d ) and Semaphorin3f ( Sema3f ) , in the epithelial cells of the gland ( Figure 1C–F ) .", "Less profound expression of the membrane-bound Semaphorin6a ( Sema6a ) was detected as well ( Figure 1G , H ) .", "There was no signal by the sense control probes ( Figure 1—figure supplement 2 ) .", "To confirm expression of Sema6a , we took advantage of the Sema6a genetrap line that allows detection of Sema6A-expressing cells by X-gal staining ( Leighton et al . , 2001 ) .", "Whole mount staining showed similar epithelial expression of Sema6A in female and male embryo mammary glands ( Figure 1I , J ) .", "Unlike TrkB . T1 expression , the expression pattern of the three Semaphorins was identical between female and male embryos .", "To determine whether BDNF-responsive neurons that innervate the mammary gland respond to Semaphorins expressed in the epithelial cells of the gland , we used an in vitro growth cone collapse assay .", "DRG explants from early E13 embryos were grown in the presence of BDNF for 48 hr , and then soluble Semaphorins were added to the media for 30 min , after which the axonal growth cones were detected by staining for actin using Phalloidin-Rhodamine .", "Under basal conditions , between 20% and 40% of the growth cones exhibited a collapsed morphology .", "Both Sema6A and Sema3D increased this fraction to about 60% of the growth cones .", "In contrast , Sema3F had no effect ( Figure 2A–F ) .", "The PlexinA4 receptor can propagate repulsive activities of both secreted and transmembrane Semaphorins ( Suto et al . , 2005; Tran et al . , 2007; Yoshida , 2012 ) .", "Sema6A can bind and signal directly through both PlexinA4 or PlexinA2 ( Suto et al . , 2005; Suto et al . , 2007 ) .", "PlexinA4 also acts as the signaling co-receptor for Sema3A in a complex with Neuropilin1 ( Nrp1 ) ( Suto et al . , 2005; Yaron et al . , 2005; Tran et al . , 2007; Yoshida , 2012 ) .", "While Sema3D was shown to bind Nrp1 ( Yazdani and Terman , 2006 ) , the specific Plexin through which it signals was not reported .", "Therefore , we tested if Sema3D and Sema6A signal through PlexinA4 in BDNF-responsive neurons by performing a collapse assay using DRGs of PlexinA4 KO mice ( Yaron et al . , 2005 ) .", "Interestingly , we detected complete loss of response both to Sema6A and Sema3D in these neurons ( Figure 2A–D ) , demonstrating that PlexinA4 serves as the signaling receptor for both Sema6A and Sema3D .", "Since Sema6A can also bind and signal through PlexinA2 ( Suto et al . , 2005; Suto et al . , 2007 ) , we tested if PlexinA2 may serve as an additional binding receptor in these neurons .", "We performed the binding assay using Sema6A-Fc on WT and PlexinA4 KO DRG neurons .", "In the WT DRGs , a strong signal was detected along the axons , however , no signal was detected in the PlexinA4 KO neurons ( Figure 2—figure supplement 1 ) , indicating that PlexinA4 is the only binding and signaling receptor for Sema6A in these neurons of early E13 embryos .", "These in vitro experiments show that PlexinA4 mediates axonal growth cone collapse by Sema3D and Sema6A in BDNF-responsive DRG sensory neurons .", "The expression pattern of the Semaphorins and their in vitro activity prompted us to examine their role in mammary gland innervation .", "We first examined the innervation of the mammary gland in WT and Sema6A KO female and male embryos at different embryonic stages .", "In female embryos , Tubulin βIII staining ( using Tuj1 antibody ) showed more axons innervating the gland in Sema6A KO than in WT embryos , beginning at late E13 and continuing to late E14 ( Figure 3 ) .", "However , male embryos showed no difference between WT and Sema6A KO in innervation density of the gland ( Figure 3—figure supplement 1 ) .", "These results support the idea that Sema6A expressed in the epithelial cells restricts innervation of the gland by the sensory axons in females , starting at E13 .", "Our findings that PlexinA4 serves as the receptor for Sema6A and Sema3D in vitro ( Figure 2 ) , and that mammary glands of Sema6A KO female embryos are hyperinnervated ( Figure 3 ) , prompted us to examine mammary gland innervation in the PlexinA4 KO embryos , as a way to look at the combined signaling from both Sema6A and Sema3D .", "Compared to WT female embryos , where the fiber density innervating the mammary gland increases gradually during development , in the PlexinA4 KO we detected hyperinnervation of the gland starting at E12 that remained constant at E13 and E14 ( Figure 4A , B ) .", "Importantly , these additional axons appear to be TrkB positive ( Figure 4—figure supplement 1 ) .", "These phenotypes were much more severe than the phenotypes we detected in the Sema6A KO ( Figure 3 ) , suggesting that PlexinA4 indeed transmits the signaling from additional Semaphorins and has a critical role in restricting sensory innervation to the mammary gland during development .", "It has been shown that BDNF produced by the mammary mesenchymal cells promotes the initial ingrowth and maintenance of sensory fibers innervating the gland ( Liu et al . , 2012 ) .", "Therefore , we next examined the interaction between these two pathways using a genetic approach .", "It has been previously demonstrated that the level of nerve growth factor ( NGF ) limits sensory innervation , such that NGF heterozygous ( Het ) animals exhibit an altered pattern of sensory projections ( Crowley et al . , 1994 ) .", "Therefore , we speculated that this is the case for BDNF in the mammary gland as well .", "We first compared innervation of the mammary gland in female embryos heterozygous for BDNF ( BDNF Het ) to WT .", "Indeed , we observed a significant reduction in mammary gland innervation at late E12 , 13 and 14 in BDNF Het embryos compared to WT ( Figure 4A , C ) .", "Next , we crossed the BDNF Het with the PlexinA4 KO .", "At late E12 , there was no effect on the innervation density of the gland compared to PlexinA4 KO ( Figure 4A , D ) , but at later stages , reduction of BDNF levels partially suppressed the hyperinnervation .", "At late E14 , the PlexinA4 KO/BDNF Het exhibited normal mammary gland innervation , indistinguishable from the WT embryos ( Figure 4A , D ) .", "These results show that innervation of the mammary gland is determined by integration of Semaphorins and BDNF pathways in female embryos .", "In male embryos , inhibition of BDNF signaling is required for pruning of the axons innervating the mammary gland ( Liu et al . , 2012 ) .", "As expected , we detected a clear developmental decrease in the innervation of WT male mammary glands between late E12 and late E14 ( Figure 5A , B ) .", "To test whether Semaphorin-PlexinA4 signaling has any role in regulating developmental axonal pruning in males , we examined the innervation of the mammary gland in the PlexinA4 KO male embryos .", "Similar to females , we detected hyperinnervation at late E12 ( Figure 5B ) , suggesting that PlexinA4 signaling restricts the initial innervation of the mammary gland in males as well .", "Interestingly , there was virtually no decrease in fiber density between late E12 and late E13 in the PlexinA4 KO .", "In comparison , in WT males , fiber density decreased by about 50% during the same time frame ( Figure 5B ) .", "Similar to our results from female embryos , these axons are TrkB positive ( Figure 5—figure supplement 1 ) .", "Between late E13 and late E14 there was a sharp decrease in fiber density in the PlexinA4 KO , but to a level which is still significantly higher than that of WT embryos .", "Importantly , the delay in pruning in the PlexinA4 KO embryos was not due to lower expression of TrkB . T1 , as we detected similar expression levels in the mesenchymal cells of WT and PlexinA4 KO embryos , using the TrkBECD antibody ( Figure 5—figure supplement 2 ) .", "Since the initial innervation in the PlexinA4 KO at late E12 , prior to pruning onset , is much higher than in the WT , we could not discriminate between two possibilities:", "1 ) the delay in pruning is due to increased innervation at the beginning of the process or", "2 ) that signaling through PlexinA4 is required for pruning .", "To address this issue , we compared the innervation of the male mammary gland on the background of BDNF Het .", "As in the females , there is a clear reduction in mammary gland innervation in the BDNF Het male embryos compared to WT at late E12 ( Figure 5A , C ) .", "Unlike in females , we detected a reduction in innervation ( ~50% ) in the PlexinA4 KO/BDNF Het male embryos compared to the PlexinA4 KO , as early as late E12 .", "Despite this greatly reduced initial innervation , there was still a strong delay in axonal elimination between late E12 and late E13 ( only 20% decrease ) .", "By late E14 , we detected similar levels of innervation in the PlexinA4 KO/BDNF Het and PlexinA4 KO embryos , which was still significantly higher than the WT ( Figure 5A , D ) .", "Overall , these results indicate that PlexinA4 is required for the normal progression of developmental axonal pruning of fibers innervating the male mammary gland .", "Our in vivo analysis suggests that balance of BDNF and Semaphorin-PlexinA4 signaling controls the innervation of the mammary gland .", "To further study this balance , we carried out in vitro growth cone collapse experiments under conditions of reduced BDNF levels .", "DRG explants of early E13 embryos were grown in the presence of BDNF for 48 hr , then the media was replaced to media containing BDNF ( +BDNF ) , or media without BDNF ( -BDNF ) , both with low concentrations of Sema6A ( 0 . 025 ng/µl ) for 30 min .", "In the presence of BDNF , this concentration of Sema6A did not induce growth cone collapse .", "However , adding Sema6A at this concentration to the -BDNF media resulted in 60% growth cone collapse ( Figure 6 ) .", "This demonstrates that at low levels of BDNF , sensory axons become hypersensitive to PlexinA4 signaling , which results in more collapsed growth cones .", "These results support the idea that BDNF inhibits the Semaphorin signaling , and may suggest that upon BDNF sequestering the axons become hypersensitive to PlexinA4 signaling .", "Since PlexinA4 does not regulate the expression of TrkB . T1 , which neutralizes BDNF in the mammary gland of male embryos ( Figure 5—figure supplement 2 ) , we tested if PlexinA4 and TrkB . T1 operate in parallel pathways .", "To assess interaction between these two pathways , we used a genetic approach in which we analyzed sensory innervation of the mammary gland in PlexinA4/TrkB . T1 double KO in late E13 and E14 embryos ( Figure 7 ) .", "In the single PlexinA4 KO embryos at late E13 and late E14 , mammary innervation is significantly greater than in the WT , but between E13 and E14 , innervation of the gland decreases to levels that are nonetheless still elevated compared to the WT .", "In the TrkB . T1 single KO at E13 , the level of innervation is higher than the WT due to decreased pruning , as previously demonstrated by Liu et al . However , at E14 , the level of innervation dropped to WT levels .", "There was no significant difference in innervation between the double KO and the PlexinA4 single KO at E13 or E14 .", "Overall , these results are consistent with the idea that TrkB . T1 and PlexinA4 operate in parallel and converge to the same axonal elimination pathway ." ], [ "The process of target innervation by sensory axons during embryonic development is largely regulated by neurotrophic factors secreted by the target in limited amounts .", "Studies of NGF have demonstrated a strong correlation between its expression and the innervation density of the skin ( Davies et al . , 1987 ) , and that overexpression of NGF results in hyperinnervation of the target organ ( Albers et al . , 1994 ) .", "However , there is growing evidence that additional target-derived cues are involved in specifying innervation , some through interaction with the neurotrophin signaling pathways ( Guha et al . , 2004; Bäumer et al . , 2014; Wheeler et al . , 2014 ) .", "The mammary gland serves as a unique model for sexually dimorphic target innervation controlled by BDNF , but whether other factors regulate this process jointly with BDNF was not known .", "Through the studies described here , we discovered a critical role for the Semaphorin-PlexinA4 signaling pathway in axonal innervation restriction and possibly pruning promotion .", "Our studies uncovered a non-dimorphic expression of several members of the Semaphorin family in the mammary gland that signal through PlexinA4 .", "Since the Androgen receptor is not expressed in DRG neurons ( Liu et al . , 2012 ) , it is likely that PlexinA4 is expressed in non-dimorphic manner as well .", "In vivo ablation of PlexinA4 caused hyperinnervation of the gland in female embryos .", "This effect of PlexinA4 ablation was reversed by genetic reduction of BDNF levels in PlexinA4 KO/BDNF Het embryos , which exhibited normal innervation of the mammary gland , indistinguishable from WT levels , at late E14 .", "These results suggest that although BDNF is expressed in limited amounts , its physiological levels are sufficient to sustain high levels of innervation , which are curbed by Semaphorin signaling .", "In male embryos , ablation of PlexinA4 resulted in initial hyperinnervation of the gland .", "Co-reduction of BDNF with PlexinA4 ablation dramatically reduced the initial hyperinnervation at late E12 , although this was not observed in females at the same stage .", "The basis for this difference is currently unclear , however , it may reflect an intrinsic hypersensitivity of the male axons to BDNF levels , which is only revealed on the background of PlexinA4 KO .", "This hypersensitivity may help to promote pruning once BDNF is sequestered .", "We detected delayed pruning of the fibers innervating the gland in the PlexinA4 KO and the PlexinA4 KO/BDNF het , supporting a model in which PlexinA4 promotes pruning independently of the initial levels of innervation .", "However , we cannot exclude the idea that the excess initial axonal innervation by itself generates a protective mechanism against pruning , for example by axon-axon adhesion as was observed in flies ( Bornstein et al . , 2015 ) .", "Lastly , while our results show that ablation of PlexinA4 does not affect the expression of TrkB . T1 , we were not able to visualize BDNF at the gland by immunostaining .", "Therefore , we cannot completely dismiss the possibility that ablation of PlexinA4 affects the expression of BDNF .", "Using in vitro growth cone collapse assays with low levels of BDNF resulted in hypersensitivity to PlexinA4 signaling , showing that inhibition of BDNF signaling renders the axons more sensitive to PlexinA4 .", "Although this assay is mainly correlated with growth inhibition by the Semaphorins and not axonal pruning , a recent study suggested that growth cone collapse is an early and critical step in axonal degeneration in response to trophic deprivation ( Unsain et al . , 2018 ) .", "Therefore , the hypersensitivity to PlexinA4-induced growth cone collapse may reflect promotion of the pruning process .", "Interestingly , while BDNF counteracts PlexinA4 effects , our results suggest that PlexinA4 and the BDNF receptor isoform TrkB . T1 operate in the same pathway of axonal elimination , since co-ablation of the two did not have any additive effect .", "This result supports previous data that TrkB . T1 antagonizes BDNF signaling in the mammary gland ( Liu et al . , 2012 ) .", "Nevertheless , neither ablation of PlexinA4 or of TrkB . T1 was able to prevent pruning at late E14 , when the innervation density drops sharply .", "During mammary organogenesis , the mammary gland of male embryos starts to regress as a result of Androgen release by the male gonads at E13 ( Robinson , 2007 ) , and this regression probably forces elimination of the axons .", "Altogether , our study shows that developmental innervation of the mammary gland is regulated by a balance between BDNF , which promotes the initial ingrowth of the sensory fibers , and constant Semaphorin signaling , which restricts innervation ( Figure 8A ) .", "Upon inhibition of BDNF-TrkB signaling , Semaphorin-PlexinA4 signaling may promote axonal pruning ( Figure 8B ) .", "A previous in vitro study has demonstrated that the class 3 Semaphorins inhibit neurotrophin signaling and that this inhibition is a part of the mechanism by which they induce growth cone collapse ( Atwal et al . , 2003 ) .", "Our results also support the idea that neurotrophins signaling suppresses the Semaphorin signaling , and once this suppression is relieved , the axons become hypersensitive to Semaphorins , as was proposed by Dontchev and Letourneau ( Dontchev and Letourneau , 2002 ) .", "Overall , our work provides the first clear evidence that the balance between these two cues plays a role in accurate wiring of the nervous system during development , and that physiologically tipping this balance may induce axonal pruning .", "The Semaphorins have been previously implicated in pruning of hippocampal axons , where developmental expression of Sema3F was proposed to largely regulate this process ( Bagri et al . , 2003 ) .", "In contrast , in the mammary gland , we did not detect any dimorphic or temporal expression of Semaphorins or PlexinA4; instead , our results suggest that their ability to prune axons in male embryos is gated by inhibition of BDNF signaling .", "Whether there are equivalent mechanisms that gate Semaphorin-dependent axonal pruning in the hippocampus remains to be discovered .", "Expression of Semaphorins and Plexins has been previously demonstrated in the developing mammary gland postnatally , and was suggested to affect ductal growth and morphogenesis of the gland ( Morris et al . , 2006 ) .", "Here we present new data that show expression of Sema3D and Sema6A in the mammary epithelium during embryonic development , and their involvement in regulation of the mammary gland innervation through the PlexinA4 receptor .", "We also detected expression of Sema3F in the mammary epithelium , but in vitro it had no effect on growth cone collapse of BDNF-responsive sensory neurons , suggesting it is not involved in target innervation .", "It may have a role in the regulation of ductal directional outgrowth and branching morphogenesis during later development .", "This is supported by the fact that Neuropilin2 , which serves as the binding receptor of Sema3F ( Yoshida , 2012 ) , promotes postnatal branching morphogenesis of the mammary gland ( Goel et al . , 2011 ) .", "Together , these results suggest that the Semaphorins play a dual role in mammary gland development , determining the extent of sensory innervation density prenatally and promoting gland morphogenesis postnatally .", "Intriguingly , the neurotrophins and the Semaphorins continue to be expressed in peripheral tissues throughout the life of the organism , and their expression may be altered in pathological conditions ( Apfel , 1999; Yamaguchi et al . , 2008; Tominaga et al . , 2009 ) .", "Our findings raise the possibility that altered expression of these cues could induce aberrant innervation in mature animals , causing either hypersensitivity or reduced sensation , depending on the balance between these pathways ." ], [ "Pregnant female ICR mice ( 8–12 week old ) were purchased from Envigo ( Rehovot , Israel ) .", "The Sema6A mutant ( Leighton et al . , 2001 ) and PlexinA4 KO ( Yaron et al . , 2005 ) lines have been described previously .", "TrkB . T1 KO mice were kindly provided by the lab of Lino Tessarollo and generated as described previously ( Dorsey et al . , 2006 ) .", "Mice heterozygous for the Bdnftm1Jae mutation ( JAX stock #002266 ) ( Ernfors et al . , 1994 ) were purchased from The Jackson Laboratory .", "All mice were of mixed strains , and colonies were bred and maintained at the Department of Veterinary Resources of the Weizmann Institute of Science , Israel , according to recommendations of the Federation of European Laboratory Animal Science Associations .", "Staged embryos were generated by interbreeding mice of the appropriate genotype in overnight mating .", "The day on which vaginal plug was observed was counted as embryonic day 0 .", "Pregnant females were sacrificed to harvest the embryos at different times of a given embryonic day: E12 , E13 or E14 .", "Early of the day was done at 9–10 and late at 16–17 as was previously described ( Liu et al . , 2012 ) .", "Embryos were genotyped by PCR using tail DNA samples , and the sex was determined by using primers of the Zfy1 gene , as was previously described ( Liu et al . , 2012 ) .", "For sensory neurons staining: embryos at different developmental stages were fixed in 4% formaldehyde overnight ( O . N ) at room temperature , followed by paraffin embedding .", "Transversal sections of 4 µm from the first pair of mammary glands were taken for immunostaining .", "The sections underwent deparaffinization with xylene and ethanol , followed by antigen retrieval using 10 mM sodium citrate buffer pH=6 at 125°C in a decloaking chamber .", "Sections were blocked with 1% bovine serum albumin ( BSA ) and 0 . 5% goat serum in PBST ( 0 . 1% Triton X-100 in PBS ) for 1 hr at room temperature , followed by O . N incubation with mouse anti-tubulin β-III antibody ( R&D , Tuj1 clone , MAB1195 , 1:500 ) in 1% BSA .", "At least five embryos were analyzed for each condition .", "For TrkBECD staining: embryos were fixed in 4% formaldehyde O . N at 4°C , followed by cryoprotection with 30% sucrose in PBS at 4°C for O . N incubation .", "Embryos were embedded in O . C . T ( Tissue Tek ) , frozen at -80°C , and sectioned at 20 µm .", "Transversal sections of the first pair of mammary glands were dried O . N at room temperature , washed with PBS and blocked with 5% donkey serum in PBST for 1 hr , followed by incubation with goat anti- TrkBECD ( R&D , AF1494 , 1:100 ) , diluted in blocking solution overnight at 4°C .", "The next day , sections were washed with PBS and incubated with secondary antibodies ( Jackson Immunoresearch , 1:200 ) and DAPI diluted in PBS for 1–2 hr , washed again with PBS , and mounted with fluoromount-G ( Southern Biotech ) .", "At least two embryos were analyzed for each sex .", "Pictures of Tuj1 staining were taken using 90i upright fluorescent microscope ( Nikon ) .", "Pictures of TrkBECD were taken with 60X oil ( UIS2 ) objective using IX81-based Olympus FluoView 1000 laser confocal scanning microscope .", "Images of transversal paraffin sections of the first pair of mammary glands stained with anti-Tuj1 and DAPI were processed and analyzed using ImageJ software .", "The measurement of fiber density was done as previously described ( Liu et al . , 2012 ) .", "In short a 15 µm band was drawn surrounding the mammary epithelium according to the DAPI staining , and the percent of Tuj1 positive pixels within this band was measured using the area fraction measurement , giving the percent of fiber density .", "Overall in this study 208 embryos were analyzed .", "Outliers were considered as data-points that are more than four standard deviations from the mean and were excluded from the data .", "Differences between groups and stages were tested with a 2-way ANOVA on log-transformed measurements .", "In cases where the interaction between group and stage was significant , we followed the ANOVA with separate t-tests per stage , to elucidate in which stage the differences between groups was significant .", "Fluorescent ISH on transversal paraffin sections of the first pair of mammary glands from ICR female and male embryos at late E13 was performed as previously described ( Shwartz and Zelzer , 2014 ) .", "The pattern of expression of different genes of the Semaphorins was detected using a digoxigenin ( DIG ) -labeled probes .", "Two embryos were analyzed for each probe with similar results .", "HEK293 and COS1 cells were originally obtained from the ATCC –The Global Bioresource Center ( no authentication was performed ) .", "Cell lines were cultured in DMEM ( Gibco ) supplemented with 10% FBS , 100 U/mL penicillin-streptomycin , 2 mM glutamine and Biomyc-3 anti-mycoplasma solution , at 37°C , 95% humidity and 5% CO2 .", "The cells were not tested for Mycoplasma .", "Cells were transfected at 70% confluency in 10 cm plates using Polyjet transfection reagent with 5 µg of the different plasmids .", "DRGs from early E13 embryos of the appropriate genotype were dissected and plated as explants in chambers coated with 10 µg/ml Poly-D-lysine ( PDL ) and 10 µg/ml Laminin , and contained Neurobasal-A growth medium supplemented with 2% B-27 , 1% Penicillin-Streptomycin , 1% L-Glutamine ( NB ) , and 50 ng/ml BDNF ( Peprotech ) .", "Twenty-four hours post plating , the medium was replaced to a one containing 30 µM Cytosine β-D-arabinofuranoside ( Ara-C ) and after additional 24 hr treated with 1 ng/µl of Sema6A-Fc/0 . 02 ng/µl of Sema3D-Fc/1 nM of AP-Sema3F for 30mins .", "The control for the HPLC-purified Semaphorins ( Sema6A-Fc and Sema3D-Fc ) was NB with BDNF , and for AP-Sema3F was conditioned medium of AP empty vector transfected cells concentrated the same way .", "For collapse assay without BDNF , 24 hr after adding the Ara-C the medium was replaced to NB or NB +50 ng/ml BDNF with or without 0 . 025 ng/µl of Sema6A-Fc for 30mins . The cultures were fixed with 4% Paraformaldehyde +30% Sucrose solution , and stained with Phalloidin-Rhodamine ( Sigma-Aldrich , 1:250 ) for visualization of F-actin filopodia .", "Growth cones with no or few filopodia were considered as collapsed .", "Percentage of collapsed growth cones was calculated for each treatment group and control .", "A minimum of three independent experiments were performed per condition , in each experiment three DRGs were analyzed per embryo .", "Percentages are means between experiments .", "Error bars represent the SEM between replicates .", "Statistical analysis was performed using one-way or two-way ANOVA followed by post hoc analysis ( Prism7 software ) .", "DRGs from early E13 embryos of the appropriate genotype were dissected and plated as explants in chambers coated with 10 µg/ml Poly-D-lysine ( PDL ) and 10 µg/ml Laminin , and contained Neurobasal-A growth medium supplemented with 2% B-27 , 1% Penicillin-Streptomycin , 1% L-Glutamine , and 50 ng/ml BDNF ( Peprotech ) .", "48 hr post plating , the DRGs were washed with binding buffer ( Hank’s- Buffered Salt Solution with 0 . 2% BSA , 0 . 1% NaN3 , 5 mM CaCl2 , 1 mM MgCl2 and 20 mM HEPES , pH=7 . 0 ) for 10 min and incubated for 90 min at room temperature with 1 ng/µl of Sema6A-Fc containing 1:1000 goat anti-human IgG ( Fc specific ) - Alkaline Phosphates ( AP ) -conjugated antibody ( A9544 , Sigma ) in binding buffer .", "After the removal of unbound ligand , DRGs were fixed with 4% PFA +30% Sucrose for 12 min and rinsed three times with 20 mM HEPES pH=7 . 0 and 150 mM NaCl .", "In order to destroy intrinsic AP activity , DRGs were heat inactivated in 65°C water bath for 30 min .", "Finally , DRGs were rinsed three times with AP buffer ( 100 mM TRIS PH=9 . 5 , 100 mM NaCl and 50 mM MgCl2 ) and the AP activity was evaluated by the formation of precipitants after an overnight incubation with 1:50 Nitro blue tetrazolium chloride/5-bromo-4-chloro-3-indolyl phosphate substrate ( NBT/BCIP , Roche ) in AP buffer .", "For whole-mount staining of β-galactosidase ( β-gal ) activity , embryos at different developmental stages were fixed in 4% formaldehyde for 2 hr in 4°C , followed by three washes in PBS .", "Staining was performed with X-gal solution ( 5 mM Potassium ferricyanide , 5 mM Potassium ferrocyanide , 2 mM MgCl2 and 0 . 5 mg/ml 5-bromo-4-chloro-3-indolylβ-D-galactopyranoside [X-gal] in PBS ) .", "Incubation time was 2–4 hr in room temperature , depending on the developmental stage , followed by three washes in PBS .", "The reaction was stopped by O . N incubation with 4% formaldehyde solution in 4°C .", "For immunohistochemical stainings the following primary antibodies were used: Goat anti-TrkBECD ( R&D , AF1494 , 1:100 ) ; Mouse anti-tubulin βIII ( R&D , TUJ-1 clone/catalog , 1:500 ) .", "Fluorescent secondary antibodies were purchased from Jackson Immunoresearch: donkey anti-goat Cy3 ( 1:200 ) , and goat anti-mouse Cy3 ( 1:200 ) .", "Phalloidin-Rhodamine was used at 1 ug/ml ( Sigma-Aldrich , P1951 ) ." ] ]

[ "The innervation of the mammary gland is controlled by brain-derived neurotrophic factor ( BDNF ) , and sexually dimorphic sequestering of BDNF by the truncated form of TrkB ( TrkB . T1 ) directs male-specific axonal pruning in mice .", "It is unknown whether other cues modulate these processes .", "We detected specific , non-dimorphic , expression of Semaphorin family members in the mouse mammary gland , which signal through PlexinA4 .", "PlexinA4 deletion in both female and male embryos caused developmental hyperinnervation of the gland , which could be reduced by genetic co-reduction of BDNF .", "Moreover , in males , PlexinA4 ablation delayed axonal pruning , independently of the initial levels of innervation .", "In support of this , in vitro reduction of BDNF induced axonal hypersensitivity to PlexinA4 signaling .", "Overall , our study shows that precise sensory innervation of the mammary gland is regulated by the balance between trophic and repulsive signaling .", "Upon inhibition of trophic signaling , these repulsive factors may promote axonal pruning ." ]

[ "Almost every action an animal can perform in its life will rely on its nervous system being wired correctly .", "Before the animal is born , nerve cells next to the spinal cord send out long fibers – called axons – to connect with different parts of its body .", "These nerves will help relay sensations to the brain .", "The ends of the nerve fibers follow trails of signals that guide them to the correct targets .", "When the axon arrives in the right place , it can receive further signals called neurotrophic factors .", "These signals keep the axon alive , but they are in short supply .", "Not every axon will receive the signal , and , without it , the nerve fiber dies back .", "This phenomenon , known as pruning , helps to make sure that nervous system does not form more connections than it needs .", "In mammals , the mammary gland is an example of a part of the body where nerve endings are pruned during development .", "Axons that connect to this milk-producing gland depend on a neurotrophic factor called BDNF to survive , and BDNF is controlled differently in males and females .", "In male mammals , another protein grabs hold of the BDNF signal and hides it away before development is complete .", "After this happens , the axons start to die , however it was not clear if other signals are also involved .", "Sar Shalom et al . have now examined if proteins called Semaphorins – which guide axons to their target locations and influence pruning too – also control how many nerves end up connected to the mammary gland .", "The experiments used nerve cells grown in the laboratory and genetically modified mice , and suggested that the nerves in the mammary gland would only develop correctly if the BDNF and Semaphorins signals were properly balanced .", "When the lab-grown nerve cells encountered Semaphorins , their growing axons collapsed .", "Yet unlike for BDNF , the levels of Semaphorins were the same in male and female mice .", "Further experiments showed that if a protein receptor that detects the Semaphorins was deleted , the nerve cells stopped responding to the signal , and their axons did not collapse .", "In mice lacking this receptor , both sexes ended up with more axons in their mammary glands .", "Too many axons grew in the female mice , while the pruning of excess axons was delayed in the males .", "Reducing the levels of BDNF in these mice helped to return axon growth to normal .", "Together , these findings suggest that a balance between the BDNF and the Semaphorins sets the correct number of nerves .", "They also suggest that once the BNDF signal is removed during the normal development of males , it is the Semaphorins that help the axons in the mammary gland to be pruned .", "Lastly , neurotrophic factors and Semaphorins are not just important during development; indeed cells make them well into adulthood .", "Altered patterns of these signals in mature animals could change the shapes of nerve networks .", "As such , future work may help scientists to understand why tissues can become too sensitive or lose sensation ." ]

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

[ "neuroscience" ]

[ [ "Myelination is well known to be important in increasing the speed of action potential propagation in long-axon connections ( Waxman and Bennett , 1972; Moore et al . , 1978 ) .", "The axons of the central nervous system ( CNS ) connecting distant brain areas are typically wrapped in myelin , as are the axons of the peripheral nervous system ( PNS ) that connect the CNS to all skeletal muscles and many sensory neurons .", "Pathologies of myelinated axons are associated with numerous neurological disorders ( Fields , 2008 ) and are fundamental to several , such as multiple sclerosis ( MS ) ( Popescu and Lucchinetti , 2012; Calabrese et al . , 2015 ) , neuromyelitis optica ( Wingerchuk et al . , 2015 ) , and leukodystrophies ( Gordon et al . , 2014 ) .", "Because myelin increases the conduction velocity of action potentials and reduces their metabolic cost ( Nave , 2010; but see Harris and Attwell , 2012 ) , it has traditionally been viewed as ‘wire insulation’ for long-range neural projections .", "Recent findings have cast myelination as a dynamic process playing an active role in normal neural circuit function and plasticity .", "For example , adult-born oligodendrocytes ( the myelin-producing cells of the CNS ) contribute new myelin to axons in an ongoing fashion ( Young et al . , 2013 ) , increased neuronal activity results in more myelination ( Gibson et al . , 2014 ) , and myelin remodeling in the CNS is required for motor skill acquisition ( McKenzie et al . , 2014 ) .", "Myelin also supplies ensheathed axons with lactate via the monocarboxylate transporter MCT1 , possibly playing an important energetic support role in the CNS ( Rinholm et al . , 2011; Lee et al . , 2012; Fünfschilling et al . , 2012; Morrison et al . , 2013 ) .", "The emerging diversity of roles for myelin calls for a more complete understanding of its distribution among neuronal cell types and along individual axonal arbors , particularly in the gray matter of the CNS .", "Excitatory pyramidal neurons in cortex are known to be a main source of long-range myelinated axons connecting the two cortical hemispheres or targeting subcortical structures .", "Foundational EM-based studies of individual Golgi- or dye-filled neurons ( Kisvárday et al . , 1986; Martin and Whitteridge , 1984; Peters and Proskauer , 1980; Tamás et al . , 1997 ) have revealed that the local axon collaterals of both excitatory and inhibitory neurons may also be myelinated .", "However , population surveys of the distribution of myelin on the axonal arbor of individual neocortical neurons have been difficult to undertake with traditional methods .", "Golgi labeling of neurons halts where the axon first enters a myelin sheath ( Peters and Proskauer , 1980; DeFelipe et al . , 1986; Fairén et al . , 1977 ) , and bulk labeling of myelin reveals a laminar patterned thicket ( Nieuwenhuys , 2013 ) , rather than individual neuronal arbors .", "Recent advances in large volume , high resolution neuroanatomy methods ( Briggman and Bock , 2012; Micheva and Smith , 2007; Economo et al . , 2016 ) now permit cortical myelin to be directly visualized on the axonal arbors of multiple individual identified neurons for all axons within the imaged volume .", "Myelin tracing in large EM datasets has revealed that myelination of the axons of pyramidal neurons in cortex is distinct from the known myelination patterns in subcortical white matter , and also differs between cortical layers ( Tomassy et al . , 2014 ) .", "About 10–20% of neocortical neurons are inhibitory and some of these neurons may also have myelinated axons .", "For example , labeling of individual neocortical neurons during in vivo electrophysiological recording , followed by light-level reconstruction and electron microscopy ( EM ) of selected sub-arbors , has revealed that inhibitory neurons may be myelinated ( DeFelipe et al . , 1986; Somogyi et al . , 1983; Kawaguchi and Kubota , 1998; Kisvarday et al . , 1987; Thomson et al . , 1996; Tamás et al . , 1997 ) .", "Complementary immunohistochemical studies have shown that parvalbumin-positive ( i . e . , putative inhibitory [Rudy et al . , 2011] ) axons may be myelinated ( McGee et al . , 2005; Chung and Deisseroth , 2013 ) .", "Given that cortical interneuron types are implicated in distinct circuit functions ( Kepecs and Fishell , 2014 ) and have distinct behavioral correlates ( Kvitsiani et al . , 2013 ) , these results raise several important questions regarding the organization of neocortical neuronal networks: how widespread is inhibitory axon myelination ?", "Is it specific to certain interneuron subtypes ?", "Does inhibitory axon myelin differ from excitatory axon myelin at the molecular level ?", "Here we combine analyses of light-microscopy-based array tomography ( AT ) data ( Micheva and Smith , 2007; Collman et al . , 2015 ) with a publicly hosted EM dataset ( Burns et al . , 2013; Martone et al . , 2002 ) generated using a custom transmission EM camera array ( TEMCA ) ( Bock et al . , 2011 ) , to obtain an integrated view of the myelination of inhibitory neurons in cortex .", "We find that an unexpectedly large proportion of myelinated axons in cortex arise from a specific subtype of inhibitory neuron: the parvalbumin-positive ( PV+ ) basket cell .", "These PV+ axons are especially prominent in the middle cortical layers .", "They constitute about half of all myelinated axons in layers 2/3 , and a quarter of myelinated axons in layer 4 .", "We also find that the inhibitory myelinated axons in cortical gray matter differ from the excitatory myelinated axons in many respects , such as their structural organization , cytoskeletal content , and myelin protein composition .", "Taken together , the abundance and cell-type selectivity of neocortical myelinated inhibitory axons described here raise fundamental questions about the role of myelination both in normal function and in disease ." ], [ "Immunofluorescence AT is particularly well suited for the study of myelinated axons in cortex .", "The abundance of myelin proteins , such as myelin basic protein ( MBP ) , and the size of myelinated axons ( >200 nm in diameter ) make them an ideal target for immunofluorescence detection ( Figure 1A , Figure 1—figure supplement 1 , Video 1 ) .", "The ultrathin AT sections provide easy access of the antibodies against MBP and do not require lipid extraction .", "Conjugate light-electron AT confirms that immunofluorescence for MBP precisely outlines the myelinated sheaths of axons ( Figure 1B , Figure 1—figure supplement 1B , Figure 1—figure supplement 2A ) .", "Overall , the MBP immunolabel is of high quality as evidenced by the consistency of the signal from section to section ( Figure 1—figure supplement 2B ) , and the low background ( mean gray value of 248 ± 6 a . u . on resin and 279 ± 2 a . u . on nuclei , vs 5754 ± 226 a . u . on myelin , mean and standard error ) .", "In addition , the use of ultrathin sections ensures that antigens can be detected equally well within myelinated and unmyelinated portions of the axon .", "For example , in our AT experiments GABA and PV immunostaining within axons is not affected by myelination ( Figures 1 , 3 ) , in contrast to preembedding immunocytochemistry where myelinated portions of axons are much more weakly stained ( Pawelzik et al . , 2002 ) . 10 . 7554/eLife . 15784 . 003Figure 1 . A large fraction of myelinated axons in upper layers of cortex contain GABA .", "( A ) An ultrathin section ( 70 nm ) through the mouse somatosensory cortex immunolabeled for MBP ( cyan ) and GABA ( red ) .", "Nuclei are stained with DAPI ( blue ) .", "Smaller regions from each layer are shown at higher magnification to the right .", "( B ) Scanning electron micrograph from layer 5 of the mouse somatosensory cortex overlaid with the corresponding immunofluorescence for MBP ( cyan ) and GABA ( red ) , and the DAPI signal ( blue ) .", "( C ) Proportion of myelinated axonal profiles containing GABA in the cortical layers of mouse somatosensory cortex .", "( D ) Density of GABA and non-GABA myelinated axons in mouse somatosensory cortex ( y-axis is in logarithmic scale to accommodate the large range of myelinated axon densities along the cortical depth ) .", "Mean from 3 animals and standard errors are shown in C and D ( number of axonal profiles analyzed per animal was 11103 , 12 , 657 and 7540 , respectively ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 15784 . 00310 . 7554/eLife . 15784 . 004Figure 1—source data 1 . Data values underlying Figure 1 . DOI: http://dx . doi . org/10 . 7554/eLife . 15784 . 00410 . 7554/eLife . 15784 . 005Figure 1—figure supplement 1 . Antibodies used in this study labeled the expected structures as assessed by IF volume reconstruction and SEM imaging .", "( A ) MBP immunofluorescence on a single ultrathin section ( 70 nm ) from layer 5 of the adult mouse cortex .", "Interruption in the MBP staining indicates a node of Ranvier ( magenta box ) ( B ) MBP staining corresponds exactly to the myelin sheath as seen in the SEM of the axon boxed in green in A . ( C ) Nodes of Ranvier ( magenta box in A ) do not stain for MBP , but the axonal path can be traced using cytoskeletal markers which persist through the nodes ( alpha tubulin in red ) .", "( D ) Volume reconstruction of myelinated axons immunolabeled with MBP ( white ) and GABA ( red ) in cortical layer 5 ( 43 sections , 70 nm each ) .", "Nuclei are stained with DAPI ( blue ) .", "SEM of layer 5 of mouse cortex overlaid with immunofluorescence for GABA ( red ) , MBP ( cyan ) , α tubulin ( green ) , glutamine synthetase ( orange ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 15784 . 00510 . 7554/eLife . 15784 . 006Figure 1—figure supplement 2 . MBP antibody performance .", "( A ) Myelin thickness as measured from IF data and SEM data .", "Myelin thickness was measured on the same sections using either immunofluorescence for MBP or ultrastructurally defined myelin on SEM images using a dataset from a previous study ( McGee et al . , 2005 ) ( N=125 axonal profiles ) .", "There is a strong correlation between the two measurements ( R2=0 . 9 ) , which allows the use of MBP immunofluorescence for more efficient sampling .", "( B ) Consistency of MBP immunolabel .", "Correlation of MBP immunofluorescence of the same myelin sheath measured on two adjacent ultrathin sections ( N=100 axonal profiles ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 15784 . 00610 . 7554/eLife . 15784 . 007Video 1 . Serial sections through mouse cortex immunostained for MBP . Stack of raw images from 59 serial sections ( 70 nm each ) from cortical layer 5 . DOI: http://dx . doi . org/10 . 7554/eLife . 15784 . 007 As expected , AT detects the presence of myelinated axons in all neocortical layers .", "Quantification of the number of MBP profiles in single sections shows that their numbers dramatically increase with cortical depth ( Figure 1D ) , consistent with previous quantitative myeloarchitectonic studies in non-rodent species ( Nieuwenhuys , 2013; Braitenberg , 1962; Hopf , 1966 ) .", "Immunostaining for the inhibitory neurotransmitter GABA reveals that a surprisingly large number of myelinated axons throughout cortex contain GABA , including more than 45% of myelinated axons in layers 2/3 ( 48 . 1 ± 3 . 2% , mean ± standard error , Figure 1C ) .", "Layers 4 and 5 exhibit similar numbers of GABA myelinated axons as layers 2/3 ( Figure 1D ) , but the deeper layers contain larger numbers of non-GABA myelinated axons , presumably of projecting pyramidal cells .", "GABA myelinated axons are relatively sparse in layers 1 , 6 , and only occasionally seen in subcortical white matter .", "In parallel with the array tomography work , and blind to its results , myelinated axons were traced within a previously acquired ( Bock et al . , 2011 ) , publicly available ( Burns et al . , 2013; Martone et al . , 2002 ) volume electron microscopy dataset from the upper layers of mouse visual cortex V1 .", "This dataset was of sufficient size and resolution that both myelinated and unmyelinated axons could be traced over hundreds of microns , allowing them to be categorized on the basis of their synaptic ultrastructure as inhibitory or excitatory .", "A section from near the middle of the EM volume was selected , and all the myelin profiles from the center portion of this section were annotated ( Figure 2A ) .", "Each of these myelinated 'seeds' ( e . g . Figure 2B , E ) was used as a starting point for further tracing of the myelinated axon through the EM volume .", "Along their length , a majority of the traced axons unmyelinated and made a number of synapses ( Figure 2C , F ) .", "Axons were classified according to the appearance of the pre- and postsynaptic densities of the synapses they made , using the classical definitions for asymmetrical ( excitatory ) and symmetrical ( inhibitory ) synapses ( Colonnier , 1968; Peters et al . , 1991 ) ( Figure 2D , G ) .", "Of the 231 myelinated 'seeds' , 73 were classified as excitatory ( 31 . 6%; Figure 2—figure supplement 1 ) , 106 as inhibitory ( 45 . 9%; Figure 2—figure supplement 2 ) , and 52 ( 22 . 5%; Figure 2—figure supplement 3 ) could not be categorized due to an absence of synapses within the volume .", "This analysis shows that inhibitory myelinated axons are abundant in cortical layer 2/3 , comprising at least 45% of myelinated axons in the mouse visual cortex . 10 . 7554/eLife . 15784 . 008Figure 2 . Volume electron microscopy shows a large fraction of myelinated axons in layer 2/3 of cortex are inhibitory .", "( A ) EM mosaic from pia through lower layer 2/3 , overlaid with the locations of myelinated axon profiles used as tracing seeds .", "Each seed is color-coded by the type of synapses the axon was determined to make after exiting its myelin sheath elsewhere in the volume ( red: symmetric; green: asymmetric; yellow , no synapses in the EM volume ) .", "Red and green rectangles indicate areas of detail in B and E , respectively .", "( B ) Detail view of area in red rectangle in A . Three inhibitory myelinated axon profiles ( false colored red ) are shown .", "( C ) A reconstructed axon arbor arising from the myelinated tracing seed at the center of B . Thick blue segments indicate myelinated internodal regions; thin red segments represent unmyelinated axon .", "Segment diameters are schematic .", "This axon was traced to its originating soma ( sphere in lower left ) and to 8 synapses ( small red dots ) .", "The arrow indicates the location on this axon’s arbor of the symmetric synapse shown in D . Note that axon arbors were traced only until their synapses could be reliably categorized as symmetric or asymmetric; therefore the arbor shown here is a small subset of the full axonal arbor arising from this inhibitory neuron .", "( D ) An unmyelinated axon profile ( red ) makes a symmetric synapse ( red triangle ) onto a postsynaptic spine ( yellow ) .", "This spine also receives an asymmetric synapse ( blue triangle ) from an excitatory axon ( green ) .", "( E ) Detail view of area in green rectangle in A , showing two excitatory myelinated axon profiles ( false colored green ) .", "( F ) A reconstructed axon arbor arising from the myelinated tracing seed at the center of E . Conventions as in C , except unmyelinated axon segments are rendered in green .", "( G ) An unmyelinated axon profile ( green ) makes three asymmetric synapses ( blue triangles ) onto three different spines .", "Scale bar in A , 50 μm; E , 3 μm ( also applies to B ) ; F , 20 μm ( also applies to C ) ; G , 1 μm ( also applies to D ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 15784 . 00810 . 7554/eLife . 15784 . 009Figure 2—figure supplement 1 . Distribution of myelin ( cyan ) on axon fragments ( green ) categorized as excitatory . Left , frontal view .", "Right , side view .", "Large green spheres: excitatory somata reached during categorization of axon fragments; medium-sized spheres indicate the position of tracing seeds of categorized axon fragments ( green: excitatory; red: inhibitory; yellow: unclassifiable ) .", "Smallest green spheres indicate the position of synapses made by the excitatory axon fragments .", "Scale bar ~50 μm . DOI: http://dx . doi . org/10 . 7554/eLife . 15784 . 00910 . 7554/eLife . 15784 . 010Figure 2—figure supplement 2 . Distribution of myelin ( cyan ) on axon fragments ( red ) categorized as inhibitory . Left , frontal view .", "Right , side view .", "Large red spheres: inhibitory somata reached during categorization of axon fragments; medium-sized spheres indicate the position of tracing seeds of categorized axon fragments ( green: excitatory; red: inhibitory; yellow: unclassifiable ) .", "Smallest red spheres indicate the position of synapses made by the inhibitory axon fragments .", "Scale bar ~50 μm . DOI: http://dx . doi . org/10 . 7554/eLife . 15784 . 01010 . 7554/eLife . 15784 . 011Figure 2—figure supplement 3 . Distribution of myelin ( cyan ) on axon fragments ( yellow ) that could not be categorized , due to an absence of synapses in the EM-imaged volume . Left , frontal view .", "Right , side view .", "Large green spheres: excitatory somata; medium-sized spheres indicate the position of tracing seeds of categorized axon fragments ( green: excitatory; red: inhibitory; yellow: unclassifiable ) .", "Scale bar ~50 μm . DOI: http://dx . doi . org/10 . 7554/eLife . 15784 . 011 Cortical GABA neurons are a highly diverse population ( Ascoli et al . , 2008; Markram et al . , 2004; Xu et al . , 2010 ) .", "About half of them contain parvalbumin ( PV ) , a calcium buffering protein .", "Array tomography ( Figure 3A , B and Figure 3—figure supplement 1 ) revealed that nearly all GABA myelinated axons ( 97 . 9 ± 0 . 8% , mean ± st . error from 3 animals ) are immunopositive for parvalbumin and therefore originate from PV interneurons .", "Correspondingly , in the examined cortical AT volumes ( Figure 3C–F ) , examples of PV and GABA-positive cell bodies forming myelinated axons are seen in layers 2/3 , 4 and 5 .", "Blind to the AT results , we conducted independent experiments in three mice expressing the fluorescent protein tdTomato driven by Cre expression in PV neurons ( Pvalb-ires-Cre;Ai9 ) ( Taniguchi et al . , 2011 ) .", "Confocal imaging of 40 μm thick Vibratome slices from these mice immunostained with MBP corroborated the AT findings by showing that about half ( 53 ± 12% ) of the myelinated axons in layer 2/3 of somatosensory cortex contain tdTomato and , therefore , PV ( Figure 4A–D ) .", "Similar experiments were also performed on mice expressing tdTomato from SOM-Cre or VIP-Cre driver lines ( Sst-ires-Cre;Ai9 , and Vip-ires-Cre;Ai9 ) ( Taniguchi et al . , 2011; Madisen et al . , 2010 ) .", "After careful inspection of selected subareas , no myelinated axons containing reporter for the non-PV cortical interneurons vasoactive intestinal polypeptide ( VIP; Figure 4E–G ) could be observed .", "A small fraction ( 4 . 4% ) of somatostatin ( SOM ) -positive myelinated axons were observed ( Figure 4H–J ) ; however , the transgenic reporter line used was recently found to have a small but consistent false-positive expression pattern , wherein up to 10% of Cre-expressing neurons are SOM-negative and PV-positive when examined by immunohistochemistry ( Hu et al . , 2013 ) .", "At least some of the SOM-positive myelinated axon profiles we observe therefore may be false positives which actually express PV , not SOM; and some may be true positives , consistent with the AT data , which show that ~2% of GABA myelinated profiles are PV negative .", "Regardless , the data obtained from the three Cre-lines support the conclusion that the great preponderance of myelinated inhibitory axon profiles in layer 2/3 arise from parvalbumin-positive axons . 10 . 7554/eLife . 15784 . 012Figure 3 . Nearly all myelinated GABA axons are parvalbumin-positive .", "( A ) A single section ( 70 nm ) from layer 2/3 of mouse cortex , immunolabeled for MBP ( white ) , GABA ( red ) and PV ( green ) ; nuclei are stained with DAPI ( blue ) .", "Note that the PV-containing neurons have weaker GABA immunoreactivity .", "( B ) The central box from A shown at a higher magnification .", "The great majority ( 97 . 9 ± 0 . 8% ) of GABA immunopositive myelinated axons ( red , left panel ) also contain PV ( green , right panel ) ; the yellow box marks an example of such an axon ( see main text for quantification ) .", "Occasionally , a GABA myelinated axon does not show detectable PV immunofluorescence as shown in the red box .", "( C ) Serial sections through the parvalbumin containing neuron boxed in A , C showing its myelinated axon .", "( D ) Volume reconstruction of the neuron in C . ( E , F ) Volume reconstructions of PV interneurons with myelinated axons from layers 4 and 5 .", "A–F , Scale bar , 10 μm . DOI: http://dx . doi . org/10 . 7554/eLife . 15784 . 01210 . 7554/eLife . 15784 . 013Figure 3—figure supplement 1 . GABA and PV immunoreactivity of myelinated axons . Top , Distribution of GABA and PV immunofluorescence within cortical myelinated axons .", "Because of the inherent variability of immunolabeling intensity and image acquisition parameters , comparisons were always done on sections from the same coverslip , which were stained and imaged at the same time .", "Measurements from layers 4 and 5 of mouse cortex from one coverslip are presented in the figure .", "The threshold for GABA and PV immunofluorescence is indicated by the blue lines .", "Bottom , Scatterplot of GABA and PV immunofluorescence from the same experiment , showing three classified populations of myelinated axons: GABA negative in blue , GABA and PV positive in red , and GABA positive but PV negative axonal profiles in green . DOI: http://dx . doi . org/10 . 7554/eLife . 15784 . 01310 . 7554/eLife . 15784 . 014Figure 4 . Immunofluorescent labeling of MBP shows half of myelin profiles ensheath PV-positive axons but no VIP- and very few SOM-expressing axons .", "( A ) Maximum intensity projection of a stack of confocal image mosaics , spanning pia ( top ) to white matter ( bottom ) of somatosensory cortex , revealing the laminar distribution of PV-expressing neurons ( red ) and immunolabeled myelin ( green ) .", "Yellow square in layer 2/3 indicates the location of the field of view portrayed in panels B–D .", "( B–D )", "Detail view of a representative subarea from a single section in layer 2/3 .", "About half of the myelinated axons are PV-positive ( yellow triangles ) .", "B shows the red channel ( PV ) only; C shows the green channel ( myelin ) only , and myelinated profiles containing PV-positive axons are false-colored cyan; and D overlays panels B and C . ( E–G ) Representative confocal images from layer 2/3 of somatosensory cortex , showing VIP-expressing neurites ( red ) , immunolabeled myelin ( green ) , and DAPI-stained nuclei ( blue ) .", "E shows only the red ( VIP ) and blue ( DAPI ) channels; F shows only the green ( myelin ) and blue ( DAPI ) channels; and G overlays panels E and F . ( H–J ) Representative confocal images from layer 2/3 of somatosensory cortex , showing SOM-expressing neurites ( red ) , immunolabeled myelin ( green ) , and DAPI-stained nuclei ( blue ) .", "H shows only the red ( SOM ) and blue ( DAPI ) channels; I shows only the green ( myelin ) and blue ( DAPI ) channels , and myelinated profiles containing SOM-positive axons are false-colored cyan; and J overlays panels H and I . No myelinated axon profiles are positive for VIP; 4 . 4% are positive for SOM .", "Scale bar in A , 100 μm; B , 10 μm ( also applies to C–D ) ; E , 10 μm ( also applies to H–J ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 15784 . 014 Among the inhibitory neurons of cortex , parvalbumin is found in basket cells , chandelier cells ( Markram et al . , 2004; Kawaguchi and Kubota , 1997; DeFelipe et al . , 1989 ) , and in a sparse sub-population of long-range inhibitory axons arising from basal forebrain ( Freund and Gulyás , 1991; Caputi et al . , 2013 ) .", "Because these different cell types are known to have distinct axonal morphology and targets , we traced the postsynaptic targets of inhibitory myelinated axons in layer 2/3 from the volume EM data set in an effort to understand their originating cell type ( Figure 5 ) .", "The postsynaptic targets were identified as inhibitory or excitatory according to previously described ultrastructural criteria ( Bock et al . , 2011 ) ( Materials and methods ) .", "Briefly , postsynaptic dendrites were classified as inhibitory if they were largely aspinous and densely coated with asymmetric ( excitatory ) synapses ( e . g . Figure 6E–F ) , while they were classified as excitatory if they were spiny and received fewer shaft excitatory synapses .", "Excitatory somata were distinguished from inhibitory somata based on cell shape , the presence of a large apical dendrite , and the absence of asymmetric contacts on the soma .", "For each synapse , the postsynaptic compartmental location and class ( excitatory or inhibitory ) was tabulated ( Table 1 ) , and postsynaptic class was used to color-code the synapses along the reconstructed axonal fragments ( Figures 5–6 ) . 10 . 7554/eLife . 15784 . 015Figure 5 . Myelinated inhibitory axons rarely make synapses upon other axons and are therefore unlikely to belong to chandelier cells .", "( A ) An inhibitory axon traced from a myelinated seed to completion within the EM volume .", "The proximal axonal arbor receives the preponderance of myelin ( thick blue arbor segments ) , whereas the distal arbor makes most of the synapses .", "The red rectangle outlines the sub-arbor shown in D . In ( A–C ) , the large red spheres indicate the position of the soma .", "Synapses are represented by dots , color-coded according to postsynaptic target class: green ( excitatory ) , magenta ( excitatory axon ) , yellow ( unclassifiable ) , and red ( inhibitory ) .", "( B ) A second inhibitory axon traced to completion .", "In this case the EM volume boundaries were reached before reaching the soma .", "A myelinated core portion of the arbor can be discerned , with most synapses at the periphery .", "( C ) All the inhibitory axon fragments traced from myelinated seeds .", "Overall , only 2% of synapses made by myelinated inhibitory axons are onto excitatory axons ( magenta spheres ) ; no synapses onto inhibitory axons were observed .", "( D ) Detail view of area shown in red rectangle in A showing one of the rare synapses made by myelinated inhibitory neurons onto the proximal axon of a pyramidal cell ( arrow ) .", "( E ) EM image of a section intersecting the pyramidal cell body and its proximal axon ( false-colored green ) , postsynaptic to the synapse indicated by the arrow in D ( false-colored red ) .", "The red rectangle indicates the area of detail shown in F . ( F ) A magnified view of the synapse shown in E . A second postsynaptic target , the spine neck of a dendrite arising from a different pyramidal cell is false-colored in yellow .", "Symmetrical synapse locations indicated by red triangles .", "Scale bar in A–C , 50 μm; E , 4 μm; F , 1 μm . DOI: http://dx . doi . org/10 . 7554/eLife . 15784 . 01510 . 7554/eLife . 15784 . 016Figure 6 . Myelinated inhibitory axons target dendritic shafts , spines and neuronal cell bodies .", "( A ) A representative inhibitory axon fragment traced from a myelinated seed profile ( arrow ) .", "Blue indicates the extent of myelin along the fragment; thin red axon segments are unmyelinated .", "The spheres indicate the location of symmetric synapses , and are color-coded red for inhibitory postsynaptic targets ( synapse 1 ) and green for excitatory postsynaptic targets ( synapses 2–5 ) .", "These five symmetric synapses were sufficient to categorize the axon as inhibitory .", "The letters above the fragment correspond to the approximate locations of later panels .", "( B ) The myelinated profile used as a tracing seed for the axon fragment in A . Myelin is false-colored blue; the axon is false colored in red .", "( C–N )", "EM micrographs through synapses 1–5 .", "The presynaptic axon is false-colored in dark red; the postsynaptic inhibitory dendrite in light red; the postsynaptic excitatory targets in green; and other excitatory boutons converging onto the same targets as the myelinated inhibitory axon , in yellow .", "( C ) Synapse 1 contacts an inhibitory dendrite , which is also shown in ( D–E ) .", "In E , the dendrite is densely coated with excitatory axonal boutons making asymmetric synapses ( blue triangles ) , a hallmark of inhibitory dendrites .", "( F ) Cross-section through synapse 2 .", "( G ) Larger field of view of the section in F , showing that the postsynaptic target is a neuronal cell body .", "( H ) A nearby section through the same neuron as in G , reveals it to be a pyramidal cell with a prominent apical dendrite .", "( I ) The myelinated axon approaching an apical dendrite .", "( J ) Synapse 3 is formed immediately after the axon unmyelinates and contacts the same apical dendrite shown in I . ( K ) The same bouton participating in synapse 3 forms synapse 4 with a spine arising from the same apical dendrite shown in I and J . ( L ) Synapse 5 targets the shaft of a dendrite , identified as excitatory by the presence of spines on near-by sections ( e . g . M–N ) .", "( M ) Synapse 5 can still be seen , and a spine neck arises from the postsynaptic dendrite .", "( N ) The spine neck in M forms a small spine head , receiving an asymmetric synapse ( blue triangle ) from an excitatory bouton from a different axon .", "Scale bar in A , ~1 μm; B , 1 μm ( also applies to C–F ) ; G , 4 μm; H , 9 μm; I , 1 μm ( also applies to J–L ) ; M , 1 μm ( also applies to N ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 15784 . 01610 . 7554/eLife . 15784 . 017Table 1 . The distribution of the postsynaptic targets . DOI: http://dx . doi . org/10 . 7554/eLife . 15784 . 017Postsynaptic targetCompletely traced axonsPartially traced axonsAllcount%count%count%Excitatory somata60*15 . 046†10 . 510612 . 6Excitatory dendritic shafts18445 . 922551 . 440948 . 7Excitatory dendritic spines11929 . 713430 . 625330 . 2Excitatory axons71 . 781 . 8151 . 8All excitatory37092 . 341394 . 378393 . 3Inhibitory dendritic shafts174 . 2204 . 6374 . 4Inhibitory dendritic spines000000Inhibitory axons10 . 210 . 1All inhibitory174 . 2214 . 8384 . 5Uncategorized dendritic spines143 . 540 . 9182 . 1Total401438839* Including 10 synapses onto somatic spines;† Including 8 synapses on somatic spines The observed distribution of postsynaptic targets of myelinated inhibitory axons was consistent with their originating from local axonal arbors of cortical basket cells , and inconsistent with their arising from either chandelier cells or long-range inhibitory afferents to cortex .", "Basket cells and chandelier cells can readily be distinguished by their morphology and synaptic targets .", "When the axons from multiple basket cells are labeled and examined at the light level , they form ‘baskets’ of synapses around neuronal somata , hence the name ( Kisvárday , 1992 ) .", "Chandelier cells have an axonal arbor which resembles a chandelier , with vertically oriented axonal cartridges that almost exclusively form synapses onto the axon initial segments of pyramidal neurons ( Somogyi et al . , 1982 ) .", "Less than 2% of the inhibitory myelinated axons in our volume electron microscopy dataset were found to make contacts on axon initial segments ( Figure 5; Table 1 ) .", "In addition , the few axonal arbors that were seen contacting axon initial segments did not have the characteristic appearance of chandelier cell axons and made single contacts with axonal initial segments , instead of several ( typically 3–5 for chandelier cells ) synapses in a row ( Figure 5A , B ) .", "Therefore we can exclude the possibility that the myelinated inhibitory axons arise from chandelier cells .", "The great preponderance ( 93% ) of postsynaptic targets of myelinated inhibitory axons in layer 2/3 of cortex were excitatory , likewise eliminating the possibility that these axons originated from the arbors of the known parvalbumin-positive long-range inhibitory afferents to cortex , all of which selectively target inhibitory neurons ( Freund and Gulyás , 1991; Gritti et al . , 2003; Henny and Jones , 2008 ) .", "The remaining possibility , that myelinated inhibitory axons arise from the arbors of local parvalbumin-positive basket cells , was well supported by the distribution of their postsynaptic targets .", "Although neocortical basket cells are best known for their synapses with cell bodies , a large ( but variable ) fraction of their synapses are with dendritic shafts and spines ( Somogyi et al . , 1983; Kawaguchi and Kubota , 1998; Kisvarday et al . , 1987; Tamás et al . , 1997 ) .", "Consistent with past surveys of basket cell synaptic targets , we found that 13% of the postsynaptic targets of the myelinated inhibitory axons were with somata , 49% were onto excitatory dendritic shafts , and 30% were onto dendritic spines ( Figure 6; Table 1 ) .", "Thus , we conclude that the majority of myelinated inhibitory axons belong to the intracortical axons of parvalbumin containing basket cells .", "Both our volume electron microscopy and array tomography data show that the axon of a parvalbumin interneuron becomes myelinated soon after exiting the cell body ( usually within 20–50 μm ) , consistent with myelination commencing shortly after the axon initial segment .", "All of the axonal arbors that were traced back to the cell body in our volume EM dataset ( n=8 ) had the same general appearance: a central core of partially myelinated axons ( whose unmyelinated stretches rarely made synapses ) , from which emerged unmyelinated branches forming numerous synapses ( Figures 2C , 5A , C ) .", "This is similar to the pattern reported for filled basket cells in both macaque somatosensory cortex ( DeFelipe et al . , 1986 ) and cat visual cortex ( Somogyi et al . , 1983 ) .", "Further analysis revealed marked differences in the myelination of GABA and nonGABA axons .", "We used array tomography and MBP immunostaining to quantify the lengths of the nodes of Ranvier , where the ion channels needed for nerve impulse transmission are concentrated ( Figure 7A–B ) .", "Only gaps in the axonal myelin sheath of less than 4 μm length were considered to be nodes .", "Throughout the cortical layers , the nodes of GABA axons were shorter than for non-GABA axons ( 0 . 98 ± 0 . 07 μm vs . 1 . 51 ± 0 . 05 μm , mean ± standard error , p<0 . 0001 , n=71 GABA and 209 non-GABA nodes of Ranvier , Mann-Whitney U Test; Figure 7C ) .", "Their internodes , the myelinated portion between two nodes of Ranvier , were also found to be shorter ( 22 . 54 ± 2 . 11 μm vs 29 . 29 ± 2 . 02 μm for non-GABA axons , mean ± standard error , p=0 . 032 , n=23 GABA axons and 32 non-GABA axons , Mann-Whitney U Test ) . 10 . 7554/eLife . 15784 . 018Figure 7 . GABA axons have shorter nodes of Ranvier .", "( A , B )", "SEM images of nodes of Ranvier ( black lines: node boundaries ) of a GABA ( A ) and non-GABA myelinated axon ( B ) , immunolabeled with MBP ( cyan ) and GABA ( red ) .", "( C ) Comparison of the lengths of the nodes of Ranvier of cortical myelinated axons . DOI: http://dx . doi . org/10 . 7554/eLife . 15784 . 01810 . 7554/eLife . 15784 . 019Figure 7—source data 1 . Data values and statistics underlying Figure 7 . DOI: http://dx . doi . org/10 . 7554/eLife . 15784 . 019 Myelin thickness is thought to be directly correlated to axon diameter ( constant 'g-ratio' ) , based mostly on studies of the peripheral nervous system ( Fraher and Dockery , 1998 ) .", "However , we found no such correlation within mouse cerebral cortex .", "Using a deconvolved array tomography dataset with MBP and GABA immunofluorescence , we found that the average myelin thickness was 0 . 13 ± 0 . 002 μm for both GABA and non-GABA axons ( mean ± standard error , n=163 GABA and 238 non-GABA axons from layers 4 and 5 , p=0 . 88 , Mann-Whitney U Test ) and it did not correlate with axon thickness ( R=0 . 008; n=163 GABA and 238 non-GABA axons from layers 4 and 5 ) .", "Greater axon diameters not accompanied by changes in myelin thickness result in the g-ratio increasing as a function of axon diameter ( Figure 8B ) .", "GABA axons are thicker than non-GABA axons ( 0 . 54 ± 0 . 001 μm vs . 0 . 45 ± 0 . 001 μm , n=163 GABA and 238 non-GABA axons , p<0 . 0001 , Mann-Whitney U Test; and Figure 8A ) , thus their average g-ratio is higher ( 0 . 66 ± 0 . 006 vs . 0 . 61 ± 0 . 006 , n=163 GABA and 238 non-GABA axons , p<0 . 0001 , Mann-Whitney U Test ) . 10 . 7554/eLife . 15784 . 020Figure 8 . Similarities and differences between the myelin of GABA and non-GABA axons .", "( A ) GABA axons have thicker axons on average .", "Frequency distribution plot of the thickness measurements of 238 non-GABA and 163 GABA axons .", "( B ) GABA and non-GABA axons have similar g-ratios ( mean ± standard error , n=238 non-GABA and 163 GABA axons , Mann-Whitney U Test ) .", "( C ) The myelin of GABA axons contains significantly more myelin basic protein ( MBP ) than non-GABA axons ( mean ± standard error , n=489 non-GABA and 254 GABA axons ) .", "( D ) There are no significant differences in the PLP content of myelinated axons .", "All the analyses for this Figure were performed in cortical layers 4 and 5 , which have high density of GABA myelinated axons .", "Asterisks indicate statistically significant differences ( **p<0 . 01 , *p<0 . 05 , Mann-Whitney U Test ) .", "( E ) and ( F ) show an example of MBP and PLP immunofluorescence on a single section from the same dataset as analyzed in C and D . DOI: http://dx . doi . org/10 . 7554/eLife . 15784 . 02010 . 7554/eLife . 15784 . 021Figure 8—source data 1 . Data values and statistics underlying Figure 8 . DOI: http://dx . doi . org/10 . 7554/eLife . 15784 . 021 Next we compared the protein composition of the myelin of GABA and non-GABA axons .", "The two major proteins in myelin are MBP and proteolipid protein ( PLP ) , which together constitute about 80% of all proteins in myelin ( Baumann and Pham-Dinh , 2001; Rosetti and Maggio , 2007 ) .", "While the average PLP immunofluorescence was very similar in both types of axons ( 2229 ± 60 a . u . vs 2282 ± 40 a . u . , for GABA and non-GABA axons respectively; mean ± standard error , n=254 GABA axons and 489 non-GABA , p=0 . 2713 , Mann-Whitney U Test , Figure 8D ) , GABA myelinated axons had almost 20% higher MBP immunofluorescence ( 5263 ± 117 a . u . vs . 4486 ± 63 a . u . , mean ± standard error , n=254 GABA axons and 489 non-GABA , p<0 . 0001 , Mann-Whitney U Test ) .", "This difference in MBP content was not related to the thickness of the axons ( Figure 8C ) .", "The higher MBP immunofluorescence of the myelin sheath of inhibitory axons was confirmed in experiments in two more animals ( A1: 3024 ± 100 a . u . vs . 2634 ± 86 a . u . , mean ± standard error , n=322 PV axons and 314 non-PV from layers 2 to 5 , p=0 . 0114 , Mann-Whitney U Test; A2: 3038 ± 117 a . u . vs . 2585 ± 120 a . u . , mean ± standard error , n=233 PV axons and 119 non-PV from layer 4 , p=0 . 0455 , Mann-Whitney U Test ) .", "Although immunofluorescence has not been calibrated to underlying MBP concentration , the observation that GABA axons have more MBP than neighboring non-GABA axons was statistically significant and qualitatively consistent across multiple experiments .", "The most striking difference between GABA and non-GABA myelinated axons is in their cytoskeleton .", "Immunostaining for the neurofilament heavy chain and for alpha tubulin revealed that myelinated GABA axons are rich in neurofilaments and have relatively low microtubule content , while the cytoskeleton of myelinated non-GABA axons is dominated by microtubules ( Figure 9 ) .", "In addition , a larger fraction of the tubulin in myelinated GABA axons appears to be acetylated .", "While GABA myelinated axons contain on average only 66% of the alpha tubulin found in non-GABA myelinated axons , they have 86% of the acetylated alpha tubulin content of non-GABA myelinated axons .", "These differences in the cytoskeletal composition of myelinated axons were observed throughout the cortical layers . 10 . 7554/eLife . 15784 . 022Figure 9 . The cytoskeletal composition of myelinated GABA axons is different from myelinated non-GABA axons , as well as from unmyelinated GABA axons .", "( A ) The reconstructed small volume ( 16 × 16 × 4 . 5 µm ) from layer 5 of the mouse barrel cortex contains GABA axons ( red ) , which are enriched in neurofilaments ( NF-H , green ) , and non-GABA axons where microtubules ( αtubulin , magenta ) predominate .", "For clarity , only immunofluorescence signal within myelinated axons ( MBP , white ) is displayed .", "Red arrow points to a GABA positive axon and the white arrow to a GABA negative axon .", "( B ) The immunofluorescence intensity ( mean ± standard error ) for neurofilament heavy chain , αtubulin and acetylated αtubulin within myelin profiles is compared between GABA ( red ) and non-GABA axons ( blue ) .", "A larger volume spanning layers 2/3 , 4 and 5 , and including the region presented in A was analyzed .", "The differences are statistically significant ( p<0 . 01 , Mann-Whitney U Test ) in all cortical layers analyzed ( layers 2/3: 80 nonGABA and 68 GABA axons; layer 4: 121 nonGABA and 65 GABA axons; and layer 5: 146 non-GABA and 119 GABA axons ) .", "( C ) Analysis of the cytoskeletal content of the two types of axons from layer 5 ( 146 non-GABA and 119 GABA axons ) .", "( D ) Comparison of myelinated vs . unmyelinated stretches of axons .", "Individual axons which contained both a myelinated and a non-myelinated portion within the dataset volume were analysed ( 11 non-GABA and 20 GABA axons from layers 2/3 , 4 and 5 ) .", "Nodes of Ranvier were excluded from the analysis .", "Statistical differences using paired t-test are reported: *p<0 . 05 , **p<0 . 01 ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 15784 . 02210 . 7554/eLife . 15784 . 023Figure 9—source data 1 . Data values and statistics underlying Figure 9 . DOI: http://dx . doi . org/10 . 7554/eLife . 15784 . 023 The differences in cytoskeletal composition of myelinated axons could be due to a general difference between GABA and non-GABA axons , regardless of their myelination status .", "To explore this possibility , we identified individual axons that contained both a myelinated and a non-myelinated portion within the dataset volume .", "Nodes of Ranvier were excluded and only non-myelinated stretches longer than 4 μm were considered .", "This analysis revealed several interesting differences ( Figure 9D ) .", "First , the cytoskeletal composition of axons is indeed related to the presence of GABA .", "Both the myelinated and the unmyelinated regions of the GABA axons have higher neurofilament content and lower microtubule content compared to non-GABA axons .", "There are also significant differences in cytoskeletal content between the different regions of GABA axons , where myelinated portions have higher neurofilament and lower microtubule content compared to the unmyelinated portions of the same axons .", "While excitatory axons show similar trends of differences in cytoskeletal composition depending on myelination , the differences are more pronounced for GABA axons , especially regarding neurofilament content ." ], [ "The present work is the first to survey the targets of myelin in neocortex using recently developed high resolution , large-volume imaging methods ( Briggman and Bock , 2012; Kleinfeld et al . , 2011; Economo et al . , 2016; Micheva and Smith , 2007 ) .", "These methods reveal that a surprisingly large fraction of myelin in cortical gray matter ensheathes the axons of a single inhibitory neuron subtype , the parvalbumin-positive basket cell .", "Based on cellular morphology and postsynaptic targets , other PV-expressing neuron types were essentially ruled out , and we found minimal evidence for the myelination of non-PV inhibitory axons .", "Although this cell-type-specificity of axon myelination was unexpected , especially since myelin has generally been thought to ensheath long-range axons of excitatory neurons , it is nevertheless consistent with earlier observations of individual myelinated inhibitory axons , including basket cell axons ( Peters and Proskauer , 1980; DeFelipe et al . , 1986; Somogyi et al . , 1983; Keller and White , 1987 ) .", "The more comprehensive survey presented in the present work was achieved through analysis of data arising from two different imaging modalities: array tomography ( Micheva and Smith , 2007; Collman et al . , 2015 ) and large-scale serial-section electron microscopy ( Bock et al . , 2011 ) .", "Work based on each method was initiated independently and in parallel , and initial annotation and analysis was performed blind to preliminary results arising from the other method .", "The results from AT and volume EM were complementary .", "The AT data spanned all cortical laminae and revealed that many myelinated axons in mouse somatosensory cortex ( almost half of layer 2/3 , and a quarter of layer 4 myelinated axons ) contain the inhibitory neurotransmitter GABA .", "Practically all of these GABA axons also contain PV .", "AT-based comparison of GABA and nonGABA myelinated axons revealed several important differences .", "The nodes of Ranvier and the internodes are significantly shorter on GABA axons .", "In addition , AT multiplex imaging detected molecular differences , such as an enrichment of MBP in the myelin of GABA axons , as well as a high neurofilament and low microtubule content of their cytoskeleton .", "The volume EM data provided independent evidence that half of the categorized myelinated axons in layer 2/3 of mouse visual cortex are inhibitory , as indicated by the symmetric pre- and postsynaptic densities of the synapses that they formed after exiting the myelin sheath ( Colonnier , 1968; Peters et al . , 1991; Gray , 1959 ) .", "Immunolabeling of myelin in a PV-Cre reporter line showed that about half of the myelin in layer 2/3 ensheathes PV+ axons , and similar experiments in VIP- and SOM-Cre lines ( which comprise almost all non-PV interneurons [Xu et al . , 2010] ) failed to show any myelinated axons arising from VIP+ neurons and very few ( if any ) from SOM+ neurons .", "Ultrastructural analysis also allowed us to deduce which of the two PV-expressing subtypes in neocortex was myelinated , by revealing that the myelinated axons’ postsynaptic targets were largely non-axonal , instead targeting the dendrites and somata of pyramidal cells .", "This finding ruled out PV-positive chandelier cells ( which synapse onto axons ) , and strongly suggested that the other predominant PV subtype , basket cells , is the myelinated subtype .", "PV basket cells comprise about half of all neocortical interneurons ( Markram et al . , 2004 ) .", "Taken together , the quantitative and qualitative consistency between the AT , volume EM , and immunohistochemistry datasets provides strong evidence that PV+ basket cell axons are the predominant myelinated inhibitory axons in neocortex .", "Our axon reconstructions showed that the distribution of myelin on basket cell axons is patchy and seemingly localized to the axon arbor near the cell body ( e . g . Figures 3D–F , 5A–C ) .", "This patchiness qualitatively resembles the distribution of myelin on the descending axons of excitatory pyramidal cells measured in the same EM data set ( Tomassy et al . , 2014 ) , and is consistent with an observation of patchy myelination of an inhibitory neuron axon in an earlier study in mouse somatosensory cortex ( Keller and White , 1987 ) .", "The concentration of myelin on the proximal arbor is also consistent with more complete reconstructions of individual filled cells in cat ( Somogyi et al . , 1983 ) and macaque ( DeFelipe et al . , 1986 ) .", "There are several caveats and limitations to this study .", "First , our experiments allow for the possibility that a small fraction of somatostatin or other non-PV axons are myelinated .", "We found by AT that ~2% of myelinated GABA axons are PV-negative , and examination of the transgenic mouse line SOM-Cre;Ai9-TdTom revealed a small percentage of TdTomato-positive myelinated axons .", "However , the lack of PV immunostaining in some myelinated axons may reflect limitations of immunodetection rather than proving that these axons are PV-negative .", "Furthermore , the SOM-Cre-line used in this study was found to have a small but consistent false-positive expression pattern , where Cre is expressed in neurons that immunohistochemically are somatostatin-negative but PV-positive ( Hu et al . , 2013 ) .", "Although additional studies of somatostatin-positive interneurons , as well as other inhibitory interneuron subtypes , will be needed to resolve this issue , we believe it does not materially affect our finding that the great preponderance of myelinated inhibitory axon profiles in layer 2/3 arise from parvalbumin positive basket cells .", "A second limitation stems from the incomplete characterization of the distribution of myelin on the full arbors of inhibitory neuron axons .", "Myelinated axon profiles were traced until they exited the myelin sheath and formed a sufficient number of synapses to categorize the axon as excitatory or inhibitory , at which point tracing was halted ( except in two cases , Figure 5A and B , where the entire arbor within the 450 × 350 × 50 μm EM volume was traced ) .", "The result was a set of axon fragments ( Figure 5C ) that cannot be positively identified according to morphological type .", "Their identification as arising from basket cells is based on a combination of immunolabeling results ( that myelinated inhibitory axons are nearly always PV+ ) with the identification of the fragments’ post-synaptic targets as being almost entirely non-axonal ( so the axons do not arise from chandelier cells , the other PV+ cell type besides basket cells ) .", "Ideally , direct confirmation of this deduction would also be obtained from complete volume EM axonal arbor reconstructions and subsequent confirmation of morphological type .", "However , the additional tracing effort to achieve this would be substantial ( Helmstaedter , 2013 ) , and , perhaps more importantly , such an investment might better be made in a larger cortical EM volume ( e . g [Lee et al . , 2016] ) , so that a greater fraction of each neuron’s axonal arbor could be reconstructed .", "Such an approach would also permit assessment of whether all PV-containing basket cells have myelinated axons .", "Considering the functional and morphological heterogeneity of PV basket cells ( Kawaguchi and Kubota , 1997; Wang et al . , 2002; Li and Huntsman , 2014 ) , it is possible that some subtypes preferentially form myelinated axons .", "Consistent with the possibility that not all basket cells ( or basket cell subtypes ) are myelinated , the fraction of somatic post-synaptic targets made by the inhibitory myelinated axons described here is at the low end of the previously reported range for cortical basket cells .", "The majority of myelinated inhibitory axons in our study contact dendritic shafts ( 53% ) and spines ( 32% ) , and only 13% of the targets are cell bodies .", "These proportions are similar to those found in a study in rat frontal cortex of four individually filled fast-spiking basket cells , which made on average 53% of synapses onto dendritic shafts , 29% onto spines , and 18% onto cell bodies ( Kawaguchi and Kubota , 1998 ) .", "However , the four cells showed wide variability in the proportion of their somatic targets , which ranged between 4% and 35% .", "Studies in cat neocortex also reveal a wide variability between individual basket cells ( between 20% and 69% of synapses targeting somata ) ( Somogyi et al . , 1983; Kisvarday et al . , 1987; Tamás et al . , 1997 ) .", "These findings in neocortex contrast with measurements in hippocampus , where more than 50% of the synaptic targets of PV basket cells are cell bodies , and dendritic spines are only occasionally targeted ( Seress and Ribak , 1990; Gulyás et al . , 1993; Halasy et al . , 1996 ) .", "The fraction of inhibitory post-synaptic targets made by the axons in this study ( 4 . 5% ) is similar to , but somewhat lower than that previously observed in rat ( 8% ) and cat ( 9% ) neocortex ( Somogyi et al . , 1983; Kawaguchi and Kubota , 1998 ) .", "This variability is consistent with a recent study in mouse visual cortex which found wide variation between individual animals in the fraction of synapses made onto inhibitory interneurons by layer 2/3 pyramidal cells ( Bopp et al . , 2014 ) .", "Overall , the anatomical literature briefly described above shows that the post-synaptic targets of a given cell type can vary widely between species , brain regions , animals , and individual neurons .", "Determining whether discrete basket cell subtypes underlay any of this variability will require further study .", "Our findings raise the question: what is the role of myelin on inhibitory interneurons in neocortex ?", "The patchy distribution of myelin along GABA axons and the lack of correlation between myelin thickness and axon diameter imply that increased conduction velocity may not be the main consequence of this myelination .", "Rather , the presence of myelin may be due to the unique cellular properties and circuit functions ( Hu et al . , 2014 ) of PV basket cells .", "Nearly all cortical PV basket cells are fast-spiking ( Kawaguchi and Kubota , 1997; Galarreta and Hestrin , 1999 , 2002; Gibson et al . , 1999 ) , and fast-spiking neurons have very high tonic activity compared to other cell types in cortex ( Gentet et al . , 2010; Hofer et al . , 2011 ) — they generate long trains of action potentials with very high frequency ( >100 Hz ) ( Kawaguchi and Kubota , 1997; Galarreta and Hestrin , 1999 , 2002; Gibson et al . , 1999 ) .", "Fast-spiking cells exhibit a number of biophysical specializations that enable high firing rates but incur an increased energy cost per action potential ( Hu and Jonas , 2014; Carter and Bean , 2009; Hasenstaub et al . , 2010 ) .", "Axonal myelination could help with these high energy demands in two ways: first , myelin is known to improve the energy efficiency of action potential propagation ( Hartline and Colman , 2007 ) .", "Secondly , myelin can provide metabolic support to the axon by supplying lactate as an energy source ( Rinholm et al . , 2011; Lee et al . , 2012; Fünfschilling et al . , 2012; Morrison et al . , 2013 ) .", "Thus , myelin could reduce the basket cells’ metabolic needs and provide an additional external energy supply from oligodendrocytes .", "At the circuit level , PV basket cells participate in regulating excitatory/inhibitory balance , in neuronal synchronization and cortical rhythm generation , and in plasticity associated with experience and learning ( Hu et al . , 2014 ) .", "Could basket-cell myelin hasten the delivery of synaptic input to their postsynaptic targets to an extent that would be functionally relevant ?", "No estimates of conduction velocities of cortical myelinated PV axons exist in the literature; however , in young ( ~P30 ) mice , PV axon conduction velocities of ~0 . 5 m/s have been recorded ( Casale et al . , 2015; Li et al . , 2014 ) .", "The majority of PV axons at this age are most likely unmyelinated , since cortical myelination is not complete until much later ( after P60 ) ( Barrera et al . , 2013; Mengler et al . , 2014 ) .", "Using this conduction velocity estimate , an unmyelinated PV axon would conduct a spike in 0 . 4 ms to synapses located 200 μm away , which was the typical path length from cell body to proximal synapses in our EM-based reconstructions .", "Thus , any reduction in the spike arrival time to proximal synapses due to myelination will inevitably be in the sub-millisecond range , likely too small a difference to affect post-synaptic dendritic integration or spike-timing-dependent plasticity , given the much slower timescales of those functions ( Dan and Poo , 2006; Feldman , 2012; Stuart and Spruston , 2015 ) .", "However , spike time arrival differences on a sub-millisecond timescale might affect the magnitude of coordinated oscillations ( Pajevic et al . , 2014 ) , which are a known circuit role of basket cells .", "The present study also bears on the genesis and dynamics of myelination .", "Cortical myelin is dynamically remodeled during learning and development ( McKenzie et al . , 2014; Bergles and Richardson , 2016; Makinodan et al . , 2012 ) and it redistributes during normal aging , where internode length decreases with age in primates ( Peters , 2009 ) .", "It is possible that these dynamics may differ between the myelin wrapping inhibitory and excitatory axons .", "This could be due to differences arising from local interactions between oligodendrocytic processes and individual axons , or from a possible allocation of distinct oligodendrocytes ( or oligodendrocyte subtypes [Tomassy et al . , 2016] ) to axons of inhibitory or excitatory neurons ( de Hoz and Simons , 2015 ) .", "Further studies will also be needed to address the interesting question of myelin diversity at the molecular level .", "Between the two major proteins of myelin , our experiments reveal that inhibitory axons have higher MBP content than excitatory axons , while the proteolipid protein ( PLP ) content appears similar .", "Because MBP is one of the potential target antigens in MS ( Wucherpfennig and Strominger , 1995; Fujinami and Oldstone , 1985; Garg and Smith , 2015 ) , this finding has important implications for pathologies of myelin .", "It is likely that other differences in the protein or lipid content of myelin exist , considering the accumulating evidence for CNS oligodendrocyte heterogeneity ( Tomassy et al . , 2016 ) .", "Characterizing these differences as a function of neuronal type will be an important aspect of future studies .", "The abundance of myelinated inhibitory axons in cortical gray matter also raises the possibility of their involvement in myelin perturbations in normal aging ( Peters , 2009 ) and disease ( Fields , 2008; Popescu and Lucchinetti , 2012; Calabrese et al . , 2015; Wingerchuk et al . , 2015; Gordon et al . , 2014; Lucchinetti et al . , 2011; Kang et al . , 2013 ) .", "For example , gray matter myelin degeneration is observed in the early stages of Alzheimer’s disease ( AD ) ( Bartzokis , 2004; de la Monte , 1989 ) , and , in relapsing remitting multiple sclerosis ( RRMS ) , the presence of cortical lesions is strongly correlated with seizure occurrence ( Calabrese et al . , 2008 ) .", "Several other diseases predicated on CNS demyelination , including neuromyelitis optica , acute disseminated encephalomyelitis , and progressive multifocal leukoencephalopathy may also display seizure activity ( Jarius et al . , 2014; Hynson et al . , 2001; Lima et al . , 2006 ) .", "Demyelination of PV interneurons may perturb the balance of large-network neuronal excitation and inhibition , biasing neocortical microcircuits toward seizure .", "By combining data from two independent efforts , we show here that a large fraction of cortical myelin is allocated to the axons of inhibitory neurons , specifically to PV basket cells .", "This work alters our understanding of the distribution of myelin in neocortical gray matter , and opens new avenues in basic neurobiology , systems neuroscience , and neurological disease research .", "Finally , this study leverages an openly available EM data set ( Burns et al . , 2013; Martone et al . , 2002 ) that has been used in several other studies of neocortex ( Tomassy et al . , 2014; Bopp et al . , 2014 ) since its publication ( Bock et al . , 2011 ) , illustrating the value of image data sharing in biomedical research ." ], [ "A publicly available ( Burns et al . , 2013; Martone et al . , 2002 ) 450 × 350 × 50 μm EM dataset from the upper layers of visual cortex ( Bock et al . , 2011 ) in an adult Thy1-YFP-H ( Feng et al . , 2000 ) mouse ( 9–14 months of age ) was used .", "The images were obtained by transmission electron microscopy of serial thin sections , resulting in a ~10 TB dataset comprised of ~4 × 4 × 45 nm voxels .", "To sample a subset of myelinated axons in the volume systematically , a section near the center of the image volume was selected , and all myelinated axon profiles were annotated in TrakEM2 with ‘seed’ points for further tracing ( Figure 2 ) .", "Myelinated profiles near the edge of the imaged area were not seeded , to reduce the probability of the axon exiting the volume without making synapses .", "To categorize each myelinated axon as arising from an inhibitory or excitatory neuron , each seed was manually traced into the EM volume until the axon lost its myelin sheath and formed at least two synapses .", "The synapses were categorized as inhibitory or excitatory depending on whether the pre- and postsynaptic densities were symmetric ( equal in thickness ) or asymmetric ( with a thicker postsynaptic density ) , respectively .", "Occasionally , individual synapses could not be definitively characterized as symmetric or asymmetric .", "This occurred most often when the section plane was oblique or parallel to the plane of the synapse .", "In these cases , tracing was continued until sufficient additional synapses were reached to make a definitive determination of axon type .", "This work was performed prior to the tracers’ knowledge that the array tomography work existed .", "Categorization of axons as inhibitory or excitatory based on synapse ultrastructure was done in the Bock laboratory prior to knowledge of the AT results from the Smith laboratory .", "All tracing of fine unmyelinated axon branches and synaptic classifications were reviewed by a highly experienced tracer ( DDB ) for correctness .", "In all cases where a traced axon reached a cell body ( Figure 2 , Figure 2—figure supplements 1 , 2 ) , the categorization of the axon as excitatory or inhibitory based on synaptic ultrastructure was found to agree with somatic structure and synaptic interactions: excitatory axons were only found to arise from pyramidal neurons receiving only inhibitory synapses on the cell body , and inhibitory axons were only found to arise from non-pyramidal neurons with both inhibitory and excitatory synapses on the cell body ( Peters et al . , 1991 ) .", "Axons that could not be classified generally failed to unmyelinate within the bounds of the EM-imaged volume and had an orientation normal to the EM section plane ( Figure 2—figure supplement 3 ) .", "On this axis , the EM image volume is only ~50 μm thick , reducing the probability that these axons could be traced to an unmyelinated , synapse-forming portion of the axonal arbor .", "The postsynaptic target at each annotated synapse was also classified as excitatory or inhibitory using ultrastructural criteria .", "The dendritic shafts of inhibitory neurons have much lower spine densities and receive many more asymmetric synapses than those of excitatory neurons ( Bock et al . , 2011; McGuire et al . , 1991 ) ( Figure 6E ) , allowing for unambiguous categorization .", "When the postsynaptic target was a dendritic shaft , categorization was therefore straightforward .", "When the target was a spine , the spine was traced through its neck to the shaft , and categorization was then based on shaft ultrastructure .", "In rare cases , the spine could not be traced to the shaft and in this case no attempt was made to categorize the postsynaptic target as excitatory or inhibitory ( Table 1 , 'Uncategorized dendritic spines' ) .", "Postsynaptic axons were identified based on a combination of the following criteria: bundled microtubules and the presence of a characteristic dense layer beneath the plasma membrane ( Peters et al . , 1991 ) ; their presynaptic relationships to other neurons ( Figure 5F ) ; and their eventual entrance into myelin sheaths .", "Postsynaptic targets were identified after pooling of data between the two laboratories had begun .", "Two myelin seeds were arbitrarily selected for complete tracing ( Figure 5A , B ) .", "Rather than halting tracing of these axons once they could be unambiguously categorized as inhibitory or excitatory , their arbors were traced to completion within the limits of the EM-imaged volume .", "In one case , the inhibitory cell body was reached ( Figure 5A ) .", "All synapses were annotated and their postsynaptic targets were categorized as with the partially traced axons .", "The distribution of postsynaptic targets was nearly identical between the completely and partially traced axons ( Table 1 ) ." ] ]

[ "Myelin is best known for its role in increasing the conduction velocity and metabolic efficiency of long-range excitatory axons .", "Accordingly , the myelin observed in neocortical gray matter is thought to mostly ensheath excitatory axons connecting to subcortical regions and distant cortical areas .", "Using independent analyses of light and electron microscopy data from mouse neocortex , we show that a surprisingly large fraction of cortical myelin ( half the myelin in layer 2/3 and a quarter in layer 4 ) ensheathes axons of inhibitory neurons , specifically of parvalbumin-positive basket cells .", "This myelin differs significantly from that of excitatory axons in distribution and protein composition .", "Myelin on inhibitory axons is unlikely to meaningfully hasten the arrival of spikes at their pre-synaptic terminals , due to the patchy distribution and short path-lengths observed .", "Our results thus highlight the need for exploring alternative roles for myelin in neocortical circuits ." ]

[ "The brain is far away from the muscles that it controls .", "In humans , for example , the brain must be able to trigger the contraction of muscles that are more than a meter away .", "This task falls to specialized motor neurons that stretch from the brain to the spinal cord , and from the spinal cord to the muscles .", "Neurons transmit information ( in the form of electrical nerve impulses ) along their length through cable-like structures called axons .", "The axons of the motor neurons are so long that , if they were ‘naked’ , it would take at least a second for nerve impulses to travel their entire length .", "Such a long delay between thoughts and actions would make rapid movement impossible .", "Nerve impulses are able to travel from the brain to the muscles much more quickly , because they are wrapped with a substance called myelin that acts like electrical insulation .", "Myelin helps the nerve impulses travel up to 100 times faster down the axon .", "Not surprisingly , diseases that damage myelin , such as multiple sclerosis , severely disrupt movement and sensation .", "Not all of the brain’s myelin is found around the long axons of motor neurons .", "The outer layer of the brain , known as the cerebral cortex , also contains myelin .", "However , most neurons within the cerebral cortex are only a few millimeters long .", "So what exactly is myelin doing there ?", "Micheva et al . have now used electron microscopy and light microscopy to study the neurons in the cortex of the mouse brain .", "This revealed that up to half of the myelin in some layers of the cortex surrounds the axons of inhibitory neurons ( which reduce the activity of the neurons they signal to ) .", "Moreover , one particular subtype of inhibitory neuron accounts for most of the myelinated inhibitory axons , namely inhibitory neurons that contain a protein called parvalbumin .", "Exactly why parvalbumin-containing neurons are myelinated remains a mystery .", "Myelin covers only short segments of the axons of these neurons , so it would speed up the transmission of signals by less than a millisecond – probably not enough to make a meaningful difference .", "Parvalbumin-containing neurons often signal frequently , and thus require large amounts of energy .", "One possibility therefore is that myelin helps to meet these energy requirements or to reduce energy consumption .", "Further research will be needed to test this and other ideas ." ]

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

[ "physics of living systems", "tools and resources", "neuroscience" ]

[ [ "Hair cells in the auditory and vestibular systems of vertebrates convert mechanical stimuli into electrical signals through the process of mechanoelectrical transduction ( Fettiplace and Kim , 2014; Martin and Hudspeth , 2021 ) .", "The mechanical receptor for such stimuli is the hair bundle , a cluster of stereocilia , or stiff enlarged microvilli , atop each hair cell .", "An extracellular molecular filament , the tip link , extends from the tip of each stereocilium to the side of its tallest neighbor in the plane parallel to the bundle's axis of symmetry .", "Mechanically gated ion channels are located at the lower end of each tip link .", "When a hair bundle pivots at its base toward its tall edge in response to stimulation , the increased tension in the tip links opens the ion channels and the ensuing ionic current depolarizes the cell ( Figure 1A ) .", "Although our understanding of the transduction process has improved significantly through the development of methods to mechanically stimulate a hair bundle , the techniques now available pose serious limitations .", "Two methods are commonly used to apply force to a hair bundle .", "The first is to deflect the bundle with a compliant glass fiber about 100 μm in length and 1 nm in diameter ( Crawford and Fettiplace , 1985; Howard and Ashmore , 1986; Howard and Hudspeth , 1988 ) .", "The fiber's tip is attached to the top of the hair bundle and its base is driven by a piezoelectric actuator .", "Because the preparation is immersed in an aqueous solution , however , the fiber is subjected to hydrodynamic drag that roughly doubles that on the bundle .", "For a typical fiber of stiffness 500 μN•m–1 and drag coefficient 150 nN•s•m–1 , the time constant of responsiveness is about 300 μs , which corresponds to a low-pass filter with a cutoff frequency near 500 Hz ( Crawford and Fettiplace , 1985; Howard and Hudspeth , 1987 ) .", "Another problem is especially acute for the stimulation of mammalian hair bundles , whose stereocilia are less cohesive than those of amphibians: when a fiber is attached at a single site in the hair bundle , the displacement of other stereocilia depends in a complex manner on elastic and hydrodynamic coupling across the bundle .", "This arrangement results in an uneven application of force to different stereocilia and can produce artifacts ( Indzhykulian et al . , 2013; Nam et al . , 2015 ) .", "The second common method of stimulation uses a fluid jet that displaces a hair bundle through the action of a piezoelectric diaphragm ( Géléoc et al . , 1997; Corns et al . , 2014 ) .", "Although the resonant frequency of fluid injection can reach 5 kHz , practical use of the method is limited to less than 1 kHz owing to uncertainties in force calibration ( Dinklo et al . , 2007 ) .", "Moreover , fluid leakage from the system might introduce a displacement bias .", "In summary , the inability of current methods to reach higher frequencies by direct stimulation limits our quantitative understanding of hair-cell mechanics over more than 95% of the range of mammalian hearing , which extends to 20 kHz in humans and at least 150 kHz in some species of bats and whales .", "What is more , the susceptibility of current approaches to artifacts has long impeded our understanding of hearing , in particular in the case of the mammalian ear ( Nam et al . , 2015 ) .", "To address these problems , we used laser irradiation to stimulate hair bundles ( Figure 1 ) .", "Because photonic force arises when photons are absorbed , reflected , or refracted upon interaction with an object , intense illumination should apply substantial force to a bundle .", "Our experiments confirmed the validity of the approach and demonstrated that the requisite irradiation does not jeopardize a bundle’s operation .", "This method allows us to probe hair-bundle physiology at previously inaccessible timescales , for the delivery time of the stimulus can accommodate the full frequency range of mammalian hearing .", "At the same time , this approach avoids the artifacts that bedevil current methods ." ], [ "The conservation of momentum entails that reflected , absorbed , and refracted photons exert force on a target .", "All these phenomena are likely to take place when light strikes an array of stereocilia in a hair bundle .", "Although an analysis based on reflection alone would indicate that a hair bundle is relatively insensitive to radiation pressure , geometric considerations reveal that multiple modes of light propagation occur in a hair bundle by virtue of the cylindrical shape of the stereocilia ( see Materials and methods ) .", "Each of these modes is capable of transferring momentum and therefore of mechanically stimulating the bundle .", "Because the diameter of each stereocilium compares to the wavelength of light , the regular spacing of stereocilia within the hair bundle might additionally give rise to complex interference effects .", "We stimulated 40 hair bundles of the bullfrog’s sacculus so that radiation pressure would push them toward their tall edges—the positive direction—and reliably elicited the expected movements ( see Appendix 1—figure 1A ) .", "The bundles followed similar trajectories at the onset of irradiation ( Figure 2 ) : the movement was approximately exponential with a time constant of 0 . 64±0 . 06⁢ms ( mean ± SEM , N = 16 ) .", "The responses , which reached displacements as great as 500 nm , encompassed the range of complex trajectories reported in the literature ( Benser et al . , 1996; Tobin et al . , 2019; Ricci et al . , 2000 ) .", "The more compliant hair bundles—those displaying initial deflections exceeding about 150 nm—displayed relatively slow movements in the direction of the photonic force , a signature of the timescale of the adaptation process that allows hair cells to reset their operating points and thus detect successive stimuli ( Figure 2A , gray arrows ) ( Howard and Hudspeth , 1987 ) .", "The twitch , a faster rebound of the hair bundle in a direction opposite to that of the stimulus , is another manifestation of the adaptation process that occurs instead in response to smaller movements of the hair bundle and whose magnitude decreases for larger deflections ( Benser et al . , 1996; Ricci et al . , 2000; Cheung and Corey , 2006 ) .", "The twitch was indeed observed in stiffer bundles with deflections of about 50 nm ( Figure 2B–C ) .", "These results indicate that photonic force is an effective means of stimulating hair bundles .", "We applied photonic stimuli to the hair bundles of both inner and outer hair cells from the cochleas of young rats .", "Consistent with previous evidence that mammalian hair bundles are stiffer than their amphibian counterparts ( Tobin et al . , 2019 ) , the recorded amplitudes of deflection were typically smaller ( Figure 3A–D ) .", "The time constants for the initial displacements were again a few hundred microseconds .", "To characterize the efficacy of photonic stimulation for rat hair bundles , we applied positive stimuli to 22 outer hair cells from three preparations .", "We deflected 13 hair bundles with amplitudes varying from 25 nm to 35 nm .", "We also deflected seven of nine bundles from inner hair cells; the response amplitudes varied from 100 nm to 75 nm and the trajectories resembled those from the frog .", "The fiber’s power delivered onto a hair bundle can be modulated by changing the laser’s power at the source .", "By combining analog and digital signals to drive the laser’s output , we were able to stimulate hair bundles with an assortment of stimuli: sine waves , frequency sweeps , step pulses of various magnitudes , and continuously ascending and descending ramps ( see Appendix 1—figure 4 ) .", "The responses of bullfrog’s hair bundles followed sinusoidal frequency sweeps at frequencies up to 2 kHz ( Figure 4 ) .", "In this case the upper boundary in the stimulus frequency was set by the ability of the hair bundle to follow , rather than by the limitations of the stimulation method .", "As a result of localized heat generation , a hair bundle from the frog can move in the positive direction in response to laser irradiation of the cellular apex from any direction ( Azimzadeh et al . , 2018 ) .", "We found that this phenomenon also occurs in hair bundles of the rat ( Figure 3E ) .", "To separate this photothermal effect from that of photonic force , we took advantage of the fact that the former requires intact tip links .", "When we disrupted the tip links with BAPTA to abolish the photothermal effect , we observed that both positive and negative stimuli evoked rapid movements in the direction of light propagation ( see Appendix 1—figure 5 ) .", "We also stimulated hair bundles along a direction perpendicular to their axis of symmetry and again found that they moved in the direction of photon flux ( see Appendix 1—figure 5C ) .", "These results indicate that bundle motion upon photonic stimulation can occur in the absence of a photothermal effect: bundle movements stem solely from optical radiation force .", "In a frog’s hair cell , the photothermal effect apparently results from light absorption by the mitochondria that accumulate around the cuticular plate at the cell’s apical surface ( Azimzadeh et al . , 2018 ) .", "Because in mammalian outer hair cells mitochondria are instead concentrated at the lateral plasma membrane ( Fuchs , 2010 ) , it was possible to isolate the photothermal effect by directing light well below the apical cell surface .", "Note that the photothermal movement was relatively slow: its time constant of 2 ms was about ten times that of the movements owing to photonic force .", "Conversely , it was possible to avert the photothermal effect by irradiating a mammalian hair bundle with intact tip links while avoiding irradiation of the cell body .", "The hair bundles of healthy hair cells from the bullfrog can oscillate back-and-forth even in the absence of external stimulation ( Martin et al . , 2003 ) .", "These spontaneous oscillations are a manifestation of the active process that these cells employ to amplify mechanical stimuli by counteracting viscous damping .", "The presence of spontaneous oscillations , which require a fully functional transduction apparatus , offers a means of assessing the viability of hair cells and the preservation of mechanotransduction following exposure to laser irradiation .", "We compared the spontaneous oscillations of six hair cells before and after subjecting them to 25 pulses of light .", "Even at laser powers sufficient to deflect hair bundles over their entire physiological range of motion , the amplitudes and frequencies of the bundles' oscillation were unaffected by laser irradiation ( Figure 5 ) .", "This experiment demonstrates that the mechanotransduction apparatus was not damaged by our stimulation method .", "To further assess the health of the hair bundles exposed to laser irradiation , we compared their uptake of FM1-43—a fluorescent dye that enters a hair cell through open mechanotransduction channels ( Gale et al . , 2001 ) —with that of the surrounding , unexposed hair cells and that of mechanically damaged bundles ( Appendix 1—figure 6 ) .", "The fluorescence signal from laser-irradiated hair bundles showed no visible difference with respect to that of the unscathed cells , whereas mechanically damaged bundles were visibly dimmer .", "This diminished fluorescence likely resulted from breakage of the tip links that reduced the opening of the mechanotransduction channels , thereby limiting the intake of the dye ." ], [ "We have used tapered optical fibers to apply both rapid and prolonged forces to individual hair bundles .", "The tip of each tapered fiber was small enough to be positioned near a hair bundle , and the ball lens at its tip restricted the divergence of the emitted light beam .", "Irradiation could be confined to a single bundle , whereupon the uniform illumination of all the stereocilia implied that each experienced a similar photonic force .", "It was also possible to irradiate only a portion of a bundle .", "Our control experiments indicated that even extensive irradiation of the magnitude necessary to evoke large displacements did not harm the hair bundles .", "There are two difficulties with photonic stimulation .", "Although a routine procedure after the assembly of the necessary facilities , fabrication of a tapered optical fiber requires specialized equipment and a safe environment for the use of etching solution .", "A second issue is calibration: unlike the force delivered by a flexible fiber , which can be calibrated through the fiber's Brownian motion , the force exerted by photonic stimulation is not easily measured .", "The force can nonetheless be estimated by the use of targets whose stiffness has been independently determined , especially glass fibers such as those used in this study , or passive hair bundles including those subjected to chemical fixation .", "The use of photonic stimulation offers at least five advantages over the currently used methods of mechanical stimulation .", "First , stimulation is rapid: in the present study , the rise time of mechanical responses was set by a hair bundle’s stiffness and drag coefficient , without any effect of the drag on a stimulus fiber or the inertia of a piezoelectric actuator .", "Second , stimulation could be made still more rapid by a process analogous to 'supercharging’ in a voltage-clamp system ( Armstrong and Chow , 1987 ) : transient irradiation with a very bright light could be used to deflect a bundle to a desired position , after which a steady force would be applied by weaker illumination during the measurement of a response .", "Because illumination can be switched off , a third virtue is that there is no possibility of an ill-defined steady-state offset in bundle position owing to mispositioning of a fiber or leakage from a fluid jet .", "The uniform illumination of the stereocilia in a bundle offers a fourth advantage , especially for mammalian hair bundles that exhibit relatively poor lateral coupling between stereocilia .", "And finally , photonic stimulation can be used in spaces too restricted to admit a flexible fiber or fluid jet .", "In particular , it should be possible to stimulate one or several hair bundles in preparations such as a hemicochlea ( He et al . , 2004 ) or an isolated cochlear segment ( Chan and Hudspeth , 2005a; Chan and Hudspeth , 2005b ) ." ], [ "Each absorbed photon imparts all of its original momentum to the absorbing object and thereby provides an impulsive force .", "A reflected photon delivers twice the momentum provided by an absorbed one , whereas a refracted photon imparts momentum dependent on the angle of refraction .", "Because reflection sets the upper limit of the force that might be delivered to a hair bundle by a particular beam of light , we begin our analysis by treating the bundle as a perfect reflector .", "Averaged over one oscillation of the electromagnetic field , the radiation pressure due to illumination striking a hair bundle at an incident angle θ to the normal of the surface is Paschotta , 2010; Hulst , 2003: ( 1 ) P=2⁢<S>c⁢c⁢o⁢s⁢θ2in which P is the radiation pressure , S is the average power of the electromagnetic wave , and c is the speed of light in vacuum .", "Equation 1 can alternatively be written in terms of irradiance I , or power per unit area , with units W⋅m-2 , and laser power ( Pwr ) : ( 2 ) P=2⁢Ic⁢c⁢o⁢s⁢θ2=2⁢P⁢w⁢rA⋅c⁢c⁢o⁢s⁢θ2 The force F produced at an angle θ is then ( 3 ) F=2⁢P⁢w⁢rc⁢c⁢o⁢s⁢θ For completely absorbed photons , this relation can be modified to ( 4 ) F=P⁢w⁢rc⁢c⁢o⁢s⁢θ In a physiological solution , the refractive index is approximately 1 . 33 , and therefore the speed of light is c/1 . 33 .", "The angle of incidence in our experiments is 20° , which is set by the physical clearance between the objective lens and the preparation .", "By Snell's law , the angle of reflection is equal to the angle of incidence .", "Therefore , in the purely reflective case , using Equation 3 , we estimate that 10 mW of laser power that impinges normal to the surface of the reflector generates approximately 80 pN of force .", "However , this upper limit of the force is not achievable because stereocilia are not perfect mirrors .", "The actual force experienced by a hair bundle depends on the difference of the refractive indices between the solution and the stereocilia , a larger difference indicating more reflected light and larger force .", "As discussed below , the angle of incidence is also important .", "In order to produce an optical fiber with a tip small enough to approach an individual hair bundle , it is necessary to thin the fiber's 60 µm-thick cladding to expose the inner core of 5 μm diameter .", "Various methods have been employed to reduce the diameter of fibers in near-field optical microscopy and in the development of optical-fiber sensors .", "One common method is to use a carbon dioxide laser to machine optical fibers ( Ozcan et al . , 2007 ) .", "Although this method is capable of creating symmetrical fibers , CO2 lasers are expensive and require complex optics .", "Two other methods used for removing material in optical fibers are femtosecond laser micromachining ( Wei et al . , 2008a; Wei et al . , 2008b; Liao et al . , 2012; Yuan et al . , 2012 ) and focused-ion-beam milling ( Kou et al . , 2010; Yuan et al . , 2011; André et al . , 2014 ) .", "Although both methods are effective , they are time-consuming and require expensive instruments .", "On the basis of previous experiments with glass fibers , we suspected that the interaction of tapered fibers with living specimens would contaminate the fibers’ tips and thus limit the use of each fiber to only a few experiments .", "Furthermore , the gradual degradation due to several hundred high-power optical pulses during an experiment would limit a fiber’s use to a few experiments .", "Both considerations required that fibers be tapered easily and cost-effectively in a typical laboratory setting .", "We created tapered optical fibers by Turner's wet chemical etching with hydrofluoric acid ( André et al . , 2014 ) .", "With this method , a fiber can be shaped in about 1 . 5 hr in any laboratory with a fume hood and few tools .", "In shaping each fiber , we started with a single-mode optical fiber 1⁢m in length and with an FC/PC connector at one end .", "The distal end of the fiber was prepared by stripping a 12⁢mm length of its polymeric jacket and the polyamide coating and cleaning it with 70% ethanol .", "We inserted the fiber’s end into a holder that allowed it to be attached to manipulators during the fabrication process .", "Etching was conducted in a fume hood ( Figure 8 ) .", "After 47 . 5 mL of concentrated ( 48% ) hydrofluoric acid had been placed in a polypropylene tube ( Corning Life Sciences , Tewksbury , MA , USA ) , 2 . 5 mL of red kerosene oil was added .", "The oil layer's purpose was twofold .", "First , it provided protection to the fiber above the surface from attack by acid vapor .", "Second , the height of the aqueous meniscus was dependent upon the diameter of the immersed fiber , and thus declined as etching proceeded .", "When etching was complete , the oil layer isolated the tip from the acid .", "The fiber’s holder was attached to a motorized linear actuator ( Nanotec Electronic GmbH and Co KG , Feldkirchen , Germany ) with 3μm positioning resolution and the height of its tip was controlled through a computer interface ( LabVIEW; National Instruments , Austin , TX ) .", "Because the diameter of the fiber’s tip at any point along its length depended on the duration of its immersion , it was critical to control the fiber’s extraction speed .", "For maximal stability during experiments , we set the length of the taper to 8⁢mm , the minimum required for reliable clearance of the objective lens .", "After coupling a 633 nm wavelength laser to the optical fiber to render its tip visible during etching , we lowered the fiber until its tip was immediately above the interface between the oil and the acid .", "Under computer control , the actuator then performed a series of insertions and extractions of the fiber .", "The initial program inserted the fiber 10 mm into the acid at 2 mm•s–1 and extracted 8mm at the same speed .", "The routine next extracted the optical fiber for 20 min at 37 . 5 μm•min–1 , reducing its diameter from 125 μm to 60 μm .", "The extraction then stopped and the fiber remained in the acid for 18 min , during which tip was etched at a steeper angle by the gradual fall of the meniscus .", "The fiber was rinsed with distilled water and then with isopropyl alcohol and air-dried in the fume hood .", "Animal experiments were conducted with the authorization and according to the protocols of the relevant institutions .", "Except where specified , sacculi from adult American bullfrog ( Rana catesbeiana ) of both sexes were dissected and maintained in artificial perilymph ( Alonso et al . , 2020 ) .", "Cochleas dissected from Sprague-Dawley rats four to twelve days of age and of both sexes were maintained in a saline solution comprising 127 . 5 mM NaCl , 10 mM sodium pyruvate , 1 . 2 mM MgCl2 , 5 mM D-glucose , 3 mM KCl , 0 . 5 mM CaCl2 , 1 mM creatine , 20 mM sucrose , 0 . 5 mM NaH2PO4 , and 2 mM Na2SO4 .", "During each experiment , the tapered optical fiber was inserted through a glass capillary placed in a custom-made electrode holder that could be affixed to a micromanipulator ( Figure 10A ) .", "This holder ensured that the fiber’s tip was stable despite possible vibrations or displacements of the remainder of the fiber .", "Under the control of a micromanipulator ( ROE 200 , Sutter Instruments , Novato , CA , USA ) , the fiber’s distal end was introduced into the experimental chamber beneath a 60X water-immersion objective lens ( LUMPlanFL N , numerical aperture 1 . 0 , Olympus , Tokyo , Japan ) .", "The incidence angle of about 20° with respect to the horizontal ensured that the fiber cleared both the upper edge of the experimental chamber and the lower rim of the lens ( Figure 10B ) .", "Light from a 600 nm light-emitting diode ( Prizmatix Ltd . , Southfield , MI , USA ) illuminated the specimen through an inverted 60X water-immersion objective lens ( LUMPlan FI/IR , numerical aperture 0 . 9 , Olympus ) that served as a condenser .", "To permit differential-interference imaging , a polarizer was positioned just above the microscope’s field diaphragm and a crossed analyzer above its tube lens , and both objective lenses were equipped with Wollaston prisms .", "A rotating quarter-wave plate above the polarizer permitted optimization of the image , and a heat filter protected the specimen from infrared damage .", "Light that had traversed the specimen , the objective lens , and the tube lens was relayed by two mirrors and projected with a total magnification of 900X onto a dual photodiode , which permitted measurements of hair-bundle movement with nanometer precision .", "A dichroic mirror imposed before the photodiode prevented contamination of the movement signal by light from the stimulating laser .", "For the selection of appropriate hair bundles , the light path could be diverted to a camera that permitted observation of the specimen on a digital monitor ." ] ]

[ "Hair cells , the receptors of the inner ear , detect sounds by transducing mechanical vibrations into electrical signals .", "From the top surface of each hair cell protrudes a mechanical antenna , the hair bundle , which the cell uses to detect and amplify auditory stimuli , thus sharpening frequency selectivity and providing a broad dynamic range .", "Current methods for mechanically stimulating hair bundles are too slow to encompass the frequency range of mammalian hearing and are plagued by inconsistencies .", "To overcome these challenges , we have developed a method to move individual hair bundles with photonic force .", "This technique uses an optical fiber whose tip is tapered to a diameter of a few micrometers and endowed with a ball lens to minimize divergence of the light beam .", "Here we describe the fabrication , characterization , and application of this optical system and demonstrate the rapid application of photonic force to vestibular and cochlear hair cells ." ]

[ "The sense of hearing relies on specialized sensory cells in the inner ear .", "Each of these hair cells converts sounds into electrical signals that the brain can interpret .", "The hair cell takes its name from the bundle of rod-like structures that protrude from its top surface , which resemble hairs under the microscope .", "The hair bundle acts as an antenna that bends in response to sound waves .", "When a hair bundle moves in a particular direction , it opens ion channels in the hair-cell membrane .", "The resulting flow of ions into the cell triggers a cascade of events that ends with an electrical signal traveling to the brain .", "Many experiments on hearing rely on being able to manipulate the movement of a hair bundle .", "Researchers typically use one of two methods to achieve this .", "In the first , a flexible glass fiber pushes against the hair bundle , whereas the second involves a jet of fluid directed against the cell .", "Neither of these techniques can move hair bundles fast enough for researchers to explore the vast range of sound frequencies that human ears can detect .", "What is more , both methods are prone to introducing errors into experiments .", "Abeytunge , Gianoli et al . have developed a new method for moving hair bundles , this time with the aid of light .", "When light interacts with objects it exerts a photonic force .", "Abeytunge , Gianoli et al . show that a tapered optical fiber with a miniscule rounded lens can focus a laser beam to deliver enough photonic force to move a hair bundle .", "The laser beam does not damage the hair bundle , but moves it fast enough to allow researchers to study a broader range of mammalian hearing , while avoiding the errors that have bedeviled previous methods ." ]

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

[ "neuroscience" ]

[ [ "Tonic pain is a common physiological consequence of injury and results in a behavioural state that favours quiescence and inactivity , prioritising energy conservation and optimising recuperation and tissue healing .", "This effect extends to cognition , and decreased attention is seen in a range of cognitive tasks during tonic pain ( Crombez et al . , 1997; Lorenz and Bromm , 1997 ) .", "However , in some circumstances , this could be counter-productive , for instance if attentional resources were required for learning some means of relief or escape from the underlying cause of the pain .", "A natural solution would be to suppress tonic pain when relief learning is possible .", "Whether and how this is achieved is not known , but it is important as it might reveal central mechanisms of endogenous analgesia .", "Two observations provide potential clues as to how a relief learning system might modulate pain .", "First , in some situations , perceived controllability has been found to reduce pain ( Salomons et al . , 2004; Salomons et al . , 2007; Wiech et al . , 2014; Becker et al . , 2015 ) , suggesting that the capacity to seek relief can engage endogenous modulation .", "Second , instructed attention has commonly been observed to reduce pain ( Bantick et al . , 2002 ) .", "Therefore , it may be that attentional processes that are internally triggered when relief is learnable might provide a key signal that controls reduction of pain .", "In general , learning involves distinct processes of prediction ( ‘state learning’ ) and control ( ‘action learning’ ) ( Mackintosh , 1983 ) , although relief learning during tonic pain has not been thoroughly investigated .", "But a quantitative model of relief learning - one that describes the computational processes that are implemented in learning centres in the brain - would allow interrogation of how an attentional process might operate to modulate tonic pain .", "In the case of phasic pain , learning can be described by reinforcement learning ( RL ) models - a well-studied computational framework for learning from experience .", "RL models describe how to predict the occurrence of inherently salient events , and learn actions to exert control over them ( maximising rewards , minimising penalties ) ( Seymour et al . , 2004 ) .", "RL models aim to provide a mechanistic ( beyond a merely descriptive ) account of the information processing operations that the brain actually implements ( Dayan and Abbott , 2001 ) , and have a solid foundation in classical theories of animal learning ( Mackintosh , 1983 ) .", "In such models , an agent learns state or action value functions through outcomes provided by interacting with the world .", "These functions can be learned by computing the error between predicted and actual outcomes , and using the error to improve future predictions and actions ( Sutton and Barto , 1998 ) .", "Experimentally , the validity of these models can be tested by comparing how well different model-generated predictors fit the actual behavioural and/or neural data ( O'Doherty et al . , 2007 ) .", "During learning , attention is thought to boost learning of predictive associations and suppress other irrelevant information .", "Computationally , this can be achieved by estimating the uncertainty as predictive associations are learned , and using this as a metric to control learning rates .", "Accordingly , high uncertainty corresponds to high attention and leads to more rapid learning ( Dayan et al . , 2000; Yu and Dayan , 2005 ) .", "One well-recognised way of formalising uncertainty in RL is by computing a quantity called the associability , which calculates the running average of the magnitude of recent prediction errors ( i . e . frequent large prediction errors implies high uncertainty/associability ) .", "The concept of associability is grounded in classical theories of Pavlovian conditioning ( the ‘Pearce-Hall’ learning rule , Le Pelley , 2004; Pearce and Hall , 1980; Holland and Schiffino , 2016 ) , and provides a good account of behaviour and neural responses during Pavlovian learning ( Li et al . , 2011; Boll et al . , 2013; Zhang et al . , 2016 ) .", "In this way , associability reflects a computational construct that captures aspects of the psychological construct of attention .", "If it is the case , therefore , that attention can be understood as an uncertainty signal that drives learning during relief-seeking , it can then be tested with it modulates tonic pain in parallel .", "Standard models of RL do not include any mechanism by which the subjective experience of outcomes is under control , although in principle endogenous modulation of tonic pain could arise from any component of the learning system , including an associability signal .", "Using an associability signal in this way would make intuitive sense , because it would reduce ongoing pain when requirement for learning was high .", "The studies presented here set two goals: to delineate the basic neural architecture of relief learning from tonic pain ( i . e . pain escape learning ) based on a state and action learning RL framework; and to understand the relationship between relief learning and endogenous pain modulation that is , to test the hypothesis that an attentional learning signal reduces pain .", "We studied behavioural , physiological and neural responses during two relief learning tasks in humans , involving", "( i ) static and", "( ii ) dynamic cue-relief contingencies .", "These tasks were designed to place a high precedence on error-based learning and uncertainty , as a robust test for learning mechanisms and dynamic modulation of tonic pain .", "Using a computationally motivated analysis approach , we aimed to identify whether behavioural and brain responses were well described as state and/or action RL learning systems and examined whether and how they exerted control over the perceived intensity of ongoing pain ." ], [ "Experiment 1 was an escape learning task ( n = 19 ) with fixed , probabilistic cue-relief contingencies ( Figure 1a ) .", "Each subject performed three instrumental sessions and three Pavlovian sessions , to allow us to compare active and passive relief learning ( Figure 1b ) .", "During each session ( lasting approximatey 5 min ) , subjects were held in continuous pain by a thermal stimulator attached to their left arm , and temporary relief ( i . e . escape ) was given by rapidly cooling the thermode for 4 s , after which it returned to the baseline tonic pain level ( Figure 1c ) .", "In instrumental sessions , subjects actively learned to select actions , a left or right button press , after viewing one of two visual cues ( fractal images on a computer screen ) .", "For one of the cues , the probability of relief was 80% for one action and 20% for the other action , and for the other cue , the action relief probabilities were 60% and 40% .", "In the Pavlovian sessions , stimulus and outcome sequences were yoked to instrumental sessions for individual subjects , and subjects were required simply to press a button to match a random direction appearing on screen 0 . 5 s after visual cue onset ( to control for motor responses ) .", "Subjective ratings of pain and relief were collected in random trials after outcome delivery , with on average eight pain and eight relief ratings per paradigm that is total 16 for each subject .", "All behavioural data including raw SCRs , choices , and ratings can be found in the manuscript data attachment .", "In Experiment 2 , 23 new subjects participated in a modified version of the instrumental escape learning task in Experiment 1 , with a number of important differences .", "First , subjects performed only instrumental sessions ( 8 sessions with 24 trials in each ) given the absence of a global effect of instrumental versus Pavlovian pain in the first experiment .", "Second , subjects were required to choose one out of three simultaneously presented visual cues to obtain relief , in which the position of each cue varied randomly from trial to trial .", "This was done to experimentally and theoretically better distinguish state-based and action-specific associability ( Figure 1d ) .", "Third , the action-outcome contingencies were non-stationary , such that the relief probability from selecting each cue varied slowly throughout the experiment duration , controlled by a random walk algorithm which varied between 20 and 80% ( Figure 1e ) .", "This ensured that associability varied constantly through the task , encouraging continued relief exploration , and allowed us to better resolve more complex models of uncertainty ( see below ) .", "It also reduced the potential confounding correlation of associability and general habituation of SCRs .", "Fourth , we increased the frequency of tonic pain ratings ( 10 per session , 80 per subject in total ) to enhance power for identifying modulatory effects on pain .", "Fifth , the rating was taken after the action but before outcome , to provide an improved assessment of ongoing tonic pain modulation without interference by the outcome .", "Finally , we also collected SCRs bilaterally , to enhance the data quality given the importance of the SCR in inferences about associability ." ], [ "Across both experiments , the results provide convergent support for two key findings .", "First , we show that relief seeking from the state of tonic pain is supported by a reinforcement learning process , in which optimal escape actions are acquired using prediction error signals , which are observed as BOLD signals in the dorsal putamen .", "Second , we show that during learning , the level of ongoing pain is reduced by the learned associability associated with state-based relief predictions .", "This signal thus reduces pain when there is a greater capacity to learn new information and is associated with BOLD responses in the pregenual anterior cingulate cortex .", "Together , these results identify a learning circuit that governs tonic pain escape learning whilst also suppressing pain according to the precise information available during learning .", "In doing so , it solves the problem of balancing tonic pain with the requirement to actively learn about behaviour that could lead to relief .", "The findings highlight the dual function of a state-based relief associability signal during tonic pain escape .", "Associability has its theoretical underpinnings in classical theories of associative learning and attention ( i . e . the Pearce-Hall theory , Pearce and Hall , 1980 ) , and its mathematical implementation here is as an approximate uncertainty quantity derived from computing the running average of the magnitude of the prediction error ( Sutton , 1992; Le Pelley , 2004 ) .", "This uncertainty signal effectively captures how predictable the environment is: when uncertainty is high ( because of lots of recent large prediction errors ) , it increases the speed of acquisition through increasing the learning rate , and so accelerates convergence to stable predicted values .", "It is therefore an effective attention-like signal for mediating endogenous analgesia , because it selectively facilitates active relief seeking by suppressing pain only when it is necessary .", "This conception of the role of uncertainty in pain may explain why uncertainty has been shown to enhance phasic pain ( Yoshida et al . , 2013 ) - where pain acts as the signal to drive learning , and suppresses tonic pain , where pain acts to reduce general cognition .", "In both instances , the role of uncertainty and attention is to facilitate learning .", "A caveat to this is that associability cannot distinguish unreliable cues - inherently poor predictors of outcomes , and so does not discriminate between reducible and irreducible uncertainty , bearing in mind there is little adaptive logic in suppressing pain for unreliable predictors .", "Over extended time-frames , it is possible that the learning system recognises this and reduces endogenous control .", "However , in rodent studies of associative learning , associability is maintained even after several days of training ( Holland et al . , 2002 ) , and it is possible that salient cues in aversive situations maintain the ability to command attention and learning longer than that would be predicted by ‘optimal’ Bayesian models .", "The localisation of the associability signal to the pgACC is consistent with a priori predictions .", "The region is known to be involved in threat unpredictability ( Rubio et al . , 2015; Nitschke et al . , 2006 ) , computations of uncertainty during difficult approach-avoidance decision-making ( Amemori and Graybiel , 2012 ) , and in the perseverance of behaviour during foraging ( McGuire and Kable , 2015; Kolling et al . , 2012 ) .", "It is distinct from a more anterior region in the ventromedial prefrontal cortex associated with action value ( FitzGerald et al . , 2012 ) .", "More importantly , it has been specifically implicated in various forms of endogenous analgesia , including coping with uncontrollable pain ( Salomons et al . , 2007 ) , distraction ( Valet et al . , 2004 ) , and placebo analgesia ( Bingel et al . , 2006; Eippert et al . , 2009 ) .", "However , an open question remains about the role of conscious awareness in driving pgACC-related endogenous control - a factor that is often important in these other paradigms .", "Whether or not the role of associability is modulated by the metacognitive awareness of uncertainty or controllability would be an important question for future studies .", "The pgACC has been suggested to be central to a ‘medial pain system’ and the descending control of pain , with its known anatomical and functional connectivity to key regions including the amygdala ( Derbyshire et al . , 1997; Vogt et al . , 2005; Salomons et al . , 2015 ) and PAG ( Stein et al . , 2012; Buchanan et al . , 1994; Vogt , 2005; Domesick , 1969 ) .", "Evidence of high level of μ-opioid receptors within pgACC ( Vogt et al . , 2005 ) , where increased occupation has been found in both acute and chronic pain ( Zubieta et al . , 2005; Jones et al . , 2004 ) , further illustrates pgACC’s potential role for cortical control of pain .", "The results provide a formal computational framework that brings together theories of pain attention , controllability and endogenous analgesia .", "Previous demonstrations of reduced pain ( albeit typically for phasic , not tonic pain ) have been inconsistent ( Becker et al . , 2015; Salomons et al . , 2004; Salomons et al . , 2007; Wiech et al . , 2014; Wiech et al . , 2006; Mohr et al . , 2012 ) .", "Our results offer insight into why - by suggesting that endogenous analgesia is not a non-specific manifestation of control , but rather a specific process linked to the learnable information .", "From the perspective of animal learning theory , the experiments here show how motivation during the persistent pain state can be understood as an escape learning problem , in which the state of relief is determined by the offset of a tonic aversive state ( Mackintosh , 1983; Solomon and Corbit , 1974 ) .", "This is theoretically distinct from the better-studied form of relief that results from omission of otherwise expected pain or punishment ( Konorski , 1967 ) , and which motivates avoidance behaviour ( Mowrer , 1960 ) .", "In our task , acquisition of dissociable behavioural responses ( SCRs and choices ) reveals the underlying theoretical architecture of the escape learning process , which involves both parallel state-outcome and action-outcome learning components .", "The action-outcome learning error signal localises to a region of the dorsolateral striatum ( dorsal putamen ) .", "Striatal error signals are seen across a broad range of action learning tasks , although the region here appears more dorsolateral than previously noted in avoidance learning ( Kim et al . , 2006; Seymour et al . , 2012; Delgado et al . , 2009 ) .", "It is not possible to definitively identify whether avoidance and escape use distinct errors , but it is well recognised that there are multiple error signals in dorsal and ventral striatum , for instance reflecting ‘model-based’ ( cognitive ) , ‘model-free’ ( including stimulus-response habits ) and Pavlovian control ( Tricomi et al . , 2009; Schonberg et al . , 2010; Yin et al . , 2004 ) .", "The reinforcement learning model we describe is a ‘model-free’ mechanism , since it learns action values but does not build an internal model of state-outcome identities and transition probabilities ( Daw et al . , 2005 ) .", "However , it is likely that a model-based system co-exists and might be identifiable with appropriate task designs ( Daw et al . , 2011 ) .", "Developing a computational account of relief learning and endogenous control may also help us understand how the brain contributes to the pathogenesis and maintenance of chronic pain ( Navratilova and Porreca , 2014 ) .", "Adaptive learning processes are thought to be important in chronic pain: learning and controllability have been proposed to play a role in the pathogenesis and maintenance of chronic pain ( Vlaeyen , 2015; Flor et al . , 2002; Apkarian et al . , 2004; Salomons et al . , 2015 ) , and brain regions such as the medial prefrontal cortex and striatum have been consistently implicated in clinical studies , for example in pain offset responses ( Baliki et al . , 2010 ) and resting functional connectivity in chronic back pain ( Baliki et al . , 2008; Baliki et al . , 2012; Fritz et al . , 2016; Yu et al . , 2014 ) .", "In addition to suggesting a possible computational mechanism that might underlie pain susceptibility in these patients , the results highlight the pgACC as a potential target for therapeutic intervention ." ], [ "Two separate groups of healthy subjects participated in the two neuroimaging experiments ( Experiment 1: n = 19 , six female , age 26 . 1±5 . 1 years; Experiment 2: n = 23 , five female , age 23 . 9±3 . 1 years ) .", "All subjects gave informed consent prior to participation , had normal or corrected to normal vision , and were free of pain conditions or pain medications .", "The two experiments were performed in different institutes , and approved by their relevant ethics and Safety committees: for the National Institute of Information and Communications Technology , Japan ( Experiment 1 ) , and the Advanced Telecommunications Research Institute , Japan ( Experiment 2 ) .", "Painful tonic thermal stimuli were delivered to the subject’s skin surface above the wrist on the left inner forearm , through a contact heat-evoked potential stimulator ( CHEPS , Medoc Pathway , Israel ) .", "The CHEPS thermode is capable of rapid cooling at 40°C/s , which made rapid temporary pain relief possible in an event-related design .", "The temperature of painful tonic stimuli was set according to the subject’s own pain threshold calibrated beforehand .", "In Experiment 1 , before the task , two series of 6 pre-set temperatures were presented in random order ( set 1: mean ± std 43 . 7±1 . 7°C; set 2: 44 . 6±0 . 6°C ) , with each temperature delivered for 8 s , after which the subject determined whether the stimulation period was painful or not ( ISI = 8 s ) .", "The higher of the two lowest painful temperatures from the two tests was used as the tonic stimulation temperature .", "In Experiment 2 , 10 temperatures were presented in each series , both were randomly generated with 44 . 4±0 . 7°C .", "After the 8 s stimulation , subjects were asked to rate their pain on a 0–10 VAS scale , which were fitted with a sigmoid function .", "The temperature was chosen from the temperature range of: 44 , 44 . 2 , 44 . 5 , 44 . 8 , 45°C , whichever closest and below the model fitted value of VAS = 8 .", "The final temperature used did not differ hugely for the two experiments despite the change in thresholding method ( Experiment 1: 44 . 3±0 . 2°C , Experiment 2: 44 . 5±0 . 4°C ) .", "The relief temperature was set constant at 13°C below threshold temperature for all subjects .", "Skin conductance responses ( SCRs ) were measured using MRI-compatible BrainAmp ExG MR System ( Brain Products , Munich , Germany ) with Ag/AgCl sintered MR electrodes , filled with skin conductance electrode paste .", "In Experiment 1 , SCR data were recorded on volar surfaces of distal phalanges of the second and fourth fingers on the left ( tonic pain side with thermode attached ) .", "In Experiment 2 , data were recorded from both hands , in the same location on the left ( with thermode ) , and on the hypothenar eminences of the palm on the right ( button press hand without thermode ) , with electrodes approximately 2 cm apart .", "The signals were collected using BrainVision software at 500 Hz with no filter .", "Off-line processing and analysis were implemented in MATLAB7 ( The MathWorks Inc . , Natick , MA ) , with the PsPM toolbox ( http://pspm . sourceforge . net/ ) .", "Data were down-sampled to 10 Hz , band-pass filtered at 0 . 0159–2 Hz ( 1 st order Butterworth ) .", "Given the variable nature of SCR onset and duration in a learning experiment , the non-linear model in PsPM was used .", "Boxcar regressors were constructed at cue onset ( duration = 3 s , cue presentation ) .", "These regressors were convolved with the canonical skin conductance response function , to estimate event-related response amplitude , latency , and dispersion ( only SCR amplitude were used in modelling ) .", "Sessions with more than 20% trials ( 4 out of 20 trials for Experiment 1 , 5 out of 24 for Experiment 2 ) with cue-evoked SCR amplitude below the threshold of 0 . 02 were labelled as not having enough viable event related SCRs .", "In Experiment 1 , 15 subjects and 50 sessions remained .", "In Experiment 2 , 19 subjects and 79 sessions remained for the left ( thermal stimulation side ) , 20 subjects and 96 sessions remained for the right ( no stimulation side ) .", "For model fitting , right side SCR reject criteria were used , since both channel’s data were included as two data sources .", "Trial SCRs were log-transformed within subject before model fitting .", "Transformed SCRs on both sides were highly correlated ( Figure 4b ) .", "Trial-by-trial choice data ( button press indicating choices ) and reaction times ( length of time taken from CS onset to button press ) of subjects were recorded as part of behavioural measurements .", "To capture relief learning we fitted behavioural responses using different learning models from previous studies ( Table 4 ) .", "Free energy ( F ) are variational Bayesian approximation of model’s marginal likelihood , table showing the sum of F for all participants to provide model absolute fit evaluation .", "Actual model comparison was conducted based on random-effect analysis .", "For instrumental learning , the reinforcement of subjects’ responses ( i . e . choices ) based on relief experience can be modelled using reinforcement learning model ( Sutton and Barto , 1998 ) .", "For Pavlovian learning , physiological responses can be used for model fitting ( Li et al . , 2011; Boll et al . , 2013; Zhang et al . , 2016 ) .", "Our prior hypothesis suggests uncertainty is a likely modulator of tonic pain perception , hence model generated uncertainty signals ( associability in Experiments 1 and 2 , with entropy and surprise added in Experiment 2 ) were used as the main pain rating predictors .", "A generalised linear model includes the uncertainty predictor , and additional terms to control for potential temporal habituation/sensitization and between-session variation: ( 15 ) Rating=β1⋅Relief+β2⋅log ( Trial ) +β3⋅Predictorwhere the ‘Relief’ term is the number of trials since the previous relief outcome , log ( Trial ) is the log of trial number within session ( 1-24 ) , ‘Predictor’ is the model generated uncertainty value using group-averaged model parameters fitted with choice/SCR data .", "All trials were used for predictor calculation , but only rated trials were included in this regression .", "For Experiment 1 , neuroimaging data was acquired with a 3T Siemens Magnetom Trio Tim scanner , with the Siemens standard 12 channel phased array head coil .", "For Experiment 2 , a 3T Siemens Prisma scanner was used , with the Siemens standard 64 channel phased array head coil .", "Scanning parameters were identical for both experiments: functional images were collected with a single echo EPI sequence ( repetition time TR = 2500 ms , echo time TE = 30 ms , flip angle = 80 , field of view = 240 mm ) , 37 contiguous oblique-axial slices ( voxel size 3 . 75 × 3 . 75×3 . 75 mm ) parallel to the AC-PC line were acquired .", "Whole-brain high resolution T1-weighted structural images ( dimension 208 × 256×256 , voxel size 1 × 1×1 mm ) using standard MPRAGE sequence were also obtained .", "Functional images were slice time corrected using SPM12 ( http://www . fil . ion . ucl . ac . uk/spm/software/spm12/ ) with individual session’s slice timing output by the scanner .", "Resulting images were then preprocessed using the fmriprep software ( build date 09/03/2017 , freesurfer option turned off , https://github . com/poldracklab/fmriprep ) , a pipeline that performs motion correction , field unwarping , normalisation , field bias correction , and brain extraction using a various set of neuroimaging tools available .", "The normalised images were smoothed using a Gaussian kernel of 8 mm using SPM12 .", "The confound files output by fmriprep include the following signals: mean global , mean white matter tissue class , three FSL-DVARS ( stdDVARS , non-stdDVARS and voxel-wise stdDVARS ) , framewise displacement , six FSL-tCompCor , six FSL-aCompCor , and six motion parameters ( matrix size: 24 × number of volumes ) .", "All event-related fMRI data were analysed with generalised linear models ( GLM ) constructed using SPM12 , estimated for each participant in the first level .", "Model generated signals used as parametric modulators were generated with one set of group-mean model parameters , obtained with behavioural data fitting as described .", "We used the mean of the fitted parameters from all participants in the imaging analysis as this provides the most stable estimate of the population mean ( taking into account the fact that individual fits reflect both individual differences and noise ) .", "For completeness , however , we also ran the analyses with individually fitted values , which led to similar results ( i . e . no change in significance level of each result ) .", "All regressors were convolved with a canonical hemodynamic response function ( HRF ) .", "We also include regressors of no interest to account for habituation and motion effects .", "Specifically , the number of trials since last receiving a relief outcome ( ‘Relief’ term in rating regression model ) , and the log of trial number within session ( log ( Trial ) term ) were included to regress out potential change in tonic pain perception simply due to prolonged stimulation .", "The resulting GLM estimates were entered into a second-level one-sample t-test for the regressors of interest to produce the random-effect statistics and images presented in Results section .", "To determine whether state-based and action-based learning involve the same brain regions during instrumental learning , we used Bayesian model selection ( BMS ) with the instrumental sessions imaging data .", "We ran Bayesian first level analysis using two separate GLMs containing the prediction error signals from TD and hybrid models ( at outcome onset time , durations = 3 s ) using unsmoothed functional imaging data , with the same regressors of no interest as other GLMs described .", "To reduce computation time , this was restricted to voxels correlated to prediction error from previous parametric modulation analysis results from our present study , within a mask of conjunction clusters from TD and hybrid prediction error analysis ( cluster formation at p<0 . 01 , k<5 ) .", "Resulting log-model evidence maps produced from each model for individual participant were first smoothed with a 6 mm Gaussian kernel , then entered into a random-effect group analysis ( Stephan et al . , 2009 ) .", "Voxel-wise comparison between models produced posterior and exceedance probability maps to show whether a particular brain region is better accounted for by one model or the other .", "Posterior probability maps were overlaid on subject-averaged anatomical scans using MRIcroGL ( https://www . nitrc . org/projects/mricrogl/ ) .", "To determine whether ROI activations to prediction errors were responding outcomes or prediction errors , we carried out ROI axiomatic analysis ( Roy et al . , 2014 ) .", "Trials were separated into relief or no relief outcomes , then into equal-size bins of ascending sorted expected relief values , calculated from TD model as we were primarily interested in instrumental/active relief learning .", "This produced four regressors ( 2 outcomes × 2 value bins ) in Experiment 1 , and six regressors ( 2 outcomes × 3 value bins ) in Experiment 2 , to be estimated at outcome time ( duration = 3 s ) when prediction error was generated .", "GLMs include button presses for choice or rating , and movement related regressors of no interest mentioned above .", "ROI masks of 8 mm spheres were generated from peak coordinates from TD model prediction error exceedance probability map calculated by BMS above ( ventral and dorsal striatum , amygdala , VMPFC and DLPFC ) .", "Mean activity were extracted from these ROI masks averaged across sessions within individual subject .", "Although the axiomatic analysis is useful for delineating outcome and prediction responses in previous reward or aversive PE studies , the continued presence of tonic pain in our study differs from the ‘no stimulation’ conditions in these studies , thus we are primarily interested in the overall BOLD activity pattern and did not include full statistics of this analysis ." ] ]

[ "Tonic pain after injury characterises a behavioural state that prioritises recovery .", "Although generally suppressing cognition and attention , tonic pain needs to allow effective relief learning to reduce the cause of the pain .", "Here , we describe a central learning circuit that supports learning of relief and concurrently suppresses the level of ongoing pain .", "We used computational modelling of behavioural , physiological and neuroimaging data in two experiments in which subjects learned to terminate tonic pain in static and dynamic escape-learning paradigms .", "In both studies , we show that active relief-seeking involves a reinforcement learning process manifest by error signals observed in the dorsal putamen .", "Critically , this system uses an uncertainty ( ‘associability’ ) signal detected in pregenual anterior cingulate cortex that both controls the relief learning rate , and endogenously and parametrically modulates the level of tonic pain .", "The results define a self-organising learning circuit that reduces ongoing pain when learning about potential relief ." ]

[ "Chronic pain lasting longer than three months is a common problem that affects about 1 in 5 people at some point in their lives .", "The lack of effective treatments has led to widespread use of a group of drugs called opioids – the best-known example is morphine .", "Opioids work by activating the brain’s natural painkilling system and are useful to relieve short-term pain , for example in trauma or surgery , or in end-of-life care .", "Unfortunately , long-term use of opioids can cause many undesirable effects , including drug dependency .", "Misuse of opioids combined with the widespread availability of prescription drugs have contributed to the current crisis of opioid addiction and overdose .", "A better understanding of how the brain’s natural painkilling system works could help scientists develop painkillers that offer relief without the harmful side effects of opioids .", "While unpleasant , pain is important for survival .", "After an injury , for example , pain saps motivation and forces people to rest and preserve their energy as they are healing .", "In a way , this sort of pain is healthy because it promotes recovery .", "There may be times when the brain might want to turn off pain , such as when an individual is seeking new ways to relieve or manage pain .", "For example , by finding a way to cool a burn .", "Now , Zhang et al . show that the brain reduces pain while individuals are trying to find relief .", "In the experiments , a metal probe was attached to the arm of healthy volunteers and heated until it became painful but not hot enough to burn the skin .", "Then , the volunteers were asked to play a game in which they had to find out which button on a small keypad cooled down the probe .", "Sometimes it was easy to turn off the heat , sometimes it was difficult .", "During the game , volunteers reported how much pain they felt and Zhang et al . used brain imaging to see what happened in their brains .", "When the subjects were actively trying to work out which button they should press , pain was reduced .", "But when the subjects knew which button to press , it was not .", "Next , Zhang et al . found that a part of the brain called the pregenual cingulate cortex was responsible for making decisions about when to turn off pain and may so trigger the brain’s natural pain killing system .", "A next step will be to see how this part of the brain decides to turn off pain and if it also controls opioid-like or other chemicals .", "This could improve the use of opioids , or even help to discover alternative treatments for chronic pain ." ]

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

[ "developmental biology", "cancer biology" ]

[ [ "Vast amount of evidence demonstrates the key role of systemic immunity in tumour progression and patient outcome .", "Efforts to manipulate the immune response to tumours are at the core of basic and translational cancer research .", "In mammals and flies , tumourigenesis triggers inflammation and activation of the immune system , leading to tumour cell death ( Cordero et al . , 2010; Parameswaran and Patial , 2010; Parisi et al . , 2014; Teng et al . , 2008 ) .", "Tumour Necrosis Factor ( TNF ) is an important player in these tumour/immune interactions and has pleiotropic effects on tumours , including induction of cell death ( Ham et al . , 2016; Parameswaran and Patial , 2010 ) .", "This function is conserved in Drosophila , where the single TNF homolog Eiger ( Egr ) , produced by tumour-associated macrophages ( TAMs ) , has been shown to drive cell death of neoplastic tumours , generated in larval imaginal discs by the loss of apico-basal complex components such as Disc large 1 , Scribble or Lethal giant larvae ( Cordero et al . , 2010; Parisi et al . , 2014; Parvy et al . , 2018 ) .", "Moreover , we have previously reported that tumours from disc large 1 Drosophila mutant larvae ( dlg40 . 2 hereafter referred to as dlg ) , activate Toll pathway in the Drosophila fat body in an Egr-dependent manner , and this immune activation is necessary for TNF-dependent tumour cell death ( Parisi et al . , 2014 ) .", "However , the mechanisms by which activation of an immune response in the fat body executes tumour cell death remain unknown ( Figure 1A ) .", "In Drosophila as in mammals , Toll pathway is well known to play a central role in the innate immune response to infection ( Lemaitre et al . , 1996 ) .", "Downstream effectors of the Toll pathway include antimicrobial peptides ( AMPs ) , which possess microbicidal activities against various pathogens .", "They display potent antimicrobial activity in vitro by disrupting negatively-charged microbial membranes .", "While intracellular activities have been reported , many AMPs kill pathogens by inserting into the lipid bilayer and disrupting the membrane integrity ( Brogden , 2005 ) .", "Host cells are instead protected from AMP as they are positively charged and contain cholesterol ( Brender et al . , 2012 ) .", "In vitro studies have revealed AMPs capacity to kill cancer cells ( Deslouches and Di , 2017 ) .", "However , whether this cancer-killing activity is a natural function of AMPs is unknown , as there are no reports on an in vivo paradigm addressing such question .", "Since the Toll pathway is activated in dlg mutant tumour bearing larvae and is required for optimal TNF-induced tumour cell death ( Parisi et al . , 2014 ) , we hypothesised that AMPs may be involved in this process .", "Here we show that Drosophila defensin is induced in the fat body and tracheal system of dlg mutant tumour bearing larvae .", "We find Defensin consistently associated to dying tumour cells .", "Critically , systemic and tissue specific knockdown of Defensin demonstrates a non-redundant role of the AMP in controlling tumour growth through the induction of tumour cell death .", "Anti-tumoural Defensin production relies on TNF-dependent activation of both Toll and Imd pathway .", "Our results demonstrate that dlg mutant tumours expose PS in response to haemocyte-derived TNF and that Defensin is present in PS enriched area on the tumour surface .", "Finally , we find that lack of TNF prevents PS exposure in tumours and makes them insensitive to the action of Defensin .", "Collectively , our results reveal an anti-tumoural role for Defensin in vivo and provide insights into the molecular mechanisms , which make tumours sensitive to the killing action of an endogenous AMP ." ], [ "In order to assess expression of several AMPs we performed RT-qPCR analysis on fat bodies dissected from wild type controls ( w1118 ) or dlg mutant larvae ( Figure 1B ) .", "Results showed consistent and statistically significant upregulation of defensin in fat bodies of dlg larvae compared to wild-type ones ( Figure 1B ) .", "Other Toll-dependent AMPs display a trend to be increased ( drosomycin and attacin A ) , even though data were highly variable amongst biological replicates , while other AMPs were not transcriptionally regulated ( drosocyn , cecropin A1 ) ( Figure 1B ) .", "Interestingly , human β-Defensin-1 displays anticancer activity in vitro ( Bullard et al . , 2008; Sun et al . , 2006 ) , and deletion of human def appears prevalent in some cancer types ( Ye et al . , 2018 ) .", "This prompted us to explore the role of Drosophila Defensin in dlg mutant tumours .", "Using dlg larvae reared on antibiotics , we confirmed that defensin upregulation was independent of the presence of microbes ( Figure 1C ) .", "Moreover , larvae bearing dlg imaginal discs tumours induced by RNAi ( en >UAS-dcr2; UAS-dlg-IR ) also displayed increased defensin expression , confirming def gene induction as a consequence of dlg-driven epithelial transformation rather than whole body dl loss ( Figure 1D ) .", "We conclude that Defensin , an AMP known for its activity against microbes , is induced , in a sterile environment , by the presence of tumours .", "We next hypothesised that Defensin may be an important mediator of anti-tumour immunity in vivo .", "To test this hypothesis , we generated null mutant alleles for the defensin ( def ) gene using the CRISPR/Cas9 system ( defsk3 and defsk4 ) ( Figure 1E ) ( Hanson et al . , 2019 ) .", "Survival analysis of defsk3 flies confirmed that this AMP contribute to resist systemic infection to certain Gram-positive bacteria ( Figure 1—figure supplement 1 ) ( Levashina et al . , 1995 ) .", "To evaluate the effect of Defensin on tumour development , we combined def and dlg loss of function alleles .", "Compared to dlg mutant animals , dlg;def double mutants displayed a significant increase in tumour size ( Figure 2A ) .", "Tumour growth is limited by apoptotic tumour cell death as revealed by Dcp1 staining ( Parisi et al . , 2014 ) .", "Interestingly , tumours from dlg;def double mutants display a very strong decrease in apoptosis ( Figure 2B–E’ ) , suggesting that increased tumour size in absence of Defensin is due to a decrease in tumour cell death .", "This was further supported by the similar proliferation rates measured in dlg and dlg , def mutant tumours as per quantification of anti-phophoHistone H3 staining ( Figure 2—figure supplement 1A–E’ ) .", "Importantly , the effect of Def on tumour size and cell death were still observed when dlg and dlg , def larvae were reared in sterile conditions ( Figure 2—figure supplement 1F–J’ ) and further confirmed upon ubiquitous knock down of def using RNA interference ( IR ) ( Figure 2F–J’ ) .", "Furthermore , fat body overexpression of def significantly rescued tumour volume and tumour cell death of dlg;defsk3 double mutant animals ( Figure 2K and L ) .", "Additionally , larval injection of a synthetic Defensin peptide increased tumour cell death of dlg or dlg;defsk3 imaginal discs ( Figure 2M ) , while it had no effect on tissues from wild-type larvae , indicating that Defensin can selectively promote cell death of tumour cells .", "Altogether , these results demonstrate that Defensin is required to control dlg-dependent tumourigenesis in vivo through induction of tumour cell death .", "Having shown that Defensin restrict tumour growth , we sought to determine the tissues that produced endogenous Defensin in the context of tumour bearing .", "Previous studies have shown that Defensin is not produced by imaginal discs or tumours ( Bunker et al . , 2015; Külshammer et al . , 2015 ) .", "In the context of infection , Defensin can be produced by the fat body as well the tracheal and gut epithelium ( Tzou et al . , 2000 ) .", "We monitored defensin expression by RT-qPCR in various immune tissues of tumour bearing animals .", "We observed that the fat body , homologue to the mammalian liver and adipose tissue , and the trachea , a network of tubes transporting oxygen to cells that resembles the mammalian vasculature , were the main sources of defensin in these animals ( Figure 3A ) .", "This was also supported by Defensin immunostaining , ( Figure 3B and C ) .", "Transcript assessment upon targeted IR knock down of defensin in the respective tissues further confirmed the domains of endogenous gene expression ( Figure 3D and I ) .", "Importantly , knocking down defensin expression specifically in the fat body or the trachea of dlg animals , resulted in increased tumour size and decreased tumour cell death ( Figure 3E–H’ and J–M’ ) confirming the non-redundant functional requirement of defensin in both tissues to efficiently promote tumour cell death .", "To investigate the possibility that Defensin produced by the fat body and the trachea can specifically target tumour cells , we made use of an inducible haemagglutinin ( HA ) tagged form of Defensin ( UAS-def-HA ) .", "We noticed leaky HA protein expression in the tracheal system ( Figure 4—figure supplement 1A ) but not in the fat body ( Figure 4—figure supplement 1B ) of animals carrying the UAS-def-HA construct only .", "Therefore , we overexpressed UAS-def-HA in the fat body using a Gal4 specific driver ( lpp >def HA ) , which resulted in significant upregulation of Defensin in the fat body ( Figure 4—figure supplement 1D ) when compared to control conditions ( Figure 4—figure supplement 1B ) , but also maintained the leaky expression of the transgene in the trachea ( Figure 4—figure supplement 1C ) .", "Strikingly , overexpression of UAS-def-HA in the fat body resulted in Defensin-HA immunostaining in transformed imaginal discs from dlg mutant animals ( Figure 4A–A’’ and B–B’’ ) but not in normal tissues from dlg heterozygous animals ( Figure 4C and C’ ) .", "Consistently , using anti-Defensin antibody , we observed endogenous Defensin staining on dlg mutant tumour ( Figure 4D–D’’ and E–E’’ ) but not in wild-type discs ( Figure 4G , G’ ) .", "Interestingly , we observed Defensin preferentially bound to tumour areas enriched with apoptotic cells ( Figure 4D–D’’ and E–E’’ ) .", "This was confirmed by quantification of the amount of Def staining colocalising with Dcp1 staining ( Figure 4F ) .", "High-resolution imaging showed Defensin enrichment at the membrane of these dying cells ( Figure 4H–H’’ ) .", "Altogether , these results show that Defensin produced by immune tissues selectively binds tumour cells to target them for apoptosis .", "defensin expression upon systemic infection relies on both Toll and Imd pathways ( Lemaitre et al . , 1996; Leulier et al . , 2000 ) .", "While we previously showed that Toll pathway activation in dlg mutant larvae is required to achieve maximal tumour cell death ( Parisi et al . , 2014 ) , the involvement of the Imd pathway in tumour bearing animals was still elusive .", "To assess the contribution of the Imd pathway to both defensin expression and tumour burden , we analysed these two phenotypes in larvae deficient for the Imd-pathway .", "We observed a 55–60% decrease in defensin expression in dlg mutants carrying a loss of function allele affecting imd ( dlg;imd1 ) ( Figure 5A ) or the gene encoding the downstream transcription factor Relish ( dlg;relE20 ) ( Figure 5F ) .", "Consistently , analysis of tumour phenotypes revealed increased tumour volume and decreased tumour cell death in dlg;imd1 and dlg;relE20 animals when compared with dlg counterparts ( Figure 5B–E’ and G–J’ ) .", "Altogether , these data demonstrate that the Imd pathway is required for defensin upregulation , impairment of tumour growth and induction of tumour cell death in dlg mutant larvae .", "Upon infection , AMP expression in the trachea exclusively relies on the Imd pathway ( Tzou et al . , 2000 ) .", "We therefore looked at defensin expression and tumour phenotype in dlg mutant larvae where imd expression had been knocked down specifically within tracheal cells ( dlg;btl >imd-IR ) .", "In this setting , defensin expression was significantly reduced in the whole larvae ( Figure 6A ) .", "Consistently , tumour volume was increased while tumour cell death was decreased ( Figure 6B–E’ ) showing the requirement of Imd pathway in the tracheal system to control tumour burden .", "In the fat body , both Imd and Toll pathways contribute to AMPs expression during infection ( Tzou et al . , 2002 ) .", "To evaluate the contribution of Toll and Imd pathways in fat body in tumour bearing larvae , we monitored defensin expression and tumour phenotype in dlg animals in which Toll or Imd pathways have been selectively knocked down in the fat body .", "Knocking down myd88 , which encodes a common adaptor to Toll receptors ( Imler and Zheng , 2004 ) , or imd in the fat body resulted in a significant decrease in defensin expression ( Figure 6F ) and , consistently , increased tumour volume and decreased tumour cell death ( Figure 6G–K’ ) .", "Moreover , concomitant overexpression of defensin and Myd88-IR or Imd-IR in the fat body of dlg larvae was sufficient to significantly rescue the tumour volume and tumour cell death phenotypes resulting from fat body knockdown of Myd88 or Imd in dlg animals ( Figure 6L–O ) .", "We conclude that , in dlg mutant animals , Toll and Imd pathways have non-redundant roles in restricting tumour growth and promoting tumour cell death through the control of defensin expression .", "Importantly , forced defensin expression in an otherwise immune-compromised animal is sufficient to reduce tumour growth and to promote tumour cell death .", "AMPs targeting of pathogens , involves the recognition of negatively charged molecules exposed on the cell surface ( Yeaman and Yount , 2003 ) .", "A key question raised by our study is how Defensin can selectively bind and kill tumour cells ( Figure 4 and Figure 2M ) .", "Selective action of cationic AMP is attributed to their ability to interact with negatively charged membrane such as those found in bacteria .", "We hypothesised that the membrane of tumour cells from dlg mutant larvae might change their electrostatic properties , making them sensitive to the action of Defensin .", "Phosphatidylserine ( PS ) is a negatively charged phospholipid , normally restricted to the inner leaflet of the cell membrane .", "However , PS can be exposed on the outer leaflet for example in apoptotic cells , which tags these cells for phagocytosis ( Birge et al . , 2016; Shklyar et al . , 2013; Tung et al . , 2013 ) .", "Moreover , PS exposure has been shown to occur independently of apoptosis in many cancer cell types ( Riedl et al . , 2011 ) .", "Therefore , we investigated whether PS externalisation could be a factor allowing specific targeting of tumour cells by Defensin .", "Our data revealed that dlg but not wild-type tissues , displayed high levels of Annexin V staining ( Figure 7A and F; compare to E ) , indicating increased exposure of PS by dlg tumours .", "We next looked at the ability of Defensin to specifically associate with tumour cells exposing PS and found that Defensin was enriched in Annexin V+ve areas on dlg tumours ( Figure 7A–A’’ and B–B’’ ) .", "This was confirmed by quantification of the amount of Def staining colocalising with Annexin V staining ( Figure 7C ) .", "Thus , Defensin produced by immune responsive tissues binds specifically to tumour cells and this ability correlated with their exposure of PS .", "Previous studies have shown that circulating macrophage-like cells in Drosophila , called haemocytes , bind to dlg mutant tumours and contribute to cell death ( Parisi et al . , 2014 ) .", "This process is mediated by the release of Egr from haemocytes ( Cordero et al . , 2010; Parisi et al . , 2014 ) , which then activates the JNK pathway in target cells to promote apoptosis ( Igaki et al . , 2009 ) .", "Moreover , Toll pathway activation in tumour bearing animals also requires Egr ( Parisi et al . , 2014 ) .", "Accordingly , we found that defensin upregulation observed in dlg animals was lost in dlg;egr3 double mutants ( Figure 7D ) .", "We next tested whether PS exposure in tumours was dependent on Eiger , and observed an almost complete loss of cell-surface PS in dlg;egr3 tumours ( Figure 7E–G ) .", "Therefore , Egr is required for PS exposure in these tumours .", "We then analysed PS exposure in dlg mutant tumours , upon specific egr knockdown in haemocytes ( dlg;hml >egr-IR ) , and observed a strong decrease in cell surface-exposed PS compared to control tumours ( dlg;hml >ctrl-IR ) ( Figure 7H–J ) .", "This indicates that PS exposure in tumour cells is likely triggered by the release of Egr from haemocytes .", "Finally , to further test the hypothesis that Def requires PS to bind to tumours , we assessed the ability of overexpressed Def-HA to bind to dlg , egr3 mutant tumours .", "Consistently with the observed basal and overexpressed Def-HA expression pattern in wild type tissues ( Figure 4—figure supplement 1 ) , Def-HA was expressed in the trachea and fat body of dlg , egr3 mutant animals ( Figure 7—figure supplement 1A , B ) .", "However , we did not find any detectable association of Def-HA to the surface of dlg , egr3 tumours ( Figure 7—figure supplement 1C , C’ ) .", "Importantly , Egr-dependent signalling and PS exposure were intact in dlg;def and dlg;imd mutants ( Figure 7—figure supplement 2 ) indicating that both events precede the action of Defensin in dlg tumours .", "To further assess whether Egr was required for Defensin-induced tumour cell death , we injected synthetic Defensin peptide into control , dlg or dlg;egr3 mutant larvae .", "While , Defensin injection was able to robustly promote tumour cell death in dlg mutant tumours , it was unable to affect tumours derived from Egr-deficient animals , further demonstrating the requirement of Egr for tumour cell death induced by Defensin ( Figure 7K ) .", "Finally , we tested whether the antitumoral action of Def extended to other tumour models .", "We observed that tumours induced by the loss of scribble ( scrib ) , another member of the same apico-basal complex as dlg , showed significant cell surface PS exposure , which was Egr dependent ( Figure 7—figure supplement 3A , A’; compare to Figure 7—figure supplement 3B , B’ ) .", "Moreover , we detected Def enrichment in areas positive for Dcp1 staining on scrib tumours ( Figure 7—figure supplement 3C–C’’ and D–D’’ ) .", "Consistently , removing def from scrib mutant animals led to increase in tumour volume and decrease in tumour cell death ( Figure 7—figure supplement 3E–H’ ) .", "Altogether , these results show that the sensitivity to Def is not restricted to dlg mutant tumours but might rather be a general feature of neoplastic growth induced by loss of cell polarity ." ], [ "Using mutants as well as ubiquitous or targeted knockdowns , our study reveals that Defensin is non-redundantly required to drive tumour cell death and restrict tumour growth in neoplastic tumours generated by loss the apico-basal determinant Dlg and Scrib .", "This was reinforced by detection of Defensin on dying tumour cells as well as genetic rescue and injection experiments showing that Defensin can actively drive tumour cell death .", "Several in vitro studies in mammals have pointed to AMPs anti-tumoural potential ( Deslouches and Di , 2017 ) .", "Amongst these suggested anticancer peptides , Human β-Defensin-1 ( hBD1 ) appears downregulated in 82% of prostate cancer and 90% of renal clear cell carcinomas ( Donald et al . , 2003 ) .", "Furthermore , expression of hBD1 induces cell death in prostate and renal cancer cells in vitro ( Bullard et al . , 2008; Sun et al . , 2006 ) .", "Moreover , a recent report shows prevalent deletion of Human defensin gene cluster in some tumour types including prostate , lung and colorectal cancers , as well as a decrease overall survival in patients carrying these deletions ( Ye et al . , 2018 ) .", "However , since the classification of a molecule as AMP is based on structural protein features , it is important to mention that Human Defensins are not homologous of , but rather have structural resemblances , to Drosophila AMPs due to convergent evolution ( Shafee et al . , 2017 ) .", "Further studies are required to determine if anticancer activity displayed by Defensins from different species is linked to their structural properties .", "As upon infection ( Tzou et al . , 2000 ) , we show that the fat body and the trachea are the main sources of Defensin in tumour bearing larvae .", "Moreover , our results indicated that Defensin’s maximal expression and anti-tumour properties rely on Imd and Toll pathways activation .", "As expected from previous studies on anti-pathogenic immunity , Imd appears to play a critical role in the tracheal system , while both Imd and Toll drive Defensin expression in the fat body ( Hoffmann and Reichhart , 2002; Tzou et al . , 2002 ) .", "Consistently , overexpressing Defensin partially rescued the effect of Imd or Toll knockdown on dlg tumours .", "However , Toll and Imd pathways are major regulators of multiple AMPs in the Drosophila fat body .", "Then , it is conceivable that AMPs other than Defensin may possess similar anti-tumoural activity .", "In fact , a recent study in Drosophila shows that ectopic expression of several antimicrobial peptides , including Defensin , can increase cell death in a haematopoietic tumour model ( Araki et al . , 2019 ) .", "Authors of that study also reported activation of Toll and Imd pathways together with increased expression of several AMPs in those tumour bearing animals .", "Together with our study , this suggests potentially general anti-tumoural properties of Drosophila AMPs .", "Importantly , we show that Defensin targets tumour cells for apoptosis while sparing normal cells .", "As their human counterparts , we show that Drosophila tumours can expose PS independently of cell death ( Riedl et al . , 2011 ) .", "Interestingly , an in vitro study showed selective anticancer activity of some synthetic peptides derived from beetle Defensin , against cancer cells exposing PS ( Iwasaki et al . , 2009 ) .", "A similar targeting mechanism has also been proposed to explain temporin-1CEa or L-K6 anticancer activities towards melanoma cells and breast cancer cells respectively ( Wang et al . , 2016; Wang et al . , 2017 ) .", "PS is a mark of apoptotic cells , which is used as an ‘eat me’ signal by phagocytes ( Shklyar et al . , 2013; Tung et al . , 2013 ) .", "In Drosophila , the phagocytic receptor Simu together with Draper contribute to the elimination of apoptotic cells through recognition of PS ( Shklyar et al . , 2013; Tung et al . , 2013 ) .", "It would be interesting to test the role of these receptors in the control of tumour progression in Drosophila .", "Our results indicate that PS exposure precedes Defensin action and is not just an ‘eat-me’ signal but likely contributes to changing the membrane of tumour cells making them sensitive to the action of AMPs .", "It is tempting to speculate that the deleterious effect of AMPs observed upon ageing or neurodegeneration may involve a similar targeting mechanism of ‘foreign-looking’ or unfit cells ( Cao et al . , 2013; Kounatidis et al . , 2017; Lezi et al . , 2018 ) .", "Noteworthy , other negatively charged molecules enriched on tumour surface such as heparan sulfates and O-glycosylated mucins may also contribute to the targeting by AMPs ( Hollingsworth and Swanson , 2004; Knelson et al . , 2014 ) .", "TNF is a major player in the tumour microenvironment by exerting pleiotropic effects on both the tumour and its surroundings ( Ham et al . , 2016; Parameswaran and Patial , 2010 ) .", "In our dlg mutant model , we observed that defensin induction requires TNF , supporting previous observations that induction of innate immune response in tumour bearing animal relied on Egr produced by the tumour ( Parisi et al . , 2014 ) .", "Indeed , Toll activation in the fat body has been proposed to be a consequence of tumour-derived TNF , which drives haemocyte proliferation leading to an increase in Toll ligand Spatzle ( Parisi et al . , 2014 ) .", "Whether Imd pathway is also indirectly activated by the changes in immune cell activity in response to tumour or by alternative mechanisms remains an open question .", "Our results show that haemocyte-derived TNF is required for PS exposure by the tumour , a key process for tumour targeting by Defensin .", "Consistently , haemocyte-derived TNF is shown to drive cell death in dlg mutant tumours ( Parisi et al . , 2014 ) .", "The precise molecular mechanisms driving PS exposure downstream of TNF remain unknown .", "However , haemocyte-derived TNF , activates JNK pathway , which triggers many changes in tumour cells including apoptosis ( Igaki et al . , 2002; Moreno et al . , 2002 ) , ROS production ( Fogarty et al . , 2016; Santabárbara-Ruiz et al . , 2015 ) , loss of cell polarity ( Zhu et al . , 2010 ) , modification of extracellular matrix ( Uhlirova and Bohmann , 2006 ) , proliferation and cell migration ( Beaucher et al . , 2007; Igaki et al . , 2006; Pastor-Pareja et al . , 2004; Srivastava et al . , 2007 ) .", "Mild activation of JNK on its own is insufficient to drive PS exposure and tissue sensitivity to Defensin-induced cell death ( data not shown ) .", "While a JNK-independent role of Egr in PS exposure and sensitivity to the AMP cannot be ruled out , TNF might also sensitise cells through activation of the JNK pathway ( Cordero et al . , 2010 ) and PS exposure , thus providing a secondary sensitisation mechanism of tumour cells to the action of Defensin .", "Further studies are needed to explore a potential link between JNK activation and PS exposure .", "Another consequence of TNF-dependent JNK activation in tumours is the increased expression of matrix metalloproteases ( Mmps ) by tumour cells ( Uhlirova and Bohmann , 2006 ) .", "Importantly , we show that Mmp-1 induction and then JNK pathway activation are still present in tumour from larvae devoided of Defensin .", "This demonstrates that TNF signalling acts upstream of Defensin .", "Mmps degrade the basal membrane facilitating metastasis of primary tumour cells ( Beaucher et al . , 2007; Pastor-Pareja et al . , 2004; Srivastava et al . , 2007 ) .", "Interestingly , a study reported that human Mmp-7 can cleave immature Defensins , promoting their activation ( Wilson et al . , 1999; Wilson et al . , 2009 ) .", "Indeed , the pro-domain present in AMPs is thought to keep their in vivo activity silent , allowing local activation of AMP upon cleavage .", "While the mechanisms of Drosophila Defensin pro-domain cleavage remain unknown , it would be interesting to explore whether the changes in the tumour microenvironment could also affect Defensin activation .", "In conclusion , our study provides the first direct in vivo demonstration of the role of an endogenous AMP as an anti-cancer agent in Drosophila .", "Our data point to a conserved mechanism of tumour control by AMPs , a potent arm of the innate immune system .", "Importantly , we identify cellular features within tumours , which may be predictive of their sensitivity to be targeted by AMPs .", "This study provides a new paradigm to decipher the molecular mechanisms influencing anti-tumoural functions of an AMP , which may extend to other non-canonical roles of AMPs , such as in ageing , long-term memory and wound healing .", "Moreover , together with the new genetic tools allowing targeting of all Drosophila AMPs ( Hanson et al . , 2019 ) , our study establishes new bases to explore in vivo a potential important natural mechanism of defence against tumours ." ], [ "Flies were reared at 25°C under 12 hr/12 hr light/dark cycles on standard oatmeal and molasses medium ( Fernandez-Ayala et al . , 2009 ) .", "Details on fly strains used in this study are presented in the key resources table and detailed genotypes are included in corresponding figure legends .", "For assessment of tumour volume and tumour cell death , flies were transferred to medium embryo collection cages and allowed to lay eggs for 8 hr at room temperature ( RT ) on agar/grape juice plates ( 2 . 1% agar , 25% grape juice , 1 . 25% sucrose , 0 . 2% methyl 4-hydroxybenzoate ) supplemented with yeast paste .", "Plates were kept at 25°C until hatching .", "200 larvae hatched within a 4 hr time window were collected and transferred to rearing bottles containing 45 mL of fly food to avoid any developmental delay and/or starvation effects due to overcrowding conditions .", "The tumour phenotype was analysed close to the normal developmental time , i . e . 7 days after hatching .", "Tumours were dissected and stained for further analysis ( see below ) .", "defsk3 and defsk4 mutants were generated using CRISPR/cas9 technology ( Hanson et al . , 2019 ) .", "Mutants were isogenised through backcrosses with a w1118 iso line as previously described in Ferreira et al . ( 2014 ) .", "Survival of defSK3 animals was compared to wild type controls ( isogenised background , iso ) .", "Male flies ( 5–7 days old ) were pricked in the thorax with a needle dipped in a concentrated pellet of fresh overnight bacterial culture .", "Infected flies were kept at 29°C .", "Listeria innocua was cultured in Brain heart infusion medium at 37°C and used at optical density of 200 nm ( OD600 ) .", "Erwinia carotovora carotovora 15 was cultured in Luria-Bertani broth at 29°C and used at OD600 200 .", "Experiments were repeated three times independently and one representative experiment is shown .", "Tissues were dissected in Phosphate Buffer Saline ( PBS ) and fixed for 20 min in 4% formaldehyde .", "Fat body samples were fixed for 30 min using the same protocol .", "Tissues were washed three times in PBS containing 1% of Triton-X100 ( PBT ) and incubated overnight at 4°C with primary antibodies .", "Tissues were washed five times in PBT and incubated for 2 hr at RT with secondary antibodies and 4’ , 6-Diamidine-2′-phenylindole dihydrochloride ( DAPI ) .", "After three washes in PBT , tissues were mounted in Vectashield using Secured-Seal spacers ( Thermo Fisher Scientific ) .", "Confocal images were captured using a Zeiss 710 or Zeiss 880 with Airyscan confocal microscope and processed with Fiji 2 . 0 . 0 or Adobe Photoshop C . S5 . 1 .", "For analysing tumour phenotype , images were acquired using optimal slice parameter and 12bits using a Zeiss 710 confocal microscope .", "Quantifications were made as previously described ( Parisi et al . , 2014 ) .", "Briefly , we used Volocity 3D imaging analysis software to quantify the total volume of tumour identified by DAPI staining and cell death visualised with anti-Dcp1 staining .", "When haltere or leg discs tumours were still associated with the wing disc , they were not considered for quantification .", "Quantifications were done in at least three independent biological replicates for each genetic setting .", "Single representative experiments are presented except for dlg;def double mutant rescue experiments ( Figure 2K–L ) for which replicates are pooled due to the few larvae available .", "In all cases the difference between control and mutant tumour were strongly reproducible between replicates .", "To quantify Def/Dcp-1 and Def/Annexin V colocalisation , we determined the intensity of Def staining in areas of positive versus negative staining for the second marker using the ImageJ macro , BatchQuantify ( Johansson et al . , 2019 ) .", "To rule out any effect of infection on the tumour phenotypes , Drosophila larvae were reared on standard food supplemented with penicillin and streptomycin when indicated in the figure .", "Total RNA from 7 to 10 whole larvae or tissues collected from 10 to 20 larvae ( 10 larvae for fat body , 20 larvae for trachea ) was extracted using TRIzol according to the manufacturer’s instructions .", "RNA was treated with Turbo DNA free Kit and RNA concentration and quality were monitored using a Nanodrop ( Thermo Fisher Scientific ) .", "The same amount of total RNA ( 1 μg for whole larvae and fat body or 100 ng for trachea ) was used to perform the three independent reverse-transcriptions using the High-Capacity cDNA Reverse Transcription Kit .", "cDNAs were pooled and qPCR was performed using PerfeCTa SYBR green following the manufacturer’s instructions .", "cDNA amplification was monitored with Applied Biosystems 7500 fast instruments .", "Serial 10-fold dilutions of an external standard were used to produce a standard curve , and RNA samples were included to control for the absence of DNA contamination .", "rpl32 expression was used to normalise expression levels of target genes .", "Data was analysed using the Ct method ( 2-∆∆ct ) .", "All qPCR experiments were carried out on three to seven independent biological replicates ( see figure legends for details ) and , larvae were sampled from the same bottle used to analyse the tumour phenotype .", "GraphPad Prism7 software was used for graphical representation and statistical analysis .", "Primer targets and sequences are presented in key resources table .", "To verify the sequence of defsk3 and defsk4 mutants , genomic DNA was extracted from single flies by grinding whole animals with a 200 μL pipette tip containing 50 μL of squishing buffer ( 10 mM Tris-HCl pH 8 . 2 , 1 mM EDTA , 25 mM NaCl , 200 μg proteinase K ) followed by incubation at 37°C for 30 min .", "Proteinase K was heat-inactivated at 95°C for 2 min . 1 μL of genomic DNA was used for PCR amplification using PfuUltra II Fusion DNA Polymerase and Eppendorf Mastercycler Ep Gradient Thermal Cycler .", "Tumours were dissected in PBS and incubated in Annexin V binding buffer containing 5% of Alexa Fluor ( 488 nm or 568 nm ) conjugated Annexin V for 10 min .", "Tissues were washed quickly in Annexin V binding buffer and fixed in 4% formaldehyde for 20 min .", "Immunostaining , mounting and imaging were then carried out as described above .", "We used Fiji 2 . 0 . 0 software to quantify Annexin V staining at the tumour surface .", "Maximal Z-projection of whole tumours were generated and the Colour Threshold tool was then used to detect the red pixels that represented Annexin V staining at the surface of tumours .", "The hue slider was set to include only the red signal , and the brightness slider was adjusted to exclude any background signal .", "This area was then selected and measured in pixels .", "The mature Defensin peptide was synthesised ( over 90% purity ) and the purity controlled by Genepep using Ultra Performance Liquid Chromatography/Mass Spectrometry .", "We subsequently compared the integrity of synthetic Defensin with authentic Defensin purified from Drosophila hemolymph using two complementary approaches , namely Matrix-Assisted Laser Desorption/Ionisation-Mass Spectrometry ( MALDI-MS ) for molecular mass determination and trypsin digestion followed by nanoLiquid Chromatography/Electrospray Ionisation tandem Mass Spectroscopy ( nanoLC/ESI MS/MS ) for molecular integrity confirmation .", "Those analyses confirmed the similar mass between natural and synthetic Defensin ( respectively provided by Bulet EIRL and Genepep ) as well the presence of disulphide bonds within synthetic Def , demonstrating it is the mature peptide ( Data not shown ) .", "For injection experiments , larvae were collected , washed three times in cold sterile PBS and injected on a fly pad upon CO2 anaesthesia using Nanoject II ( Drummond ) and glass capillaries 3 . 5 inch ( Drummond ) .", "Larvae were injected with 69 nL of PBS containing 7 . 5 nmol of synthetic Defensin or 69 nL of PBS only as control .", "Larvae were then gently transferred into agar/grape juice plates and kept at 25°C .", "Tumours were dissected 4 hr after injection and stained for visualisation of nuclei and tumour cell death as describe above .", "All data are presented as mean ± SD and n values are indicated in the figure legends .", "Statistical analyses were carried out using GraphPad Prism7 software and only significant differences are indicated in dot-plots .", "Survival upon infection was analysed using Log-rank test .", "qPCR data were analysed using unpaired t-test with a two-tailed p value .", "Tumour volume and tumour cell death comparing two samples were analysed using Mann-Whitney test with a two-tailed p value and the one comparing three genotypes were analysed using One-way ANOVA followed by Turkey’s multiple comparisons test .", "Tumour volume from injected larvae was analysed using Two-way ANOVA followed by Turkey’s multiple comparisons test and only significant statistical differences between ctrl and Defensin injected larvae for each genotype tested are indicated .", "All raw data is available through http://dx . doi . org/10 . 5525/gla . researchdata . 834 ." ] ]

[ "Antimicrobial peptides ( AMPs ) are small cationic molecules best known as mediators of the innate defence against microbial infection .", "While in vitro and ex vivo evidence suggest AMPs’ capacity to kill cancer cells , in vivo demonstration of an anti-tumour role of endogenous AMPs is lacking .", "Using a Drosophila model of tumourigenesis , we demonstrate a role for the AMP Defensin in the control of tumour progression .", "Our results reveal that Tumour Necrosis Factor mediates exposure of phosphatidylserine ( PS ) , which makes tumour cells selectively sensitive to the action of Defensin remotely secreted from tracheal and fat tissues .", "Defensin binds tumour cells in PS-enriched areas , provoking cell death and tumour regression .", "Altogether , our results provide the first in vivo demonstration for a role of an endogenous AMP as an anti-cancer agent , as well as a mechanism that explains tumour cell sensitivity to the action of AMPs ." ]

[ "Animals have a natural defence system – the immune system – that is needed to fight off disease-causing microbes , known as pathogens .", "One way the immune system attacks pathogens is by producing small microbe-killing molecules called antimicrobial peptides .", "These antimicrobial peptides carry a positive charge , which allows them to interact with and disrupt the negatively charged cell surfaces of many microbes .", "Healthy animal cells do not have these negatively charged components on their cell surface , which means they are invisible to antimicrobial peptides .", "Studies have reported that antimicrobial peptides can attack cancer cells grown in a dish .", "However , it was unclear whether they could fight cancer cells in a live animal .", "Parvy et al . have now addressed this issue by studying tumours in the larvae of fruit flies .", "Flies with tumours made an antimicrobial peptide called Defensin , which normally helps to fight infections .", "When Parvy et al . deleted the gene coding for Defensin , less tumour cells were dying and the tumours became bigger .", "This result indicated that Defensin was protecting the fruit flies from tumours .", "Examining the tumours under the microscope showed that Defensin protein interacted with the membranes of tumour cells .", "Defensin was not , however , interacting with healthy cells .", "Further analysis revealed that a negatively charged component of cell membranes called phosphatidylserine , which normally faces the inside of healthy cells , is exposed to the outer surface of tumour cells .", "This negatively charged molecule renders cancer cells visible to positively charged Defensin .", "Importantly , the exposure of the phosphatidylserine is mediated by the fly equivalent of a protein called Tumour Necrosis Factor , a key player in cancer biology .", "Defensin binding to tumour cells leads to their death .", "These experiments in the fruit fly highlight key molecular mechanisms that allow antimicrobial peptides to fight cancer cells in a living organism .", "Because human tumour cells can also expose phosphatidylserine , these latest findings may open up the possibility of a new kind of anti-cancer therapy for patients ." ]

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

[ "neuroscience" ]

[ [ "New memories do not form instantly at the moment of an experience , but rather undergo a stabilization period during which they are gradually consolidated into a stable , long-term memory ( Asok et al . , 2019 ) .", "Later , recall may cause the memory to return to an unstable state ( i . e . destabilized ) where it can be modified .", "Importantly , the memory must undergo a re-stabilization process called reconsolidation in order to persist ( Haubrich and Nader , 2016 ) .", "This process of destabilization-reconsolidation allows memories to adaptively change .", "Importantly , manipulations targeting the reconsolidation process can permanently alter a memory trace , by enhancing , impairing or modifying it , offering great treatment potential to clinicians ( Beckers and Kindt , 2017; Nader et al . , 2013; Phelps and Hofmann , 2019; Soeter and Kindt , 2011 ) .", "For instance , one can induce the destabilization of a maladaptive memory and then block reconsolidation pharmacologically– preventing the memory from returning to a stable state and leading to memory impairment ( Nader et al . , 2000; Suzuki et al . , 2004 ) .", "Memory content can also be updated during reconsolidation , allowing it to be modified to a less aversive form ( Haubrich et al . , 2015; Lee et al . , 2017; Monfils et al . , 2009; Popik et al . , 2020; Schiller et al . , 2010 ) .", "However , extreme fear learning can result in pathological memories that are resistant to change through reconsolidation ( Suzuki et al . , 2004; Wang et al . , 2009 ) , making them difficult to treat ( Zhang et al . , 2018 ) .", "In order , for reconsolidation interventions to work , memory destabilization must be triggered first .", "However , retrieval does not always induce destabilization .", "In rodent experiments , this is particularly the case in memories generated by high intensity fear conditioning or protocols that induce asymptotic levels of learning ( García-DeLaTorre et al . , 2009; Gazarini et al . , 2015; Lee , 2010; Morris et al . , 2006; Winters et al . , 2009 ) .", "For instance , we ( Finnie and Nader , 2020; Wang et al . , 2009 ) and others ( Holehonnur et al . , 2016 ) have found that while auditory fear memories created with one tone-shock pairing ( 1P ) do undergo destabilization and can be attenuated by reconsolidation blockade , a stronger , 10 tone-shock pairings ( 10P ) protocol creates an intense fear memory that is resistant to this treatment ( Figure 1 ) .", "Thus , the strength of the memory serves as a boundary condition to determine whether memory destabilization will occur after retrieval or not ( Zhang et al . , 2018 ) .", "This boundary condition is thought to be mediated in part by mechanisms that initialize destabilization .", "A well described mechanism for destabilization is GluN2B-containing NMDA receptor activation ( Ben Mamou et al . , 2006 ) .", "GluN2B-NMDA receptors are reduced in reconsolidation-resistant memories ( Wang et al . , 2009 ) and blocking these receptors prevents destabilization ( Milton et al . , 2013 ) .", "Another important mechanism involves the expression of GluA2-containing AMPA receptors , which are important for LTP maintenance ( Henley and Wilkinson , 2016 ) and have been linked with memory strength and stability ( Migues et al . , 2014; Migues et al . , 2010 ) .", "In order for destabilization to occur , a transient reduction of GluA2-AMPA receptor synaptic expression is necessary ( Clem and Huganir , 2010; Ferrara et al . , 2019; Hong et al . , 2013; Rao-Ruiz et al . , 2011 ) .", "The fact that strong training leads to memories that do not reconsolidate likely reflects changes triggered at the time of memory encoding .", "These changes likely determine the future plasticity of the memory by altering mechanisms needed for destabilization , such as GluN2B-NMDA and GluA2-AMPA receptors expression .", "However , the specific links between memory encoding and reconsolidation have yet to be determined .", "It is well-established that emotionally arousing experiences cause increased release of noradrenaline in the amygdala ( Quirarte et al . , 1998 ) that modulates memory formation through the activation of β-adrenergic receptors ( McGaugh , 2000 ) .", "The locus coeruleus is the main source of noradrenergic projections in the brain ( Giustino and Maren , 2018 ) , and although it acts on many targets , its influence in the amygdala is critical to mediate the impact of stress on memory processes ( Giustino and Maren , 2018; Johansen et al . , 2011; Kaouane et al . , 2012 ) .", "For instance , overactivation of projections coming from the locus coeruleus ( LC ) to the amygdala have been implicated in encoding traumatic memories ( Hurlemann , 2008 ) and these projections promote fear learning ( Uematsu et al . , 2017 ) .", "This evidence suggests that the noradrenaline-locus coeruleus system ( NOR-LC ) likely contributes to the formation of maladaptive , reconsolidation-resistant fear memories .", "If so , NOR-LC signaling may trigger changes that determine future plasticity and destabilization , such as altered GluN2B and GluA2 expression .", "Here , we compared how animals fear conditioned with a 1P or a 10P training protocol differ regarding plasticity mechanisms in the amygdala and the induction of destabilization .", "Using pharmacologic and chemogenetic interventions , we also tested the hypothesis that the overactivation of the NOR-LC system during severe fear encoding underlies the formation of reconsolidation-resistant memories with limited plasticity ." ], [ "To study the difference between mild and strong fear memories , our first aim was to assess how they differ at the behavioral level .", "Animals were trained in two different auditory fear conditioning tasks: to create a mild fear memory , we trained rats with one tone-shock pairing ( 1P ) , whereas strong fear memories were created using 10 tone-shock pairings ( 10P ) .", "The next day , fear memory was assessed in a test session where one unreinforced tone was presented ( Figure 2A ) .", "Animals trained with 10 shocks displayed higher freezing to the tone than rats trained with one shock ( Independent samples t-test t ( 28 ) = 2 . 308 , p=0 . 028 ) .", "The difference in freezing during the test did not seem large due to a ceiling effect in the 10P group .", "To better visualize the differences in memory strength , fear-conditioned rats underwent extinction with 20 unpaired tone presentations and were tested for extinction retention in the next day ( Figure 2B ) .", "A mixed ANOVA with training protocol as a between-subjects variable and bin as a within-subjects variable revealed a significant main effect between groups , with rats in the 10P group freezing more during the entire extinction session and at test ( F1 , 13 = 58 . 18 , p<0 . 001 ) .", "Tukey’s post hoc test revealed that only animals trained with 1P displayed extinction acquisition , with significant fear suppression within the extinction session ( 1-to-5 tone vs 16-to-20 tone: 1P group , t ( 52 ) = 3 . 65 , p=0 . 02; 10P group , t ( 52 ) = 2 . 43 , p>0 . 05 ) .", "Also , extinction retention 24 hr later was observed only in the 1P group ( 1-to-5 tone vs Test: 1P group , t ( 52 ) = 3 . 45 , p=0 . 03; 10P group , t ( 52 ) = 0 . 85 , p>0 . 05 ) .", "Therefore , in contrast with 1P , fear memories created with 10P exhibit impaired extinction learning , indicating a considerable difference in memory strength .", "Next we assessed reconsolidation in 1P and 10P memories as previously described by Wang et al . , 2009 .", "One day after 1P or 10P training , a 1-tone test was conducted to reactivate the fear memory .", "The protein synthesis inhibitor anisomycin ( 125 µg/µl; 0 . 5 µl per hemisphere ) was infused in the basolateral amygdala ( BLA ) immediately after to block reconsolidation .", "The effectiveness of the treatment was then evaluated in a test 1 day later .", "A mixed ANOVA with training and drug as between-subjects variables and session as a within-subjects variable indicated that there was a significant interaction between training , drug , and session ( F1 , 35 = 18 . 27 , p<0 . 001 ) .", "At test , post-reactivation anisomycin impaired performance in animals trained with one shock ( Tukey’s post hoc test , t ( 55 ) = 5 . 59 , p<0 . 001 ) but had no effect in strongly trained rats ( Tukey’s post hoc test , t ( 55 ) = −0 . 26 , p>0 . 05 ) .", "This shows that retrieval rendered the 1P memory labile , necessitating reconsolidation shortly afterwards .", "On the other hand , retrieval did not render the 10P memory vulnerable to anisomycin , and hence it can be considered a reconsolidation-resistant memory .", "We evaluated the expression of molecules implicated with synaptic plasticity , GluN2B ( Zhang et al . , 2018 ) and GluA2 ( Anggono and Huganir , 2012 ) , between animals trained in the 1P and 10P protocols .", "Animals were fear conditioned in the 1P or 10P protocol , tested the next day , and their brains collected 1 hr or 24 hr later for western blot analysis of BLA tissue .", "Controls were kept in their home cages during the entire behavioral procedure ( Home cage controls , HC ) .", "This control , although not addressing the role of shock or context alone , informs the baseline levels of the targeted proteins when no learning occurs .", "A one-way ANOVA indicated significant group differences in GluN2B expression at the BLA postsynaptic density ( PSD ) ( Figure 3A , left; F2 , 11 = 7 . 34 , p=0 . 009 ) .", "The 1P group displayed an upregulation of GluN2B ( Tukey’s post hoc test , HC vs 1P: t ( 11 ) = −2 . 99 , p=0 . 031 ) , indicating that the formation of a reconsolidation-permissive memory coincides with an increase in this receptor critical for reconsolidation induction .", "However , 10P trained rats displayed GluN2B equivalent to HC levels ( Tukey’s post hoc test , t ( 11 ) = −0 . 09 , p>0 . 05 ) .", "This shows that unlike 1P memories that do reconsolidate , strong reconsolidation-resistant memories are formed without GluN2B upregulation .", "Next , we looked at how GluA2-containing AMPARs in the BLA PSD vary between 1P reconsolidation-permissive and 10P reconsolidation-resistant memories ( Figure 3A , right ) .", "There was a significant difference between groups ( One-way ANOVA , F2 , 19 = 17 . 98 , p<0 . 001 ) .", "In comparison with HC animals , GluA2 levels of animals trained with 1P were upregulated one day after testing ( Tukey’s post hoc test , t ( 19 ) = −2 . 87 , p=0 . 025 ) , but an even higher increase was found in rats trained with 10P ( Tukey’s post hoc test , HC vs 10P: t ( 19 ) = −5 . 97 , p<0 . 001; 1P vs 10P: t ( 19 ) = −3 . 81 , p=0 . 003 ) .", "Hence , GluA2 levels increase as a function of training strength , and the high levels found in the strong training group might reflect a resistance to memory destabilization .", "Lastly , we investigated GluA2 trafficking by comparing GluA2 levels 1 hr or 24 hr after recall at both the PSD and at extrasynaptic fractions of the BLA .", "These time-points fall inside ( 1 hr ) and outside ( 24 hr ) the reconsolidation window , allowing us to capture GluA2 transient downregulation following destabilization and its levels after it has been normalized after reconsolidation ( Ferrara et al . , 2019; Rao-Ruiz et al . , 2011 ) .", "One-way ANOVA revealed a significant difference between groups ( Figure 3B , left; F2 , 26 = 7 . 38 , p=0 . 003 ) .", "Tukey’s post hoc test indicated that in rats trained with 1P , synaptic GluA2 levels were increased 24 hr after recall but were downregulated to HC levels shortly after recall ( HC vs 1 hr: t ( 24 ) = 0 . 002 , p>0 . 05; HC vs 24 hr: t ( 24 ) = 2 . 97 , p=0 . 018; 1 hr vs 24 hr: t ( 24 ) = 3 . 45 , p=0 . 006 ) .", "GluA2 levels also differed between groups at the extrasynaptic fractions ( Figure 3B , right; One-way ANOVA , F2 , 17 = 5 . 74 , p=0 . 014 ) , with higher GluA2 expression 1 hr after recall compared to HC animals ( Tukey’s post hoc test , HC vs 1 hr: t ( 15 ) = −2 . 85 , p=0 . 031; HC vs 24 hr , t ( 15 ) = −0 . 38 , p>0 . 05; 1 hr vs 24 hr , t ( 15 ) = 2 . 97 , p=0 . 024 ) .", "This indicates that shortly after recall GluA2 is transiently removed from the synapse , rendering the 1P memory labile .", "This pattern of GluA2 trafficking , however , was not observed in 10P reconsolidation-resistant animals .", "GluA2 levels were unchanged by recall at both the synaptic ( Figure 3C , left; One-way ANOVA , F2 , 21 = 5 . 46 , p=0 . 012; Tukey’s post hoc test , HC vs 1 hr: t ( 21 ) = −2 . 95 , p=0 . 02; HC vs 24 hr: t ( 21 ) = −2 . 96; p=0 . 02 , 1 hr vs 24 hr: t ( 21 ) = −0 . 01 , p>0 . 05 ) , and extrasynaptic fractions ( Figure 3C , right; One-way ANOVA , F2 , 12 = 0 . 024 , p>0 . 05 ) .", "This shows that strong memories that fails to become labile upon recall display increased GluA2 synaptic expression .", "Overall , this set of data shows that , unlike reconsolidation-permissive 1P memories , memories created by 10P are reconsolidation-resistant with have attenuated plasticity , explaining why reconsolidation-blockade is ineffective in strong memories .", "Here , we investigated whether β-adrenergic receptor activation during strong training plays a role in the expression of pro-plasticity molecules .", "After showing that strong training results in memories with decreased GluN2B and increased GluA2 postsynaptic expression in the BLA , we investigated if the β-adrenergic inhibitor propranolol would affect such outcomes .", "Animals were injected with either propranolol ( 10 mg/mL/kg , i . p . ) or vehicle and 20 min later were trained in the 10P fear conditioning protocol .", "Rats were tested one day later and sacrificed 24 hr post-test when their brains were collected for western blot analysis .", "In comparison with the vehicle groups , propranolol treatment increased GluN2B ( Figure 4B , left; Independent samples t-test t ( 17 ) = 2 . 47 , p=0 . 025 ) and decreased GluA2 ( Figure 4A , right; Independent samples t-test t ( 9 ) = 2 . 41 , p=0 . 039 ) in the BLA .", "Therefore , blocking β-adrenergic signaling during 10P training modulates GluN2B and GluA2 toward the levels found in 1P memories that do reconsolidate .", "Importantly , no direct comparison with the 1P and HC groups were made .", "Thus , in respect to 1P memories and HC levels , the extent to which propranolol before strong training modulates GluN2B and GluA2 remains to be established .", "Next , we assessed if blocking β-adrenergic signaling during strong training would affect a memory’s susceptibility to undergo reconsolidation later on .", "Again , animals were injected with propranolol or vehicle and trained in a 10P fear conditioning protocol .", "Afterwards , reconsolidation blockade was conducted , and its effectiveness evaluated as described above .", "A three-way ANOVA indicated a significant interaction between treatment 1 , treatment two and session ( Figure 4B; F1 , 54 = 4 . 14 , p=0 . 047 ) .", "During reactivation , animals that received vehicle and propranolol displayed the same freezing levels , indicating that propranolol did not impair strong fear learning ( Tukey’s post hoc test , t ( 54 ) = −0 . 57 , p>0 . 05 ) .", "In vehicle-injected animals , post-reactivation anisomycin infusion had no effect on fear expression at the test ( Tukey’s post hoc test , veh + veh vs veh + anisomycin: t ( 54 ) = 0 . 98 , p>0 . 05 ) .", "Thus , the strong fear memory was reconsolidation-resistant .", "Pre-training propranolol treatment , however , rendered animals susceptible to post-reactivation anisomycin , resulting in amnesia at test ( Tukey’s post hoc test , propranolol + veh vs propranolol + anisomycin: t ( 54 ) = 4 . 56 , p<0 . 001 ) .", "These data reveal that reconsolidation-resistance induced by 10P training requires the activation of β-adrenergic receptors during initial learning .", "We hypothesized that the LC-to-BLA projection could mediate the formation of reconsolidation-resistant memories in 10P training .", "First , we quantified the expression of the activity-regulated gene c-fos in the LC 90 min after 1P and 10P training ( Figure 5A ) .", "We observed that 10P training resulted in higher c-fos activity ( Figure 5A; Independent samples t-test t ( 4 ) = 4 . 35 , p=0 . 01 ) , indicating that the LC is overly engaged during the formation of strong fear memories .", "Next , we used a chemogenetic approach to silence LC to BLA projections during strong training ( Figure 5B–C ) .", "Virus ( pAAV-hSYN-DIO-hM4Di-mCHerry ) expressing the inhibitory DREADD receptor hM4Di was infused in the LC .", "Controls were infused with viruses not expressing hM4Di ( pAAV-hSYN-tdTomato ) .", "After 3 months , there was robust somatic expression in the LC and terminal expression in the BLA ( Figure 5C ) .", "To silence LC inputs to the BLA during strong fear learning , we infused the DREADD agonist C21 in the BLA 5 min before 10P training .", "One day later , rats were infused in the BLA with anisomycin or vehicle after retrieval to determine if reconsolidation could occur ( Figure 5D ) .", "There was a significant interaction between DREADD , drug , and session ( Three-way ANOVA , F1 , 54 = 8 . 26 , p=0 . 006 ) .", "During reactivation , all groups exhibited similar freezing behavior indicating that silencing LC-BLA projections did not impair strong fear learning ( Tukey’s post hoc test , tdTomato vs hM4Di: t ( 54 ) = 1 . 05 , p>0 . 05 ) .", "Control animals infused with anisomycin did not show a memory impairment on a subsequent test ( Tukey’s post hoc test , tdTomato + veh vs tdTomato + anisomycin: t ( 54 ) = 1 . 68 , p>0 . 05 ) , indicating that reconsolidation did not occur in these animals .", "When the LC to BLA projections were silenced during strong memory formation , however , recall did enable reconsolidation since anisomycin caused amnesia in these animals ( Tukey’s post hoc test , hM4Di + veh vs hM4Di + anisomycin: t ( 54 ) = 5 . 64 , p<0 . 001 ) .", "These results show that inputs from the LC to the BLA during fear learning promote memory formation into a fixed state that is resistant to change .", "If these projections are silenced , even the memory of a strong fear experience can become labile upon recall and be modulated through reconsolidation ." ], [ "Previous studies revealed that unlike mild fear memories , very strong ones do not destabilize upon recall ( Holehonnur et al . , 2016; Wang et al . , 2009 ) .", "Some of the mechanisms required to induce destabilization have been identified ( Haubrich and Nader , 2016; Zhang et al . , 2018 ) , but not much is known regarding how severe fear experiences end up forming reconsolidation-resistant memories .", "In the current study we investigated the hypothesis that strong memories are different than mild ones due to the action of the NOR-LC system on encoding .", "Our results show that strong fear conditioning involves elevated β-adrenergic receptor activation which modulates encoding to produce memories that do not destabilize .", "At the systems level , reconsolidation-resistant memory formation entails inputs from the LC to the BLA during strong fear learning , and if these projections are silenced , even the memory of a strong fear experience can later undergo destabilization and reconsolidation .", "This work builds on previous studies showing that the induction of reconsolidation involves the presence of specific cellular mechanisms in the amygdala , such as the activation of GluN2B-containing NMDA receptors ( Wang et al . , 2009 ) and transient reduction of GluA2-containing AMPA receptors upon retrieval ( Rao-Ruiz et al . , 2011 ) .", "Here , we first replicated the seminal findings of Wang et al . , 2009 by showing that , while memories created with one tone-shock pairing destabilize upon recall , memories created with 10 pairings do not .", "Also , memories created with 10 pairings are much more robust and display impaired extinction learning , a hallmark of posttraumatic stress disorder ( Pitman et al . , 2012 ) .", "When looking at GluN2B expression , we confirmed previous reports showing that strongly-trained animals display lower GluN2B levels than mildly trained ones ( Holehonnur et al . , 2016; Wang et al . , 2009 ) .", "However , here we included a home cage control group in our analysis which revealed that GluN2B is not actually downregulated by strong training .", "In fact , GluN2B is normally upregulated by fear learning , which is hampered when the experience is very intense .", "We also examined GluA2 expression , which is central for synaptic stability and must be endocytosed after recall for memory destabilization ( Hong et al . , 2013; Rao-Ruiz et al . , 2011 ) .", "By comparing GluA2 levels of trained animals with home cages , we found a moderate increase in rats trained with one pairing and a significantly higher increase in rats trained with 10 pairings , indicating increased stability of strong memories .", "Retrieval transiently reduced synaptic GluA2 to home cage levels only in the mild memory , further indicating that the strong memory was overly stable and unable to destabilize .", "Overall , these data show that strong memories are stored in a state with different metaplastic properties that makes them resistant to reconsolidation .", "Most studies concerning the boundary conditions of reconsolidation focus on the mechanisms implicated at the time of retrieval , while those at initial encoding have not received much attention .", "Here we show that conditions during encoding are crucial to defining the memory’s future plasticity .", "Emotionally arousing experiences cause increased release of noradrenaline from the locus coeruleus to the amygdala ( Quirarte et al . , 1998 ) , which increases its activation ( Rodríguez-Ortega et al . , 2017 ) and promotes fear learning ( Gazarini et al . , 2013; Uematsu et al . , 2017 ) .", "Moreover , the overactivation of this system has been implicated with traumatic memory formation in humans ( Hendrickson and Raskind , 2016; Rombold et al . , 2016 ) .", "To investigate the role of noradrenaline signaling on the formation of reconsolidation resistant memories , we used pharmacologic and chemogenetic approaches .", "First , we pharmacologically blocked β-adrenergic receptors during strong fear learning and re-assessed plasticity mechanism in the BLA and reconsolidation induction .", "Propranolol before strong training modulated GluN2B and GluA2 expression towards levels seen in weakly trained animals— increasing GluN2B and decreasing GluA2 synaptic expression .", "Likewise , these strong memories formed under β-adrenergic receptors blockade can be destabilized following retrieval .", "Next we investigated the role of projections from the LC to BLA during strong fear memory formation .", "To this end , LC neurons were infected with the inhibitory DREADD receptor HM4Di and its projections to the BLA were silenced during strong training with the local infusion of the DREADD agonist C21 .", "When these specific LC to BLA projections were silenced during strong training , recall was successful in triggering destabilization .", "As a result , infusion of a protein synthesis inhibitor blocked reconsolidation , leading to amnesia .", "Importantly , both pharmacologic and chemogenetic manipulations of noradrenaline signalling did not alter rats’ freezing compared to control animals .", "This suggests that noradrenaline blockade did not reduce memory strength per se .", "Overall , this reveals that the activation of LC-BLA pathway during severe fear learning is necessary for memories to encode in a fixed state and do not destabilize in the future ( Figure 6 ) .", "If this pathway is silenced , however , even the memory of a very aversive event displays plasticity and can undergo destabilization upon recall .", "Reconsolidation is a critical biological feature to maintain memory’s relevance to guide future behavior ( Lee , 2009; Lee et al . , 2017 ) .", "Further , reconsolidation-targeted treatments provide a unique opportunity to weaken pathological fear memories ( Monfils and Holmes , 2018; Phelps and Hofmann , 2019 ) .", "Nonetheless , the intensity of the experience is an important parameter which can modulate whether reconsolidation can happen in the future , curbing the efficacy of some treatments .", "Thus , in order to better understand and treat disorders implicated in severe fear , it is crucial to elucidate how mild and strong memories differ .", "The current work underscores the widely accepted— but understudied— possibility that mild and strong fear memories are neurobiologically distinct .", "We have described how the NOR-LC system shapes memory encoding towards a maladaptive state that is resistant to change .", "This is achieved by triggering molecular modifications that increase memory stability at the expense of plasticity .", "The identification of this mechanism advances our understanding of the boundary conditions to reconsolidation .", "Moreover , this knowledge will help guide future research to understand why strong memories are so implacable , and how resistance to treatment can be overcome ." ], [ "Male Sprague Dawley rats ( 275–300 g at arrival; Charles River , Quebec , Canada ) were housed in pairs in plastic cages in a temperature-controlled environment ( 21–23°C ) with ad libitum access to food and water and maintained on a 12 hr light/dark cycle ( lights on at 7:00 A . M . ) .", "All experiments were conducted during the light phase .", "In all experiments animals were randomly assigned to each behavioral condition .", "Sample size estimates were determined based on effect sizes observed in previous reports using similar assays ( Hardt et al . , 2010; Holehonnur et al . , 2016; Wang et al . , 2009 ) resulting in statistical power estimates between 70% and 90% .", "Each rat was handled for at least 4 days before the behavioral procedures .", "All procedures were approved by McGill's Animal Care Committee ( Animal Use Protocol #2000–4512 ) and complied with the Canadian Council on Animal Care guidelines .", "Animals were anesthetized with a mixture of ketamine ( 50 mg/ml ) , xylazine ( 3 mg/ml ) , and Dexdomitor ( 0 . 175 mg/ml ) injected intraperitoneally .", "Analgesic treatment was administered subcutaneously before surgery ( carprofen; 5 mg/ml ) .", "Stainless-steel 22-gauge cannulae were bilaterally implanted in the basolateral amygdala ( AP , −3 . 0 from Bregma; ML , + / − 5 . 1 from the midline; DV , −8 . 0 from the skull surface ) .", "The cannulae were kept in place by dental cement tightly fixed to the skull with three stainless-steel screws .", "Obturators were then inserted into the cannulae to prevent blockage .", "An intraperitoneal injection of Antisedan ( 0 . 5 mg/ml ) was administered after surgery to reverse the anesthesia , and the animals were placed in individual cages on heating pads until they woke up .", "The rats were then monitored daily during a 1 week recovery period , before the beginning of the behavioral procedures .", "For viral injections in the LC , animals were anesthetized as above and a 33-gauge stainless steel injector was inserted into the brain ( coordinates , from bregma: A/P −9 . 8 mm , L ± 1 . 3 mm , D/V −7 . 5 mm from the skull surface ) .", "The injector was attached by polyethylene tubing ( Intramedic #427406 ) to 10 uL Hamilton syringes and driven at 0 . 4 μL/min by a microinfusion pump ( KD Scientific; model 780220 ) .", "A volume of 0 . 7 uL/side of pAAV-hSYN-DIO-hM4D ( Gi ) -mCHerry ( 1 . 5 × 1013 GC/mL , Neurophotonics Centre - ULAVAL ) or pAAV-hSYN-tdTomato ( 1 . 5 × 1012 GC/mL , Neurophotonics Centre - ULAVAL ) was infused at a rate of 0 . 05 uL/min and the injector was withdrawn 15 min afterwards .", "To target LC terminals in the BLA , 7 weeks after viral infusions stainless steel cannulae were bilaterally implanted in the BLA as described above but using the following coordinates: A/P −3 . 0 mm , L ± 5 . 1 mm , D/V −9 . 0 mm from bregma .", "Anisomycin ( 125 μg/μl; Sigma-Aldrich ) was dissolved in equimolar HCl and sterile saline .", "Propranolol ( 10 mg/mL; Sigma-Aldrich ) was dissolved in sterile saline .", "C21 ( 2 μg/μL , Hello Bio ) was dissolved in sterile water .", "The pH-value of each solution was adjusted to 7 . 2–7 . 5 .", "Propranolol was administered intraperitoneally at a volume of 1 mL/kg 15 min before training .", "Anisomycin ( immediately after reactivation ) and C21 ( 5 min before training ) were infused bilaterally into the amygdala using a 23 gauge injectors connected to Hamilton syringes via 20 gauge plastic tubes .", "A total volume of 0 . 5 μl per side was infused by a microinfusion pump at a rate of 0 . 125 μl/min .", "Injectors were left in place for an additional minute to ensure proper drug diffusion .", "For experiments requiring Western blots , rats were anesthetized with isoflurane either 1 hr or 24 hr after test and were decapitated , and their brains were removed and frozen at −80°C until further use .", "The basolateral amygdala was dissected from each frozen brain in the cryostat using a neuro punch ( 1 mm; Fine Science Tools ) and homogenized in ice-cold Tris-HCl buffer ( 30 mm , pH 7 . 4 ) containing 4 mm EDTA , 1 mm EGTA , and a protease inhibitor cocktail ( Complete; Roche ) .", "The subcellular fractionation procedure performed was described previously ( Migues et al . , 2010 ) .", "Briefly , the amygdala homogenates were centrifuged at 3 , 000 × g for 10 min at 4°C to remove the nuclei .", "The supernatant was then centrifuged at 100 , 000 × g ( Beckman Coulter ) for 1 hr at 4°C .", "The pellets were resuspended in 50 μl of 0 . 5% Triton X-100 homogenization buffer and incubated for 20 min on ice , before being layered over 100 μl of 1 M sucrose solution and centrifuged at 100 , 000 × g for 1 hr at 4°C .", "The layer remaining above the sucrose , which contained the extrasynaptic receptors , was collected , and the Triton X-100-insoluble material that sedimented through the sucrose layer , containing the postsynaptic densities , was resuspended in 40 μl homogenization buffer and stored at −80°C .", "Total protein concentration was determined by the BCA protein assay kit ( Pierce ) .", "Western blots were performed with 8% SDS-PAGE .", "The proteins were then transferred overnight onto nitrocellulose membranes .", "The membranes were incubated for 1 hr in blocking solution [0 . 1% Tween 20% and 5% BSA in Tris-buffered saline ( TBS ) ] , rinsed with TBS , and then incubated with polyclonal GluN2B ( 1:250; 71–8600 Thermo Fischer ) , GluA2 ( 1:2000; AB10529 Millipore ) , GAPDH ( 1:10 , 000; 6C5 Abcam ) or β-Tubulin ( 1:10 , 000; T 8328Sigma ) overnight .", "After TBS washes , the membranes were then incubated for 1 hr with a secondary antibody ( goat anti-rabbit horseradish peroxidase-linked IgG [NA934V GE Healthcare] or sheep anti-mouse horseradish peroxidase-linked IgG [NA931V GE Healthcare] ) and revealed with the Pierce ECL 2 Western Blotting Substrate ( Thermo Fischer ) .", "The membranes were then scanned on a Storm Laser scanner ( Molecular Dynamics ) and the signals quantified using image analysis software ( ImageLab , BioRad ) .", "The raw values obtained were normalized to the loading control values and expressed as a percentage of the control group .", "To analyze c-fos expression , coronal brain slices were incubated for 1 hr in blocking solution at room temperature ( 3% NGS , 0 . 3% Triton X-100 ) and then for 20 hr with anti-c-fos primary rabbit antibody ( 1:500 , 226 . 003; Synaptic Systems , Göttingen , Germany ) for 24 hr .", "Sections were washed and incubated with anti-rabbit Alexa-488 secondary antibody ( 1:500 , Jackson Immunoresearch , West Grove , PA ) for 2 hr at room temperature .", "Afterwards , sections were washed , mounted on slides and immediately coverslipped with Fluoromount-G with DAPI ( Thermo Fischer ) .", "Images were examined by fluorescence microscopy ( Leica DM 5000 B ) and c-fos positive cells were counted bilaterally from two LC coronal slices for each animal with ImageJ .", "To identify cannulae placements , brains were removed and post-fixed in 10% formalin-saline , 20% sucrose solution and cryo-sectioned at 50 μm thickness .", "The slides were examined by bright-field light microscopy ( Olympus IX81 ) by an experimenter blind to the group assignments .", "Only animals with injector tips bilaterally positioned within the BLA were included in the data analysis .", "Rats with extensive damage were excluded from analysis .", "To verify viral expression , brains were post-fixed in paraformaldehyde 4% , phosphate buffer 0 . 2M for 3 days followed by 10% formalin-saline , 20% sucrose solution for another 3 days and were then cryo-sectioned at 80 μm thickness .", "Slices were mounted onto slides with Fluoromount-G , with DAPI ( Thermo Fischer ) and examined by fluorescence microscopy ( Leica DM 5000 B ) .", "Only animals with somatic viral expression in the LC were included in the data analysis , and rats with extensive damage were excluded .", "Across all experiments , a total of 32 animals with cannula placements or viral expression outside the targeted areas , blocked cannulae , and/or with tissue damage were excluded from analysis .", "We used two-tailed independent-samples t test , one-way , two-way and three-way independent , repeated measures and mixed ANOVA for data analysis .", "Tukey’s post hoc tests were further used to identify the critical differences that contributed to significant interaction .", "Type-one error rate was set at 0 . 05 ." ] ]

[ "Memory reconsolidation is a fundamental plasticity process in the brain that allows established memories to be changed or erased .", "However , certain boundary conditions limit the parameters under which memories can be made plastic .", "Strong memories do not destabilize , for instance , although why they are resilient is mostly unknown .", "Here , we investigated the hypothesis that specific modulatory signals shape memory formation into a state that is reconsolidation-resistant .", "We find that the activation of the noradrenaline-locus coeruleus system ( NOR-LC ) during strong fear memory encoding increases molecular mechanisms of stability at the expense of lability in the amygdala of rats .", "Preventing the NOR-LC from modulating strong fear encoding results in the formation of memories that can undergo reconsolidation within the amygdala and thus are vulnerable to post-reactivation interference .", "Thus , the memory strength boundary condition on reconsolidation is set at the time of encoding by the action of the NOR-LC ." ]

[ "New memories must go through a period of consolidation to become stable and long-lasting in the brain .", "Recalling memories can make them unstable again , so that they need reconsolidating .", "Treatments in which the reconsolidation process is interrupted have been used to help weaken traumatic fear memories .", "However , memories of severe trauma , such as in post-traumatic stress disorder , are particularly resistant to reconsolidation treatments .", "Haubrich et al . used rats to study how trauma shapes memory formation and what biological mechanisms are involved in preventing the destabilization/reconsolidation cycle .", "The rats were exposed to a sound at the same time as receiving a mild electric shock .", "Half of the rats experienced the shock once , creating a ‘weak’ memory .", "The other half experienced it ten times , creating a ‘strong’ memory .", "The rats’ memory of the electric shock was measured by seeing how they responded when they heard the sound again without the shock .", "Some of the rats were given the drug anisomycin , an antibiotic that stops cells from making new proteins and is known for producing amnesia , to block reconsolidation of the memory after hearing the sound again .", "Treatment with the drug reduced future responses in the rats that had experienced the shock once , but had no effect on the rats that had experienced it ten times , demonstrating that the stronger memories were resistant to reconsolidation therapy .", "The rats with the strong memories also had lower levels of proteins in the brain that are involved in plasticity – the ability of the brain to change and adapt .", "Haubrich et al . hypothesized that the stability of the strong memories could be caused by signaling from the locus coeruleus , a region of the brainstem involved in the response to stress .", "When the signaling from the locus coeruleus was blocked in the strong-memory rats , they became responsive to reconsolidation therapy with anisomycin .", "These results help to better understand how traumatic memories become engrained , potentially leading to new treatment options for people with post-traumatic stress disorder ." ]

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

[ "neuroscience" ]

[ [ "Human genetic evidence and studies in App-knock-out ( KO ) mice indicate that the amyloid precursor protein ( APP ) plays an important physiological role in the central nervous system .", "In humans , a polymorphism in APP that reduces APP processing protects from sporadic Alzheimer’s disease ( AD ) and normal aging-dependent cognitive decline ( De Strooper and Voet , 2012 , Jonsson et al . , 2012 ) .", "In contrast , mutations in APP and in genes that regulate APP processing – such as PSENs and BRI2/ITM2B – cause familial AD ( FAD ) and the AD-like familial British dementia and familial Danish dementia ( De Strooper , 2007 , De Strooper et al . , 2010 , De Strooper and Voet , 2012 , Garringer et al . , 2010 , Matsuda et al . , 2005 , Giliberto et al . , 2008 , Matsuda et al . , 2009 , Tanzi , 2012 , Vidal et al . , 1999 , 2000 ) .", "Analysis of App-KO mice has also suggested a synaptic role for APP ( Korte et al . , 2012 , Marcello et al . , 2012 , Octave et al . , 2013 , Randall et al . , 2010 , Turner et al . , 2003 , Venkitaramani et al . , 2007 ) .", "For example , hippocampal App-KO neurons in culture show enhanced α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid ( AMPA ) and N-methyl-D-aspartate ( NMDA ) receptor-mediated synaptic transmission and increased size of the readily releasable synaptic vesicle pool ( RRP ) .", "In addition , long-term potentiation ( LTP ) , a form of synaptic plasticity that underlies memory formation , is impaired in App-KO mice; and these LTP deficits are clearly noticeable in aged but not in juvenile App-KO mice ( Dawson et al . , 1999 , Korte et al . , 2012 , Priller et al . , 2006 , Venkitaramani et al . , 2007 ) .", "Although the evidence indicating that APP plays a role in several processes controlling synaptic function is strong , the molecular mechanisms are poorly understood .", "A confounding factor when studying APP function is the complex APP metabolism .", "The best-characterized cascade involves a sequential β/γ-secretase cleavage of APP .", "In this pathway , APP is cleaved by β-secretase/BACE1 into a soluble ectodomain ( sAPPβ ) and a membrane-bound COOH-terminal fragment of 99 amino acids ( C99 or β C-terminal fragment [CTF] ) .", "C99/β-CTF is cleaved by the γ-secretase to produce Aβ peptides , which are believed to be the main culprit of AD , and the APP intracellular domain ( AID/AICD ) .", "Alternatively , α-secretase cleaves APP in the Aβ sequence into sAPPα and a membrane-bound COOH-terminal fragment of 83 amino acids ( C83 or α-CTF ) .", "C83/α-CTF is cleaved by the γ-secretase to produce a short peptide comprising the COOH-terminus of Aβ , called P3 , and AID/AICD ( Annaert et al . , 2000 , De Strooper , 2003 , Selkoe and Kopan , 2003 , Sisodia and St George-Hyslop , 2002 , Vassar et al . , 1999 ) .", "Another processing pathway , potentially important yet less studied , involves cleavage of APP in the cytoplasmic domain – between D664 and A665 , following the numbering of the main brain APP isoform of 695 amino acids – by caspase-6 , -3 , and -8 .", "Caspases can in theory process all APP metabolites comprising this intracellular motif , such as full-length APP , C99/β-CTF , C83/α-CTF , and AID/AICD .", "So far , cleavages of full-length APP and AID/AICD by caspases have been described ( Bertrand et al . , 2001 , Gervais et al . , 1999 , Lu et al . , 2000 , Madeira et al . , 2005 , Pellegrini et al . , 1999 , Weidemann et al . , 1999 ) .", "Processing of full-length APP generates a membrane bound NH2-terminal fragment called APP-Ncas and a COOH-terminal intracellular peptide called APP-Ccas or C31 ( Bertrand et al . , 2001 , Gervais et al . , 1999 , Lu et al . , 2000 , Madeira et al . , 2005 , Pellegrini et al . , 1999 , Weidemann et al . , 1999 ) .", "Caspase cleavage of AID/AICD splits the cytoplasmic domain of APP into two soluble cytosolic peptides: the aforementioned Ccas/C31 and JCasp .", "The latter comprises the NH2-terminal peptide segment of the APP intracellular domain ( Bertrand et al . , 2001 , Madeira et al . , 2005 ) .", "Recently , a new secretase activity ( called η-secretase ) has been described ( Willem et al . , 2015 ) .", "Cleavage by η-secretase occurs primarily at amino acids 504–505 of APP695 and releases a ~500 amino acid-long ecto-domain and a membrane-bound fragment called CTF-η .", "Interestingly , CTF-η is further processed by α- and β-secretases to release a long and a short Aη peptide ( Aη-α and Aη-β , respectively ) .", "Many of the APP metabolites produced by these cleavages are biologically active .", "AID/AICD modulates programmed cell death , gene transcription and Ca2+ homeostasis ( Baek et al . , 2002 , Cao and Südhof , 2001 , Checler et al . , 2007 , Cupers et al . , 2001 , Hamid et al . , 2007 , Kim et al . , 2003 , Leissring et al . , 2002 , Liu et al . , 2007 , Madeira et al . , 2005 , Pardossi-Piquard et al . , 2005 , Passer et al . , 2000 , von Rotz et al . , 2004 ) .", "The caspase-derived APP fragments Ccas/C31 and JCasp ( Bertrand et al . , 2001 , Lu et al . , 2000 , Madeira et al . , 2005 ) also possess toxic activities .", "Aβ both regulates normal synaptic transmission and prompts deficits in synaptic plasticity ( Fogel et al . , 2014 , Mota et al . , 2014 , Puzzo and Arancio , 2013 , Puzzo et al . , 2008 , Shankar et al . , 2007 , Sivanesan et al . , 2013 , Um et al . , 2012 ) .", "sAPPα has neurotrophic functions and is involved in synaptic plasticity ( Hick et al . , 2015 , Milosch et al . , 2014 ) .", "sAPPβ promotes axon pruning and neurodegeneration ( Nikolaev et al . , 2009 ) .", "Aη-α can reduce LTP ( Willem et al . , 2015 ) .", "β-CTF mediates long-term synaptic plasticity deficits , behavioral deficits and neurodegeneration ( Neve et al . , 1996 , Neve and Robakis , 1998 , Oster-Granite et al . , 1996 , Tamayev et al . , 2012b , 2012c ) .", "The presence of the functionally redundant APP-like protein-1 and -2 ( APLP1 and APLP2 ) is another confounding factor .", "APLP1 and APLP2 are processed like APP ( Heber et al . , 2000 , Herms et al . , 2004 , Müller and Zheng , 2012 , Scheinfeld et al . , 2002 , von Koch et al . , 1997 ) and release intracellular peptides – called APLP2-intracellular domain ( ALID ) 1 and ALID2 , respectively – that , like AID/AICD , can potentially regulate transcription ( Scheinfeld et al . , 2002 , 2003 ) .", "The evidence that Aplp2-KO and App-KO mice are viable , whereas combined App/Aplp2-dKO mice develop neuromuscular junction deficits and die shortly after birth ( Wang et al . , 2005 ) vividly illustrates the functional redundancy of APP and APLP2 .", "The robust phenotype of App/Aplp2-dKO mice can be used to map the functional domains of APP by expressing App knock-in mutations on an Aplp2-KO genetic background .", "This in vivo functional mapping has identified an essential role for the highly conserved APP intracellular domain in the patterning of neuromuscular junction and survival ( Barbagallo et al . , 2011a , 2011b , Li et al . , 2010 ) , indicating that the intracellular region of APP is functionally important in vivo The APP cytoplasmic domain is very short ( ~50 amino acids ) and does not possess recognizable enzymatic domains , raising the possibility that APP functions could be dictated by molecular interaction of the APP cytoplasmic domain with intracellular proteins .", "Using an unbiased proteomic approach , we found that the brain APP intracellular domain-interactome includes synaptic vesicles trans-membrane proteins , proteins associated with synaptic vesicles and the presynaptic active zone ( Del Prete et al . , 2014 ) .", "Many of these proteins , such as cis- and trans-SNAREs , complexin , and the Ca2+ sensors synaptotagmins , function as key regulators of synaptic vesicles exocytosis ( McMahon and Boucrot , 2011 , Rizzoli , 2014 , Royle and Lagnado , 2010 , Südhof , 2012 , 2013 , Südhof and Rizo , 2011 , Xu et al . , 2009 ) .", "Remarkably a similar brain APP–presynaptic interactome was identified in transgenic mice overexpressing human APP ( Kohli et al . , 2012 , Norstrom et al . , 2010 ) .", "These findings are consistent with the evidence that APP in the brain is found at presynaptic terminals and synaptic vesicles ( Del Prete et al . , 2014 , Groemer et al . , 2011 , Laßek et al . , 2014 , Wilhelm et al . , 2014 , Yang et al . , 2005 ) .", "The fact that APP is present at synaptic vesicles and the presynaptic active zone – specialized region of the presynaptic membrane where neurotransmitter release occurs – and that the APP intracellular domain interacts with the neurotransmitter release machinery suggests that APP regulates transmitter release .", "Here , we investigated this proposition .", "By recording neuronal activity in hippocampal brain slices while using and APP-derived peptide that interferes with the APP-neurotransmitter release machinery interaction and acts as a dominant negative inhibitor of APP , we implicate APP in the physiological regulation of the synaptic release of glutamate at hippocampal excitatory synapses .", "Moreover , we found that APLP2 can compensate for loss of APP function and that genetic ablation of both APP and APLP2 protein expression produces glutamatergic synaptic transmission deficits similar to those caused by JCasp , confirming that JCasp acts as a dominant negative inhibitor of some functions of APP in wild-type ( WT ) animals .", "Our results indicate that APP and APLP2 fine-tune synaptic vesicle release likely through the interaction with , and regulation of , the molecular machinery that controls synaptic vesicle exocytosis ." ], [ "Given that the intracellular domain of APP interacts with proteins that mediate synaptic vesicle exocytosis ( Del Prete et al . , 2014 ) , APP could regulate transmitter release .", "To test this hypothesis , one should target those functions associated with these presynaptic APP interactions , possibly without directly interfering in the function of other APP domains/regions , such as Aβ or the long APP extracellular domain and derived products ( sAPPα and sAPPβ ) .", "As an initial step toward the generation of biological tools to test the hypothesis , we mapped the APP intracellular domain that interacts with the exocytosis machinery , and used a proteomic approach to identify the brain proteins that interact with the N-terminus ( JCasp ) and C-terminus ( Ccas ) sub-domains of the APP intracellular region .", "JCasp and Ccas are two naturally occurring fragments that are generated by a double γ-secretase/caspases cleavage of APP ( Figure 1A and B ) ( Bertrand et al . , 2001 , Gervais et al . , 1999 , Lu et al . , 2000 , Madeira et al . , 2005 , Pellegrini et al . , 1999 , Weidemann et al . , 1999 ) .", "It is therefore conceivable that this physiological proteolytic event has been selected to separate two functionally distinct intracellular regions of APP .", "In line with this thought , presynaptic interactors of APP bind to the NH2-terminal intracellular region JCasp .", "Other APP-binding proteins , such as the APP interactor Ddb1 ( Watanabe et al . , 1999 ) and the Ddb1-interacting proteins Cul4A and Cul4B ( O’Connell and Harper , 2007 ) , specifically bind the COOH-terminal APP intracellular region Ccas ( Table 1 , Source Data were uploaded to the PRIDE repository – European Bioinformatics Institute ( EBI ) , Cambridge , UK – with Proteome Xchange identifier #48018 ) , underscoring the reliability of the mapping method .", "In addition , JCasp , but not ScJCasp , which contains the same amino acids of JCasp but in a scrambled order , competes for the binding of Syp , Vamp2 , and Syt2 to St-AID , supporting the notion that JCasp contains the binding domain of APP for these presynaptic proteins ( Figure 1B and C ) . 10 . 7554/eLife . 09743 . 003Figure 1 . The AID–presynaptic interactome binds to JCasp .", "( A ) The APP intracellular domain , composed by the two sub-regions JCasp and Ccas , is always cytosolic whether APP molecules are localized at synaptic vesicles or the active zone .", "( B ) Sequences of AID , JCasp , and Ccas used as baits in the proteomic experiments .", "( C ) Brain lysates were incubated for 30 min with 10 μM of either JCasp or ScJCasp .", "After incubation , the lysates were affinity purified on StrepTactin columns bound to the indicated St-peptides .", "Bound proteins were analyzed by Western blot .", "JCasp reduces binding of Syp , Vamp2 , and Syt2 , but not Ddb1 , to St-AID .", "( D ) Triplicate experiment showing that JCasp , but not ScJCasp , significantly reduces binding of Syp , Vamp2 , and Syt2 to St-AID .", "( E ) Quantification of the data shown in ( D ) .", "( F ) Hippocampal slices from 8-week-old WT or App-KO mice were stained with an αNeuN ( red , left panels ) and an αAPP ( green middle panel ) antibody .", "The images were merged in the right panels .", "APP is widely distributed in the hippocampus .", "The strongest staining is seen in the Hi , MF , CA3 , and CA1 .", "The staining for APP is specific since there is no signal in App-KO hippocampi .", "( G ) Staining for APP ( red , left panels ) and Bassoon ( green middle panel ) show that APP is expressed in the Stratum radiatum where it partially co-localizes with Bassoon ( arrows in the merged image , right panel ) .", "Again , the staining for APP and the co-localization spots are specific as shown by their absence in App-KO hippocampi .", "AID , APP intracellular domain; APP , amyloid precursor protein; WT , wild type .", "DOI: http://dx . doi . org/10 . 7554/eLife . 09743 . 00310 . 7554/eLife . 09743 . 004Table 1 . Proteins that bind JCasp are in redbold .", "Proteins that bind Ccas are in bluethe other entries .", "Source Data containing all search results , coverage maps , peptide lists and product ion data were uploaded to the PRIDE repository ( European Bioinformatics Institute ( EBI ) , Cambridge , UK ) with Proteome Xchange identifier #48018 .", "Details of protein identification data related to the proteins discussed in this manuscript can be found in Table1 .", "The table file contains: the list of proteins identified ( Column 1 ) ; the database accession numbers ( Column 2 ) ; the spectral counts ( SpC , Columns 3-7 ) ; Spectral Abundance Factor ( SAF , Columns 8-12 ) ; subsequent Normalized Spectral Abundance Factor ( NSAF , Columns 13-17 ) .", "This was based on the equation: NSAF = ( SpC/MW ) /Σ ( SpC/MW ) N , where SpC = Spectral Counts , MW = Protein MW in kDa and N = Total Number of Proteins , NSAF contains NSAF values without alone . DOI: http://dx . doi . org/10 . 7554/eLife . 09743 . 004ST ST-AID ST ST-Ccas ST-JCasp ST ST-AID ST ST-Ccas ST-JCasp ST ST-AID ST ST-Ccas ST-JCasp Prot . Acc .", "Num . SpC SpC SpC SpC SpC SAFSAFSAFSAFSAFNSAFNSAFNSAFNSAFNSAFDdb1 sp|Q3U1J4| 0 418 0 730 0 0 . 0000 3 . 2913 0 . 0000 5 . 7480 0 . 0000 0 . 0000 0 . 0139 0 . 0000 0 . 0366 0 . 0000Syt1sp|P46096|088001130 . 00001 . 87230 . 00000 . 00002 . 40430 . 00000 . 00790 . 00000 . 00000 . 0129Stxbp1sp|O08599|04408460 . 00000 . 64710 . 00000 . 11760 . 67650 . 00000 . 00270 . 00000 . 00070 . 0036Cul4a sp|Q3TCH7| 0 40 0 36 0 0 . 0000 0 . 4545 0 . 0000 0 . 4091 0 . 0000 0 . 0000 0 . 0019 0 . 0000 0 . 0026 0 . 0000 Sv2bsp|Q8BG39|05500660 . 00000 . 71430 . 00000 . 00000 . 85710 . 00000 . 00300 . 00000 . 00000 . 0046Syngr3sp|Q8R191|04006790 . 00001 . 60000 . 00000 . 24003 . 16000 . 00000 . 00680 . 00000 . 00150 . 0170Cul4b sp|A2A432| 0 19 0 15 0 0 . 0000 0 . 1712 0 . 0000 0 . 1351 0 . 0000 0 . 0000 0 . 0007 0 . 0000 0 . 0009 0 . 0000 Stx1bsp|P61264|02300310 . 00000 . 69700 . 00000 . 00000 . 93940 . 00000 . 00290 . 00000 . 00000 . 0050Vamp2sp|P63044|02300460 . 00001 . 76920 . 00000 . 00003 . 53850 . 00000 . 00750 . 00000 . 00000 . 0190Syngr1sp|O55100|0800300 . 00000 . 30770 . 00000 . 00001 . 15380 . 00000 . 00130 . 00000 . 00000 . 0062Sv2asp|Q9JIS5|01101230 . 00000 . 13250 . 00000 . 01200 . 27710 . 00000 . 00060 . 00000 . 00010 . 0015Snap25sp|P60879|016012350 . 00000 . 69570 . 00000 . 52171 . 52170 . 00000 . 00290 . 00000 . 00330 . 0082Stx1asp|O35526|0200210 . 00000 . 06060 . 00000 . 00000 . 63640 . 00000 . 00030 . 00000 . 00000 . 0034Stx7sp|O70439|030040 . 00000 . 10000 . 00000 . 00000 . 13330 . 00000 . 00040 . 00000 . 00000 . 0007Syt2sp|P46097|01500360 . 00000 . 31910 . 00000 . 00000 . 76600 . 00000 . 00130 . 00000 . 00000 . 0041Syt12sp|Q920N7|0300100 . 00000 . 06380 . 00000 . 00000 . 21280 . 00000 . 00030 . 00000 . 00000 . 0011Stx12sp|Q9ER00|060050 . 00000 . 19350 . 00000 . 00000 . 16130 . 00000 . 00080 . 00000 . 00000 . 0009 These interactions suggest a presynaptic function of APP in vivo .", "This hypothesis is substantiated by a number of studies showing localization of APP at presynaptic terminals and synaptic vesicles .", "Biochemical studies have shown that endogenous APP is present in fractions highly enriched in synaptic vesicles and the active zone ( Del Prete et al . , 2014 , Groemer et al . , 2011 , Laßek et al . , 2013 , Lundgren et al . , 2015 ) .", "Immune-precipitation of APP from mouse brains showed interaction of APP with key regulators of synaptic vesicles exocytosis .", "Although the possibility that these interactions happen post-cellular lysis cannot be formally discarded , these data are consistent with a presynaptic localization of APP in vivo .", "Immuno-electron microscopy experiments ( performed using the specific Y-188 anti-APP antibody , see below ) have indicated that APP is localized at presynaptic terminals and in synaptic vesicles in the hippocampus ( Del Prete et al . , 2014 ) .", "Finally , localization of APP at presynaptic terminals has been reported also in primary neuronal cultures where it can regulate synaptic transmission functioning as a presynaptic receptor for Aβ ( Fogel et al . , 2014 ) .", "The data discussed above support a presynaptic function of APP .", "Next , we analyzed the pattern of expression of APP in the hippocampus , a brain structure critically involved in memory formation .", "We revisited this question in view of a recent report showing that most of the antibodies that have been used to determine the localization of endogenous APP are non-specific as they label App-KO and WT cells/tissues in a similar fashion ( Guo et al . , 2012 ) .", "The authors showed that Y188 was the only APP-specific antibody .", "The evidence that Y188 does not stain App-KO hippocampi confirmed the specificity of this antibody ( Figure 1E and F ) .", "In addition , low magnification analysis shows that APP is expressed , albeit at different levels , in all hippocampal circuits , including the CA1 neurons and the stratum radiatum containing the CA3 projections ( Schaffer collaterals , or SC ) to CA1 neurons ( Figure 1E ) .", "Higher magnification analysis confirms that APP staining is stronger in CA1 pyramidal neurons as compared to the stratum radiatum ( Figure 1F ) .", "Several factors may explain these differences:", "1 ) APP is synthesized and undergoes maturation in the endoplasmic reticulum ( ER ) and Golgi that are highly concentrated in cell bodies;", "2 ) APP may be expressed at synapses at levels that are , albeit functionally important , too low to be clearly detected by Y188;", "3 ) Y188 recognizes the C-terminal epitope of APP , and this epitope may be functionally engaged in multi-molecular interactions at presynaptic terminals and may therefore be scarcely accessible to Y188 .", "Nevertheless , the staining shown in Figure 1F indicates that APP is indeed expressed in the stratum radiatum , where it partially co-localizes with the presynaptic protein Bassoon .", "This presynaptic APP-interacting network can stem from APP molecules localized at synaptic vesicles , presynaptic membranes or both , and it is likely formed via the direct interaction of the intracellular domain of APP with some release machinery protein .", "The other components are most likely indirectly associated to APP ( Figure 2A ) .", "Since JCasp interferes with these interactions in vitro , if delivered inside neurons , JCasp could hamper the function of this presynaptic APP–interacting complex in vivo ( Figure 2B ) and reveal the function of this intracellular APP domain . 10 . 7554/eLife . 09743 . 005Figure 2 . Pen1-JCasp is delivered intra-neuronally . Models of assembly of the amyloid precursor protein ( APP ) presynaptic interactome and mechanism of action of Pen1-JCasp .", "( A ) SV = synaptic vesicles; AZ = active zone .", "SV proteins and proteins associated to SV and to the AZ bind via direct and indirect interactions with the JCasp region of APP molecules localized to either SV or the AZ .", "The interactions depicted here are hypothetical .", "( B ) Pen1-JCasp is delivered intracellularly and can interfere with the interaction of APP with presynaptic proteins .", "( C ) Immune-fluorescence experiment showing that Pen1 delivers the JCasp peptide in hippocampal neurons . DOI: http://dx . doi . org/10 . 7554/eLife . 09743 . 005 Thus , we systematically characterized the effect of JCasp on excitatory synaptic transmission at SC–CA1 pyramidal cell synapses .", "To provide intracellular delivery , we synthesized JCasp and the negative control peptide ScJCasp linked to the cell-penetrating peptide ( Davidson et al . , 2004 ) Penetratin1 ( Pen1 ) at the N-terminus ( Figure 2B ) , a strategy that we have previously used to deliver peptides to murine hippocampal neurons in vivo ( Tamayev et al . , 2012c ) .", "First , we verified whether Pen1-JCasp efficiently delivers JCasp at hippocampal SC–CA1 synapses .", "To this end , we incubated hippocampal slices with 1 μM of either biotinylated JCasp or biotinylated Pen1-JCasp .", "Later , 5 hr after incubation , slices were stained for the peptide using Streptavidin .", "As shown in Figure 2C , Pen1-JCasp , but not JCasp , is detected in neurons of the CA1 pyramidal neurons and in the stratum radiatum , which contains the SCs .", "Thus , we determined whether Pen1-JCasp alters synaptic transmission at SC–CA1 synapses .", "The experiments were performed as follows: soon after preparation the brain slices were incubated in 200 ml of artificial cerebrospinal fluid ( ACSF ) containing the indicated concentrations of either Pen1-JCasp or the negative control peptide Pen1-ScJCasp .", "Recordings were performed between ~5 and 8 hr after preparation of slices and incubation with Pen1 peptides .", "We found that Pen1-JCasp impaired basal synaptic transmission , as indicated by a reduction in the input/output ( I/O ) function ( Figure 3A ) .", "These effects were dose-dependent and specific since the negative control peptide Pen1-ScJCasp did not alter synaptic transmission . 10 . 7554/eLife . 09743 . 006Figure 3 . Pen1-JCasp impairs synaptic plasticity .", "( A ) CA1 recordings of hippocampal slices incubated with either artificial cerebrospinal fluid ( ACSF ) , 1 μM Pen1-ScJCasp or Pen1-JCasp at the indicated concentrations .", "The synaptic input/output ( I/O ) relationship was obtained by plotting the fiber volley amplitude against the initial slope of the evoked field excitatory postsynaptic potential ( fEPSP ) .", "Representative traces are shown on top .", "The slope values of I/O recording for each group were compared for statistical assessments .", "It has been found that 1 μM Pen1-JCasp significantly reduces synaptic transmission as compared to ACSF ( * ) , Pen1-ScJCasp ( * ) , 10 nM ( * ) , and 100 nM Pen1-JCasp ( *p ) .", "Representative traces are shown .", "( B ) Average paired-pulse facilitation ( PPF ) ( 2nd fEPSP/1st fEPSP ) plotted as a function of the inter-stimulus interval .", "Representative traces of fEPSPs evoked at 40 ms inter-stimulus interval are shown .", "Pen1-JCasp increases PPF .", "( C ) Synaptic facilitation elicited by stimulus trains at 1 , 5 , 10 , and 20 Hz .", "fEPSP slopes are normalized to the slope of the first fEPSP of the stimulus train .", "Representative traces of fEPSPs evoked at 20 Hz are shown .", "Stimulus artifacts are removed for clarity .", "Pen1-JCasp increases frequency facilitation ( FF ) in a frequency and dose-dependent manner .", "Statistical assessments were performed by: one-way analysis of variance ( ANOVA ) followed by Tukey’s multiple comparisons test for slopes of I/O curves; two-way repeated measures ( RM ) ANOVA followed by Tukey’s multiple comparisons test for PPF and FF ( *p < 0 . 05; **p < 0 . 01; ***p < 0 . 001; ****p < 0 . 0001 ) .", "In FF , 10 nM Pen1-JCasp increased facilitation in a statistically significant manner only at the 10th stimulation ( indicated with ( * ) and ( * ) ) .", "The number of recordings and the number of mice analyzed for each group are shown in ( C ) .", "All data represent means ± SEM .", "The complete statistical analyses are shown in the attached Excel file . DOI: http://dx . doi . org/10 . 7554/eLife . 09743 . 00610 . 7554/eLife . 09743 . 007Figure 3—source data 1 . Source data for statistical analysis of input/output curves in Figure 3A . DOI: http://dx . doi . org/10 . 7554/eLife . 09743 . 00710 . 7554/eLife . 09743 . 008Figure 3—source data 2 . Source data for statistical analysis of paired-pulse facilitation in Figure 3B . DOI: http://dx . doi . org/10 . 7554/eLife . 09743 . 00810 . 7554/eLife . 09743 . 009Figure 3—source data 3 . Source data for statistical analysis of frequency facilitation at 1 Hz in Figure 3C . DOI: http://dx . doi . org/10 . 7554/eLife . 09743 . 00910 . 7554/eLife . 09743 . 010Figure 3—source data 4 . Source data for statistical analysis of frequency facilitation at 5 Hz in Figure 3C . DOI: http://dx . doi . org/10 . 7554/eLife . 09743 . 01010 . 7554/eLife . 09743 . 011Figure 3—source data 5 . Source data for statistical analysis of frequency facilitation at 10 Hz in Figure 3C . DOI: http://dx . doi . org/10 . 7554/eLife . 09743 . 01110 . 7554/eLife . 09743 . 012Figure 3—source data 6 . Source data for statistical analysis of frequency facilitation at 20 Hz in Figure 3C . DOI: http://dx . doi . org/10 . 7554/eLife . 09743 . 012 To test whether the reduction in I/O function could be due to presynaptic impairment , we first estimated the probability of release ( Pr ) by analyzing two forms of short-term synaptic plasticity: paired-pulse facilitation ( PPF ) and synaptic frequency facilitation ( FF ) in response to a stimulus train ( 10 stimuli at either 1 , 5 , 10 , or 20 Hz ) .", "These forms of short-term synaptic plasticity are determined , at least in part , by changes in Pr , such that a decrease in Pr leads to an increase in facilitation and vice versa ( Zucker and Regehr , 2002 ) .", "Pen1-JCasp but not Pen1-ScJCasp significantly increased both PPF ( Figure 3B ) and FF ( Figure 3C ) in a dose-dependent manner .", "In addition , Pen1-JCasp reduced miniature excitatory postsynaptic current ( EPSC ) ( mEPSC ) frequency ( Figure 4A ) , but had no effect on mEPSC amplitude ( Figure 4B ) or waveform ( Figure 4C ) , suggesting a presynaptic mechanism of action .", "We also delivered a high-frequency train of stimuli to deplete the RRP .", "The rate of RRP depletion , as measured by the suppression of synaptic transmission during repetitive stimulation , is proportional to the initial Pr ( i . e . , a reduction in Pr would decrease this rate and vice versa , an increase in Pr would accelerate it ) .", "Indeed , Pen1-JCasp slowed down the rate of synaptic suppression induced by a 500 stimuli , 28 Hz train ( Figure 5A and B ) .", "After depletion , the recycling of synaptic vesicles replenishes the RRP .", "We found that the recovery phase after depletion – measured by stimulating at 2 Hz – was unaffected by Pen1-JCasp ( Figure 5C and D ) , arguing against an alteration of synaptic vesicle recycling as a cause of the reduced synaptic strength .", "Taken together , our findings strongly suggest that Pen1-JCasp reduces basal synaptic transmission by interfering with glutamate release . 10 . 7554/eLife . 09743 . 013Figure 4 . Pen1-JCasp reduces frequency of excitatory miniature currents .", "( A ) Cumulative probability of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor ( AMPAR ) mediated miniature excitatory postsynaptic current ( EPSC ) ( mEPSC ) inter-event intervals .", "( B ) Cumulative probability of AMPAR-mediated mEPSC amplitudes .", "Insets in cumulative probability graphs represent average mEPSC frequency ( A ) and amplitudes ( B ) .", "mEPSC frequency was significantly reduced by Pen1-JCasp .", "( C ) Average mEPSC of the two groups were not significantly different .", "( D ) Representative recording traces of miniature EPSCs are shown .", "The number of recordings and the number of mice analyzed for each group are shown in ( A ) .", "All data represent means ± SEM .", "Statistical assessments were performed by paired t test ( **p < 0 . 01 ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 09743 . 01310 . 7554/eLife . 09743 . 014Figure 4—source data 1 . Source data for statistical analysis of mEPSCs frequency in Figure 4A . DOI: http://dx . doi . org/10 . 7554/eLife . 09743 . 01410 . 7554/eLife . 09743 . 015Figure 5 . Pen1-JCasp reduces the rate of depletion of glutamatergic vesicles .", "( A ) Artificial cerebrospinal fluid ( ACSF ) vs Pen1-JCasp , p < 0 . 05; Pen1-SCJCasp vs . Pen1-JCasp , p < 0 . 01; ( B ) ACSF vs . Pen1-JCasp , p< 0 . 01; Pen1-SCJCasp vs . Pen1-JCasp , p < 0 . 01 .", "Recovery kinetics ( C and D ) are not affected .", "The experiments were performed in 3-month-old C57Bl/6j ( A and C ) and C57Bl/6j-129 hybrid mice ( B and D ) .", "All experiments shown in this paper were performed in C57Bl/6j mice , except for the experiments shown in Figure 7; App-KO and wild-type ( WT ) littermate animals were on a C57Bl/6j-129 hybrid genetic background .", "Data represent means ± SEM .", "Statistical assessments were performed by nonlinear regression curve fit , one phase decay . DOI: http://dx . doi . org/10 . 7554/eLife . 09743 . 01510 . 7554/eLife . 09743 . 016Figure 5—source data 1 . Source data for statistical analysis of decay rate shown in Figure 5A . DOI: http://dx . doi . org/10 . 7554/eLife . 09743 . 01610 . 7554/eLife . 09743 . 017Figure 5—source data 2 . Source data for statistical analysis of decay rate shown in Figure 5B . DOI: http://dx . doi . org/10 . 7554/eLife . 09743 . 017 Given that several APP metabolites have been involved in a wide range of neuronal functions , including synaptic plasticity , Pen1-JCasp could reduce synaptic transmission indirectly by altering APP processing .", "To address this possibility , we tested the effect of Pen1-JCasp on APP processing .", "Several soluble APP metabolites resulting from APP processing , such as APPβ , sAPPα , and Aβ , are diluted into the large volume ( 200 ml ) of ACSF used for culturing hippocampal slices during recordings .", "Therefore , it is difficult to accurately measure the levels of these soluble APP metabolites .", "However , other APP metabolites , that is , β-CTF and α-CTF , remain membrane bound and can be measured by Western blot , together with full-length APP .", "Pen1-JCasp does not change levels of full-length APP , β-CTF , and α-CTF ( Figure 6A and B ) , arguing against the possibility that Pen1-JCasp alters synaptic plasticity via modulation of APP processing . 10 . 7554/eLife . 09743 . 018Figure 6 . Pen1-JCasp does not alter amyloid precursor protein ( APP ) processing .", "( A ) Western blot analysis of Actin , APP , β C-terminal fragment ( CTF ) and α-CTF in hippocampal slices incubated for 8 hr with either 1 μM Pen1-JCasp or 1 μM Pen1-ScJCasp .", "β-CTFp and α-CTFp are phosphorylated forms of β-CTF and α-CTF , respectively .", "( B ) Quantification of the data: for each protein , the average signal obtained with 1 μM Pen1-ScJCasp is arbitrarily considered 1; the average signal obtained with 1 μM Pen1-JCasp is expressed as a percent of the control .", "The * indicates an aspecific band , which is also present in App-KO lysates ( not shown ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 09743 . 018 Based on our findings , we hypothesized that JCasp reduces transmitter release by interfering with the intracellular interactions of APP with key regulators of vesicle exocytosis .", "To test this hypothesis we compared the effects of Pen1-JCasp and JCasp side-by-side .", "Pen1-JCasp , but not JCasp , which lacks the cell penetrating properties of Pen1 and is not detected inside neurons ( Figure 2C ) , reduced basal synaptic transmission ( Figure 7A ) and increased PPF ( Figure 7B ) and FF ( Figure 7C ) .", "Thus , to suppress transmitter release , the JCasp peptide must access the intracellular compartment . 10 . 7554/eLife . 09743 . 019Figure 7 . Pen1-JCasp impairs excitatory synapses via an intracellular mechanism . Pen1-JCasp , but not JCasp , reduces basal synaptic transmission ( A ) paired-pulse facilitation ( PPF ) ( B ) and frequency facilitation ( FF ) at 20 Hz ( C ) .", "Representative traces are shown .", "Stimulus artifacts are removed from the FF traces for clarity .", "The number of recordings and of mice analyzed are shown in ( B ) .", "Statistical assessments were performed by: one-way analysis of variance ( ANOVA ) followed by Tukey’s multiple comparisons test for slopes of input/output ( I/O ) curves; two-way repeated measures ( RM ) ANOVA followed by Tukey’s multiple comparisons test for PPF and FF .", "*Pen1-JCasp 1 μM vs . artificial cerebrospinal fluid ( ACSF ) , *Pen1-JCasp 1 μM vs . JCasp; *p < 0 . 05; **p < 0 . 01; ***p < 0 . 001; ****p < 0 . 0001 .", "The complete statistical analyses are shown in the attached Excel file .", "All data represent means ± SEM . DOI: http://dx . doi . org/10 . 7554/eLife . 09743 . 01910 . 7554/eLife . 09743 . 020Figure 7—source data 1 . Source data for statistical analysis of input/output curves in Figure 7A . DOI: http://dx . doi . org/10 . 7554/eLife . 09743 . 02010 . 7554/eLife . 09743 . 021Figure 7—source data 2 . Source data for statistical analysis of paired-pulse facilitation in Figure 7B . DOI: http://dx . doi . org/10 . 7554/eLife . 09743 . 02110 . 7554/eLife . 09743 . 022Figure 7—source data 3 . Source data for statistical analysis of frequency facilitation at 20 Hz in Figure 7C . DOI: http://dx . doi . org/10 . 7554/eLife . 09743 . 022 A recent report shows that Aβ in cultured neurons triggers APP dimerization that , by interacting via the intracellular domain with Gαoβ1γ2 protein , increases Ca+2 influx through Cav2 channels , thereby enhancing Pr ( Fogel et al . , 2014 ) .", "Thus , Pen1-JCasp could reduce Pr by inhibiting this pathway and reducing Ca+2 influx .", "However , we found that Pen1-JCasp was equally effective in the presence of 100 μM Cd+2 , a manipulation that non-selectively blocks Ca+2 influx through voltage-gated Ca+2 channels ( Figure 8 ) . 10 . 7554/eLife . 09743 . 023Figure 8 . Pen1-JCasp reduces the frequency of miniature excitatory postsynaptic currents ( EPSCs ) ( mEPSCs ) independently of alterations of Ca+2 influx .", "( A ) Cumulative probability of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor ( AMPAR ) mediated mEPSC inter-event intervals .", "( B ) Cumulative probability of AMPAR-mediated mEPSC amplitudes .", "Insets in cumulative probability graphs represent average mEPSC frequency ( A ) and amplitudes ( B ) .", "mEPSC frequency was significantly reduced by Pen1-JCasp in the presence of Cd+2 .", "( C ) Average mEPSC of the two groups were not significantly different .", "( D ) Representative recording traces of miniature EPSCs are shown .", "The number of recordings and the number of mice analyzed for each group are shown in ( A ) .", "All data represent means ± SEM .", "Statistical assessments were performed by paired t test ( *p < 0 . 05 ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 09743 . 02310 . 7554/eLife . 09743 . 024Figure 8—source data 1 . Source data for statistical analysis of mEPSCs frequency in Figure 8A . DOI: http://dx . doi . org/10 . 7554/eLife . 09743 . 024 Next , we determined whether JCasp has any effect on SC–CA1 synaptic transmission in App-KO and WT littermates .", "Interestingly , Pen1-JCasp reduced I/O function ( Figure 9A ) and increased PPF ( Figure 9B ) and FF ( Figure 9C ) in WT but not in App-KO animals , suggesting that Pen1-JCasp does not alter transmission at APP-lacking synapses . 10 . 7554/eLife . 09743 . 025Figure 9 . Amyloid precursor protein ( APP ) is required for the inhibitory effect of Pen1-JCasp on synaptic transmission .", "( A ) Analysis of slopes of each input/output ( I/O ) recording by one-way analysis of variance ( ANOVA ) followed by Tukey’s multiple comparisons test showed no significant differences among the groups .", "However , uncorrected Fisher’s LSD test shows that Pen1-JCasp reduces basal synaptic transmission in wild-type ( WT ) hippocampal slices as compared to WT slices treated with Pen1-ScJCasp .", "Pen1-JCasp increases paired-pulse facilitation ( PPF ) ( B ) and frequency facilitation ( FF ) ( C ) in WT but not App-KO excitatory Schaffer collateral ( SC ) synapses .", "Representative traces are shown on the right of summary plots .", "The number of recordings and of mice analyzed for each group are shown in ( A ) .", "All data represent means ± SEM .", "Statistical assessment by two-way repeated measures ( RM ) ANOVA shows significant differences in PPF and FF between WT mice treated with Pen1-JCasp and the other three experimental groups ( * , vs . WT+Pen1-SCJCasp; * , vs . App-KO+Pen1-SCJCasp; * , vs . App-KO+Pen1-JCasp; *p < 0 . 05; **p < 0 . 01; ***p < 0 . 001; ****p < 0 . 0001 ) .", "The complete statistical analyses are shown in the attached Excel file . DOI: http://dx . doi . org/10 . 7554/eLife . 09743 . 02510 . 7554/eLife . 09743 . 026Figure 9—source data 1 . Source data for statistical analysis of input/output curves in Figure 9A . DOI: http://dx . doi . org/10 . 7554/eLife . 09743 . 02610 . 7554/eLife . 09743 . 027Figure 9—source data 2 . Source data for statistical analysis of paired-pulse facilitation in Figure 9B . DOI: http://dx . doi . org/10 . 7554/eLife . 09743 . 02710 . 7554/eLife . 09743 . 028Figure 9—source data 3 . Source data for statistical analysis of frequency facilitation at 20 Hz in Figure 9C . DOI: http://dx . doi . org/10 . 7554/eLife . 09743 . 028 The fact that Pen1-JCasp-mediated suppression of synaptic transmission requires APP expression not only indicates that Pen1-JCasp acts as a dominant negative inhibitor of endogenous APP but also suggests that endogenous APP regulates glutamate release at excitatory synapses .", "However , we found no differences in synaptic facilitation between App-KO and WT hippocampi ( Figure 9B and C ) , suggesting that compensatory mechanisms operate in App-KO mice to counterbalance the loss of APP function .", "Good candidates for these redundant functions are APLP1 and APLP2 , the other two members of the APP protein family; particularly APLP2 since functional redundancy between APP and APLP2 has already been demonstrated ( Barbagallo et al . , 2011b , Dawson et al . , 1999 , von Koch et al . , 1997 , Wang et al . , 2005 ) .", "Of note , APLP1 and APLP2 have also been localized to presynaptic terminals ( Laßek et al . , 2013 ) , further supporting a compensatory function at presynaptic terminals .", "As a first step to determine whether APLP2 shares this presynaptic function of APP , we characterized the brain interactome of the ALID2 using the same unbiased proteomic approach used for Ccas and JCasp .", "We synthesized two peptides: control Strep-tag only peptide and St-ALID2 .", "Interestingly , the brain interactome of ALID2 comprises most of the key proteins of the presynaptic release machinery that bind to JCasp and AID ( Table 2 , Source Data were uploaded to the PRIDE repository – European Bioinformatics Institute ( EBI ) , Cambridge , UK – with Proteome Xchange identifier #58725 ) . 10 . 7554/eLife . 09743 . 029Table 2 . Interaction of ALID2 with presynaptic protein .", "Source Data containing all search results , coverage maps , peptide lists and product ion data were uploaded to the PRIDE repository ( European Bioinformatics Institute ( EBI ) , Cambridge , UK ) with Proteome Xchange identifier #58725 .", "The table file contains: the list of proteins identified ( Column 1 ) ; the database accession numbers ( Column 2 ) ; the spectral counts ( SpC , Columns 3-7 ) ; Spectral Abundance Factor ( SAF , Columns 8-12 ) ; subsequent Normalized Spectral Abundance Factor ( NSAF , Columns 13-17 ) .", "This was based on the equation: NSAF = ( SpC/MW ) /Σ ( SpC/MW ) N , where SpC = Spectral Counts , MW = Protein MW in kDa and N = Total Number of Proteins , NSAF contains NSAF values without alone . DOI: http://dx . doi . org/10 . 7554/eLife . 09743 . 029ST ST-ALID2 ST ST-ALID2 ST ST-ALID2 Prot . Acc .", "Num . SpCSpCSAFSpCNSAFSpCSyt1sp|P46096|0290 . 00000 . 32350 . 00000 . 0045Stxbp1sp|O08599|0220 . 00000 . 64710 . 00000 . 0027Sv2bsp|Q8BG39|0150 . 00000 . 19480 . 00000 . 0034Syngr3sp|Q8R191|070 . 00000 . 280 . 00000 . 0067Stx1bsp|P61264|060 . 00000 . 12120 . 00000 . 0029Vamp2sp|P63044|0110 . 00000 . 84610 . 00000 . 0203Syngr1sp|O55100|060 . 00000 . 88460 . 00000 . 0055Sv2asp|Q9JIS5|0140 . 00000 . 40190 . 00000 . 0023Syt2sp|P46097|0180 . 00000 . 38290 . 00000 . 0053Syphsp|Q62277|080 . 00000 . 23530 . 00000 . 0049 The preceding data are consistent with the idea that APLP2 , via its intracellular domain , can compensate the function of the cytoplasmic domain of APP in App-KO synapses .", "Yet , the evidence that Pen1-JCasp does not alter synaptic transmission in App-KO synapses ( Figure 9 ) indicates that Pen1-JCasp acts as an inhibitor of the intracellular domain of APP but not APLP2 in vivo .", "Consistent with this possibility JCasp and the corresponding APLP2 region ( RKRQYGTISHGIVEVD ) are only 63% identical , and JCasp does not interfere with the interaction of ALID2 with Vamp2 , Syt2 , and Syp ( Figure 10 ) .", "Thus , Pen1-Jcasp is a tool to selectively study the function of APP . 10 . 7554/eLife . 09743 . 030Figure 10 . JCasp does not compete for the interaction of amyloid precursor protein ( APP ) like protein-2 ( APLP2 ) intracellular domain ( ALID2 ) with Syt2 , Syp , and Vamp2 . Brain lysates were incubated for 30 min with 10 μM of either JCasp or ScJCasp .", "After incubation , the lysates were affinity purified on StrepTactin columns bound to the indicated St-peptides .", "Bound proteins were analyzed by Western blot .", "JCasp does not reduce binding of Syp , Vamp2 , and Syt2 to St-ALID2 .", "The graphs at the bottom of the figure show quantitative analysis of the Western blot data . DOI: http://dx . doi . org/10 . 7554/eLife . 09743 . 030 To directly test whether APLP2 can compensate for loss of APP function , we monitored the effect of constitutive APP , APLP2 , and APP+APLP2 deletion on excitatory synaptic transmission at SC–CA1 synapses in acute brain slices .", "Because App/Aplp2-dKO mice die within the first 4 weeks of age , for comparison reasons all experiments were performed on 16- to 24-day-old mice of both sexes .", "Animals were on a mixed C57BL/6J and 129 genetic background .", "Aplp2-KOand App/Aplp2-dKO showed reduced basal synaptic transmission as compared to both WT and App-KO mice; however , these differences reached statistical significance only when compared to App-KO samples ( Figure 11A ) . 10 . 7554/eLife . 09743 . 031Figure 11 . Amyloid precursor protein ( APP ) like protein-2 ( APLP2 ) compensates for loss of APP function .", "( A ) Input/output ( I/O ) recording showed significant differences between different genotypes ( App-KO vs . Aplp2-KO , **p < 0 . 01; App-KO vs . App/Aplp2-dKO , *p < 0 . 05 ) .", "PPR ( B ) and frequency facilitation ( FF ) ( C ) are significantly increased in App/Aplp2-dKO as compared to all other genotypes .", "Representative traces are shown on the right of summary plots .", "The number of recordings and of mice analyzed for each group are shown in ( A ) .", "Statistical assessment by two-way repeated measures analysis of variance ( RM ANOVA ) followed by Tukey’s multiple comparisons tests .", "All data represent means ± SEM ( *p < 0 . 05; **p < 0 . 01; ***p < 0 . 001 ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 09743 . 03110 . 7554/eLife . 09743 . 032Figure 11—source data 1 . Source data for statistical analysis of input/output curves in Figure 11A . DOI: http://dx . doi . org/10 . 7554/eLife . 09743 . 03210 . 7554/eLife . 09743 . 033Figure 11—source data 2 . Source data for statistical analysis of paired-pulse facilitation in Figure 11B . DOI: http://dx . doi . org/10 . 7554/eLife . 09743 . 03310 . 7554/eLife . 09743 . 034Figure 11—source data 3 . Source data for statistical analysis of frequency facilitation at 20Hz in Figure 11C . DOI: http://dx . doi . org/10 . 7554/eLife . 09743 . 034 The App/Aplp2-dKO mice showed an increase in PPF ( Figure 11B ) and FF ( Figure 11C ) , suggesting a reduction in Pr ( Zucker and Regehr , 2002 ) .", "The magnitude PPF and FF in App-KO and Aplp2-KO SC–CA1 synapses was not significantly different than in WT mice ( Figure 11B and C ) .", "Next we monitored mEPSCs .", "Interestingly , we found that mEPSC frequency was decreased by genetic ablation of APP and APLP2 expression , with APLP2 deletion having a greater effect as compared to APP deletion ( Figure 12A , WT>App-KO>Aplp2-KO>App/Aplp2-dKO ) .", "However , these differences reached statistical significance only between WT Vs . Aplp2-KO and WT Vs . App/Aplp2-dKO samples .", "Thus , our results showing changes in short-term plasticity and mEPSC activity strongly suggest that while APP and APLP2 have redundant effects on glutamate release , APLP2 compensates for loss of APP function in App-KO mice more efficently than APP compensates loss of APLP2 function in Aplp2-KO hippocampi .", "Remarkably , the synaptic deficits observed in App/Aplp2-dKO SC–CA1 excitatory synapses are strikingly similar to those induced by JCasp in WT hippocampi , supporting the notion that a common mechanism underlies these deficits . 10 . 7554/eLife . 09743 . 035Figure 12 . Reduced frequency of miniature excitatory postsynaptic currents ( EPSCs ) ( mEPSCs ) in Aplp2-KO and App/Aplp2-dKO CA1 pyramidal neurons .", "( A ) Cumulative probability of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor ( AMPAR ) -mediated mEPSC inter-event intervals .", "( B ) Cumulative probability of AMPAR-mediated mEPSC amplitudes .", "Insets in cumulative probability graphs represent average mEPSC frequency ( A ) and amplitudes ( B ) .", "mEPSC frequency was significantly reduced in Aplp2-KO and App/Aplp2-dKO CA1 pyramidal neurons as compared to wild-type ( WT ) but not App-KO littermates .", "( C ) Average mEPSC of the four groups were not significantly different .", "( D ) Cumulative probability of AMPAR-mediated mEPSC decay time with insets representing average mEPSC decay time .", "mEPSC decay time was significantly increased in App/Aplp2-dKO CA1 pyramidal neurons as compared to WT , App-KO , and Aplp2-KO littermates .", "( E ) Representative recording traces of miniature EPSCs are shown .", "The number of recordings and the number of mice analyzed for each group are shown in ( A ) .", "All data represent means ± SEM .", "Statistical assessment was performed using ordinary one-way ANOVA followed by uncorrected Fisher’s least significant difference ( LSD ) multiple comparisons test ( *p < 0 . 05; **p < 0 . 01; ***p < 0 . 001; ****p < 0 . 0001 ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 09743 . 03510 . 7554/eLife . 09743 . 036Figure 12—source data 1 . Source data for statistical analysis of mEPSCs frequency in Figure 12A . DOI: http://dx . doi . org/10 . 7554/eLife . 09743 . 03610 . 7554/eLife . 09743 . 037Figure 12—source data 2 . Source data for statistical analysis of mEPSCs decay time in Figure 12B . DOI: http://dx . doi . org/10 . 7554/eLife . 09743 . 037 We also found that the mEPSC decay time was increased in App/Aplp2-dKO as compared to WT , App-KO , and Aplp2-KO ( Figure 12C , D ) , suggesting that APP and APLP2 may possess some redundant postsynaptic function , consistent with previous observations indicating a postsynaptic role for some APP metabolites , such as Aβ ( Gu et al . , 2009 , Hsieh et al . , 2006 ) .", "Interestingly , Pen1-JCasp does not prompt this sharp decline in mEPSCs’ decay , suggesting that the JCasp domain of APP is primarily involved in the presynaptic functions of APP ." ], [ "Using an unbiased proteomic methodology and competition experiments , we found that the NH2-terminal activation-induced cytidine deaminase ( AID ) region , JCasp , is the APP domain that binds presynaptic proteins regulating synaptic transmission .", "We have used a peptide encompassing this JCasp region to test whether the interaction of APP with proteins that regulate synaptic vesicles exocytosis enables APP to tune synaptic vesicles release .", "Strikingly , intracellular delivery of JCasp caused a strong reduction in basal synaptic transmission .", "Consistent with a presynaptic action , this reduction was associated with an increase in PPF and FF , as well as in mEPCS frequency but not amplitude , rise time , and decay time .", "These effects are evident in WT synapses but absent at APP-lacking synapses .", "In addition , Pen1-JCasp reduced the rate of synaptic vesicles depletion without affecting recycling .", "Taken together , these results indicate that JCasp acts as a dominant negative inhibitor of APP , and that APP facilitates the release of excitatory synaptic vesicles , likely via the interaction with and functional regulation of the molecular machinery that controls synaptic vesicles exocytosis .", "The evidence that glutamatergic synaptic vesicles release is normal in the hippocampus of young App-KO mice suggests that compensatory mechanisms counterbalance the loss of APP function .", "Given the functional redundancy of APP and APLP2 in neuromuscular junctions patterning and survival ( Barbagallo et al . , 2011b , Li et al . , 2010 , Wang et al . , 2005 ) , we thought that APLP2 could compensate for loss of APP function at hippocampal synapse .", "Consistent with this hypothesis , we found that the intracellular domain of APLP2 , ALID2 , interacts with key regulators of synaptic vesicle exocytosis .", "A systematic characterization of SC–CA1 synaptic transmission revealed that APP and APLP2 have redundant functions in the hippocampus as indicated by the following observations:", "1 ) App-KO animals showed normal synaptic transmission at SC–CA1 excitatory synapses;", "2 ) Aplp2-KO mice presented a significant reduction in mEPSCs frequency;", "3 ) App/Aplp2-dKO mice showed increased PPF and FF , which was not observed in single App-KO and Aplp2-KO mice , as well as reduced mEPSCs frequency .", "Overall , the synaptic phenotype observed in App/Aplp2-dKO mice is very similar to that induced by Pen1-JCasp in WT animals , suggesting a primary functional role for the intracellular domains of APP and APLP2 in tuning glutamate release .", "A notable exception is represented by an increase in mEPSC decay time – present in App/Aplp2-dKO mice but not in hippocampal slices incubated with Pen1-JCasp .", "This observation suggests that Pen1-JCasp can unveil the role of the JCasp domain of APP without directly affecting the function of other APP region , such as , for example , Aβ or the APP extracellular domain .", "Our results indicate that JCasp is an inhibitor of APP but not APLP2 .", "This specificity of JCasp for APP allows to study the function of the APP intracellular domain in WT adult animals , eliminating the confounding effects associated with both constitutive and conditional App-KO models .", "In fact , it permits acute inhibition of the functions of this APP domain in WT adult animals , circumventing developmental issues and compensatory mechanisms , such as those mediated by APLP2 and/or APLP1 .", "The results presented here indicate that APP and APLP2 facilitate the release of excitatory synaptic vesicles , likely via the interaction with and functional regulation of the molecular machinery that controls synaptic vesicle exocytosis .", "How exactly these presynaptic APP interactions tune vesicles exocytosis and how this function is regulated remain to be elucidated .", "While a recent study in primary neuronal cells has shown that Aβ can increase Pr by enhancing presynaptic Ca+2 influx ( Fogel et al . , 2014 ) , our findings support a Ca+2 influx-independent mechanism .", "These two mechanisms of action of APP are not mutually exclusive and could act synergistically to control neurotransmitter release .", "Inherited or acquired factors leading to altered APP metabolism and Aβ levels could negatively impact this physiological function of APP and , eventually , contribute to the pathogenesis of AD .", "It is worth noting that AID/AICD and JCasp are natural proteolytic products of APP .", "The mechanisms by which cleavage of APP can regulate synaptic transmission are twofold;", "1 ) severing the APP–presynaptic multi-molecular complex from membranes; and", "2 ) liberating cytosolic proteolysis fragments of APP , such as AID/AICD and JCasp , which could act , as we show for Pen1-JCasp , as dominant negative inhibitors of APP .", "AID/AICD and JCasp have very short half-lives and are hardly detectable in vivo ( Cupers et al . , 2001 , Giliberto et al . , 2010 , 2008 , Passer et al . , 2000 ) .", "It therefore appears that evolution has selected mechanisms , which are perhaps expensive in terms of genetic information and energy usage , to tightly regulate the levels of AID/JCasp , suggesting that AID/AICD and JCasp are biologically active and need to be strictly controlled .", "APP plays a central role in the pathogenesis of both sporadic AD and FAD .", "Many previous studies investigating synaptic dysfunction induced by Aβ , which derives from APP processing and is considered the main mediator of AD pathogenesis , have suggested that early pathogenic changes in AD were driven by postsynaptic impairments ( Cissé et al . , 2011 , Ferreira et al . , 2012 , Li et al . , 2009 , Mucke and Selkoe , 2012 , Parameshwaran et al . , 2008 , Roberson et al . , 2011 , Shankar et al . , 2007 , Um et al . , 2012 ) .", "However , the presynaptic function of APP described here , together with evidence that the other familial Alzheimer’s proteins PS1 and PS2 regulate glutamate release in mature neurons by a presynaptic mechanism ( Fogel et al . , 2014 , Wu et al . , 2013 , Xia et al . , 2015 , Zhang et al . , 2009 ) , support the possibility that deficits in neurotransmitter release represents a convergent mechanism involved in AD ." ], [ "Mice were handled according to the Ethical Guidelines for Treatment of Laboratory Animals of the NIH .", "The procedures were described and approved by the Institutional Animal Care and Use Committee ( IACUC ) .", "The App-KO ( Stock No: 004133 ) and Aplp2-KO ( Stock No: 004142 ) mice were originally purchased from The Jackson Laboratory on a C57BL/6J background .", "Mice were crossed to obtain App+/-/Aplp2+/- animals .", "App+/-/Aplp2+/- mice were crossed to 129 mice to select App+/-/Aplp2+/- animals on a C57BL/6J/129 mixed background .", "App+/-/Aplp2+/- animals on a C57BL/6J/129 were crossed and WT , App-KO , Aplp2-KO , or App/Aplp2-dKO were used for experiments .", "C57BL/6J mice were generated at the Einstein animal facility .", "The brain samples used for proteomic experiments were prepared as follows .", "Brains were homogenized in 5 mM ( 4- ( 2-hydroxyethyl ) -1-piperazineethanesulfonic acid ( HEPES ) /NaOH pH 7 . 4 , 1 mM ethylenediaminetetraacetic acid ( EDTA ) , 1 mM ethylene glycol tetraacetic acid ( EGTA ) , 0 . 25 M sucrose supplemented with protease and phosphatase inhibitors ( Thermo Scientific ) .", "Brain homogenates were centrifuged at 800g for 10 min .", "Supernatant was collected and centrifuged at 9200g for 10 min to obtain the pellet ( P2 ) and the supernatant ( S2 ) fractions .", "The P2 fraction , which contains synaptosomes , was solubilized in 0 . 32 M sucrose , 20 mM Tris-base pH 7 . 4 , 1 mM EDTA , incubated for 30 min at 4°C and centrifuged at 21000g for 15 min .", "The supernatant ( LS1 ) was collected .", "The S2 and LS1 fractions were combined and used for the proteomic experiments .", "For Western blot analysis on hippocampal slices treated with either Pen1-JCasp or Pen1-ScJCasp ( Figure 5 ) , P2 fractions were prepared and solubilized in 1% TritonX100 RIPA buffer for 30 min at 4°C and centrifuged at 21000g .", "The triton soluble supernatant fractions were separated on a 16 . 5% Tris-Tricine sodium dodecyl sulfate polyacrylamide gel electrophoresis ( SDS-PAGE ) , to obtain a better separation of APP-CTF species , and transferred onto nitrocellulose membranes .", "For Western blot analysis of pull-downs pre-incubated with JCasp or ScJCasp ( Figure 1D ) , samples were separated on a 4–20% SDS-PAGE and transferred onto nitrocellulose membranes .", "The following antibodies were used in Western blots: anti-APP C-terminal AbD ( Zymed ) , anti-APP N-terminal 22C11 ( Millipore ) , anti-Actin ( Cell Signaling ) , anti-Syp ( Synaptic Systems , Goettingen , Germany ) , anti-Vamp2 ( Synaptic Systems ) , anti-Syt2 ( Synaptic Systems ) and anti-Ddb11 ( Cell Signaling ) .", "The StrepTag peptides were immobilized on StrepTactin column ( IBA , St . Louis , MO ) , incubated with StrepTactin pre-cleared WT brain S2+LS1 fractions , and washed and eluted with desthiobiotin .", "The following analysis was performed by MSBioworks ( Ann Arbor , Michigan ) .", "To prepare samples for mass spectrometry , the volume of each sample was reduced to 50 μL by vacuum centrifugation , 20 μL of each concentrated sample was processed by SDS-PAGE using a 10% Bis-Tris NuPAGE gel ( Invitrogen ) with the 2- ( N-morpholino ) ethanesulfonic acid ( MES ) buffer system , the gel was run approximately 2 cm .", "The mobility region was excised into 10 equal sized segments and in-gel digestion was performed on each using a robot ( ProGest , DigiLab ) with the following protocol: washed with 25 mM ammonium bicarbonate followed by acetonitrile .", "Reduced with 10 mM dithiothreitol at 60°C followed by alkylation with 50 mM iodoacetamide at RT .", "Digested with trypsin ( Promega ) at 37°C for 4 hr .", "Quenched with formic acid and the supernatant was analyzed directly without further processing .", "Each digest was analyzed by nano liquid chromatography–mass spectrometry ( LC/MS/MS ) with a Waters NanoAcquity High Performance Liquid Chromatography ( HPLC ) system interfaced to a Thermo Fisher Q Exactive mass spectrometer .", "Peptides were loaded on to a trapping column and eluted over a 75 μm analytical column at 350 nL/min; both columns were packed with Jupiter Proteo resin ( Phenomenex ) .", "The mass spectrometer was operated in data-dependent mode , with MS and MS/MS performed in the Orbitrap at 70 , 000 and 17 , 500 full width at half maximum ( FWHM ) resolution , respectively .", "The 15 most abundant ions were selected for MS/MS .", "The mice were anesthetized by intraperitoneal injection ( IP ) of Avertin and perfused with ~20–30 ml of Soresan’s Buffer followed by 30–50 ml of 4% paraformaldehyde ( PFA ) fixative .", "After the perfusion brains were quickly removed and fixed in 4% PFA in Soresan’s Buffer overnight at 4°C .", "Brains were sectioned ( 50 μm thick ) with a vibratome; slices were washed with phosphate buffer saline ( PBS ) and incubated for 90 min with pre-blocking agent containing 10% normal goat serum ( NGS ) in PBS , 0 . 02% saponin , and 0 . 1% Triton .", "Slides were washed in PBS and incubated overnight with primary antibodies Neun ( 1:300 , Millipore ) , APP Y188 ( 1:200 , Abcam ) , and Snap25 ( 1:300 , Synaptic Systems ) prepared in pre-blocking agent solution .", "After washes , sections were incubated with secondary Alexa 488 and 594 fluorescent antibodies ( Molecular Probes ) at room temperature for 2 . 5 hr .", "After washes , slices were fixed on glass slides and incubated with the antifade ( Prolong Gold ) .", "Images were captured using SP5 confocal microscope ( Leica ) , under 10X and 63X magnification .", "We used C57BL/6J WT mice aged 3–12 weeks of both sex ( experiments shown in Figures 3 , 4 , 5 , 7 , 8 ) , as well as 16–28-day-old C57BL/6J/129 hybrid mice , which were either WT , App-KO , Aplp2-KO , or App/Aplp2-dKO ( experiments shown in Figures 9 , 11 , 12 ) .", "The experimenter was blind to genotype and/or treatment .", "Coronal brain slices containing the hippocampal formation were prepared as previously described ( Talani et al . , 2011 ) .", "In brief , animals were anesthetized with isoflurane , following the University of California San Diego Institutional Animal Care and Use Committee ( IACUC ) approved protocol .", "Brains were rapidly removed from the skull and placed in an ice-cold modified ACSF solution containing ( in mM ) : 215 sucrose , 2 . 5 KCl , 1 . 6 NaH2PO4 , 4 MgSO4 , 1 CaCl2 , 4 MgCl2 , 20 glucose , 26 NaHCO3 ( pH 7 . 4 equilibrated with 95% O2 and 5% CO2 ) .", "Coronal brain slices ( 400 μm thick ) were prepared with a Vibratome VT1200S ( Leica Microsystems , Germany ) and then incubated at room temperature in a physiologic ACSF , containing ( in mM ) : 120 NaCl , 3 . 3 KCl , 1 . 2 Na2HPO4 , 26 NaHCO3 , 1 . 3 MgSO4 , 1 . 8 CaCl2 , 11 Glucose ( pH 7 . 4 equilibrated with 95% O2 and 5% CO2 ) .", "The hemi-slices were transferred to a recording chamber perfused with ACSF at a flow rate of ~2 ml/min using a peristaltic pump; experiments were performed at 28 . 0 ± 0 . 1°C .", "All recordings were performed using a multiclamp 700B amplifier ( Molecular Devices ) .", "For extracellular field recordings ( fEPSP recordings ) , a patch-type pipette was fabricated on a micropipette puller ( Sutter Instruments , Novato , CA , USA ) , filled with 1M NaCl , and placed in the middle third of stratum radiatum in area CA1 .", "Field excitatory postsynaptic potentials ( fEPSPs ) were evoked by activating SCs with a patch-type pipette ( monopolar stimulation ) filled with ACSF and placed in the middle third of stratum radiatum 150–200 μm away from the recording pipette and at approximately the same slice depth ( 150–200 μm ) .", "Square-wave current pulses ( 60 μs pulse width ) were generated by a stimulus generator ( Master 8 , AMPI ) and delivered through a stimulus isolator ( Isoflex , AMPI ) .", "I/O function was generated by stimulating in 0 . 1 mA steps from 0 . 1 to 1 . 0/1 . 2 mA .", "Paired-pulse ratio ( PPR ) was measured by delivering two stimuli at 20 , 40 , 80 , 200 , 500 , and 1000 ms inter-stimulus intervals .", "Synaptic facilitation was examined by repetitive stimulation ( 10 stimuli ) at 1 , 5 , 10 , and 20 Hz .", "Depletion of synaptic transmission was elicited by a long train ( 500 stimuli , 28 Hz ) in 5 mM extracellular Ca2+ and in the presence of the NMDA receptor antagonist 2-amino-5-phosphopentanoic acid ( DL-AP5 ) ( 100 µM ) .", "Recovery was assessed with nine stimuli at 2 Hz .", "Depletion and recovery plots were generated by normalizing responses to the peak amplitude of the fEPSP in the depletion train .", "The stimulus strength was adjusted so that the basal synaptic amplitude was similar ( e . g . , 0 . 5–1 . 0 mV ) across experiments .", "To monitor miniature excitatory postsynaptic currents ( mEPSCs ) , patch-clamp whole-cell recordings were performed from CA1 pyramidal cells , and we used an infrared differential interference contrast microscope ( Nikon eclipse E600FN , Morrel Instrument Company Inc , Melville New York ) .", "The cells were voltage-clamped at -65 mV , and the patch pipette ( resistance 3–6 MΩ ) was filled with an internal solution containing ( in mM ) : 131 CsCl , 8 NaCl , 1 CaCl2 , 10 EGTA , 10 Glucose , 10 HEPES ( pH = 7 . 3 , 286 mmol/Kg ) .", "Series resistance ( 5 and 15 MΩ ) was constantly monitored throughout the experiments by delivering a -5 mV , 80 ms voltage step , and cells with >10% change in series resistance were excluded from analysis .", "mEPSCs were recorded in the presence of 1 μM TTX and 15 μM 1 ( S ) , 9 ( R ) ( − ) bicuculline methiodide , to block action potentials and GABA-A receptors , respectively .", "To calculate average mEPSCs recorded from at least three ( Figures 4 and 8 ) and five ( Figure 12 ) CA1 pyramidal neurons ( between ~1000 and 600 events each ) were normalized , aligned , and averaged .", "Data acquisition and statistical analysis .", "Output signals were acquired at 5 kHz , filtered at 2 . 4 kHz , and stored online using pCLAMP 10 . 3 Electrophysiology Data Acquisition and Analysis Software ( Molecular Devices ) .", "mEPSCs were analyzed using Mini Analysis software ( Synaptosoft ) .", "In all cases the experimenter was blind to genotype and/or treatment .", "Data are presented as means ± SEM .", "Statistical comparisons of pooled data were performed by unpaired t-test or ANOVA ( one- or two-way ) using Prism software ( GraphPad , San Diego , CA , USA ) .", "A p value of <0 . 05 was considered statistically significant .", "Drugs were obtained from Sigma-Aldrich 1 ( S ) , 9 ( R ) ( − ) bicuculline methiodide , Biotium ( TTX ) and Enzo Life Science ( DL-AP5 ) .", "Stock solutions were prepared in water and dimethyl sulfoxide ( DMSO ) and added to the ACSF as needed ." ] ]

[ "The amyloid precursor protein ( APP ) , whose mutations cause familial Alzheimer’s disease , interacts with the synaptic release machinery , suggesting a role in neurotransmission .", "Here we mapped this interaction to the NH2-terminal region of the APP intracellular domain .", "A peptide encompassing this binding domain -named JCasp- is naturally produced by a γ-secretase/caspase double-cut of APP .", "JCasp interferes with the APP-presynaptic proteins interaction and , if linked to a cell-penetrating peptide , reduces glutamate release in acute hippocampal slices from wild-type but not APP deficient mice , indicating that JCasp inhibits APP function . The APP-like protein-2 ( APLP2 ) also binds the synaptic release machinery .", "Deletion of APP and APLP2 produces synaptic deficits similar to those caused by JCasp .", "Our data support the notion that APP and APLP2 facilitate transmitter release , likely through the interaction with the neurotransmitter release machinery .", "Given the link of APP to Alzheimer’s disease , alterations of this synaptic role of APP could contribute to dementia ." ]

[ "Alzheimer’s disease has been linked to mutations in a gene encoding the amyloid precursor protein ( APP ) .", "Mutations in this gene cause early onset Alzheimer’s disease in some families .", "Studies in animals suggest this mutant form of the protein may interfere with the messages sent between brain cells .", "But it remains unclear how this protein works even when it isn’t mutated .", "One reason it has been difficult to figure out how APP works is because it is a long protein and is often cut into smaller , but still functional proteins .", "It is unclear which parts of the protein are involved in sending messages between brain cells , but previous research has allowed scientists to zero in on one stretch of the protein containing just 50 amino acids .", "Now , Fanutza , Del Prete et al . reveal that this short stretch of APP interacts with the proteins that control the release of glutamate , one of the brain’s most important excitatory neurotransmitter .", "This stretch is cut from the full protein and released into the cell as a protein fragment ( or peptide ) .", "Further experiments showed that this naturally occurring peptide interferes with the activity of APP , and in doing so reduces the release of glutamate from brain cells in mice .", "However , there was no effect on glutamate release when this peptide was introduced inside brain cells from mutant mice that lacked APP .", "Together , the results show that this 50-amino acid stretch of APP promotes glutamate release between brain cells , and that a peptide containing the same stretch of the protein interferes with this process .", "Altered glutamate neurotransmission could explain the memory loss and thinking problems seen in early Alzheimer’s disease .", "Therefore , drug treatments that aim to restore normal glutamate transmission may prove a beneficial therapeutic strategy in Alzheimer’s disease ." ]

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

[ "neuroscience" ]

[ [ "To localize sound sources in the horizontal plane , humans and many other species use submillisecond timing differences in the signals arriving at the two ears ( Ashida and Carr , 2011 ) .", "The ear closer to the source receives the sound earlier than the other .", "These interaural time differences ( ITDs ) are encoded in the auditory brainstem by binaural neurons , which are tuned to both frequency and ITD .", "An influential theory proposes that ITD is represented by the activity pattern of cells with heterogeneous tunings , a pattern code for sound location ( Jeffress , 1948 ) .", "In a stronger version , ITD is represented by the identity of the most active cell in each frequency band , a labeled line code for sound location .", "Although this theory has proved successful in barn owls ( Konishi , 2003 ) , discrepancies have been observed in mammals .", "In particular , at low frequencies , many cells have best delays ( BDs ) larger than the physiological range of ITDs experienced by the animal ( McAlpine et al . , 2001 ) .", "In a labeled line code , these cells would not have any function .", "An alternative theory was proposed , in which ITD is coded not by the firing of individual cells , but by the relative summed activity of each hemisphere , a summation code for sound location ( Stecker et al . , 2005; Grothe et al . , 2010 ) .", "The nature of the neural code for ITD in mammals is still contentious because it is not known whether the auditory system sums activity or uses cell identity in decoding responses .", "In favor of the summation code hypothesis , cells with large BDs maximize ITD sensitivity of firing rate in the physiological range , whereas they are useless in a labeled line code .", "However , most of the cells with BDs inside the physiological range ( most cells in cats; Kuwada and Yin , 1983; Yin and Chan , 1990a ) actually degrade a summation code because their rates do not vary monotonically with ITD .", "In simple situations where there is a single acoustical stimulus ( e . g . , tone ) with unknown ITD , theoretical arguments show that a summation code is optimal at low frequencies ( Harper and McAlpine , 2004 ) .", "Previous studies have also shown that with simple stimuli , taking into account cell identity rather than simply summing all responses does not improve performance ( Lesica et al . , 2010; Lüling et al . , 2011 ) .", "However , what is optimal in a simple world may not be optimal in an ecological environment .", "In a simple situation where only the ITD varies , the optimal code is the most sensitive one .", "In complex situations where other dimensions also vary , there is a trade-off between sensitivity and robustness , so the optimal code is not the most sensitive one ( Brette , 2010 ) .", "In fact , theory predicts that in complex situations , the heterogeneity of ITD tunings is critical to produce robust estimates .", "To address this , we studied the performance of different decoders in increasingly complex situations , including variations in spectrum , background noise , and head-related acoustic filtering .", "We found that summing cell responses is strongly suboptimal and that heterogeneity in tunings is information rather than noise ." ], [ "Previous studies have tested the performance of simple decoders based on single-unit cell responses to acoustical stimuli ( Fitzpatrick et al . , 1997; Hancock and Delgutte , 2004; Stecker et al . , 2005; Devore et al . , 2009; Miller and Recanzone , 2009; Lesica et al . , 2010; Lüling et al . , 2011 ) .", "However , this approach is limited to a small number of acoustical stimuli and cells .", "Here , we wanted to test the performance of different decoders based on the response of a large population ( up to 480 cells ) to a large variety of sounds totaling 11 hr of sound per cell .", "Obtaining this amount of data from electrophysiological recordings is not feasible because it would correspond to more than 7 months of single-unit recordings .", "We therefore decided to base our comparison on responses generated by a standard computational model , fitted with empirical data , which has been shown to produce realistic responses ( ‘Materials and methods’ ) .", "First , we sampled many cells from a distribution of BD vs best frequency ( BF ) ( Figure 1A , left ) .", "For guinea pigs , the distribution was defined by the measured mean and variance of BD as a function of BF ( McAlpine et al . , 2001 ) .", "For cats , we fitted a distribution to a set of measurements of BDs and BFs in 192 cells ( the source data was in terms of characteristic frequency rather than BF , but these are equivalent for the linear model used here ) ( Joris et al . , 2006 ) .", "The cells are then modeled as generalized cross-correlators with an internal delay BD ( Yin et al . , 1987 ) ( Figure 1A , right ) .", "Figure 1B illustrates the details of the model .", "We first model the acoustical propagation of the sound to the two ears .", "In the first part of this study , we consider only fixed ITDs , ignoring diffraction effects .", "In the second part , we move toward more realistic cues , using measured head-related transfer functions ( HRTFs ) .", "The signals are then band-pass filtered around the cell’s BF using gammatone filters and normalized ( representing the saturation seen in bushy cells; Kuenzel et al . , 2011 ) and then crosscorrelated with an internal delay equal to the cell’s BD ( ‘Materials and methods’ ) .", "The result is the firing rate of the cell , and we generate spikes with Poisson statistics .", "Figure 1C displays the responses of 480 cells of the guinea pig model to white noise ( left column ) and to a natural sound ( right column ) at two different ITDs ( top and bottom ) .", "We will then estimate the sound’s ITD from these population responses , using various decoders . 10 . 7554/eLife . 01312 . 003Figure 1 . Overview of model .", "( A ) The distribution of best delay vs best frequency for cells in the guinea pig model ( left ) , with the physiological range of ITDs shown in gray , and a sample tuning curve ( right ) .", "( B ) Illustration of the model: a sound source is filtered via position-dependent HRTFs to give left and right channels .", "For each best frequency on each channel , the signal undergoes cochlear filtering using gammatone filters .", "An internal delay is added , and the two channels are combined and sent to a binaural neuron model that produces Poisson distributed spikes .", "( C ) The response of the cells to sounds at two different ITDs ( rows ) for white noise ( left column ) and a natural sound ( right column ) .", "The ITD is indicated by the black dashed line .", "Each cell is surrounded by a disc with a color indicating the response of that neuron ( hot colors corresponding to strong responses ) .", "When two or more discs overlap , each point is colored according to the closest cell .", "The strongest responses lie along the line of the ITD . DOI: http://dx . doi . org/10 . 7554/eLife . 01312 . 003 Figure 2A illustrates the peak and hemispheric decoders .", "A 100-ms sound is presented at 200 µs ITD .", "The peak decoder picks the most active cell and reports its BD as the estimated ITD .", "We observe already that although we chose cells with BFs in a narrow frequency band ( 640–760 Hz ) , the peak decoder performs poorly because of the noise in spiking .", "Therefore , we introduce a smoothed peak decoder .", "We first discard the information about BF , and simply consider the spike count of cells as a function of their BD .", "This relationship is smoothed to reduce noise , and we take the BD at the peak of the smoothed curve ( one of the possible variations of crosscorrelation models; Stern and Trahiotis , 1995 ) .", "Smoothing could be neurally implemented by pooling the activity of cells with similar tuning .", "This decoder is less noisy .", "Finally , we consider the hemispheric decoder , in which we also discard information about BD in each hemisphere .", "To simplify , we define each hemisphere as the set of cells with BDs of the same sign .", "We calculate the total spike count for each hemisphere ( yellow and orange rectangles ) and compute the difference , normalized by the total activity .", "This gives a value between −1 and 1 , the hemispheric difference , which varies systematically with ITD ( Figure 2A , right ) .", "Therefore , from the observation of the difference , one can invert the relationship and infer the ITD .", "Note , however , that this relationship depends on the frequency band in which the hemispheric difference is computed .", "In blue , cells are picked with BFs around 700 Hz and the hemispheric difference varies almost linearly with ITD .", "In purple , cells are picked with BFs around 1300 Hz and the curve is a sigmoid .", "More importantly , ambiguities start to occur at these high BFs: for example , a hemispheric difference of −0 . 8 is consistent with ITDs of both 100 and 300 µs .", "This occurs when the physiological range of ITD represents more than one period of the auditory filter’s center frequency . 10 . 7554/eLife . 01312 . 004Figure 2 . Decoders in single frequency bands .", "( A ) Peak and hemispheric decoders .", "Left: response of binaural neurons to sound at ITD = 0 . 2 ms ( dashed line ) , in a narrow frequency band .", "The size of points is proportionate to spike count , and the crossed point corresponds to the highest spike count .", "Middle: the same cell responses are displayed as best delay vs spike count ( note the different horizontal axis ) .", "The solid black line is the Gaussian smoothed spike count , whose peak ( circle ) is the ITD estimate .", "The maximally responsive neuron is also indicated with a circle for comparison .", "The yellow and orange bars give the mean response of neurons with positive and negative best delays , respectively , from which the normalized hemispheric difference is computed .", "Right: the hemispheric difference as a function of ITD at 700 Hz ( blue ) and 1 . 3 kHz ( purple ) .", "At 1 . 3 kHz , the difference shown by the dashed line gives an ambiguous estimate of the ITD .", "( B ) Mean error for the guinea pig and cat , for the peak ( blue , dashed ) , smoothed peak ( blue , solid ) , hemispheric ( red ) , and pattern match ( green ) decoders .", "The distribution of BD vs BF is shown in the inset .", "( C ) Illustration of the pattern match decoder and a neural circuit that implements it .", "The response ( left ) is compared to two patterns A and B , corresponding to two different ITDs ( right ) .", "Each response neuron is connected to a pattern-sensitive neuron with weights proportional to the stored response of each pattern .", "When the weights match the responses , the output of the pattern-sensitive neuron is strongest . DOI: http://dx . doi . org/10 . 7554/eLife . 01312 . 004 We now systematically test the estimation error of these three decoders for cells whose BFs are in narrow frequency bands within the range 100–1500 Hz ( Figure 2B ) .", "Stimuli are white noise bursts lasting 100 ms . For the hemispheric decoder , we use the hemispheric difference curve calculated in the same frequency band in which it is tested .", "Thus , there is a specific decoder for each frequency band tested , which is the most favorable scenario .", "As expected , the peak decoder performs very poorly , both for the guinea pig and the cat models .", "The two animal models differed by the BD distributions and by the physiological range of ITDs ( 300 µs for the guinea pig model , Sterbing et al . , 2003; 450 µs for the cat model , Tollin and Koka , 2009a ) .", "Using the smoothed peak decoder improves substantially on these results .", "The hemispheric decoder performs better than both decoders for the guinea pig model at all frequencies , but for the cat , it is only better than the smoothed peak decoder for frequencies below 600 Hz .", "Thus , it appears that even for this simple scenario , the hemispheric decoder is a very poor decoder of ITD for the cat model , except at very low frequency .", "The fact that the estimation error of the hemispheric decoder starts increasing at a lower frequency for the cat than for the guinea pig model was expected based on the larger head size of the cat ( Harper and McAlpine , 2004 ) .", "The reasons for the limitations of the different decoders are simple .", "Because the peak decoder selects a single cell , its estimation error reflects the level of noise in individual responses , which is high .", "The smoothed decoder improves on this matter , but still mostly uses the responses of cells with similar tuning .", "In addition , at low frequencies , both estimators rely on the responses of a small pool of cells with BDs inside the physiological range .", "The hemispheric decoder sums all responses , which reduces noise but also discards all information about BF and BD .", "We introduce the pattern match decoder , a simple decoder that addresses both problems ( Figure 2C ) .", "We calculate the average response of cells to sounds presented at each ITD to be identified .", "This population response , which we call a pattern , is stored in a vector ( w1 , … , wn ) , normalized to have length 1 .", "When a sound is presented , the cell responses are compared with the patterns by computing a normalized dot product between the responses and the patterns , varying between 0 ( perfectly dissimilar ) and 1 ( perfectly similar ) ( ‘Materials and methods’ for formulae ) .", "This can be implemented by a single-layer neural network in which the output neurons encode the preferred ITD and the synaptic weights represent the patterns .", "The reported ITD is the ITD associated to the most similar pattern , that is , with the highest dot product .", "Figure 2B also shows the performance of the pattern matching decoder .", "As for the hemispheric decoder , patterns were computed in the same frequency band in which the decoder is tested .", "The pattern match decoder performs better than both the other decoders , both for the guinea pig and the cat models .", "The difference with the hemispheric decoder is very large for the cat model , but for the guinea pig model , it only starts being substantial above 1 kHz .", "The pattern match decoder combines the advantages of the hemispheric and peak decoders: it averages spiking noise over all cells , but it still uses individual information about BF and BD .", "The purpose of introducing this decoder is not to suggest that the auditory system extracts information about sound location in this exact way , but rather to estimate how much information can be obtained from the heterogeneous responses of these neurons .", "We also tested several other standard decoders from machine learning , including optimal linear decoding , maximum likelihood estimation , and nearest neighbor regression , but the pattern match decoder outperformed them in all cases and so we do not present the results of these decoders here ( although see Figure 3—figure supplement 1 for a sample of these results ) .", "The estimation task in Figure 2 was very simple because we trained and tested the decoders in the same narrow frequency bands .", "In Figure 3 , we investigate the issue of frequency integration .", "All decoders are now trained with white noise at various ITDs , considering all cells with BFs between 100 Hz and 1 . 5 kHz .", "For the hemispheric decoder , this means that we pool the responses of all cells in the same hemisphere , for all BFs , and we use a single broadband hemispheric difference curve to estimate ITDs .", "Decoder performance is then tested with white noise .", "For this more realistic task , it appears that the error made by the pattern match decoder is about half the error of the hemispheric decoder for the guinea pig model .", "For the cat models , this difference is even larger .", "In fact , it turns out that for the cat , the smoothed peak decoder performs better than the hemispheric decoder .", "To understand why , we now test the decoders on band-pass noises , as a function of the center frequency , while the decoders are still trained with broadband noise ( Figure 3B ) .", "This test addresses the robustness of these decoders to changes in sound spectrum .", "We make two observations .", "First , all decoders perform much worse than when decoders are trained and tested in the same frequency bands ( compare with Figure 2B; the unsmoothed peak decoder performs very poorly and is not shown ) .", "This means that frequency integration is indeed an issue .", "Second , the hemispheric decoder performs worse than the two other decoders above 700 Hz for the guinea pig models and above 500 Hz for the cat .", "This was expected for two reasons: ( 1 ) the hemispheric difference is ambiguous at high frequency ( above about 1200 Hz for both animals ) , and ( 2 ) the hemispheric difference depends not only on ITD but also on frequency ( Figure 2A , right ) .", "We attempt to solve the first problem by discarding all cells with BF higher than a specified cutoff frequency ( Figure 3C ) .", "Performance is tested again with white noise .", "Both for the guinea pig and the cat models , the error of the hemispheric decoder starts increasing when cells with BF above 1 . 2 kHz are included .", "For this reason and because the hemispheric difference becomes ambiguous above 1 . 2 kHz in both models , we restrict to cells with BF <1 . 2 kHz in the rest of this study .", "We note , however , that the error of the pattern match decoder continues decreasing as cells with high frequency are added . 10 . 7554/eLife . 01312 . 005Figure 3 . Integration across frequencies .", "( A ) Mean error in estimating ITD for white noise using the smoothed peak ( blue ) , hemispheric ( red ) , and pattern match ( green ) decoders , as a function of the number of binaural cells .", "Training and testing of the decoders are both performed using white noise .", "( B ) Mean error as a function of frequency band when decoders are trained on white noise but tested on band-pass noise centered at the given frequency .", "Notice the different vertical scale between ( A and B ) .", "( C ) Performance when cells with a frequency above the cutoff are discarded .", "( D ) Mean error and bias to center in the decoders for guinea pig ( with a maximum frequency of 1 . 2 kHz ) when trained on white noise and tested on colored noise . DOI: http://dx . doi . org/10 . 7554/eLife . 01312 . 00510 . 7554/eLife . 01312 . 006Figure 3—figure supplement 1 . Comparison with standard machine learning decoders . As for Figure 3 , except that the main decoders are shown in light colors , and there are two additional decoders: ridge regression ( magenta ) and nearest neighbor regression ( black ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 01312 . 006 We have seen that estimation error depends on the frequency band of the presented sound ( Figure 3B ) .", "This observation extends to broadband sounds that vary in spectrum .", "We tested the estimation performance for the guinea pig models when the decoders are trained on white noise and tested with 1/fα noise: from white noise ( α = 0 ) to pink ( α = 1 ) and brown ( α = 2 ) ( Figure 3D ) .", "The hemispheric decoder is not robust to changes in spectrum , as the error increases with noise color α .", "The pattern match decoder shows the same trend , but the error remains constant on a larger range .", "The error of the smoothed peak decoder does not depend on sound spectrum .", "The main reason for the lack of robustness of the hemispheric decoder is shown in Figure 3D ( bottom ) .", "As α increases , the estimate of the ITD becomes more and more biased to the center .", "This is because with high α , every cell receives more low frequencies than high frequencies compared to the white noise case , and therefore the hemispheric difference curve changes and becomes flatter ( Figure 2A , right ) .", "Note that this happens not by the recruitment of more low-frequency cells but also by the change in the hemispheric difference for all cells .", "We now attempt to improve frequency integration in the hemispheric decoder by taking into account the change in hemispheric difference with frequency ( Figure 4A ) .", "The ITD tuning curves have different shapes , depending on the cell’s BF ( left ) .", "As a result , the hemispheric difference in each frequency band varies with the center frequency ( middle ) .", "The curves are shallower in low frequency and sharper in high frequency ( right ) .", "In fact , the slope is expected to be proportional to frequency: the hemispheric difference is determined by the interaural phase difference , which is the product of frequency and ITD .", "Therefore , we fix this issue by normalizing the cell responses by their BF in the calculation of the hemispheric difference ( ‘Materials and methods’ ) .", "This produces hemispheric differences with similar slopes in all frequency bands .", "Note that there are constant biases due to the fact that the cells’ BDs are not exactly symmetrical between the two hemispheres ( only their distribution is ) . 10 . 7554/eLife . 01312 . 007Figure 4 . Frequency-dependent improvements .", "( A ) Comparing hemispheric differences across frequency channels .", "In each plot , color indicates frequency with red being high frequency and blue being low frequency .", "Left: tuning curves for a few binaural neurons .", "Middle: hemispheric difference ( L − R ) / ( L + R ) .", "Right: frequency-dependent hemispheric difference ( 1/f ) ( L − R ) / ( L + R ) .", "( B ) Mean error as a function of frequency band in the guinea pig model , for hemispheric ( red ) and pattern match ( green ) decoders ( dashed lines ) , and frequency-dependent hemispheric and pattern match ( solid ) decoders .", "The shaded regions show the difference between the simple and frequency-dependent versions .", "The dotted lines show the mean error for band-pass noise if the decoder is trained and tested on the same frequency band , as shown in Figure 2B ( guinea pig ) .", "This represents the lower bound for the error . DOI: http://dx . doi . org/10 . 7554/eLife . 01312 . 007 This frequency-dependent correction indeed improves ITD estimation when the decoder is trained on broadband noise and on band-passed noise ( Figure 4B ) .", "However , the error still remains higher than in the simple case when the decoder is trained and tested in the same frequency band .", "In the same way , we improved frequency integration for the pattern match decoder by calculating intermediate estimates in each frequency band and combining the results ( ‘Materials and methods’ ) .", "This correction improves the performance above 600 Hz , where it is close to the performance obtained in the simple case .", "In the remainder of this study , we only consider these two frequency-corrected decoders .", "We then test the decoders in increasingly realistic situations .", "First , we consider the effect of background noise on performance ( Figure 5 ) .", "Interaural correlation is decreased by adding dichotic background noise to the binaural signals , and the estimation error is computed as a function of signal-to-noise ratio ( SNR ) .", "All decoders were trained in quiet .", "In all cases , the pattern match decoder performs best , but for the guinea pig models , it substantially outperforms the hemispheric decoder at low SNR , whereas it showed similar performance in quiet .", "Interestingly , the smoothed peak decoder also outperforms the hemispheric decoder at SNR below 10 dB , both for the guinea pig and the cat models .", "Indeed , although this decoder performs worst in quiet , it proves more robust than the hemispheric decoder .", "The poor performance of the hemispheric decoder can again be accounted for by a bias problem .", "At high SNR , the hemispheric difference curves become shallower , which implies that ITD estimation is biased toward the center .", "The problem is less present for the pattern match decoder .", "Note that the smoothed peak decoder tends to be biased away from the center .", "This is simply because BDs are more represented away from the center . 10 . 7554/eLife . 01312 . 008Figure 5 . Background acoustic noise .", "( A ) Illustration of protocol: a binaural sound is presented with a given ITD , with independent white noise added to the two channels .", "( B ) Mean error ( left column ) and central bias ( right column ) at different signal to noise levels .", "Decoders are smoothed peak ( blue ) , hemispheric ( red ) , and pattern match ( green ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 01312 . 008 Previously , we considered a simplistic model of sound propagation , in which sounds are simply delayed .", "In reality , sounds are diffracted by the head .", "A better description of this process is that sounds arriving at the two ears are two filtered versions of the original sound , with filters depending on source direction .", "These are called HRTFs and can be measured in anechoic chambers .", "We measured high-resolution HRTFs of a stuffed guinea pig in a natural posture ( Figure 6A ) .", "It is known that diffraction produces ITDs that depend on frequency for the same source direction , with larger ITDs in low frequency ( Kuhn , 1977 ) .", "We find the same pattern in our measurements ( Figure 6B ) , and the range of ITDs is similar to previously reported measurements in live guinea pigs ( Sterbing et al . , 2003 ) .", "For the cat model , we used HRTFs measured in an anesthetized cat ( Tollin and Koka , 2009a ) . 10 . 7554/eLife . 01312 . 009Figure 6 . Realistic head-related transfer functions .", "( A ) Photograph of stuffed guinea pig used for HRTF recordings , and three pairs of left/right ear impulse responses corresponding to the directions marked on the photograph .", "( B ) Frequency dependence of ITD for the three azimuths shown in panel ( A ) , in guinea pig and cat HRTFs .", "( C ) Mean response of the model to white noise stimuli at the same azimuth ( 90° ) for both animals , the frequency-dependent ITD curve is shown for this azimuth ( dashed ) .", "( D ) Performance of the model as a function of the number of cells for hemispheric ( red ) and pattern match ( green ) decoders . DOI: http://dx . doi . org/10 . 7554/eLife . 01312 . 009 Figure 6C displays the cell responses for sounds presented at 90° azimuth , where we used the HRTFs to filter the sound in an acoustically realistic way .", "We then test the estimation error in azimuth , rather than in ITD , for white noise presented in quiet ( Figure 6D ) .", "For both animals , the pattern match decoder is substantially better than the hemispheric decoder .", "Indeed , since the hemispheric decoder discards all information about BF and BD , it cannot take advantage of the frequency variation of ITDs , whereas the pattern match decoder does .", "The difference is particularly striking for the cat due to its larger head size .", "We have argued that the hemispheric decoder performs poorly because it discards the information present in the heterogeneity of ITD tunings of the cells .", "We demonstrate this point in Figure 7A by varying the amount of heterogeneity in the BDs of the guinea pig model .", "The standard deviation of the BD is multiplied by a ‘spread factor’: below 1 , the BD distribution is less heterogeneous than in the original distribution; above 1 , it is more heterogeneous .", "We then test the estimation error for white noise as a function of spread factor .", "When the BDs are less heterogeneous , there is little difference between the performance of the hemispheric and pattern match decoder .", "But as heterogeneity increases , the pattern match decoder performs better , whereas the hemispheric decoder shows little change in performance .", "Therefore , heterogeneity of tunings is indeed useful to estimate the ITD , and pooling the responses discards this information .", "Performance remains stable when the distribution is made more heterogeneous than the actual distribution ( spread factor >1 ) . 10 . 7554/eLife . 01312 . 010Figure 7 . Effect of heterogeneity and lesions .", "( A ) Mean error for the hemispheric ( red ) and pattern match ( green ) decoders in the guinea pig model , depending on the spread of the best delays , for white noise presented with acoustic noise ( SNR between −5 and 5 dB , no HRTF filtering ) .", "For every frequency , the standard deviation of BDs is multiplied by the spread factor: lower than 1 denotes less heterogeneous than the original distribution ( top left ) , greater than 1 denotes more heterogeneous ( top right ) .", "Dashed lines represent the estimation error for the original distribution .", "( B ) Mean error for the pattern match ( green ) and smooth peak ( blue ) decoders before ( dashed ) and after ( solid ) lesioning one hemisphere in the guinea pig , as a function of presented ITD .", "The model is retrained after lesioning .", "The error curves are Gaussian smoothed to reduce noise and improve readability . DOI: http://dx . doi . org/10 . 7554/eLife . 01312 . 010 Lesion studies in cats ( Jenkins and Masterton , 1982 ) and in humans ( Litovsky et al . , 2002 ) show that when one inferior colliculus is removed , the sound localization performance in the contralateral field drops but remains almost intact in the ipsilateral field .", "This is not compatible with an ITD estimation based on the comparison between the activity of the two hemispheres .", "We simulated a hemispheric lesion in the pattern match and smoothed peak decoders ( Figure 7B ) , by removing all cells with negative BDs .", "The performance for positive ITDs is essentially unchanged , whereas it is highly degraded for negative ITDs , especially for the smoothed peak decoder .", "Lesion data indicate that sound localization performance is greatly degraded in the contralateral hemifield , but not completely abolished , which would discard the smoothed peak decoder—although lesions might not have been complete , and those were free-field experiments involving other cues than ITD .", "Finally , we test the estimation performance in barn owls and humans , for white noise filtered through measured HRTFs ( ‘Materials and methods’ ) ( Figure 8 ) .", "For barn owls , we used a previously measured distribution of BD vs BF ( Wagner et al . , 2007 ) .", "Barn owls are sensitive to ITDs in very high frequency ( about 2–8 kHz ) , and therefore , as expected , the hemispheric decoder performs very badly compared to the pattern match decoder ( Figure 8A ) .", "For humans , the distribution of BD vs BF is unknown .", "Some indirect evidence from MRI and EEG studies suggests that BD is not uniformly distributed ( Thompson et al . , 2006; Briley et al . , 2013 ) .", "We tested three possibilities: uniformly distributed BD within the pi-limit , similar to what is observed in birds ( Figure 8B ) , BD distribution of the guinea pig model ( Figure 8C ) , and BD distribution of the cat model ( Figure 8D ) .", "In all cases , the estimation error of the hemispheric decoder is very high , an order of magnitude larger than human sound localization acuity ( on the order of 3° ) ( Carlile et al . , 1997 ) . 10 . 7554/eLife . 01312 . 011Figure 8 . Humans and owls .", "( A ) Mean error for the pattern match ( green ) and hemispheric ( red ) decoders for the barn owl model , with sounds presented through measured HRTFs .", "( B ) Performance in the human model with uniformly distributed best interaural phase differences .", "( C ) Performance in the human model with best delays distributed as in the guinea pig model .", "( D ) Performance in the human model with best delays distributed as in the cat model . DOI: http://dx . doi . org/10 . 7554/eLife . 01312 . 011 Our results can be compared with behavioral performance measured in psychophysical experiments .", "One may immediately object that the pattern decoder is in fact too accurate compared to natural performance , in particular for humans ( Figure 8 ) .", "Therefore , we must stress again that the performance obtained by a decoder in a given context is always overestimated , compared to the same decoder adapted to a more general context , ‘even when tested with the same sounds’ .", "To give an example , all decoders are much more accurate when trained and tested on a single narrow frequency band ( Figure 2B ) than when trained with broadband sounds and tested with exactly the same narrowband sounds ( Figure 3B ) .", "Additional imprecision is introduced by other uncontrolled sources of variability in the sounds , many of which we have not considered:1 .", "Sound locations differ not only by azimuth but also elevation and distance , both of which impact binaural cues2 .", "Reflections on the ground and objects impact ITDs ( Gourévitch and Brette , 2012 ) 3 .", "There are often multiple sound sources4 .", "Sound sources are generally not point sources and are directional5 .", "Natural sounds have widely diverse spectrotemporal properties A sound localization system adapted for the full range of ecological variability necessarily performs worse in a narrower range of conditions than a system specifically optimized for that narrow range .", "In addition , acoustical cues must be associated with sound location through some feedback mechanism , and therefore sound localization acuity is constrained by the precision of this feedback .", "Indeed a comparative study across 23 mammalian species shows that sound localization acuity is best explained by the width of the field of best vision ( Heffner and Heffner , 1992 ) .", "Therefore , our model results should be understood as a lower bound for the accuracy of these decoders .", "In particular , the poor performance of the hemispheric decoder is actually optimistic , all the more so as we made specific efforts to enhance it by taking into account nonlinearities ( Figure 2A ) and by applying frequency-dependent corrections ( Figure 4 ) .", "Most psychophysical studies in animals have focused on the measurement of the minimum audible angle ( MAA ) , which is a discrimination threshold for sources near the midline .", "In cats , the MAA is about 5° for broadband noises longer than 40 ms ( defined as the speaker separation given 75% correct responses ) ( Casseday and Neff , 1973; Heffner and Heffner , 1988a ) .", "Tollin et al . ( 2005 ) measured accuracy in an absolute localization study in the −25° to 25° range .", "When the cat’s head is unrestrained , direction estimates show little bias and the standard deviation is 3–4° , which corresponds to a mean unsigned error ( the measure used in this article ) of 2 . 4–3 . 2° ( assuming normally distributed responses ) .", "In Moore et al . ( 2008 ) , the mean unsigned error was directly reported ( although only for directions near the midline ) and was in the 2–4° range .", "In a behavioral study in which cats were trained to walk to a target speaker in the −90° to 90° range , the animals could do the task with nearly 100% accuracy , with no apparent dependence on speaker azimuth ( Malhotra et al . , 2004 ) —but speakers were spaced by 15° .", "In Figure 6D , we report a mean unsigned error of 5° for the optimized hemispheric model ( including frequency-dependent and nonlinear corrections; error for the pattern decoder was nearly 0° ) .", "The model was trained and tested in quiet with broadband noises , with sources constrained to the horizontal plane .", "Therefore , it is a very optimistic estimate , especially given that the sound localization tasks mentioned above were two-dimensional ( i . e . , the elevation had to be estimated as well ) .", "It could be argued that the behavioral task included additional cues , in particular interaural intensity differences , because the sounds were broadband .", "However , Moore et al . ( 2008 ) showed for sources near the midline that sound localization accuracy in the horizontal plane is very similar for broadband and low-pass filtered noises ( <5 kHz ) , which do not include these cues .", "In cats , the just noticeable difference in ITD is similar for tones of 500 Hz and 1 kHz , about 25 µs ( Wakeford and Robinson , 1974 ) .", "The performance of the pattern decoder is generally not strongly dependent on frequency in the 500–1200 Hz range ( Figures 2–4 ) , whereas the performance of the hemispheric decoder consistently increases with frequency ( between about 500 and 1 kHz in Figure 2 ) .", "Unfortunately , there are no behavioral studies in guinea pigs .", "In the gerbil , another small mammal with low-frequency hearing , the MAA is 27° ( Heffner and Heffner , 1988b ) .", "This makes sound localization acuity in gerbils one of the worst of all mammalian species in which it has been measured ( Heffner and Heffner , 1992 ) .", "Given that the maximum ITD is about 120 µs ( Maki and Furukawa , 2005 ) , the threshold ITD should be about 54 µs ( using Kuhn’s formula; Kuhn , 1977 ) .", "Given that this threshold is so high , and in the absence of absolute localization studies in these two species , it is difficult to discard any model on the basis of the existing behavioral data alone .", "We note however that , for a given accuracy , the hemispheric decoder requires many more neurons than the pattern match decoder ( Figure 3A ) .", "In owls , ITD is a cue to azimuth , whereas interaural level difference is a cue to elevation ( Takahashi et al . , 1984; Moiseff , 1989 ) .", "We found that the mean unsigned error with the hemispheric decoder was greater than 30° , when trained and tested in quiet with HRTFs ( Figure 8A ) .", "Behaviorally , barn owls localize broadband sounds with azimuthal error smaller than about 10° at all azimuths ( Knudsen et al . , 1979 ) , and a large part of this error is due to an underestimate of eccentric azimuths that can be accounted for by a prior for frontal directions ( Fischer and Peña , 2011 ) .", "In humans , behavioral estimates of azimuth are largely dominated by low-frequency ITDs ( Wightman and Kistler , 1992 ) , and the mean unsigned error with broadband noise bursts ( open-loop condition ) is about 5° in the frontal hemifield ( 2–10° depending on azimuth ) in a two-dimensional absolute localization task ( Makous and Middlebrooks , 1990 ) .", "In contrast , the hemispheric decoder has an average error of about 10° in the most favorable scenario ( Figure 8B ) , although sources are constrained to the horizontal plane .", "In summary , our results with the hemispheric decoder appear inconsistent with behavioral data for cats , humans , and owls .", "In contrast , the results obtained by the pattern match decoder imply that there is enough information in the activity of binaural neurons to account for the sound localization accuracy of these species .", "There is insufficient behavioral data in the guinea pig model to distinguish between different decoders ." ], [ "Our results appear to contradict the previous studies showing that hemispheric codes for ITD are optimal ( Harper and McAlpine , 2004 ) and that response patterns do not provide more information than simply summing ( Lesica et al . , 2010; Lüling et al . , 2011 ) .", "However , these studies focused on a simple task , in which only the ITD was allowed to vary .", "This is an elementary task for a decoder because any variation in the pattern of responses can be attributed to a change in sound location .", "It is much more difficult to estimate location independently of irrelevant dimensions found in ecological situations , such as level , spectrum , and background noise .", "This point is related to the concept of ‘overfitting’ in statistical learning theory: an estimator may be very accurate when trained and tested with the same data , while in fact very poor when tested on new data .", "This is precisely what happens with the hemispheric decoder .", "When tested with the same sounds used to calibrate the decoder , its performance is indeed very good for the guinea pig model ( Figure 2B ) , consistently with previous results .", "However , when the decoder is calibrated for broadband sounds and tested with the same sounds as before , performance degrades drastically ( Figure 3B ) .", "Thus , our results directly demonstrate that indiscriminate pooling of the activity in each hemisphere is a poor way to decode information about sound location .", "Figure 7A also directly contradicts the claim that the optimal code for ITD consists of two populations of identically tuned neurons ( Harper and McAlpine , 2004 ) .", "On the contrary , heterogeneity of tunings is critical for robust estimation , consistently with theoretical arguments ( Brette , 2010 ) .", "The discrepancy with previous arguments in favor of hemispheric or ‘slope’ coding seems to stem from a confusion between accuracy and acuity ( Heffner and Heffner , 2005; Tollin et al . , 2005 ) : accuracy measures how well one can estimate the correct value ( absolute localization ) ; acuity measures how easily one can distinguish between two values ( discrimination ) .", "Acuity can be directly related to ITD sensitivity of neural responses ( favoring a ‘slope code’ ) , but accuracy is in fact the relevant ecological concept for the animal .", "It may be objected that the focus on optimality may be irrelevant because animals only need to be accurate enough , given the ecologically relevant tasks .", "However , our results also imply that for a given level of accuracy , the hemispheric decoder requires many more neurons than the pattern match decoder ( Figures 3A and 6D ) , and therefore it is energetically inefficient .", "Although there may be little evolutionary pressure for very accurate sound localization in some species , the same argument does not apply to energy consumption in the brain ( Attwell and Laughlin , 2001 ) .", "Nevertheless , there are also physiological constraints on the way information can be extracted , which we examine in the section ‘Physiological mechanisms’ below .", "It is known that the structure of neural correlations can be critical to optimal decoding .", "There are different sources of correlations: anatomical divergence ( overlap in the sets of presynaptic neurons ) , shared variability due to feedback or lateral connections , and stimulus-dependent variability ( e . g . , changes in level ) .", "In the medial superior olive ( MSO ) , the earliest nucleus with ITD-sensitive neurons , correlations due to anatomical divergence are probably limited because frequencies are narrowly tuned in these binaural neurons and receive inputs from few monaural neurons ( Couchman et al . , 2010 ) .", "The second source of correlations is also likely to be weak because there are no identified lateral connections within the MSO and little evidence of feedback connections , although GABAergic receptors have been recently characterized ( Couchman et al . , 2012 ) .", "Thus , neural correlations in this early sound localization circuit are presumably mainly due to the shared acoustic stimulus .", "When these correlations are neglected and neurons are assumed to fire independently , conditionally to the ITD , then the optimal code is the most sensitive one ( Harper and McAlpine , 2004 ) , and reliable estimates can be obtained by simply pooling the estimates obtained from individual responses .", "This conclusion is wrong in general when there are stimulus-dependent correlations ( Brette , 2010 ) .", "In this case , as we have shown , the structure of neural correlations contains useful information that can be exploited by simple decoders .", "For example , changes in various aspects of the sound induce shared variability in individual responses , which results in the same variability in pooled responses , but little variability in the relative activity of neurons ( used by the pattern decoder ) .", "Another mechanism to reduce the impact of shared stimulus-dependent variability is divisive normalization ( Carandini and Heeger , 2011 ) .", "Level normalization was in fact included in all the models we tested , so as to focus on ITD cues ( rather than interaural level differences ) .", "Previous studies have assessed the performance of pattern decoders in estimating sound location from the responses of ensembles of cortical neurons .", "These decoders included artificial neural networks using spike counts or relative spike timing ( Furukawa et al . , 2000; Stecker et al . , 2005; Lee and Middlebrooks , 2013 ) and maximum likelihood estimation ( Miller and Recanzone , 2009 ) , which is close to the pattern decoder used in this study .", "It is generally found that good performance can be achieved provided that the set of neurons is large enough .", "A previous study also found good performance with an opponent channel model , similar to the hemispheric model we tested in our study ( Stecker et al . , 2005 ) .", "However , these studies tested the decoders on responses to a single type of sound ( white noise ) , although with several levels , and as we have shown , substantial differences between the performances of decoding mechanisms only arise when sounds are allowed to vary in dimensions other than the dimension being estimated ( e . g . , spectrum ) .", "In a recent experimental study , a maximum likelihood decoder was found to outperform a hemispheric decoder in estimating sound location from responses of neurons in the inferior colliculus ( Day and Delgutte , 2013 ) , which is consistent with our study .", "However , as in previous studies , only responses to a single type of sound were used , which implies that performance in more realistic scenarios was overestimated .", "The pattern decoder is essentially a perceptron ( Dayan and Abbott , 2001 ) : spatially tuned neurons are formed by simply pooling neural responses with different weights , and then the maximally active neuron indicates source location .", "These weights reflect the average activity pattern for the preferred location , and thus could be learned by standard Hebbian plasticity mechanisms .", "The smoothed peak decoder is essentially the same , except the weights are not learned .", "In the cat model , most low-frequency neurons in the central nucleus of the inferior colliculus are spatially tuned , with preferred azimuth homogeneously distributed in the contralateral hemifield ( Aitkin et al . , 1985 ) .", "These neurons receive excitatory inputs from ITD-sensitive neurons in the MSO .", "In addition , when one inferior colliculus is removed , sound localization performance drops only in the contralateral field .", "Both the pattern and smooth peak decoders are in line with these findings .", "The hemispheric decoder pools neural activity from each hemisphere , and then calculates the normalized difference , which are both simple operations .", "However , two remarks are in order .", "First , contrary to the other decoders , the presence of both functional hemispheres is required to estimate sound direction in either hemifield .", "Second , these operations produce a graded estimate of sound direction , not a spatially tuned response .", "Therefore , producing spatially tuned responses requires an additional step , with neurons tuned to a specific ratio of hemispheric activity .", "The firing rate of these neurons must then depend nonmonotonically on the activity of each side .", "Thus , the hemispheric decoder appears more complex , in terms of neural circuitry , than any of the other decoders .", "It could be argued that creating spatially tuned responses is in fact not necessary for sound localization behavior .", "For example , movements toward the sound source could be generated with activity in the two hemispheres controlling opposite muscles ( Hancock and Delgutte , 2004 ) .", "However , in addition to the fact that there are spatially tuned neurons in the inferior colliculus ( Aitkin et al . , 1985 ) , this idea does not fit with what is known of the physiology of eye movements .", "Cats orient their gaze toward a briefly presented sound , a behavioral response that has been used to measure sound localization accuracy ( Tollin et al . , 2005 ) .", "Eye movements are controlled by neurons in the superior colliculus ( SC ) , which form a map ( a ‘place code’ ) : stimulation of neurons in the SC produces saccades whose amplitude and direction depend on the site of stimulation , but not on intensity or frequency of stimulation ( Sparks and Nelson , 1987 ) .", "Some of these neurons are tuned to sound location ( Populin et al . , 2004 ) .", "The anatomy and physiology of the ITD processing pathway are very similar across mammalian species ( Grothe et al . , 2010 ) .", "However , while hemispheric decoding might be consistent with behavioral data in small mammals , it is not with data in cats and humans .", "Therefore , if there is a common mechanism for ITD processing in mammals , it cannot be based on pooling neural activity on each hemisphere .", "A traditional argument in favor of the hemispheric model of ITD processing or ‘slope coding’ is that in small mammals , there are many binaural neurons with large BDs , which is contradictory with a labeled line code .", "However , it should be noted that the exact symmetrical argument applies as well: there are many binaural neurons with small BDs ( within the ecological range ) , both in small and large mammals , which is contradictory with the slope coding hypothesis .", "Traditionally , physiological studies of ITD processing have focused on the question of sensitivity: how responses vary along the dimension to be estimated ( ITD ) , which is typically measured by recording ITD selectivity curves .", "Indeed , there can be no information about ITD in responses that are insensitive to ITD .", "But sensitivity is only a necessary condition .", "To understand how ITD is extracted , one must identify those aspects of neural responses that are specific to ITD .", "In other words , one must analyze not only what varies with ITD but also what is invariant when properties other than ITD vary .", "This point is related to the difference in behavioral studies between acuity , a measure of discriminability between stimuli , and accuracy , a more ecologically relevant measure of how well the animal can reach a target ( Heffner and Heffner , 2005; Tollin et al . , 2005 ) .", "Indeed , the computationally challenging task for a sensory system is not to discriminate between two signals , but to extract meaningful information in face of the tremendous diversity of sensory inputs in ecological environments .", "Such an analysis requires recording neural responses to a large variety of sounds .", "For practical reasons , we based our study on model responses .", "In principle , the same analysis could be done experimentally by recording the responses of a large number of cells to a broad set of sounds and levels presented at different locations .", "Given the large number of stimuli , such a study might require imaging or multielectrode recordings .", "An initial approach could be to look for invariant properties in the response of a subset of neurons to natural sounds presented at the same spatial location ." ], [ "The basic model consists of the following stages , illustrated in Figure 1B .", "A sound S has a location θ , which could be an ITD or azimuth .", "Sounds are either", "( i ) white noise ,", "( ii ) band-passed white noise , or", "( iii ) colored noise with a 1fα spectrum with color parameter α between 0 and 2 ( 0 = white noise , 1 = pink noise , 2 = brown noise ) .", "The signal received at the two ears is this sound transformed by a pair of HRTFs for that location .", "We consider two HRTF models:", "( i ) no diffraction , that is , frequency-independent ITDs , and", "( ii ) HRTFs measured in an anechoic chamber ( Figures 6 and 8 ) .", "In addition to the target sound , each ear can receive an acoustic noise ( Figure 5 ) .", "The signal received at each ear is then monaurally filtered by a gammatone filter bank ( Glasberg and Moore , 1990; Slaney , 1993 ) with center frequencies and bandwidths defined by the animal model ( see below ) .", "The equivalent rectangular bandwidth ( ERB ) Q factor of a filter is defined as QERB ( f ) =fERB ( f ) where ERB ( f ) is the width of a rectangular filter that would pass as much power as the filter ( for white noise ) .", "Following Shera et al . ( 2002 ) , we use the formula QERB=β ( fkHz ) α , where α and β are parameters specific to the animal model ( see below ) .", "Each binaural neuron receives two monaurally filtered inputs , one from each side , with an internal delay defined by the animal model .", "The firing rate response of the binaural neuron is given by the formula ∫ ( L+R ) k , with a constant k defined by the animal model , and L ( t ) and R ( t ) are delayed and normalized versions of the gammatone filtered signals at the left and right ears at time t .", "The normalization factor is proportional to |∫ ( L+R ) k|1k , and chosen for a target maximal firing rate F of the binaural neuron .", "This binaural model is a generalization of two previous models that were found to produce good fits for the delay–response curves of the guinea pig ( Harper and McAlpine , 2004 ) and owl models ( Fischer et al . , 2008 ) .", "Here , we generalized it to include cats and humans , and checked that the delay–response curves give good fits to published data for the cat ( Joris et al . , 2006 ) .", "The result is the output response r of the binaural neuron , and the spike count is drawn from a Poisson distribution with mean r ( so that r/T is the firing rate of the neuron for duration T ) .", "All simulations were performed using the ‘Brian’ simulator ( Goodman and Brette , 2008 , 2009 ) with the ‘Brian hears’ auditory periphery library ( Fontaine et al . , 2011 ) .", "Each binaural neuron then is specified by a BF and a BD so that the left channel is delayed by BD/2 and the right channel by −BD/2 .", "The distribution of these parameters , as well as the bandwidths for the monaural filters , is defined separately for each animal model .", "For all models , we used a range of 480 BFs ERB spaced between 100 Hz and 1 . 5 kHz , with the exception of the cat model with HRTFs ( as the recorded HRTFs were not reliable below 400 Hz ) and the owl ( which uses higher frequencies for ITD processing ) .", "The firing rate of the binaural neurons was calibrated to have a peak of F = 200 Hz .", "As firing is Poisson , smaller or larger values would only increase or decrease neuronal noise .", "Parameters for all models are summarised in Table 1 . 10 . 7554/eLife . 01312 . 012Table 1 . Summary of animal modelsDOI: http://dx . doi . org/10 . 7554/eLife . 01312 . 012NameITD sourceITD range , μsBest delays ( BD ) Best frequencies ( BF ) αβkGuinea pigArtificial± 300Measured100–1500 Hz0 . 354 . 08Guinea pigHRTF± 250Measured100–1500 Hz0 . 354 . 08CatArtificial± 400Measured100–1500 Hz0 . 375 . 04CatHRTF± 450Measured400–1500 Hz0 . 375 . 04HumanHRTF± 950Uniform within π-limit100–1500 Hz0 . 375 . 04HumanHRTF± 950Guinea pig distribution100–1500 Hz0 . 375 . 04HumanHRTF± 950Cat distribution100–1500 Hz0 . 375 . 04OwlHRTF± 260Measured2–8 kHz0 . 504 . 32 The decoding problem is to compute an estimate θ^ of θ , given the vector of responses r of the binaural neurons .", "We define a training set and a testing set of data .", "The acoustical inputs can be different between the two sets , for example , training with white noise and testing with colored noise ( Figure 3D ) .", "The training set is used to set the parameters of the decoder , and the testing set is used to compute the errors and biases of the decoder ( ‘Analysis’ ) .", "We consider the following decoders , all of which can be straightforwardly implemented with a simple neural circuit: We analyze the decoders based on their errors and biases .", "The error is computed as E[|θ^−θ|] , where the expectation is taken over the testing data .", "The bias is computed by taking a linear regression through the points ( θi , θ^i ) with the restriction that the line must pass through ( 0 , 0 ) .", "The bias b is given as a percentage bias toward the center from the slope g of the best fit line via b=100 ( 1−g ) .", "To get a better estimate , we compute multiple values of the error and bias over 25 different shuffles of the data , and compute the mean and standard deviation of these values over the multiple shuffles .", "We generate 6400 total data , and to form each shuffled set of data , we take the following steps:", "( i ) choose a subset of the full set of cells to consider ( in those analyses where the number of cells was varied ) ,", "( ii ) choose a random subset of the data as training data , usually 400 data ,", "( iii ) choose a nonoverlapping random subset of the data as testing data , usually 800 data .", "This procedure was chosen to minimize biases introduced by the random sampling while keeping total computation times to a reasonable level ( total computation time on an 8-core Intel i7 desktop was approximately 1 week ) ." ] ]

[ "The activity of sensory neural populations carries information about the environment .", "This may be extracted from neural activity using different strategies .", "In the auditory brainstem , a recent theory proposes that sound location in the horizontal plane is decoded from the relative summed activity of two populations in each hemisphere , whereas earlier theories hypothesized that the location was decoded from the identity of the most active cells .", "We tested the performance of various decoders of neural responses in increasingly complex acoustical situations , including spectrum variations , noise , and sound diffraction .", "We demonstrate that there is insufficient information in the pooled activity of each hemisphere to estimate sound direction in a reliable way consistent with behavior , whereas robust estimates can be obtained from neural activity by taking into account the heterogeneous tuning of cells .", "These estimates can still be obtained when only contralateral neural responses are used , consistently with unilateral lesion studies ." ]

[ "Having two ears allows animals to localize the source of a sound .", "For example , barn owls can snatch their prey in complete darkness by relying on sound alone .", "It has been known for a long time that this ability depends on tiny differences in the sounds that arrive at each ear , including differences in the time of arrival: in humans , for example , sound will arrive at the ear closer to the source up to half a millisecond earlier than it arrives at the other ear .", "These differences are called interaural time differences .", "However , the way that the brain processes this information to figure out where the sound came from has been the source of much debate .", "Several theories have been proposed for how the brain calculates position from interaural time differences .", "According to the hemispheric theory , the activities of particular binaurally sensitive neurons in each of side of the brain are added together: adding signals in this way has been shown to maximize sensitivity to time differences under simple , controlled circumstances .", "The peak decoding theory proposes that the brain can work out the location of a sound on the basis of which neurons responded most strongly to the sound .", "Both theories have their potential advantages , and there is evidence in support of each .", "Now , Goodman et al . have used computational simulations to compare the models under ecologically relevant circumstances .", "The simulations show that the results predicted by both models are inconsistent with those observed in real animals , and they propose that the brain must use the full pattern of neural responses to calculate the location of a sound .", "One of the parts of the brain that is responsible for locating sounds is the inferior colliculus .", "Studies in cats and humans have shown that damage to the inferior colliculus on one side of the brain prevents accurate localization of sounds on the opposite side of the body , but the animals are still able to locate sounds on the same side .", "This finding is difficult to explain using the hemispheric model , but Goodman et al . show that it can be explained with pattern-based models ." ]

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

[ "neuroscience" ]

[ [ "Our capacity to learn and reliably execute motor skills underlies much of what we do .", "Motor skill learning is characterized by high initial motor variability that is gradually reduced as performance improves and skills are consolidated ( Figure 1A ) ( Lee et al . , 1999; Park et al . , 2013 ) .", "Variability in motor output can be beneficial early in learning as it allows the motor system to explore a range of actions and selectively reinforce ones that improve performance ( Sutton and Barto , 1998; Tumer and Brainard , 2007; Wu et al . , 2014 ) .", "But as viable solutions are found ( a good tennis serve , for example ) , variability in motor output can become detrimental for expert performance and is often reduced .", "This capacity of the nervous system to regulate variability and plasticity in motor output as a function of learning or skill level enables new skills to be acquired and those already mastered to be stably expressed and maintained .", "However , the neural circuit mechanisms that regulate motor variability as a function of learning have not been identified . 10 . 7554/eLife . 03697 . 003Figure 1 . Probing the neural mechanisms underlying the regulation of motor variability in songbirds .", "( A ) A strong coupling between performance improvements and variability reduction is a hallmark of most forms of motor skill learning .", "( B ) Spectral derivatives of songs from a single zebra finch at different stages of song learning ( dph-days post hatch ) show a reduction in vocal variability as a function of learning .", "( C ) The neural circuits associated with the acquisition and execution of song .", "HVC ( red ) and RA ( purple ) constitute the cortical part of the vocal motor pathway ( VMP ) and control the learned song; the anterior forebrain pathway ( AFP , green ) , is essential for inducing vocal variability and guiding the song learning process .", "( D ) Presumed functional organization of the motor pathway in which HVC represents time in the song ( t ) in the form of a synaptic chain network and RA neurons control specific muscles .", "Learning in the motor pathway is thought to be driven by plasticity in RA that is guided by input from LMAN , the output of the AFP . DOI: http://dx . doi . org/10 . 7554/eLife . 03697 . 003 Addressing this question means linking learning-related changes in the motor circuits underlying skilled performance to a reduction in the variability of their action-related dynamics .", "While it is known that motor practice induces structural and functional changes in motor control circuits ( Rioult-Pedotti et al . , 1998; Xu et al . , 2009; Wang et al . , 2011; Fu et al . , 2012 ) , it is not clear how such modifications influence overall network function or how they lead to reduced neural and behavioral variability ( Peters et al . , 2014 ) .", "Moreover , studies of plasticity in motor circuits have tended to focus on connectivity within anatomically confined motor regions ( Sanes and Donoghue , 2000; Adkins et al . , 2006 ) , leaving open the question of how motor learning modifies connections between functionally distinct motor areas .", "Consequently , the logic by which motor skills are acquired and consolidated in neural circuitry and how this may contribute to reduced variability in motor output has remained elusive .", "The zebra finch , a songbird , presents an experimentally tractable model in which to address these questions ( Brainard and Doupe , 2002; Mooney , 2009; Ölveczky and Gardner , 2011 ) .", "Male birds learn a complex motor sequence—their courtship song—by first memorizing the song of a tutor , then engaging in trial-and-error learning to match their vocalizations to a stored template of their tutor's song ( Immelmann , 1969; Tchernichovski et al . , 2001 ) .", "As with human motor skill learning , birdsong learning is characterized by gradual improvements in performance ( similarity to the tutor song ) and decreased motor variability ( Tchernichovski et al . , 2001; Ölveczky et al . , 2011 ) ( Figure 1A , B ) .", "Two main pathways are involved in the acquisition and production of the bird's song .", "The Vocal Motor Pathway ( VMP ) , which comprises HVC ( used as proper name ) and the robust nucleus of the arcopallium ( RA ) ( Figure 1C , D ) , encodes and controls learned vocalizations ( Simpson and Vicario , 1990; Yu and Margoliash , 1996; Leonardo and Fee , 2005 ) .", "The Anterior Forebrain Pathway ( AFP ) , a song-specialized basal-ganglia-thalamo-cortical circuit , provides input to the VMP at the level of RA and is necessary for vocal exploration and learning ( Bottjer et al . , 1984; Scharff and Nottebohm , 1991; Kao et al . , 2005; Ölveczky et al . , 2005 ) .", "During singing , RA-projecting HVC neurons fire sparse and song-locked spike bursts that are thought to control song timing ( Hahnloser et al . , 2002; Long and Fee , 2008; Ali et al . , 2013 ) .", "Projection neurons in RA drive brainstem motor neurons that innervate muscles involved in singing ( Vicario , 1991; Wild , 1997 ) .", "Given this functional architecture , song learning has been cast as the process of transforming the timing signal in HVC into effector-specific RA motor commands by establishing connections between time-keeper neurons in HVC and muscle-related neurons in RA appropriate for producing the desired vocal output ( Figure 1D ) ( Fee et al . , 2004; Fiete et al . , 2004 , 2007; Fee and Goldberg , 2011 ) .", "Inputs to RA from the lateral magnocellular nucleus of the anterior neopallium ( LMAN ) , the cortical outflow of the AFP , is thought to guide the learning process within RA by adding variability to the motor program ( Kao et al . , 2005 , 2008; Ölveczky et al . , 2005 , 2011; Thompson and Johnson , 2007 ) and mediating auditory feedback-based error-correction ( Brainard and Doupe , 2000; Andalman and Fee , 2009; Warren et al . , 2011; Charlesworth et al . , 2012 ) .", "Thus , the capacity for exploration and learning is promoted by inputs to RA from LMAN , while the consolidation of learned song occurs within the HVC-RA network .", "Early in song learning , when vocal exploration is needed to drive trial-and-error learning , the RA motor program is dominated by LMAN input ( Aronov et al . , 2008; Ölveczky et al . , 2011 ) , but as learning proceeds and exploitation of the learned vocal patterns becomes essential for successful courtship , the temporally precise inputs from HVC take over and the premotor role of LMAN decreases ( Ölveczky et al . , 2005; Aronov et al . , 2008 ) .", "What are the circuit-level changes that underlie this shift in premotor control and hence reduction in vocal variability ?", "Is it age-dependent attenuation of LMAN input to RA , developmental changes in HVC input , or both ?", "Addressing this question requires a thorough circuit-level characterization of the developmental changes in both HVC and LMAN input to RA .", "While prior studies have examined anatomical correlates of song development in RA and shown significant age-related changes in both the spine density of RA neurons ( Herrmann and Arnold , 1991; Kittelberger and Mooney , 1999 ) and HVC terminal bouton frequency ( Kittelberger and Mooney , 1999 ) , how these anatomical changes translate into functional changes in VMP connectivity is not clear .", "And though the relationship between HVC fiber stimulation and evoked postsynaptic potentials in RA neurons is known to be age-dependent ( Kittelberger and Mooney , 1999 ) , the detailed nature of the underlying changes in circuitry has not been revealed .", "Similarly , our understanding of how LMAN inputs to RA neurons change during song development—essential for understanding regulation of motor variability—is lacking .", "Here , we examine changes in the distribution and relative number of HVC and LMAN input to RA neurons as a function of song development .", "We show that while HVC-RA connections undergo significant strengthening and pruning , inputs from LMAN remain largely unchanged throughout .", "This suggests that LMAN's effectiveness in inducing motor variability and promoting plasticity is curtailed by the strengthening and pruning of HVC-RA connections .", "A simple network model shows how strengthening and pruning of action-specific connections in motor control circuits makes the dynamics in those circuits less sensitive to external sources of variability ( e . g . LMAN ) and neural ‘noise’ .", "This identifies a simple and general circuit-level mechanism for learning-related regulation of motor variability ." ], [ "To characterize how inputs to RA change as a function of sensorimotor learning , we used birds of ages corresponding to three distinct phases of song learning ( ‘Materials and methods’ , Figure 1B ) : ( 1 ) Subsong ( 40–45 days post-hatch , dph ) —the earliest stage of sensorimotor learning characterized by highly variable songs driven largely by LMAN ( Aronov et al . , 2008 ) ; ( 2 ) Plastic song ( 60–65 dph ) —an intermediate stage of development characterized by recognizable but variable song elements or syllables that are subject to further change; ( 3 ) Crystallized song ( 90–130 dph ) —adult stage at which a stereotyped and stable version of the bird's courtship song has developed ( Immelmann , 1969; Price , 1979 ) .", "RA has two major cell types: excitatory projection neurons and inhibitory interneurons ( Spiro et al . , 1999 ) .", "Adult vocalizations are driven by the precise burst firing of RA projection neurons ( Simpson and Vicario , 1990; Yu and Margoliash , 1996; Leonardo and Fee , 2005 ) that are thought to be largely triggered by inputs from time-keeper neurons in HVC ( Hahnloser et al . , 2002; Hahnloser , 2006 ) .", "Activity in RA projection neurons is further influenced by inputs from LMAN ( Ölveczky et al . , 2005; Kao et al . , 2008 ) , which induce variability in the RA motor program during singing ( Ölveczky et al . , 2011 ) .", "This organization makes the connections between HVC and RA projection neurons a likely site of learning and memory ( Doya and Sejnowski , 1995; Fee et al . , 2004; Fiete et al . , 2007 ) and the inputs from LMAN to RA an important locus for driving motor variability and plasticity ( Figure 1D ) ( Kao et al . , 2005; Ölveczky et al . , 2011 ) .", "Thus , our current study focuses on RA projection neurons and how their inputs from HVC and LMAN , as well as their intrinsic properties , change during learning .", "Experiments were done in acute brain slices that included a large fraction of RA ( Figure 2A; ‘Materials and methods’ ) .", "Because LMAN and HVC fibers enter RA in different planes ( Mooney and Konishi , 1991; Mooney and Rao , 1994 ) , we used two different slices to probe inputs from the respective nuclei , optimizing for the number of input fibers for each experiment ( ‘Materials and methods’ ) .", "Input strength was probed by stimulating the fiber tract entering RA from HVC or LMAN at different stimulation intensities ( Figure 2B , ‘Materials and methods’ ) and recording evoked currents in RA projection neurons under whole-cell voltage clamp ( Figure 2C , D ) . 10 . 7554/eLife . 03697 . 004Figure 2 . Experimental approach for probing inputs to RA projection neurons from LMAN and HVC during different stages of song development .", "( A ) Bright-field image of an acute brain slice encompassing RA and incoming fibers from HVC ( parasaggital slice ) .", "The fibers are stimulated and evoked currents from RA projection neurons recorded in voltage clamp .", "( B ) Using different slices we can interrogate the number and strength of HVC ( red ) and LMAN ( green ) inputs to RA projection neurons .", "( C–D )", "Increasing the stimulation intensity activates increasingly more input fibers , allowing us to measure single fiber ( SF ) currents and maximal ( MAX ) currents .", "Examples of HVC fiber-evoked currents in a plastic-song bird .", "( E ) Single fiber currents were measured at stimulus intensities that produced both failures ( grey traces ) and evoked EPSCs of reliable amplitude ( black traces , see also ‘Materials and methods’ ) .", "Data from stimulating a putative single HVC fiber in a plastic-song bird .", "In C and E , red arrow denotes time of stimulation .", "Stimulus artifacts removed . DOI: http://dx . doi . org/10 . 7554/eLife . 03697 . 004 Drawing on the characterization of synaptic refinement at the retinogeniculate synapse ( Hooks and Chen , 2006; Noutel et al . , 2011 ) , we used the metrics of Single Fiber ( SF ) and Maximal ( MAX ) current to estimate the relative strength and number of LMAN and HVC inputs .", "SF currents from individual fibers were recorded at a stimulus intensity that produced a mixture of evoked unitary EPSCs of consistent peak amplitude and failures ( Figure 2E , ‘Materials and methods’ ) .", "The peak of the single fiber-evoked EPSC ( SF current ) was used to gauge the strength of inputs from single HVC or LMAN neurons .", "MAX currents , estimated from the peak EPSC at saturating stimulus intensities ( Figure 2C , D; ‘Materials and methods’ ) , were used to approximate the overall drive from HVC or LMAN fibers to single RA projection neurons .", "We first characterized the strength of single HVC inputs to RA projection neurons across the three age categories by recording HVC-fiber-evoked SF currents in 176 RA cells in 86 birds ( Figure 3A ) .", "By stimulating the HVC fiber tract using an array of stimulation electrodes ( ‘Materials and methods’ ) , we were often able to characterize more than one SF input to a single cell ( on average 1 . 65 ± 0 . 81; mean ± SD ) . 10 . 7554/eLife . 03697 . 005Figure 3 . Inputs from HVC to RA neurons change throughout song development .", "( A ) Schematic highlighting the inputs being probed ( red box ) .", "( B ) Distributions of SF currents for the three age groups tested .", "Red line represents the log-normal fit to the data ( ‘Materials and methods’ ) .", "( C ) Cumulative SF current distributions .", "( D ) Box and whisker plots showing the median ( white line ) , IQR ( grey box ) , mean ( + ) , and the 10th and 90th percentile of the SF current distributions ( whiskers ) .", "( E ) Distributions of MAX currents for the three age groups .", "( F ) Average MAX currents .", "( G ) Average number of HVC inputs to an RA projection neuron in our slice preparation .", "Error bars in F and G denote standard deviations . DOI: http://dx . doi . org/10 . 7554/eLife . 03697 . 005 While HVC input to RA is mediated by both AMPA and NMDA receptors ( Mooney and Konishi , 1991; Mooney , 1992 ) , the NMDA component is not thought to be essential for driving song in either juvenile ( Ölveczky et al . , 2005 ) or adult ( Charlesworth et al . , 2012 ) birds , and we thus characterized the strength of HVC-RA connections by recording AMPA receptor ( AMPAR ) -mediated currents at a holding potential ( Vh ) of −70 mV ( Stark and Perkel , 1999 ) .", "The shapes of the SF current distributions were strikingly similar to synaptic weight distributions found in other systems ( Song et al . , 2005; Barbour et al . , 2007 ) , and could be well approximated by a log-normal fit for all three age groups ( Figure 3B ) .", "SF currents shifted to higher values with age , indicating a gradual strengthening of HVC inputs throughout learning ( Figure 3B–D ) .", "For example , while only 3% [2/72] of the SF inputs were stronger than 100 pA in subsong birds , this fraction increased to 8% [5/60] in plastic-song birds and to 21% [25/120] in adults .", "The median SF current also increased from 22 . 5 pA ( IQR: 22 . 5 , n = 72 SFs in 55 cells ) in subsong birds ( n = 24 birds ) to 40 . 2 pA ( IQR: 28 . 4; n = 60 SFs in 42 cells; p = 3 × 10−7 ) in plastic-song birds ( n = 23 birds ) and 59 . 8 pA ( IQR: 65 . 0 , n = 120 SFs in 79 cells ) in adult birds ( n = 39 birds , p = 0 . 02 comparing second and third age categories , Figure 3D ) .", "Consistent with a prior study ( Kittelberger and Mooney , 1999 ) , the input resistance of RA projection neurons did not change significantly with age ( Table 1 ) , an indication that our measurements of SF currents were not confounded by changes in intrinsic cell properties .", "The strengthening of HVC SF inputs across age categories was also accompanied by a reduction in the variability ( CV ) of single fiber EPSCs across repeated stimulations ( Table 1 ) . 10 . 7554/eLife . 03697 . 006Table 1 . HVC–RA synaptic propertiesDOI: http://dx . doi . org/10 . 7554/eLife . 03697 . 006BirdsAge ( dph ) Input resistance ( MΩ ) Capacitance ( pF ) Spontaneous firing rate ( Hz ) SF peak amplitude ( pA ) SF CVSF latency to peak ( ms ) MAX peak amplitude ( nA ) MAX CVMAX latency to peak ( ms ) Subsong Juvenile ( 24 ) 40–45135 . 84 ± 95 . 0996 . 80 ± 22 . 776 . 74 ± 4 . 7929 . 52 ± 2 . 580 . 30 ± 0 . 219 . 24 ± 2 . 940 . 53 ± 0 . 260 . 05 ± 0 . 048 . 57 ± 2 . 13 ( 55 ) ( 55 ) ( 55 ) ( 72 ) ( 72 ) ( 72 ) ( 29 ) ( 29 ) ( 29 ) Plastic-song Juvenile ( 23 ) 60–65123 . 95 ± 93 . 2695 . 48 ± 27 . 477 . 25 ± 5 . 1049 . 60 ± 3 . 920 . 19 ± 0 . 148 . 39 ± 2 . 541 . 31 ± 0 . 610 . 05 ± 0 . 047 . 47 ± 1 . 50 ( 42 ) ( 42 ) ( 42 ) ( 60 ) ( 60 ) ( 60 ) ( 24 ) ( 24 ) ( 24 ) *p < 0 . 001†*p < 0 . 001**p < 0 . 001**p < 0 . 05Crystalized-song adult ( 39 ) 90–130105 . 80 ± 76 . 0586 . 71 ± 24 . 927 . 95 ± 5 . 5073 . 56 ± 5 . 490 . 18 ± 0 . 157 . 68 ± 2 . 840 . 80 ± 0 . 430 . 05 ± 0 . 047 . 08 ± 1 . 70 ( 80 ) ( 80 ) ( 80 ) ( 120 ) ( 120 ) ( 120 ) ( 52 ) ( 52 ) ( 52 ) *p < 0 . 05**p < 0 . 001†*p < 0 . 001**p < 0 . 001**p < 0 . 001**p < 0 . 01***p < 0 . 05†**p < 0 . 001*Values are mean ± SD *Versus Subsong Juvenile; **versus plastic-song juvenile; statistically significant differences in bold .", "*Two-tail Student's t Test .", "†Wilcoxon Rank–Sum Test: used when one or more of the distributions under comparison were significantly non-parametric , as determined by the Kolmogorov–Smirnov .", "In addition to stronger SF inputs , song development was accompanied by marked but non-monotonic changes in the overall input drive from HVC to RA neurons ( Figure 3E , F; Table 1 ) .", "While MAX currents more than doubled from 0 . 55 ± 0 . 26 nA ( n = 29 cells; mean ± SD ) in subsong to 1 . 31 ± 0 . 61 nA in plastic-song ( n = 24 cells , p = 3 × 10−6 ) , the trend reversed and MAX currents subsequently decreased by ∼40% over the last month of song learning to 0 . 80 ± 0 . 43 nA in crystallized adults ( n = 52 cells , p = 7 × 10−4 as compared to plastic-song , Figure 3F ) .", "This suggests very significant pruning of HVC inputs during later stages of sensorimotor learning .", "To address the change in the number of functional HVC inputs to RA projection neurons in our slice , we estimated the relative number of inputs by dividing the MAX current for each cell by the age-matched mean SF current ( ‘Materials and methods’ ) .", "This revealed a 1 . 4-fold increase in the average number of HVC inputs during early sensorimotor learning from 18 . 7 ± 8 . 9 in subsong birds to 26 . 4 ± 12 . 2 in plastic-song birds ( p = 0 . 01 ) , followed by a 2 . 4-fold decline to 10 . 8 ± 5 . 8 in crystallized-song adults ( p = 2 × 10−6 between plastic-song and adult , Figure 3G ) .", "We next examined how inputs from LMAN to RA change with learning ( Figure 4A ) .", "Despite a reduction in the size of LMAN with development ( Bottjer et al . , 1985; Bottjer and Sengelaub , 1989 ) , the number of RA-projecting LMAN neurons remains largely unchanged during sensorimotor learning ( Nordeen et al . , 1992 ) , as does the topography of LMAN projections to RA ( Iyengar et al . , 1999 ) .", "Yet whether and how the functional properties of these inputs change during song learning is not known .", "To address this , we employed the same experimental strategy as for HVC-RA connections , but in coronal slices that isolated LMAN inputs to RA ( ‘Materials and methods’ ) , measuring LMAN-fiber evoked EPSCs at both +40 mV and −70 mV ( Figure 4B ) .", "Input strength was characterized at the depolarized holding potential because , unlike HVC inputs , inputs from LMAN are mediated predominantly by NMDA receptors ( NMDARs ) ( Mooney and Konishi , 1991; Mooney , 1992; Stark and Perkel , 1999 ) , which are blocked by Mg2+ at −70 mV ( Mayer et al . , 1984; Nowak et al . , 1984 ) . 10 . 7554/eLife . 03697 . 007Figure 4 . Inputs from LMAN to RA neurons remain largely unchanged throughout song development .", "( A ) Schematic highlighting the inputs being probed ( green box ) .", "( B ) Currents evoked in response to stimulating the LMAN fiber tract with an intensity resulting in either failures ( grey ) or EPSCs of consistent amplitudes ( black ) .", "Top: at a holding potential ( Vh ) of +40 mV , where both AMPA- and NMDA receptor-mediated currents are measured .", "Bottom: holding potential of −70 mV , where AMPAR-mediated currents dominate .", "Stimulus artifacts removed .", "( C ) Distributions of SF currents at Vh = +40 mV for the three age groups tested .", "( D ) Cumulative SF current distributions .", "( E ) Box and whisker plots showing the median ( white line ) , IQR ( grey box ) , mean ( + ) , and the 10th and 90th percentile of the SF current distributions ( whiskers ) .", "( F ) The mean ratio of SF currents evoked at Vh = +40 mV ( AMPA and NMDA receptor mediated ) and −70 mV ( AMPAR mediated ) .", "( G ) Average MAX currents .", "( H ) Average number of LMAN inputs to an RA projection neurons in our slice preparation .", "Error bars in F , G , and H denote standard deviations . DOI: http://dx . doi . org/10 . 7554/eLife . 03697 . 007 In contrast to HVC SF inputs , the distribution of LMAN SF EPSCs did not change significantly across the three age categories ( Figure 4C , D ) .", "The median SF peak EPSC was 138 . 32 pA ( IQR: 202 . 0 pA , n = 40 SFs in 24 cells ) in subsong birds ( n = 7 birds ) , 90 . 73 pA ( IQR: 143 . 5 pA , n = 45 SFs in 30 cells ) in plastic-song birds ( n = 13 birds ) , and 94 . 41 pA ( IQR: 136 . 5 pA , n = 38 SFs in 33 cells ) in adult birds ( n = 12 birds; p > 0 . 1 for all pairwise Mann-Whitney-Wilcoxon test comparisons , Figure 4E ) .", "Though the mean strength of SF LMAN input was near identical in plastic-song and adult birds ( 146 . 1 and 142 . 3 pA respectively , p = 0 . 75 ) , these numbers were ∼25% lower than the mean SF input in subsong birds .", "Though this difference did not reach statistical significance ( p > 0 . 12 ) , we cannot rule out a slight developmental weakening of LMAN SF input early in sensorimotor learning .", "There was also no significant change in the shape of the SF distributions across the different age categories ( p > 0 . 1 for all pairwise Kolmogorov–Smirnov test comparisons ) .", "The CV of the peak EPSC amplitude for successive stimulations of the same SF ( Table", "2 ) was also similar across the age groups ( p > 0 . 2 for all pairwise Student's t test comparisons ) .", "The ratio of peak SF EPSC at a holding potential ( Vh ) of −70 mV vs 40 mV ( Figure 4F ) , however , decreased significantly in plastic-song birds ( 0 . 10 ± 0 . 09 , n = 45 ) and adult birds ( 0 . 09 ± 0 . 08 , n = 38 ) as compared to subsong birds ( 0 . 17 ± 0 . 09 , n = 40 , p < 0 . 001 for both pairwise comparisons ) , suggesting a small developmental decrease in the relative contribution of AMPA vs NMDA receptors at LMAN-RA synapses during early development ( Table 2 ) .", "Interestingly , assuming a reversal potential of 0 mV for AMPA and NMDA currents , our results indicate an NMDA:AMPA ratio of 9:1 in subsong birds ( ‘Materials and methods’ ) , consistent with a prior study ( Stark and Perkel , 1999 ) . 10 . 7554/eLife . 03697 . 008Table 2LMAN-RA synaptic propertiesDOI: http://dx . doi . org/10 . 7554/eLife . 03697 . 008BirdsAge ( dph ) SF peak amplitude Vh = +40 mV ( pA ) SF CV Vh = +40 mVSF latency to peak Vh = +40 mV ( ms ) Ratio SF peak amplitude at Vh = −70 mV to that at Vh = +40 mVMAX peak amplitude Vh = +40 mV ( nA ) MAX CV Vh = +40 mVMAX latency to peak Vh = +40 mV ( ms ) Ratio MAX peak amplitude at Vh = −70 mV to that at Vh = +40 mVSubsong Juvenile ( 7 ) 40–45191 . 30 ± 162 . 240 . 10 ± 0 . 0611 . 06 ± 3 . 900 . 17 ± 0 . 090 . 42 ± 0 . 210 . 08 ± 0 . 0412 . 97 ± 1 . 710 . 32 ± 0 . 28 ( 40 ) ( 40 ) ( 40 ) ( 40 ) ( 18 ) ( 18 ) ( 18 ) ( 18 ) Plastic-song Juvenile ( 13 ) 60–65147 . 41 ± 132 . 100 . 12 ± 0 . 0510 . 66 ± 4 . 290 . 10 ± 0 . 090 . 48 ± 0 . 280 . 07 ± 0 . 0511 . 56 ± 0 . 730 . 28 ± 0 . 21 ( 45 ) ( 45 ) ( 45 ) ( 45 ) ( 18 ) ( 18 ) ( 18 ) ( 18 ) *p < 0 . 01*Crystalized-song adult ( 12 ) 90–130141 . 24 ± 126 . 400 . 10 ± 0 . 0610 . 18 ± 4 . 200 . 11 ± 0 . 080 . 39 ± 0 . 170 . 05 ± 0 . 0311 . 23 ± 3 . 380 . 30 ± 0 . 17 ( 38 ) ( 38 ) ( 38 ) ( 38 ) ( 15 ) ( 15 ) ( 15 ) ( 15 ) *p < 0 . 001**p < 0 . 01*Values are mean ± SD *Versus Subsong Juvenile; **Versus plastic-song juvenile; statistically significant differences in bold .", "*Two-tail Student's t Test . Wilcoxon Rank–Sum Test: used when one or more of the distributions under comparison were significantly non-parametric , as determined by the Kolmogorov–Smirnov test .", "In contrast to significant developmental changes in the HVC drive to RA neurons ( Figure 3F ) , the total input from LMAN to RA projection neurons did not show a significant change across the age categories we tested ( Figure 4G ) .", "The average MAX LMAN current was 0 . 42 ± 0 . 21 nA ( mean ± SD , n = 18 cells in five birds ) in subsong birds , 0 . 48 ± 0 . 28 nA ( n = 18 cells in seven birds ) in plastic-song birds , and 0 . 38 ± 0 . 17 ( n = 15 cells in five birds ) in adults ( p > 0 . 25 for all pairwise comparisons , Figure 4F ) .", "The ratio of MAX current to mean SF current suggests that the number of LMAN inputs to an RA cell is also largely unchanged across sensorimotor learning ( p > 0 . 05 for all pairwise comparisons ) , remaining relatively sparse throughout ( Figure 4H ) .", "In addition to changes in synaptic strength , learning and development have been shown to produce changes in the intrinsic excitability of neurons ( Brons and Woody , 1980; Sourdet et al . , 2003; Zhang and Linden , 2003; Zhang , 2004 ) .", "As the song-related firing frequency of RA neurons increases throughout sensorimotor learning ( Ölveczky et al . , 2011 ) , we sought to examine whether changes in the excitability of RA projection neurons in response to depolarizing input may contribute to such changes ( Figure 5A ) . 10 . 7554/eLife . 03697 . 009Figure 5 . Intrinsic properties of RA projection neurons do not change significantly with song development .", "( A ) To test intrinsic excitability of RA neurons as a function of age we injected current into RA cells and measured the membrane voltage in current clamp .", "( B ) Membrane voltage in an RA projection neuron as a function of injected current .", "Example shown is from a 60 dph bird .", "( C ) Average instantaneous firing frequency ( IFF ) throughout the 0 . 5-s current injection for the cell in B ( n = 3 current sweeps ) .", "Traces correspond to differing intensities of injected current ( −0 . 2–2 nA in increments of 0 . 2 nA ) .", "( D ) Average IFF during the initial ( FI ) and final ( FF ) 5 ms of the current injection for the cell in B . ( E ) Spike frequency adaptation ( FI/FF ) in RA neurons for the three age categories as a function of stimulation intensity ( n = 9 cells from subsong , 9 from plastic-song , and 10 from adult birds ) .", "( F ) F-I curves for the same population of RA neurons as in E . p > 0 . 1 for all pairwise comparisons across age categories of the slope of the linear portion of the F-I curve . DOI: http://dx . doi . org/10 . 7554/eLife . 03697 . 009 The recordings were performed in whole-cell current clamp at 35°C .", "This allowed us to capture the current-to-firing rate transformations in RA neurons with better control over leak currents and at more physiological temperatures , and hence higher instantaneous firing rates , than prior studies ( Mooney , 1992; Kittelberger and Mooney , 1999 ) .", "RA projection neurons were stimulated by injecting 0 . 5 s current pulses ranging in intensity from −200 pA to 2 nA in 200 pA steps ( Figure 5B ) .", "Experiments were performed in parasagittal slices from subsong ( n = 9 cells in two birds ) , plastic-song ( n = 9 cells in two birds ) , and adult birds ( n = 10 cells in two birds ) .", "We calculated the average instantaneous firing frequency ( IFF ) in response to three repeated current injections ( Mooney , 1992; Kittelberger and Mooney , 1999 ) ( Figure 5C ) .", "RA projection neurons showed marked spike-frequency adaptation that increased with increased current stimulation ( Figure 5D , E , R = 0 . 97 , p = 10−7 , ‘Materials and methods’ ) .", "The magnitude of adaptation ( initial vs steady-state IFF ) was similar across the different age groups ( p > 0 . 05 ) for all pairwise comparisons at each stimulus intensity , a finding consistent with a previous study ( Kittelberger and Mooney , 1999 ) .", "The input/output gain of RA projection neurons , estimated from the linear part of the F-I curve , was also similar across the age groups , consistent with previous reports ( Mooney , 1992; Kittelberger and Mooney , 1999 ) .", "For current injections ≤1 . 4 nA , the average slope of the linear fit was 163 . 37 Hz/nA for subsong , 185 . 33 Hz/nA for plastic-song , and 201 . 50 Hz/nA for adult birds ( R2 ≥ 0 . 99; Figure 5F ) .", "Though there was a trend towards a small increase in the overall gain from subsong birds to adults , this did not reach significance in our data set ( p > 0 . 11 ) .", "Additionally , IFF at a given stimulus intensity was not significantly different across age categories ( Figure 5F , p > 0 . 05 ) .", "Previous studies have shown that song development , and hence the reduction in vocal variability , is accompanied by a relative decrease in LMAN's premotor influence ( Brainard and Doupe , 2000; Ölveczky et al . , 2005; Aronov et al . , 2008; Kao et al . , 2008 ) in favor of an increased role for HVC ( Aronov et al . , 2008; Fee and Goldberg , 2011 ) .", "But the intuitive circuit-level explanation—a learning-related increase in overall HVC drive to RA coupled with a decrease in LMAN input—was not observed .", "On the contrary , our results revealed that the LMAN input remains largely unchanged throughout song learning ( Figure 4 ) , while the overall HVC drive to RA actually decreases ( Figure 3F ) .", "How to account for the developmental switch in premotor drive from LMAN to HVC in light of these results ?", "One possibility is that the strengthening and pruning of HVC-RA connections makes LMAN inputs to RA less effective .", "To probe this idea further and , more generally , to examine the neural mechanisms that can regulate motor variability , we constructed a simple computational model of the HVC-RA-LMAN network ( Figure 6A ) .", "We used this model to evaluate how song-related firing patterns of simulated RA neurons change when HVC-RA synapses are strengthened and pruned and how this process is impacted by other possible changes to the circuit , thus dissecting the potential contributions of various neural mechanisms to the regulation of motor variability . 10 . 7554/eLife . 03697 . 010Figure 6 . A simple model relates strengthening and pruning of connections in a motor control network ( here: HVC-RA ) to reduced motor variability .", "( A ) Top: the early phase of song learning is characterized by high vocal variability ( left panel ) .", "This is associated with relatively larger number of weaker inputs from HVC to RA neurons ( middle panel ) .", "A simple model of this circuit organization ( ‘Materials and methods’ ) , in which RA neurons integrate input from precise HVC time-keeper neurons and variable LMAN neurons , produces variable firing patterns in RA ( right panel ) .", "Bottom: song learning is associated with a strengthening and pruning of HVC-RA connections and a concomitant decrease in song variability .", "Pruning the relative number of active HVC-RA inputs in our model while strengthening the remaining ones ( middle panel ) dramatically decreases the variability in song-related RA firing ( right panel ) .", "The simulations of RA spike trains were done using model parameters consistent with our experimental data from age groups 2 ( top , plastic song ) and 3 ( bottom , crystallized song ) , respectively .", "Note that the simulations for the two neurons are independent ( i . e . , the ‘older’ is not derived from strengthening and pruning connections of the ‘younger’ ) .", "See ‘Materials and methods’ for further details .", "( B ) The average cross-correlation ( CC ) between spike trains during different ‘song’ renditions of the simulated RA neuron increases with the degree of strengthening and pruning of HVC-RA synapses .", "The open circles correspond to the age groups 2 and 3 in our data set .", "Solid black line represents simulations using ‘standard’ model parameters chosen to conform to our experimental data ( see ‘Results’ , Figures 3–5 , and ‘Materials and methods’ ) .", "Dashed lines show simulations where inputs from HVC were either only strengthened ( red ) or pruned ( blue ) , relative to the ‘standard’ model at age group 2 .", "The x-axis ( bottom labels ) shows the fraction of active HVC inputs to RA ( ‘Materials and methods’ ) , as well as the average strength of HVC inputs ( top labels ) .", "( C–E )", "Effects on variability in RA firing from: ( C ) strengthening/weakening LMAN input by up to 50% , ( D ) changing NMDA:AMPA ratio at the LMAN-RA synapse , and ( E ) changing the gain ( F-I relationship ) of the RA neuron .", "All changes are relative to the ‘standard’ model ( black lines ) in ‘B’ .", "( F–H )", "Effects on variability in RA firing stemming from changes to LMAN firing patterns .", "Changes are relative to the ‘standard’ model ( black lines ) .", "( F ) Different LMAN spike trains tested in our model ( for 50 ‘song renditions’ ) .", "Top: Poisson spike train .", "Middle: Poisson spike train with 30% of the spikes in the form of Poisson bursts ( see ‘Materials and methods’ ) .", "Bottom: time-varying Poisson firing .", "Blue curve shows the average instantaneous firing rate as a function of time in song with 50% modulation compared to the baseline rate .", "( G ) Effect of altering the burstiness of LMAN neurons .", "Here , Poisson burst were added to the normal Poisson spike train .", "( H ) Effect of increasing the song-locking of LMAN firing . DOI: http://dx . doi . org/10 . 7554/eLife . 03697 . 010 The gradual strengthening and pruning of HVC inputs to RA in our model were parameterized to fit our experimental observations ( Figures 3 , 4; ‘Materials and methods’ ) .", "In particular , we focused on changes that occur from plastic song to adult song , as variability in both song and RA firing patterns has been quantified and seen to decrease during this phase of learning ( Ölveczky et al . , 2011 ) .", "The combination of strengthening and pruning HVC input to RA led to a dramatic reduction in LMAN's capacity to drive rendition-to-rendition variability in RA neurons ( Figure 6A , B ) .", "Despite the simplicity of the model , these changes mirrored almost perfectly what has been observed in vivo ( Ölveczky et al . , 2011 ) , meaning that the strengthening and pruning of HVC-RA synapses can , on their own , explain much of the learning-related reduction in song variability .", "Our simulations also showed that strengthening and pruning of HVC-RA connections is far more effective in reducing LMAN-induced variability in RA neurons than either strengthening or pruning on its own ( Figure 6B ) , suggesting that the two processes act synergistically to reduce variability .", "While our experiments did not reveal any significant learning-related changes in LMAN inputs to RA , small modifications at this synapse , particularly early in learning , cannot be ruled out .", "To explore the consequences of such changes , we simulated both strengthening and weakening of LMAN inputs to RA relative to the ‘standard’ model derived from our measurements ( Figure 6C , ‘Materials and methods’ ) .", "When paired with the strengthening and pruning of HVC-RA connections , however , weakening LMAN input by as much as 50% explained less than 20% of the overall decrease in the rendition-to-rendition variability of RA firing .", "Thus age-related changes in LMAN-RA connectivity are unlikely to play a major role in reducing song variability during learning .", "Our results also suggested that the NMDA:AMPA ratio at LMAN-RA synapses increases modestly during early development ( between subsong and plastic-song; Figure 4F ) .", "To probe whether changes in the receptor composition of LMAN-RA synapses impact LMAN's capacity to drive variability in RA , we ran simulations with different NMDA:AMPA ratios .", "Eliminating the AMPA component completely ( 100% NMDA ) only decreased the variability in RA neurons by a few percent ( Figure 6D ) .", "But even if a small fraction of the early reduction in vocal variability can be explained by a developmental decrease in AMPAR-mediated currents at the LMAN-RA synapse , this is not a plausible mechanism for reducing variability during later phases of sensorimotor learning ( from plastic-song to adult ) when receptor composition at the LMAN-RA synapses remains largely unchanged ( Figure 4F ) .", "Though we did not find any major age-related differences in the intrinsic properties of RA projection neurons ( Figure 5 ) , there was a slight but non-significant trend towards a steeper F-I curve with age ( ∼10% steeper from subsong to plastic-song , and then again from plastic-song to adult birds , Figure 5F ) .", "Similar small changes in the gain of RA neurons , however , did not materially impact RA variability in our model ( Figure 6E ) .", "Our simulations thus far assumed Poisson-like input from LMAN to RA ( Figure 6F , top panel; ‘Materials and methods’ ) , an approximation based on recordings from RA-projecting LMAN neurons in learning birds ( Ölveczky et al . , 2005 ) .", "However , LMAN neurons are known to fire intermittent high-frequency bursts that may become more prominent in adults ( Kao et al . , 2008; Kojima et al . , 2013 ) .", "This raises the question of whether an age-related increase in the burstiness of RA-projecting LMAN neurons ( Figure 6F , middle panel ) could contribute to reducing vocal variability .", "Our simulations suggest that it does not; on the contrary , burstier LMAN spiking increases variability ( Figure 6G; ‘Materials and methods’ ) .", "While song-aligned firing patterns of identified RA-projecting LMAN neurons in juvenile birds ( corresponding to age groups two and three in our experiments ) show no significant rendition-to-rendition correlation ( Ölveczky et al . , 2005 ) , recordings in older adults , albeit from unidentified LMAN neurons , show significant song-locking ( Kao et al . , 2008 ) .", "Not surprisingly , our simulations showed that less random ( i . e . , more song-locked ) LMAN firing yields less variable RA firing ( Figure 6F [bottom panel] and Figure 6H ) .", "Whether there is indeed a learning-related increase in the song-locking of RA-projecting LMAN neurons still needs to be experimentally tested .", "Importantly , all our simulations showed that strengthening and pruning HVC input to RA decreases variability in RA firing irrespective of whether there are other concomitant changes in the circuit ( Figure 3 ) .", "Moreover , changes in HVC-RA connectivity could account for much , if not all , of the reduction in variability seen during song learning ( Ölveczky et al . , 2011 ) .", "Though we cannot rule out that other mechanisms , including modifications to the spiking output of LMAN neurons ( Figure 6F–H ) , LMAN-RA connectivity strength ( Figure 6C ) , and the receptor composition at LMAN-RA synapses ( Figure 6D ) , contribute to regulating variability , our results suggest that reorganization in HVC-RA connectivity is the dominant mechanism underlying learning-related reduction in vocal variability ." ], [ "Our results also speak to the logic of connectivity within the song system .", "Because we recorded from RA neurons in slices where not all inputs from HVC and LMAN may have been faithfully preserved , the number of inputs we report ( Figures 3G and 4H ) should be seen as lower bounds .", "In these estimates each RA neuron receives , on average , an age-dependent 11–26 inputs from HVC and an age-independent 2–3 inputs from LMAN .", "The relative ratio of LMAN to HVC inputs is in agreement with structural studies , which found RA neurons to receive more synaptic contacts from HVC than from LMAN ( Canady et al . , 1988; Herrmann and Arnold , 1991 ) .", "That there are more HVC than LMAN inputs to RA neurons is also consistent with the hypothesized function of these inputs .", "If the role of LMAN inputs is to ‘experiment’ on HVC-RA connections , as is widely assumed ( Fiete et al . , 2004 ) , having each RA neuron receive only a few relatively strong inputs from LMAN ensures that single LMAN neurons can drive changes in RA firing and that the interference from different ‘experimenters’ is limited .", "Inputs from HVC , on the other hand , are required to drive on average 12 bursts per song motif in adult birds ( Leonardo and Fee , 2005 ) , and hence more HVC inputs may be needed .", "Whether RA bursts are driven by single HVC neurons , or whether multiple concurrent HVC inputs are required to trigger bursting in RA remains to be explored .", "The circuit-level changes that accompany song learning and the associated reduction in vocal variability were in many ways surprising and counterintuitive .", "Inputs to RA from LMAN , the main source of vocal variability in juvenile birds ( Ölveczky et al . , 2005 , 2011 ) , remain largely unchanged , while the overall input drive from HVC , which provides RA with the precise timing input that ultimately drives adult stereotyped song ( Hahnloser et al . , 2002; Long and Fee , 2008; Long et al . , 2010 ) , actually decrease with song development .", "We show that this decrease is driven entirely by the elimination of HVC inputs; remaining inputs from HVC continue to get stronger ( Figure 3 ) .", "Pruning connections from HVC to RA may serve to decrease the number of depolarizing events in RA neurons , thereby reducing RA neurons' sensitivity to LMAN input .", "This is because NMDARs , which dominate at the LMAN-RA synapse ( Mooney and Konishi , 1991; Mooney , 1992; Stark and Perkel , 1999 ) , require additional depolarizing input to be effective ( Mayer et al . , 1984; Jahr and Stevens , 1990 ) .", "Thus if AMPAR-mediated HVC inputs to RA are being eliminated with learning ( Figure 3G ) , such depolarizing events will become less frequent and thereby limit the times at which LMAN's can drive motor variability .", "Increased hyperpolarization of RA projection neurons over the course of development ( Ölveczky et al . , 2011 ) could further curtail LMAN's capacity to drive spiking in RA neurons at times when there is no other depolarizing input .", "Strengthening individual HVC inputs to RA , on the other hand , could decrease LMAN's effectiveness by saturating RA neurons .", "During singing , RA projection neurons tend to fire bursts of spikes ( Leonardo and Fee , 2005; Ölveczky et al . , 2011 ) , the instantaneous frequency of which increases with development ( Ölveczky et al . , 2011 ) .", "This trend is likely driven , in part at least , by the increased strength of individual HVC inputs ( Figure 3 ) .", "Thus at times when RA neurons are released from hyperpolarization and NMDAR block , they will be increasingly saturated by their HVC input , thus reducing the capacity of LMAN to further modulate their firing .", "A simple model inspired by the HVC-RA-LMAN network ( Figure 6 ) formalized the above intuition and showed that strengthening and pruning of action-specific connections in a motor control network , such as the VMP , reduces its sensitivity to external sources of variability ( e . g . , LMAN ) .", "Though strengthening and pruning can contribute independently , we found that the reduction in variability is accentuated when the processes co-occur ( Figure 6B ) .", "Intriguingly , the reorganization of HVC-RA synapses we observed experimentally ( Figure 3 ) was sufficient to explain the developmental decrease in motor variability seen in vivo ( Ölveczky et al . , 2011 ) .", "We did not find any evidence that developmental changes in the strength or number of LMAN-RA connections contribute to this process ( Figure 4 ) .", "Yet even if there are modest changes , the effect on motor variability is likely to be small compared to those induced by changes at the HVC-RA synapse ( Figure 6C ) .", "Furthermore , our simulations suggest that changes in HVC and LMAN input to RA are largely additive in terms of their effects on motor variability , suggesting independent mechanisms through which the song system can regulate variability .", "We also explored whether and how changes to the firing patterns of LMAN neurons may impact variability and found that increased burstiness increases rendition-by-rendition variability in RA ( Figure 6G ) .", "Interestingly , female-directed ‘performance’ song , which is associated with less bursty LMAN firing ( Kao et al . , 2008 ) , is significantly more stereotyped than undirected ‘practice’ song ( Stepanek and Doupe , 2010 ) .", "This suggests that modulating the burstiness of LMAN neurons , a basal ganglia-dependent process ( Kojima et al . , 2013 ) , could be an effective mechanism for fast and context-dependent regulation of vocal variability .", "Another aspect of LMAN firing patterns that can impact song variability is the degree to which LMAN neurons are locked to song ( Figure 6H ) .", "While activity of RA-projecting LMAN neurons in juvenile birds shows no significant rendition-to-rendition correlation ( Ölveczky et al . , 2005 ) , recordings from non-identified LMAN neurons in adult birds have shown a relatively high degree of song-locking ( Kao et al . , 2008 ) .", "How rendition-to-rendition variability in LMAN firing is regulated , and whether it contributes to learning-related changes in motor variability , remains to be explored .", "Though our simulations show that several circuit-level mechanisms may contribute to regulating motor variability ( Figure 6 ) , our experimental and modeling results taken together suggest that strengthening and pruning of HVC-RA connections is the dominant mechanism during song learning .", "Learning-related changes in action-specific circuits , like the ones we describe ( Figure 3 ) , have been demonstrated also in the mammalian cortex ( Silva et al . , 2009; Xu et al . , 2009; Wang et al . , 2011; Caroni et al . , 2012; Fu et al . , 2012 ) , raising the possibility that the mechanism for coupling motor variability and skill learning suggested by our experiments may apply more broadly , including to mammalian motor learning .", "Whether there is an LMAN-equivalent in mammals , or how exploratory variability is induced and regulated in mammalian motor control circuits more generally , remains to be explored .", "But regardless of whether the source of motor variability is intrinsic neural noise or a dedicated LMAN-equivalent input , our simulations suggest that learning-related strengthening and pruning of action-specific connections in motor control circuitry is , under the assumptions of our model , sufficient to ensure reduction in motor variability ( Figure 6 ) .", "Having the source of variability in motor control networks be mediated predominantly via NMDARs , as in songbirds , makes the variability in these circuits ( e . g . , RA ) more sensitive to the learning-related reorganization of their connectivity ( Figure 6D ) .", "Inducing variability through NMDARs also ensures that the resulting motor exploration is focused on instances when there are other depolarizing inputs to the neuron , that is , when there are active connections in the control network that can be experimented with and modified .", "Having learning-related reorganization of action-specific connections regulate motor variability lends considerable flexibility to the process of motor skill learning .", "Consider the case of learning multiple actions ( e . g . , syllables in a song ) .", "Assuming that learning an action leads to synaptic strengthening and pruning in the neuronal assemblies that encode and control that action ( Xu et al . , 2009; Wang et al . , 2011; Fu et al . , 2012 ) , then , in the absence of other mechanisms limiting plasticity , the network should retain its native capacity to learn new actions as long as those are not contingent on network connections associated with already acquired ones .", "In the songbird system , a tight correspondence between specific actions ( i . e . , syllables ) and HVC-RA connections is assured , since one HVC neuron only contributes to one time-point in the song ( Hahnloser et al . , 2002 ) .", "A specific prediction from this conceptual model as it relates to birdsong is that syllables that have not yet been fully formed ( i . e . , ones still far from matching the ‘template’ ) should be more influenced by LMAN , and hence be more variable , than syllables that are already a close match .", "Intriguingly , this is exactly what was reported in a recent study ( Ravbar et al . , 2012 ) .", "The authors found a strong correlation between the distance of a syllable from its target and its variability .", "Thus the variability of two actions ( syllables ) was a function of how well they had been learned .", "The mechanism we propose for regulating the effect of LMAN provides a simple circuit-level explanation for this observation .", "The circuit-level changes we observed during song development may impact the capacity for learning beyond reducing exploratory variability .", "Since HVC-RA connections are a likely substrate for song learning , having an abundance of these connections , as is the case during the height of sensorimotor learning ( Figure 3G ) , means that the learning system has a relatively large number of connections to explore and modify .", "But as the system ‘learns’ which HVC inputs to strengthen and which to eliminate , the number of connections decreases , thus also shrinking the substrate for plasticity and learning , making the acquired behavior more robust to change .", "This general motif of initial hyper-innervation , followed by synaptic strengthening and pruning is also seen in other developing circuits , including the mammalian cortex ( Huttenlocher , 1979; Rakic et al . , 1994; Katz and Shatz , 1996; Hashimoto and Kano , 2003; Walsh and Lichtman , 2003 ) .", "In summary , our study characterized circuit-level changes that accompany song development and identified a simple , general , and adaptive mechanism for coupling motor skill learning and variability in a way that does not compromise the capacity for future learning and plasticity in motor control circuits .", "This offers a circuit-level explanation for one of the most ubiquitous features of motor skill learning , namely the action-specific and learning-related reduction in motor variability ." ], [ "The 124 male zebra finches ( Taeniopygia guttata ) used for this study were obtained from our breeding colony .", "The care and experimental manipulation of the animals were carried out in accordance with guidelines of the National Institutes of Health and were reviewed and approved by the Harvard Institutional Animal Care and Use Committee .", "Since our aim was to characterize how inputs to RA change as a function of sensorimotor learning , which in zebra finches takes place between ∼35–90 dph ( Immelmann , 1969 ) , we focused our experiments on birds in this age range .", "Our experimental subjects were divided into three age groups corresponding to three distinct stages of song learning:", "( i ) subsong juveniles ( 40–45 dph ) ,", "( ii ) plastic-song juveniles ( 60–65 dph ) , and", "( iii ) crystallized-song adults ( 90–130 dph ) .", "Birds , kept on a 14-/10-hr light/dark cycle , were brought up from the aviary on the day of the experiment , anesthetized with isoflurane and subsequently decapitated .", "Brains were harvested and placed in 4°C oxygenated ( bubbled with 0 . 4 liters per minute 95% O2/5% CO2 ) artificial cerebrospinal fluid ( ACSF ) containing ( in mM ) 119 NaCl , 2 . 5 KCl , 1 . 3 MgCl2 , 0 . 5 CaCl2 , 1 NaH2PO4 , 26 . 2 NaHCO3 , 11 D-glucose , in which equimolar choline chloride replaced NaCl to limit excitotoxicity .", "Osmolarity of all ACSF solutions was elevated to 350 mOsm with sucrose ( Bottjer , 2005 ) .", "All reagents were purchased from Sigma–Aldrich ( St . Louis , MO ) .", "Experiments were performed in 300 μm thick acute brain slices ( Mooney and Konishi , 1991 ) cut using a vibrating microtome ( Leica VT1000 S , Germany ) .", "For experiments characterizing HVC inputs to RA , parasagittal slices were sectioned at a 26° angle relative to the interhemispheric fissure by cutting the brain down the midline and gluing each hemisphere to platforms angled at 26° with the dorsal surface facing down .", "We found that this slice preserves more HVC inputs to RA than the straight parasagittal slice , yielding larger HVC fiber stimulation-evoked MAX currents .", "The slice containing the largest fraction of RA ( diameter of nucleus in the cut plane >600 µm ) and HVC afferent fibers , as judged visually through an IR-DIC microscope ( Figure 2A ) , was used .", "For experiments characterizing LMAN inputs to RA , the optic tecta and brain stem were removed , and the hemispheres laid flat on their ventral surface .", "The brain was then cut in half along the coronal plane and the posterior half glued onto its freshly cut anterior face and sectioned .", "300 μm coronal slices were cut and the one containing the largest fraction of RA ( diameter of nucleus in the cut plane >400 µm ) and afferent fibers from LMAN was used .", "After cutting , slices were transferred to a chamber containing ACSF at 37°C , in which 50% of the NaCl was replaced with choline chloride .", "The slices were allowed to recover for 30 min , before being transferred to ACSF without choline chloride for an additional 30+ minutes before experiments commenced .", "Solutions in both chambers were allowed to cool to room temperature ( 20–23°C ) .", "Whole-cell electrophysiological recordings in RA were performed under IR-DIC visual guidance .", "Voltage-clamp recordings were carried out at room temperature ( 20–23°C ) using electrodes of 2 . 5–3 . 5 MΩ resistance , filled with an internal solution containing ( in mM ) : 35 CsF , 100 CsCl , 10 EGTA , and 10 HEPES , with pH adjusted to 7 . 32 with CsOH .", "50 μM picrotoxin was added to the external solution to block fast feed-forward GABAergic inhibition ( Kittelberger and Mooney , 1999; Stark and Perkel , 1999 ) .", "Extracellular divalents ( both Mg2+ and Ca2+ ) were elevated to 4 mM in order to dampen excitability and reduce spontaneous activity .", "HVC-evoked EPSCs were recorded at −70 mV in order to minimize contamination from predominantly NMDAR-mediated LMAN inputs ( Mooney and Konishi , 1991; Mooney , 1992; Stark and Perkel , 1999 ) by means of hyperpolarization induced Mg2+ block .", "LMAN-evoked EPSCs were recorded at +40 mV in addition to −70 mV in order to characterize both NMDA and AMPA receptor mediated components ( Figure 4B ) .", "Current-clamp recordings were carried out at 35°C ( TC-324B , Warner Instruments , Hamden , CT ) with 2 . 5–3 . 5 MΩ electrodes containing ( in mM ) : 130 K-Gluconate , 0 . 2 EGTA , 4 KCl , 2 NaCl , 10 HEPES , 2 Mg-ATP and 0 . 5 Na-GTP , with pH adjusted to 7 . 25 with KOH .", "RA projection neurons were identified based on their spontaneous tonic activity and characteristic spike waveform recorded in cell-attached configuration prior to breaking into the cell ( Mooney , 1992; Spiro et al . , 1999 ) .", "The data presented are from identified projection neurons .", "Electrophysiological data were recorded with a MultiClamp 700B ( Molecular Devices , Sunnyvale , CA ) using mafPC software ( courtesy of MA Xu-Friedman ) and custom macros in Igor Pro ( WaveMetrics , Portland , OR ) .", "Signals were digitized at 100 kHz and Bessel filtered at 10 kHz .", "Input resistance was measured throughout the experiment in response to a 10 ms long 10 mV hyperpolarization step .", "Series resistance was not compensated , but was monitored throughout the recording , and not allowed to vary by more than 10% .", "If it did , the cell was excluded from the analysis .", "Inputs were stimulated either with a pair of saline-filled glass electrodes ( Figure 2A ) or a pair of tungsten electrodes ( MicroProbes , Gaithersburg , MD ) selected from an array of four evenly spaced electrodes .", "Using the array allowed us to switch the electrodes across which stimulation was delivered and thus activate different parts of the fiber tract independently without moving the electrodes and risk losing the cell .", "Stimulation currents were delivered using an ISO-Flex Stimulator ( A . M . P . I . , Israel ) .", "The current pulse was 0 . 2 ms in width and varied in amplitude ( see below ) .", "HVC and LMAN afferent fibers enter RA along different tracts , allowing isolated activation of these inputs to RA ( Mooney and Konishi , 1991 ) .", "For HVC experiments the stimulating electrodes were placed ∼1 mm dorsal to RA on the fiber tract connecting HVC to RA ( tractus archistriatalis ) ( Figure 2A ) .", "For LMAN experiments , the stimulating electrodes were placed ∼1 mm lateral to RA along the fiber tract connecting LMAN and RA ( Stark and Perkel , 1999 ) .", "The stimulus intensity was gradually increased from 10 μA to 1 mA .", "At the lowest intensities no current was typically evoked , but as stimulation intensity increased , the evoked postsynaptic currents ( EPSCs ) also increased in amplitude , until reaching a maximum ( Figure 2C , D ) .", "Currents were included only if they showed a smooth rise to peak indicative of monosynaptic input .", "For current-clamp recordings , cells were stimulated with a 0 . 5 s direct current injection at intensities ranging from −200 to 2000 pA ( Figure 5B ) .", "The recorded current traces were smoothed using a 1 ms sliding window .", "The threshold for detecting a unitary EPSC single fiber ( SF ) input was two times the RMS of the signal acquired without any stimulation .", "The RMS ( or ‘noise’ ) of our recordings was 2–5 pA , meaning that SF currents <4 pA were not registered .", "SF currents were measured at a minimal stimulus intensity that produced 25–75% failures ( a pre-set criteria ) relative to EPSCs of consistent amplitude .", "The SF was defined as the average peak EPSC ( N > 3 ) evoked at that minimal stimulus intensity ( Figure 3B ) .", "In 107 out of 264 recorded projection neurons , we were able to measure up to three SFs by stimulating across different neighboring stimulus–electrode pairs of the 4-electrode array .", "If two SF currents measurements were made in the same cell by stimulating contiguous pairs of stimulation electrodes , the second observation was only included if the evoked currents in the two cases differed significantly in peak amplitude ( p < 0 . 05 , Student's t test ) .", "MAX currents were characterized at the stimulus intensity ( MAX-stim ) where the EPSC peak amplitude no longer increased despite a greater than threefold increase in stimulus intensity ( Figures 2E and 4B ) .", "MAX current was the average peak response evoked at MAX-stim ( N > 3; CV ≤ 0 . 2 ) .", "MAX currents were recorded by stimulating across the electrode pair that evoked the largest EPSC .", "The relative number of inputs to a cell ( Figures 3G and 4G ) was estimated from the ratio of the MAX current of a cell to the mean SF current for the given age-category .", "This calculation assumes that inputs to RA neurons sum linearly , and that the average SF input to an RA neuron can be estimated from the average SF current across the population of cells at the given age .", "An alternative method for approximating the number of inputs to a cell originally introduced to quantify number of inputs at the retinogeniculate synapse ( Hooks and Chen , 2006 ) , uses the SF current ( s ) recorded in a cell as the estimate of mean input strength .", "The fiber fraction ( SF current/MAX current ) for each input can then be estimated and averaged across the SFs in an age group to get the mean fiber fraction .", "The inverse of this quantity provides an alternative estimate for the number of inputs to cells in a given age group .", "We note that the two estimates yield very similar numbers .", "Estimating the number of HVC inputs using the mean SF current across the population as a proxy for any given cell's mean input , that is , the method we use for reporting the number of inputs in the text , yields 19 , 26 , and 11 inputs for age groups 1 , 2 , and 3 respectively , whereas the original method using fiber fractions ( Hooks and Chen , 2006 ) yields 19 , 28 , and 8 inputs .", "Importantly , these numbers represent lower bounds on the average number of inputs to a cell at a given age .", "F-I curves were generated by calculating the instantaneous firing frequency ( IFF ) evoked by 0 . 5 s long direct current injections , ranging from −200 pA to 2 nA in 200 pA steps ( Figure 5B ) ( McCormick et al . , 1985 ) .", "Current injections were 30 s apart and the sweep from −200 pA to 2 nA repeated three times .", "The IFFs for a given current injection were then averaged for each cell ( Figure 5C ) .", "Spike-frequency adaptation was characterized both in terms of magnitude , by comparing the average IFF during the first and last 5 ms of the stimulus for each cell ( Figure 5D ) , and kinetics , by fitting a single exponential to the first 100 ms of the decay of the average IFF curve at each stimulus intensity for each cell .", "SF data did not follow a normal distribution , as determined by the Kolmogorov–Smirnov test .", "For these distributions statistical significance was assessed using the non-parametric Mann–Whitney–Wilcoxon test .", "Box and whisker plots are shown as medians ( white lines ) , with 25th to 75th percentile range boxes and 10th and 90th percentile whiskers .", "The other data were normally distributed ( K–S test ) and differences across age groups were tested using the parametric Student's t test .", "Statistical significance is indicated on graphs: *p < 0 . 05; **p < 0 . 01; ***p < 0 . 001 .", "If nothing is indicated on the graphs , p > 0 . 05 .", "Values reported are mean ± SD , unless otherwise noted .", "The log-normal distribution has the form:p ( x ) =e ( logx−μ ) 2/2σ2xσ2π , with parameters μ and σ related to the mean and variance as 〈x〉=eμ+σ2/2 and 〈x2〉−〈x2〉= ( eσ2−1 ) e2μ+σ2 , respectively .", "The maximum-likelihood estimates of the parameters μ and σ , given n observed data points xk ( single fiber currents in our data set ) are:μ^=∑klogxkn , σ^2=∑k ( logxk−μ^ ) 2n .", "We estimated the parameters of the log-normal distribution for each of the three age groups separately ( Figure 3 ) , according to the above equations .", "We constructed a simple feed-forward version of a prior network model of the HVC-RA-LMAN circuit ( Fiete et al . , 2007 ) with the aim of identifying general mechanisms that contribute to regulating motor variability as a function of learning and development ( Figure 5A ) .", "At the top of our hierarchical model is the timekeeper circuit ( HVC ) , with neurons active only once during the ‘song’ ( Hahnloser et al . , 2002 ) .", "These neurons , which produce the same pattern of activity on each rendition , project downstream to muscle-related neurons that drive the behavior ( RA ) .", "Variability is introduced at the level of RA by an external source of variable input ( LMAN ) .", "The RA neuron is modeled as a leaky integrate-and-fire neuron with the membrane potential V obeying the following equation:τmdVdt= ( VR−V ) +R IHVC+R ILMAN−VINH , where τm = 20 ms is the membrane time constant , VR = −70 mV is the resting membrane potential , IHVC is the excitatory input from HVC neurons , ILMAN is the excitatory input from LMAN , and VINH is the tonic inhibitory input .", "R = 260 MΩ is the input resistance of the RA neuron .", "Note that we measured the input resistance to be around 130 MΩ ( Table 1 ) , but to allow for two HVC inputs at any given time point in the song and to compensate for possible loss of HVC and LMAN inputs in our slice recordings , we multiplied this by a factor of two .", "The qualitative results we obtained , however , were not sensitive to this choice .", "If the membrane potential V reaches a threshold Vth = −50 mV an RA spike is triggered and V is subsequently reset to VR .", "The membrane potential V remains clamped at VR for a refractory period of 1 . 5 ms , after which it resumes its dynamics according to the above equation .", "There are NHVC = 100 HVC neurons in the model .", "Each HVC neuron produces a single burst of spikes in the 1000 ms long ‘song motif’ .", "The duration of each burst is 10 ms consisting of five spikes 2 ms apart .", "The onset of the burst of the i -th HVC neuron is ( i − 1 ) × 10 ms from the beginning of the song , such that the HVC bursts tile the whole interval of the song regularly and in a non-overlapping fashion .", "The input from HVC neurons to the RA neuron obeys the following equation:dIHVCdt=− IHVCτs+∑i=1NHVCWiHVC∑j=1tji<tδ ( t−tji ) , where τs = 5 ms is the synaptic time constant , WiHVC is the strength ( peak EPSC ) of the synapse from the i -th HVC neuron to the RA neuron , and tji is the time of the j -th spike produced by the i -th HVC neuron .", "Each model RA neuron receives input from two LMAN neurons ( Figure 4G ) , each with a firing rate around 40 Hz ( Ölveczky et al . , 2005 ) .", "We modeled the LMAN spike train as a Poisson point processes with a rate of 80 Hz .", "The input from LMAN to RA is composed of AMPA and NMDA receptor mediated components .", "The AMPAR component is modeled as:dILMANAMPAdt=− ILMANAMPAτs+r WLMAN∑j=1tj'<tδ ( t−tj' ) , where 0 < r < 1 is the contribution of AMPAR mediated current at LMAN-RA synapses , WLMAN = 120 pA is the strength ( peak EPSC ) of the input from LMAN to the RA neurons , compatible with our measurements ( Figure 4E ) , and tj' is the time of the j -th spike produced by LMAN .", "The NMDAR mediated component is modeled as:dILMANNMDAdt=− ILMANNMDAτNMDA+ ( 1−r ) WLMAN∑j=1tj'<tG ( tj' ) δ ( t−tj' ) .", "Here , τNMDA = 100 ms is the synaptic time constant ( Stark and Perkel , 1999 ) , and G ( t ) the voltage-dependent modulation of the NMDA R mediated current ( Jahr and Stevens , 1990 ) :G ( t ) = ( 1+[Mg]3 . 57mM exp ( −V ( t ) /16 . 13mV ) ) −1 , where [Mg] = 0 . 5 mM is the intracellular Mg2+ concentration .", "The total input from LMAN to the RA neuron is ILMAN=ILMANAMPA+ILMANNMDA .", "Unless otherwise noted ( see below and Figure 6D ) we assumed 90% NMDA and 10% AMPA at the LMAN-RA synapse ( Stark and Perkel , 1999 ) .", "The tonic inhibition VINH was set to be proportional to the HVC drive , that is:VINH=RINH m ρ , where m is the mean HVC-RA connection strength , ρ is the ratio of active HVC-RA input ( see below ) and RINH = 800 MΩ is the ‘inhibitory input resistance’ , that is , the proportionality constant determining the amount of inhibition as a function of HVC input .", "Its high value reflects the fact that VINH represents the inhibitory counterpart of 100 excitatory HVC inputs .", "With the above set of parameters , the firing rate of the RA neuron is maintained at around 50 Hz given the HVC-RA connectivity profile specified below , which is consistent with in vivo recordings ( Ölveczky et al . , 2011 ) .", "The strengths of inputs from HVC to RA are drawn from a log-normal distribution as suggested by our experimental results ( Figure 3B ) .", "In order to probe age-related strengthening ( Figure 6B ) , the mean and standard deviation of the distribution were linearly inter- and extrapolated between the values observed for our second and third age groups .", "We also pruned the HVC inputs by a fraction of 1 − ρ ( i . e . , their corresponding WiHVC values are set to zero ) as suggested by our experimental results .", "In order to probe age-related pruning , we linearly changed ρ from 1 to 0 . 2 along with changes in HVC-RA synaptic distribution ( Figure 6B ) .", "The changes in the parameters of input strength distribution and pruning were aligned such that the points ρ = 0 . 9 and ρ = 0 . 37 ( Figure 6B , open circles ) represent the connectivity profile measured in our second and third experimental groups , respectively , that is , a 2 . 4-fold decrease in the number of active inputs , and an increase in mean input strength from 50 pA to 70 pA , with the standard deviation concomitantly changing from 35 pA to 70 pA .", "To assess how modifications to the strength of LMAN inputs to RA would affect RA firing patterns , we increased/decreased WLMAN in our model by up to 50% ( Figure 6C ) .", "A prior study showed LMAN-RA synaptic input , when averaged across juvenile and adult birds , to be composed of 90% NMDA and 10% AMPA ( Stark and Perkel , 1999 ) .", "Though we did not measure the NMDA/AMPA currents directly , our results conform to this ratio .", "LMAN input currents measured at +40 mV holding potential ( Mg2+ block is removed ) reflect both AMPA and NMDA receptor mediated components , while , at a holding potential of −70 mV , the synaptic current is mainly mediated by AMPARs .", "Therefore , the ratio between peak synaptic currents given the driving force at these two holding potentials ( Figure 4F ) can be used to estimate the relative contribution of NMDA and AMPA receptor mediated currents at LMAN-RA synapses using the following formula:Single fiber amplitude at−70mVSingle fiber peak amplitude at+40 mV=gAMPA ( E+70 ) ( gAMPA+gNMDA ) ( E−40 ) , where gAMPA is the peak conductance of the AMPAR mediated component , gNMDA the peak conductance of the NMDAR mediated component , and E the reversal potential of the excitatory synapse .", "Assuming E = 0 mV , our data ( Figure 4F ) results in r=gAMPAgAMPA+gNMDA=0 . 09 in age group 1 and r = 0 . 06 in age groups two and three .", "To probe how changes in relative receptor composition may affect RA spike patterns , we varied r in the range of 0–0 . 2 and , in addition , simulated pure AMPAR input ( Figure 6D ) .", "The slope of the F-I curve of our model leaky integrate-and-fire neuron is proportional to 1/ ( τm Vth ) .", "Our recordings revealed a slight developmental increase in the slope of the F-I curve of RA neurons ( Figure 5F ) .", "To address the effect that such changes may have on the firing patterns of RA neurons , we altered the membrane time constant of the integrate-and-fire model neuron in the range of τm = 16 − 25 ms ( Figure 6E ) .", "This corresponds to ±20% change in the slope of the F-I curve of the neuron , compared to the τm = 20 ms case .", "To explore how burstiness in LMAN neurons influences variability in RA neurons , we added Poisson bursts to LMAN spike trains while keeping the rate of the LMAN firing fixed at 40 Hz ( Figure 6F , middle panel , Figure 6G ) .", "The onsets of these bursts were themselves modeled as a Poisson point process , and each individual burst event was composed of a stereotypical pattern of 5 spikes , 2 ms apart ( i . e . , the duration of each burst was 10 ms ) .", "Therefore , in order to generate LMAN spikes trains with the rate of 40 Hz and fraction b of the spikes in bursts , the rate of the tonic Poisson spikes was set to 40 ( 1 − b ) Hz , and the rate of burst events was set to ( 40 b/# spikes per burst ) = 8b Hz .", "To explore the extent to which song-locking of LMAN firing influences variability in RA , we modulated the rate of the Poisson spike trains with a single sine wave profile along the 1000 ms interval of simulation ( Figure 6F , lower panel , Figure 6H ) .", "All the simulation results in Figure 6 were obtained by averaging 5000 realizations of the network with the given parameters .", "The time constant of integration in the simulations was 0 . 2 ms . In order to quantify the rendition-to-rendition variability , first the RA spike train for the i-th rendition was converted into instantaneous firing rate Ri ( t ) as follows:Ri ( t ) =1tk+1i−tki , tki<t<tk+1i , where tki is the time of k-th RA spike in i-th rendition .", "The obtained instantaneous firing rates were then convolved with a 10 ms Gaussian function , to get a smoothed firing rate function ri ( t ) .", "As a measure of rendition-to-rendition variability , the correlation coefficient ( CC ) was calculated between firing rates Ri ( t ) for all pairs of spike trains as follows:CC=1N ( N−1 ) ∑i=1N∑j>iNCCi , j , CCi , j=〈ri¯ ( t ) rj¯ ( t ) 〉t〈ri¯2 ( t ) 〉〈r¯2 ( t ) 〉t , where the averaging 〈 .", "〉t is performed over the length of renditions ( 1000 ms ) , N is the number of renditions and ri¯ ( t ) is the mean-subtracted version of ri ( t ) .", "For the simulations in Figure 6 , the number of renditions , N , was 200 ." ] ]

[ "Motor skill learning is characterized by improved performance and reduced motor variability .", "The neural mechanisms that couple skill level and variability , however , are not known .", "The zebra finch , a songbird , presents a unique opportunity to address this question because production of learned song and induction of vocal variability are instantiated in distinct circuits that converge on a motor cortex analogue controlling vocal output .", "To probe the interplay between learning and variability , we made intracellular recordings from neurons in this area , characterizing how their inputs from the functionally distinct pathways change throughout song development .", "We found that inputs that drive stereotyped song-patterns are strengthened and pruned , while inputs that induce variability remain unchanged .", "A simple network model showed that strengthening and pruning of action-specific connections reduces the sensitivity of motor control circuits to variable input and neural ‘noise’ .", "This identifies a simple and general mechanism for learning-related regulation of motor variability ." ]

[ "‘Practice makes perfect’ captures the essence of how we learn new skills .", "When learning to play a musical instrument , for example , it often takes hours of practice before we can play a single piece of music properly for the first time .", "And as we get better , the variability in our performance—which is an advantage during the early stages of learning—becomes less .", "Likewise , songbirds need lots of practice in order to master the intricate songs they need to sing to attract mates .", "Studies in songbirds show that the neural circuits in the brain that are responsible for producing song and for generating vocal variability both converge on a motor control region called the robust nucleus of the arcopallium ( or RA for short ) .", "However , the details of how learning a song leads to reduced variability in vocal performance are poorly understood .", "Now Garst-Orozco et al . have investigated the relationship between learning and variability by studying brain slices of zebra finches .", "Their experiments reveal that the inputs received by RA neurons from a higher-order brain region that controls song change with practice , with some inputs becoming stronger and others being eliminated as the birds' singing ability improves .", "However , inputs received by RA neurons from the circuit that generates vocal variability do not change despite the song becoming increasingly precise .", "Using a computer simulation , Garst-Orozco et al . show that the sensitivity of RA neurons to variable or ‘noisy’ input is reduced when inputs from the brain region that controls song are adaptively strengthened and eliminated .", "This ensures that when the notes and syllables that make up the bird's song have finally been learned , they will be uttered with high fidelity and precision .", "Intriguingly , motor skill learning in mammals have been associated with neural connectivity changes very similar to those described by Garst-Orozco et al . , suggesting that insights from songbirds may lead to a better understanding of how ‘practice makes perfect’ also works in humans ." ]

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

[ "neuroscience" ]

[ [ "Remembering previous events is one of the hallmarks of human cognition , with memory retrieval contributing to , and depending on , many other cognitive abilities .", "Memory retrieval involves a complex set of processes that activate a large network of brain regions ( Rugg and Vilberg , 2013; Spaniol et al . , 2009; Kim , 2010 ) .", "Recently , it has been suggested that the memory retrieval network can be divided into two sub-systems ( Ranganath and Ritchey , 2012 ) , both anchored on the hippocampus: an anterior temporal system , comprising the amygdala , temporopolar cortex , orbitofrontal cortex and perirhinal cortex , and a posterior medial system , including regions such as the posterior cingulate , precuneus , angular gyrus ( AnG ) , medial prefrontal cortex , and parahippocampal cortex .", "According to this framework , the anterior temporal system is more related to semantic memory , familiarity judgments , and event salience , whereas the posterior medial system contributes to episodic memory and recollection .", "Despite such attempts to characterize sub-systems within the retrieval network , and suggestions about the roles of some of its constituent regions , functional specializations within the memory retrieval network remain poorly understood , in part due to common co-activation during most retrieval tasks .", "It is likely that memory retrieval tasks typically activate such a wide network of regions because the components of successful retrieval can reflect the contribution of a number of different properties of memory representations , such as their subjective vividness , objective accuracy , and the objective level of detail , specificity , or precision with which they are retrieved .", "To better understand how memory judgments are derived , it is important to consider that the outcome of a retrieval attempt is typically not ‘all-or-none’ , but that the amount and quality of information we retrieve can vary considerably ( Parks and Yonelinas , 2007 ) .", "In order to distinguish memory retrieval components and the role of regions within the retrieval network , it is necessary to use sensitive measures that can detect such variations in retrieval performance .", "Consistent with this approach , some research has studied the specificity with which memories can be retrieved as an alternative to simple 'old/new' decisions or other categorical judgments such as ‘remember/know’ .", "For example , source memory tasks , requiring participants to remember the context in which an item was studied , have revealed that participants sometimes remember 'partial source' information ( such as the gender of a speaker ) , but not necessarily all specific details ( Simons et al . , 2004 ) .", "The idea that memory representations can vary in quality has prompted researchers to measure recollection objectively using more graded measures , exploring how many details can be remembered ( Horner and Burgess , 2014; Qin et al . , 2011; Vilberg and Rugg , 2009 ) .", "Although such measures are important advances in capturing differences in retrieval outcome , they remain essentially categorical .", "Memory is not only defined by the number of details that are remembered , but also by the fidelity with which those details can be recalled ( Brady et al . , 2013 ) .", "Recent developments in the study of short-term memory have demonstrated that memory representations vary along a continuous scale of memory precision ( Bays and Husain , 2008; Zhang and Luck , 2008; Fougnie et al . , 2012; van den Berg et al . , 2012; Bays , 2014 ) , highlighting the flexibility of memory representations in terms of both quantity and quality ( Ma et al . , 2014 ) .", "These influential methods from the working memory literature have started to be applied to behavioral studies of long-term memory ( Brady et al . , 2013 ) .", "Importantly , it is possible to separate behaviorally the probability of recollection success from the precision with which information is retrieved ( Harlow and Donaldson , 2013; Harlow and Yonelinas , 2016 ) , which can be differentially influenced by attention and retrieval practice ( Fan and Turk-Browne , 2013 ) .", "Furthermore , a recent EEG study supports the idea that variations in long-term memory precision may be associated with distinct neural signatures , finding the well-established left parietal old/new effect to be graded according to recollection precision ( Murray et al . , 2015 ) .", "The observation that retrieval success and retrieval precision can be manipulated independently suggests that using continuous measures to differentiate such components will lead to a more detailed understanding of episodic memory retrieval at both behavioral and neural levels .", "Retrieval success and retrieval precision both constitute objective measures of memory performance .", "However , increasing research has additionally considered subjective or metacognitive measures of memory performance , such as confidence ( Yonelinas , 1994 ) or the vividness with which retrieved memories are experienced ( Kuhl and Chun , 2014; St-Laurent et al . , 2015 ) .", "Research has found that subjective and objective measures of memory performance can diverge ( Chua et al . , 2012; Harlow and Yonelinas , 2016 ) .", "Most notably , patients with parietal cortex lesions exhibit impairment in subjective memory reports even though their objective performance appears to be intact ( Simons et al . , 2010 ) , and lateral parietal transcranial magnetic stimulation has been found to selectively reduce memory confidence but not retrieval success ( Yazar et al . , 2014 ) .", "One possibility is that the subjective and objective memory components that may drive mnemonic decisions are separable , with subjective aspects of retrieval , such as vividness , and objective aspects of retrieval , such as success and precision , relying on largely independent processes .", "Alternatively , it is possible that performance on subjective measures of memory retrieval used to date can be fully explained by subtle variations in objective memory , such as the precision with which a memory is recalled , which previous objective measures may not have been sensitive enough to detect .", "To summarize , memory retrieval involves a wide network of brain regions and may reflect the contribution of both objective and subjective memory components , with objective memory success being distinguishable from objective memory precision and both being distinguishable from subjective memory vividness .", "To understand the roles of brain regions within the retrieval network and to dissociate memory retrieval components , more sensitive , continuous measures of memory performance are needed .", "In the current study , we combined continuous long-term memory measures in an fMRI paradigm with model-based approaches derived from the working memory literature .", "For each retrieval trial we obtained a binary measure of retrieval success ( successful versus unsuccessful ) , a continuous measure of the precision with which color , orientation , and location features of visual objects were remembered ( based on the error between a target value and the participant’s response ) , as well as a subjective vividness rating , on a continuous scale .", "These measures allowed us to characterize behavioral and neural mechanisms that distinguish between recollection success , precision , and the vividness with which information is retrieved ." ], [ "Model-based analyses were first conducted on the error values obtained for each memory retrieval trial .", "The distribution of errors for all 5724 trials across all participants and features ( see Materials and methods ) can be seen in Figure 1 .", "Three models were fitted to the error data ( Bays et al . , 2009 ) , both across all trials as well as separately for each participant , and model fit was assessed via Akaike information criterion ( AIC ) and Bayesian information criterion ( BIC ) .", "Modeling the errors with a von Mises ( circular normal ) distribution alone ( model", "1 ) fit the data less well than a mixture model ( model", "2 ) that also included a uniform component ( AIC difference: 1882 . 2; BIC difference: 1875 . 6; also true across subjects , AIC: t ( 19 ) = 9 . 01 , p < 0 . 001; BIC: t ( 19 ) = 8 . 63 , p < 0 . 001 ) and a third model in which non-target binding errors were additionally accounted for ( AIC difference: 1880 . 2; BIC difference: 1866 . 9; also true across subjects , AIC: t ( 19 ) = 8 . 96 , p < 0 . 001; BIC: t ( 19 ) = 8 . 20 , p < 0 . 001 ) .", "Model 2 provided a better fit than model 3 ( AIC difference: 2 . 0; BIC difference: 8 . 7; also true across subjects , AIC: t ( 19 ) = 4 . 61 , p < 0 . 001; BIC: t ( 19 ) = 16 . 48 , p < 0 . 001 ) , suggesting that non-target errors did not account for a significant amount of variance in performance on this long-term memory task .", "Therefore , the distribution of errors was best characterized by a model with one parameter capturing the proportion of successfully retrieved features ( responses within the von Mises distribution relative to the uniform distribution ) and another parameter capturing the precision ( concentration ) of successfully retrieved memories .", "As seen in Figure 1 , errors clustered around the target value , indicating that participants often successfully retrieved information about the probed feature .", "Moreover , a certain percentage of trials resulted in errors evenly distributed between −180 and 180 degrees , consistent with responses due to forgetting .", "Using model 2 ( von Mises + uniform ) , the estimated mean proportion of trials successfully retrieved was 0 . 62 ( SD = 0 . 16 ) , and estimated mean precision ( assessed by the concentration parameter of the von Mises distribution ) was 10 . 05 ( SD = 3 . 41 ) .", "The distribution of vividness responses across all subjects and trials can also be seen in Figure 1 .", "We calculated the mean vividness across all 48 displays for each subject ( mean = 48 . 23 , SD = 15 . 83 ) , which indicated that subjects' memories were , on average , moderately vivid , and responses were widely spread between 0–100 . 10 . 7554/eLife . 18260 . 003Figure 1 . Behavioral responses for all retrieval trials .", "( a ) Distribution of errors ( difference between reported feature value and target feature value ) , across all 5724 trials across all participants , also visualized in circular space by wrapping the distribution around a circle .", "( b ) Distribution of vividness responses across all 954 vividness ratings for all participants . DOI: http://dx . doi . org/10 . 7554/eLife . 18260 . 00310 . 7554/eLife . 18260 . 004Figure 1—source data 1 . Data associated with the error distribution and vividness rating analyses . DOI: http://dx . doi . org/10 . 7554/eLife . 18260 . 004 We hypothesized that our three indices of memory retrieval ( retrieval success , precision and vividness ) measure different components of retrieval performance .", "If so , we would expect them to correlate , but only to a moderate degree .", "To compare the covariance of retrieval success , precision , and vividness , across-subject pairwise correlation analyses and separate regression analyses predicting each retrieval measure were conducted .", "As expected , each pairwise correlation revealed a moderate positive correlation of similar magnitude , including a relationship between retrieval success and precision ( r = 0 . 527 , p = 0 . 017 ) , retrieval success and vividness ( r = 0 . 543 , p = 0 . 013 ) , and precision and vividness ( r = 0 . 519 , p = 0 . 019 ) ( see Figure 2 ) .", "However , regression analyses highlighted that a substantial proportion of variance in each retrieval measure remained unexplained when predicted by the other two measures: For retrieval success , 62 . 3% of the variance was unexplained after accounting for vividness and precision ( R2 = 0 . 377 , F ( 2 , 17 ) = 5 . 14 , p = 0 . 018 , with no significant individual effect of vividness , β = 0 . 369 , t ( 17 ) = 1 . 65 , p = 0 . 118 , or precision , β = 0 . 336 , t ( 17 ) = 1 . 50 , p = 0 . 152 , when entered in the same model ) .", "Similarly , 64 . 5% of variance in retrieval precision was unexplained by the other two variables ( R2 = 0 . 355 , F ( 2 , 17 ) = 3 . 67 , p = 0 . 024 , with no significant effect of vividness , β = 0 . 330 , t ( 17 ) = 1 . 42 , p = 0 . 173 , or retrieval success , β = 0 . 348 , t ( 17 ) = 1 . 50 , p = 0 . 152 ) .", "Lastly , 63% of variance in retrieval vividness was unexplained by retrieval success and precision ( R2 = 0 . 370 , F ( 2 , 17 ) = 4 . 98 , p = 0 . 020 , with no significant effect of retrieval success , β = 0 . 373 , t ( 17 ) = 1 . 64 , p = 0 . 118 , or precision , β = 0 . 322 , t ( 17 ) = 1 . 42 , p = 0 . 173 ) .", "The behavioral analyses , therefore , support the relative separability of retrieval success , precision , and vividness at a cognitive level . 10 . 7554/eLife . 18260 . 005Figure 2 . Relationship between the three measures of retrieval performance . Correlation between", "( a ) retrieval success and precision ,", "( b ) retrieval success and vividness ratings , and", "( c ) precision and vividness ratings .", "Shaded areas indicate the standard error of the correlation . DOI: http://dx . doi . org/10 . 7554/eLife . 18260 . 00510 . 7554/eLife . 18260 . 006Figure 2—source data 1 . Data associated with the pairwise correlation analyses . DOI: http://dx . doi . org/10 . 7554/eLife . 18260 . 006" ], [ "By capitalizing on the use of continuous measures of objective and subjective retrieval performance , our study provides novel insights into dissociable components of memory retrieval .", "We demonstrate that regions of the episodic memory network that are normally co-activated during retrieval can be dissociated and linked to specific aspects of retrieval performance .", "Using the current approach to study and analyze behavioral and fMRI memory data , future studies will be able to characterize more accurately the mechanisms involved in different retrieval tasks , and to assess specific variations in memory performance in target groups , such as patients and older adults ." ], [ "Twenty-one subjects ( 9 female; mean age = 29 . 81 ) participated in the current experiment .", "Subjects were 18–45 years of age , right-handed , fluent English speakers , had normal or corrected-to-normal vision , and had no history of psychiatric or neurological disorders .", "Subjects were recruited via participant databases at the Memory Laboratory , Cambridge University , as well as social media adverts .", "Informed consent was obtained according to procedures approved by the Cambridge Psychology Research Ethics Committee and subjects were paid a standard honorarium for their time .", "One male participant was excluded from all analyses due to very low , chance-level behavioral performance ( more than 2 SD below the group mean for all parts of the memory test ) , resulting in a final sample of 20 subjects .", "For one additional subject , the first scan run out of 8 had to be excluded due to a technical error during scanning .", "Stimuli consisted of 48 background scenes and 144 object pictures .", "The background scenes consisted of scene photographs ( including naturalistic scenes , buildings , and other landmarks ) , excluding any images showing people or distinct objects .", "The object stimuli were a subset of photographs of everyday objects used by Brady et al . ( 2013 ) , obtained from http://timbrady . org/stimuli/ColorRotationStimuli . zip .", "Objects showing rotational symmetry or that were strongly associated with a specific color were excluded .", "Background scenes and object pictures were randomly combined to create 48 stimulus displays ( 750 × 750 pixels ) , each consisting of a unique background scene with 3 unique superimposed objects varying in color , orientation , and location ( on an invisible circle ) .", "Colors , orientations , and locations of all objects were selected at random from circular parameter spaces with 360 increments ( appearing continuous ) , but with the following constraint: a minimum distance of 62 . 04 degrees was enforced between the same feature of different objects in the same display .", "This was the minimum distance required to ensure that the objects would never overlap in their locations and , for consistency , the same minimum distance in feature space was enforced for all features .", "All object-background assignments were randomized , but the displays were only generated once and were then kept constant across subjects , for whom the order of presentation was randomized .", "Upon arrival , participants completed a short training session to familiarize themselves with task requirements , and the continuous response options .", "Stimuli used in this training session were unique and did not overlap with those used in the main experiment .", "The fMRI session involved 8 scan blocks , each of which contained an encoding phase and a retrieval phase .", "The trial structure of the encoding and retrieval phases is displayed in Figure 5 .", "A video of an example retrieval trial can be accessed online ( Video 1 ) . 10 . 7554/eLife . 18260 . 010Figure 5 . Task design .", "( a ) Examples of displays learnt during encoding and", "( b ) part of the sequence of retrieval questions for a single display illustrating the manipulation of the objects with the continuous dial .", "During retrieval , the order in which features ( color/orientation/location ) were tested was counterbalanced .", "Two out of the three objects associated with an encoded display were tested during retrieval .", "A video of an example retrieval trial can be accessed online ( Video 1 ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 18260 . 010 In each encoding phase , subjects were presented with 6 stimulus displays , each of which was displayed for 12 s .", "Subjects were instructed to try to learn the appearance of the objects on the display , including their color , orientation , and location on the background , as best as they could .", "Displays were separated by a fixation cross of varying duration ( 400 to 2800 ms , approaching a Poisson distribution , with a mean of 961 . 1 ms ) .", "A 10 s delay followed the encoding phase before the retrieval phase started .", "In each retrieval phase trial , participants were presented first with only the background scene of a display and were asked to rate how vividly they remembered the appearance of the objects associated with the presented background , based on their memory for the identity and features of all three objects .", "The background was presented for 2 s with the question “How vividly do you remember this display ? ” in white font .", "After 2 s , a slider was added with the labels ‘not vividly’ and ‘very vividly’ at the end points .", "Subjects had 6 s to respond using their index and middle finger to indicate the vividness of their memory on a continuous scale of 0 ( 'not vividly' ) to 100 ( 'very vividly' ) .", "They confirmed their responses by pressing a third button with their thumb .", "In cases where participants did not respond within the first 4 s , the text turned red to indicate to participants that they had 2 s left in which they could respond .", "The vividness scale was represented by a 500 pixel wide line , on which participants could move a cursor up or down by holding down one or the other of two buttons .", "Each ‘press’ of the button moved the cursor by 5 pixels , resulting in the 100-point scale .", "The vividness rating was requested from participants before they recreated any of the object features so that their subjective vividness judgment would not be influenced by their experience of their performance on that trial .", "Participants were then tested on their memory for two of the three objects that were associated with a given display , with the same two objects tested for all participants .", "Each tested object was initially presented in a random color , location , and orientation .", "The participants’ task was to sequentially recreate the objects’ original features ( color/orientation/location ) using a continuous dial ( 360 increments ) over 6 judgments ( 3 features for 2 objects ) .", "For each judgment , a cue word in the center of the screen instructed the participant which object feature they were being asked to recall ( ‘Color’ , ‘Orientation’ , or ‘Location’ ) .", "Participants had 6 s to recreate these object features as precisely as they could using their index and middle finger to change the features on a continuous dial .", "Similar to the vividness rating , the color of the cue word changed from white to red if no response was made within the first 4 s of the 6 s response window .", "The order in which the features were tested was counterbalanced , such that ( 1 ) each of the 6 possible orders of the feature questions was tested equally and ( 2 ) the same feature was not tested more than 4 times in a row at the same position ( first , second , or third ) .", "Once participants recreated one of the object features , the feature value chosen ( from 0–360 around the circular space ) would be carried over to the subsequent questions for that object .", "A fixation cross of varying length ( 400 to 2800 ms , approaching a Poisson distribution , with a mean of 961 . 1 ms ) was displayed after the vividness question as well as after each of the object feature questions .", "To generate estimates of retrieval success and precision , three models were fitted to the distribution of errors ( absolute difference between the correct response from 0–360 and the participant’s response on each trial ) across all the feature questions within each subject ( see Figure 6b–d ) : ( 1 ) a von Mises distribution; ( 2 ) a von Mises + uniform distribution , and ( 3 ) a von Mises + uniform distribution + von Mises distributions capturing non-target errors ( Bays et al . , 2009; Zhang and Luck , 2008; Harlow and Donaldson , 2013; model analysis code available at http://www . paulbays . com/code/JV10/ ) .", "Model 1 assumes participants' memories continuously vary in precision from 0–180 degrees ( with no guessed responses ) , model 2 assumes that participants either remember a feature with varying precision or guess , resulting in a uniform distribution of responses around the circle , and model 3 assumes that , in addition to model 2 , participants also commit binding errors where they remember the color/orientation/location that was present in one of the two other objects in the display , but not the target object .", "Retrieval success was estimated as the proportion of responses that fell within the von Mises distribution , and precision was estimated as the concentration of the von Mises distribution .", "Importantly , using this operationalization , retrieval success and precision can vary independently , so that high retrieval success does not necessarily imply a participant will also have high retrieval precision ( see Figure 7 for simulated data that illustrates this dissociation ) .", "Of note , when the three models were fitted within each feature separately , assessment of the model fit revealed the same pattern as for all features . 10 . 7554/eLife . 18260 . 011Figure 6 . Models fitted to the error distribution .", "( a ) Components used to model the error data .", "( b–d )", "Illustrations of the three models compared to analyze participants’ error distributions , including von Mises alone", "( b ) , von Mises + uniform", "( c ) , and von Mises + uniform + additional von Mises distributions modeling non-target errors", "( d ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 18260 . 01110 . 7554/eLife . 18260 . 012Figure 7 . Simulated data illustrating the possibility of statistical independence of retrieval success ( proportion of responses in the von Mises distribution vs . the uniform distribution ) and retrieval precision ( the concentration of the von Mises distribution ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 18260 . 01210 . 7554/eLife . 18260 . 013Video 1 . Example of a retrieval trial . The video demonstrates the use of the continuous response options for the vividness question ( 00:01–00:09 ) , and the three feature questions for the first ( 00:09–00:29 ) , as well as the second object ( 00:30–00:51 ) .", "Participants were able to adjust their responses continuously by moving around the slider ( vividness question ) or the circular dial ( feature questions ) .", "Once they were happy with their response , they locked their response with a button press .", "For the first five feature questions in the example the participant entered their response within the first 4 s of the question , and therefore the font color of the cue word ( 'Orientation'/'Colour'/'Location' ) remains white .", "For the last feature question , the color of the cue changes to red as no response was given within the first 4 s of the question interval , indicating to the participant that 2 s remain to respond . DOI: http://dx . doi . org/10 . 7554/eLife . 18260 . 013" ] ]

[ "A network of brain regions have been linked with episodic memory retrieval , but limited progress has been made in identifying the contributions of distinct parts of the network .", "Here , we utilized continuous measures of retrieval to dissociate three components of episodic memory: retrieval success , precision , and vividness .", "In the fMRI scanner , participants encoded objects that varied continuously on three features: color , orientation , and location .", "Participants’ memory was tested by having them recreate the appearance of the object features using a continuous dial , and continuous vividness judgments were recorded .", "Retrieval success , precision , and vividness were dissociable both behaviorally and neurally: successful versus unsuccessful retrieval was associated with hippocampal activity , retrieval precision scaled with activity in the angular gyrus , and vividness judgments tracked activity in the precuneus .", "The ability to dissociate these components of episodic memory reveals the benefit afforded by measuring memory on a continuous scale , allowing functional parcellation of the retrieval network ." ]

[ "Remembering is something we do countless times each day .", "The detail and vividness with which we can remember is part of what makes memories so precious .", "Given the significance and complexity of memories , it is perhaps unsurprising that several parts of the brain are needed for us to experience them .", "Indeed , the brain regions involved in memory all work so closely together that it is a challenge to identify what role each region plays .", "Richter , Cooper et al . aimed to design a memory task that could separate key characteristics of remembering , which would allow them to study links between each aspect and the different brain regions involved in memory .", "The resulting test involved showing people images of different objects whilst they were in an MRI medical imaging scanner .", "The people taking the test were asked to remember several objects that could vary in color , position and orientation .", "Participants were asked to rate how vividly they remembered the objects and then tried to precisely recreate their color , orientation and position .", "The test allowed Richter , Cooper et al . to link specific parts of the brain to certain aspects of remembering .", "The hippocampus , an area known to be important in memory processing , indicated whether or not information had been remembered .", "More vivid memories were linked to greater activity in a region called the precuneus , which plays a role in imagination .", "Lastly , activity in a third region – the angular gyrus – indicated the precision of each memory .", "Being able to study different aspects of memory using tests like this that collect detailed measurements could be important in identifying memory problems , for example , in people with brain diseases or head injuries , or after a stroke .", "Specifically , the methods developed by Richter , Cooper et al . could provide sensitive tools for detecting memory difficulties at an early stage .", "This may help more people to get treated sooner , potentially minimizing lasting complications ." ]

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

[ "neuroscience" ]

[ [ "The precise coordination of movements on the two sides of the body is an essential feature of locomotion in animals with bilateral symmetry ( Grillner and Jessell , 2009; Arber , 2012; Moult et al . , 2013; Kiehn , 2016 ) .", "The left-right coordination during locomotion can vary in a context-dependent manner to produce alternating , e . g . walking in limbed animals or swimming in fish , or synchronous , e . g . hopping movements in limbed animals .", "This is largely achieved by regulating the activity of populations of commissural interneurons connecting local interacting circuits on the two sides of the spinal cord ( Soffe et al . , 1984; Dale , 1985; Buchanan , 1999a , 1999b; Butt and Kiehn , 2003 ) .", "The V0 interneurons , which originate from the p0 progenitor domain , represent a major commissural neuronal population controlling the left-right alternation of locomotor movements .", "The molecular mechanisms of differentiation of the V0 interneurons display striking similarities between zebrafish and mice .", "This interneuron population can be subdivided into two broad classes; one is excitatory and located ventrally early during development ( V0v ) , and the other is inhibitory and occupies a dorsal position ( V0d ) ( Moran-Rivard et al . , 2001; Pierani et al . , 2001; Lanuza et al . , 2004 ) .", "In addition , a detailed birth-date analysis in zebrafish has revealed three different subclasses of excitatory V0v interneurons arising in an order that correlates with their morphology and axonal projections , arguing for a further subdivision of this class of interneurons ( Satou et al . , 2012 ) .", "The current view of commissural V0 function in mediating the left-right alternation of locomotor movements derives from studies in immature motor systems ( newborn mice and zebrafish larvae ) and without detailed information about the activity at the single neuron level during locomotion .", "In newborn mice , a pattern of recruitment of the V0 interneurons has been proposed based on results from genetic ablations of the inhibitory ( V0d ) or the excitatory ( V0v ) interneurons .", "The two classes of V0 interneurons are thought to be differentially recruited to mediate the left-right alternation at different locomotor speeds .", "The inhibitory V0d interneurons are recruited first , during slow locomotion , and mediate direct inhibition .", "The excitatory V0v interneurons are recruited only at higher frequencies and mediate alternation by activating contralateral inhibitory interneurons ( Talpalar et al . , 2013; Bellardita and Kiehn , 2015 ) .", "By contrast , in larval zebrafish , a morphologically defined subclass of V0v interneurons , the multipolar commissural descending ( MCoD ) , has been shown to be recruited only at slow swimming speeds and become inhibited at faster swimming frequencies ( McLean et al . , 2007; McLean and Fetcho , 2009 ) .", "The apparent discrepancy between the proposed roles of V0v interneurons in zebrafish versus in mice could be the result of a functional switch during evolution that could account for the difference in the speed-dependent recruitment of the V0v population in these two species .", "Alternatively , the V0v interneurons could represent a functionally heterogeneous population with separate subclasses being engaged with increased locomotor speed .", "While there is mounting evidence for such a heterogeneity within the spinal interneuron classes ( e . g . V2a and V1 ) ( Kimura et al . , 2008; McLean and Fetcho , 2009; Ausborn et al . , 2012; Satou et al . , 2012; Griener et al . , 2015; Bikoff et al . , 2016 ) , a direct characterization of V0v interneuron diversity has remained elusive .", "Here , we have examined the functional heterogeneity of V0v interneurons in the adult zebrafish spinal cord , a feat so far intractable in the adult mouse .", "We sought to determine if the V0v interneurons form one class engaged only within a specific swimming frequency range or if they are subdivided into functional sub-classes differentially recruited at specific speeds .", "The results show that this is a functionally heterogeneous sub-class of interneurons composed of three sub-types recruited and active at specific speeds during the swimming motor program , and one additional sub-type that does not display any locomotor-related activity .", "Furthermore , we show that the V0v interneurons which are active during locomotion are engaged at slow , intermediate or fast swimming frequencies and that their recruitment thresholds are determined by the scaling of their synaptic drive and intrinsic properties .", "Our work thus reveals that the V0v interneurons are not homogenously engaged at a defined locomotor speed , but conform to a modular organization that allows them to participate in the elaboration of the locomotor pattern at all speeds ." ], [ "The distribution of the V0v interneurons in the spinal cord of adult zebrafish was examined by crossing the Tg[vglut2:loxP-DsRed-loxP-GFP] with the Tg[dbx1b:Cre] line which results in GFP expression in the excitatory V0v interneurons ( Satou et al . , 2012 , 2013 ) .", "These interneurons have axons that initially project ventrally from the somata before they cross the midline ( Figure 1A , B ) .", "The axonal projections of the V0v interneurons were carefully examined in preparations mounted with the dorsal side up ( Figure 1C ) .", "These interneurons first project their axons laterally before turning to cross the midline ( Figure 1C ) .", "The V0v interneurons were evenly distributed along the rostro-caudal axis in the spinal cord ( Figure 1D ) .", "These interneurons were also distributed along the dorso-ventral axis with a large proportion occupying the middle of the spinal cord ( Figure 1D ) .", "There were 21 ± 6 V0v interneurons per hemi-segment ( n = 6 preparations ) .", "The distribution of V0v interneurons in the spinal cord as well as their number per hemi-segment are comparable to those reported for the excitatory V2a interneurons ( 20 per hemi-segment ) , while motor neurons are more numerous ( 40 per hemi-segment ) ( Ampatzis et al . , 2013 ) . 10 . 7554/eLife . 18579 . 003Figure 1 . Distribution of V0v interneurons in the spinal cord .", "( A , B )", "Lateral views of a spinal segment from two different preparations showing the distribution of GFP-expressing V0v interneurons .", "Images are the maximum intensity projections from 164 and 193 optical sections respectively .", "Most V0v interneurons project ventrally before crossing the midline as indicated in expansions ( right panels ) .", "( C ) Dorsal view of the spinal cord showing two V0v interneurons and their crossing axonal projections ( neurons are colored and expanded in the right panels for clarification ) .", "The image is the maximum intensity projections from 90 optical sections ( D ) Graph showing the dorso-ventral and rostro-caudal distribution of V0v interneurons within one hemisegment .", "Data are from three different preparations ( black , grey and open circles ) .", "The ventral edge is defined as 0 and the dorsal edge as 1 .", "V0v interneurons are evenly distributed along the rostro-caudal axis , with an average of 21 ± 6 neurons per hemisegment ( mean ± SEM ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 18579 . 003 The available data from larval zebrafish and newborn mouse suggest that the V0v interneurons are not active at all speeds of locomotion but are only engaged during slow or fast speeds in zebrafish and mice , respectively .", "However , in larval zebrafish , recordings were only limited to a single morphologically defined sub-class of V0v interneurons ( MCoDs ) , while in mice the V0v interneuron activity has thus far not been possible to evaluate during locomotion .", "Therefore , we sought to determine the pattern of activity of the V0v interneurons during the swimming motor pattern in adult zebrafish .", "To this end , we used the ex-vivo brainstem-spinal cord preparation in which fictive swimming activity is induced by stimulation of descending inputs and monitored by recording from a motor nerve ( Gabriel et al . , 2011; Kyriakatos et al . , 2011 ) , while whole-cell patch-clamp recordings are obtained from V0v interneurons expressing GFP .", "The V0v interneurons ( n = 119 from 87 preparations ) were heterogeneous and displayed a range of activity patterns during fictive swimming ( Figure 2 ) out of which two broad types emerged .", "The first includes V0v interneurons displaying rhythmic synaptic inputs coordinated with the locomotor activity recorded in the motor nerve ( Figure 2A–C ) , and the second comprises interneurons showing tonic and non-patterned synaptic activity ( Figure 2D ) . 10 . 7554/eLife . 18579 . 004Figure 2 . Activity of V0v interneurons during locomotion . Slow ( red ) , intermediate ( green ) , fast ( light blue ) and non-patterned ( purple ) V0v interneurons show different activity patterns during locomotion .", "( A ) Recording from a V0v interneuron that was active already at a slow swimming frequency and remained recruited at high frequencies .", "( B ) Recording from a V0v interneuron that was not active at slow swimming frequencies and became recruited only at intermediate and high frequencies .", "( C ) Recording from a V0v interneuron that only displayed subthreshold membrane potential oscillations that did not reach the firing threshold at slow and intermediate swimming frequencies .", "( D ) Recording from a V0v interneuron whose activity was non-patterned by swimming activity .", "( E ) Recruitment frequency was significantly different between the three subtypes of V0v interneurons with patterned membrane potential oscillations during swimming activity ( one-way ANOVA , F ( 2 , 28 ) = 123 . 3 , p<0 . 0001; followed by Tukey’s procedure as post hoc; graph displays means and SEM; n = 17 from 16 preparations , 9 from 8 preparations and 5 from 5 preparations for slow , intermediate and fast , respectively ) .", "( F ) The amplitude of the membrane potential oscillations of the three ( slow , intermediate and fast ) rhythmically active V0v interneurons varied with swimming frequency .", "( G ) The membrane oscillation amplitude increased significantly between slow ( <3 Hz ) and intermediate ( >5 Hz ) swimming frequencies for the V0v interneurons recruited at intermediate and fast swimming frequencies ( Student’s paired t-test; p=0 . 035 for slow , p=0 . 0029 for intermediate , p=0 . 018 for fast; n = 5 from 5 preparations , 8 from 8 preparations , and 9 from 7 preparations for slow , intermediate and fast , respectively ) .", "( H ) There is no topographic organization of the somata of the different V0v interneurons in the spinal motor column .", "There was no significant difference ( one-way ANOVA , F ( 3 , 115 ) = 2 . 129 , p>0 . 05 ) between the soma size of the different sub-classes ( slow: 40 . 4 ± 1 . 5; intermediate: 44 . 5 ± 3 . 0; fast: 41 . 8 ± 1 . 2; and unpatterned: 53 . 3 ± 9 . 9 ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 18579 . 004 The V0v interneurons that displayed rhythmic activity could be further subdivided based on their activity pattern , recruitment frequency and the amplitude of their synaptic inputs .", "Based on these criteria , three sub-classes emerged that conform to the same organization previously found for the motor neurons and the V2a interneurons ( Gabriel et al . , 2011; Ausborn et al . , 2012; Ampatzis et al . , 2013 ) .", "One sub-class of V0v interneurons bore characteristics previously found in motor neurons and V2a interneurons belonging to the slow microcircuit module .", "These neurons are recruited at the onset of the locomotor activity and fire action potentials throughout a swim episode ( Figure 2A ) .", "These slow V0v interneurons always received suprathreshold excitation that allowed them to be recruited already at slow swimming speed ( range: 1 . 18–2 . 35 Hz; 1 . 74 ± 0 . 10 Hz; Figure 2E ) , which corresponds to the lowest frequency obtained in these experiments .", "The second sub-class of V0v interneurons was recruited only when the swimming activity fell within an intermediate frequency span , a feature of neurons belonging to the intermediate microcircuit module ( Figure 2B ) .", "These intermediate V0v interneurons received subthreshold synaptic excitation at slow speeds ( <3 Hz ) and were recruited at a minimum locomotor frequency of 3 . 64 ± 0 . 40 Hz ( range: 2 . 33–5 . 39; Hz; Figure 2E ) .", "Finally , the third sub-class displayed only subthreshold rhythmic activity and did not fire action potentials at the slow and intermediate swimming frequencies elicited in our preparation , a characteristic feature of neurons belonging to the fast microcircuit module ( Figure 2C ) .", "The minimum recruitment frequency of fast V0v interneurons was estimated to 13 . 78 ± 1 . 46 Hz ( range: 10 . 50–17 . 60 Hz; Figure 2E ) by linearly extrapolating the change in their synaptic inputs to their firing threshold .", "These neurons are likely to display a larger increase in the amplitude of the synaptic input they receive when the swim frequency approaches their recruitment threshold ( see [Ampatzis et al . , 2013] ) .", "The three sub-classes of V0v interneurons could also be clearly distinguished based on the change in the amplitude of their rhythmic synaptic inputs as a function of the locomotor frequency ( Figure 2F ) .", "Synaptic inputs were measured from the peak to the through of the oscillations .", "At locomotor frequencies below 3 Hz , the slow V0v interneurons received large synaptic inputs ( red; 8 . 0 ± 1 . 35 mV ) followed by the intermediate ones ( green; 7 . 9 ± 1 . 2 mV ) while fast interneurons received the weakest inputs ( blue; 2 . 7 ± 0 . 3 mV ) .", "The amplitude of the synaptic inputs decreased somewhat for the slow , but increased in the intermediate and fast V0v interneurons when the locomotion reached intermediate frequencies ( 5–10 Hz ) reaching an amplitude of 6 . 13 ± 0 . 8 mV in slow; 14 . 5 ± 2 . 0 mV in intermediate and 3 . 3 ± 0 . 8 mV in fast V0v interneurons ( Figure 2G ) .", "The different functional sub-classes of V0v interneurons did not show any difference in their soma size , nor did they occupy a specific location in the spinal cord , and were hence not organized in a topographic manner ( Figure 2H ) .", "The lack of a topographic organization is contrary to what we have previously found for the motor neurons ( McLean et al . , 2007; Gabriel et al . , 2011 ) , but in accordance with the V2a interneurons ( Ausborn et al . , 2012 ) .", "Since the activity of a neuron during locomotion is very likely to be determined by the balance between its intrinsic properties and the synaptic input it receives , we decided to investigate the nature of the synaptic input to the V0v interneurons in more detail .", "We recorded the excitatory and inhibitory currents received by the V0v interneurons in voltage clamp at −65 mV ( reversal potential of inhibition ) and 0 mV ( reversal potential of excitation ) , respectively .", "All V0v interneurons , except those receiving non-patterned inputs , displayed rhythmic and alternating excitatory and inhibitory currents during swimming activity ( Figure 3A–C; n = 8 from 8 preparations for slow , n = 8 from 7 preparations for intermediate , and n = 20 from 18 preparations for fast ) .", "Current amplitude varied with swimming frequency , such that the neurons received larger currents at higher swimming frequencies ( Figure 3A–C ) .", "This observation corresponded well with the variation of oscillation amplitude measured in current-clamp ( see Figure 2 ) , suggesting that neurons displaying high amplitude oscillations in current-clamp receive large excitatory and inhibitory currents .", "The excitatory current underlies the on-cycle excitation controlling the recruitment of the V0v interneurons , while the inhibitory current is mostly controlling the mid-cycle inhibition .", "Therefore , we focused our analysis on the excitatory current and how it controls the activity of the V0v interneurons at different swimming frequencies .", "However , there was no correlation between the amplitude of voltage oscillations and excitatory synaptic current received by the three sub-classes of V0v interneurons ( Figure 3D ) .", "Interestingly , slow and intermediate V0v interneurons could be segregated from the fast interneurons based on the amplitude of rhythmic inputs ( voltage oscillations ) they received during locomotion , the former always displaying oscillation amplitudes above 5 mV at slow/intermediate swimming frequencies ( i . e . , 4–5 Hz; Figure 3D ) .", "In addition , there was also a separation between the firing thresholds of slow/intermediate and the fast V0v interneurons at around −40 mV ( Figure 3E ) .", "These results indicate that the recruitment of the different V0v interneurons is not solely defined by the excitatory drive they receive , but also by their firing properties . 10 . 7554/eLife . 18579 . 005Figure 3 . Recruitment order is determined by synaptic input and intrinsic properties .", "( Ai–Ci )", "The patterned activity of the V0v interneurons was mediated by rhythmic and alternating excitatory and inhibitory currents .", "( Aii–Ciii )", "Both the excitatory and inhibitory current amplitude was dependent on the swim frequency , but did not vary significantly between the three V0v interneuron subtypes for excitatory currents ( one-way ANOVA F ( 2 , 26 ) = 2 . 13 , p>0 . 05; Tukey’s procedure as post hoc; n = 8 from 8 preparations , 6 from 5 and 15 from 15 preparations for slow , intermediate and fast respectively ) and only between intermediate and fast for the inhibitory currents ( one-way ANOVA F ( 2 , 24 ) = 5 . 21 , p<0 . 05; Tukey’s procedure as post hoc; n = 8 from 8 preparations , 6 from 5 preparations , and 13 from 13 preparations for slow , intermediate and fast , respectively ) .", "( D ) There was no correlation between the amplitude of the membrane potential oscillations and the excitatory currents .", "Slow/intermediate neurons ( red and green ) differed significantly from fast neurons ( blue ) in oscillation amplitude ( Student’s t-test; p<0 . 0001; n = 14 and 10 for slow/intermediate and fast neurons , respectively ) .", "Slow/intermediate neurons always had oscillation amplitudes above 5 mV and fast always below 5 mV ( as indicated by the dashed line ) .", "( E ) There was a correlation between the amplitude of the membrane potential oscillations and the firing thresholds of the different V0v interneurons .", "Firing thresholds of slow/intermediate ( red and green ) neurons were significantly different from those of fast ( blue ) neurons ( Student’s t-test; p<0 . 0001; n = 26 and 28 for slow/intermediate and fast neurons , respectively ) .", "Most slow/intermediate neurons had a firing threshold below −40 mV and most fast neurons a firing threshold above −40 mV ( indicated by the dashed line ) .", "( F ) There was no correlation between the amplitude of the excitatory current and the input resistance of the different V0v interneurons .", "( G ) The amplitude of the membrane potential oscillations was strongly correlated with the calculated excitatory drive received by the different V0v interneurons ( linear regression , R2 = 0 . 52 ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 18579 . 005 Furthermore , the order of recruitment of the different V0v interneurons did not correlate with the input resistance since there was a large overlap of the input resistance of the three V0v interneuron subclasses .", "In addition , no correlation was found between the amplitude of the excitatory current and the input resistance of V0v interneurons ( Figure 3F ) .", "These results indicate that neither the excitatory current nor the input resistance alone can account for the resulting rhythmic excitation setting the recruitment order of the V0v interneuron subclasses .", "Another possibility is that the synaptic inputs received by the V0v interneurons during swimming results from a combined contribution of their excitatory currents and their input resistance .", "In this scenario , each sub-class would always receive optimal excitation by compensating for a weak current with a high input resistance or a strong current with a low input resistance .", "To determine if this is the case , we calculated the excitation received by each V0v interneuron by multiplying the measured excitatory currents with the input resistance of the neurons .", "There was a strong correlation between the calculated excitation and the measured rhythmic inputs recorded during swimming in the different V0v interneurons ( Figure 3G; R2 = 0 . 52; p<0 . 001 ) .", "This indicates that the amount of excitation received by each V0v interneuron is the result of a scaling of the excitatory current and input resistance .", "These results show that the recruitment of the V0v interneurons is determined by a combination of their intrinsic properties and the received excitatory drive .", "To determine if the different sub-classes of V0v interneurons display specific firing patterns , current pulses of different amplitudes were applied and the resulting active responses examined .", "The slow V0v interneurons displayed a tonic or bursting firing pattern with little or no adaptation ( Figure 4A ) .", "The intermediate V0v interneurons also displayed a tonic or bursting firing pattern ( Figure 4B ) .", "However , the fast and non-patterned V0v interneurons displayed adaptation and in some cases only fired maximally a single action potential in response to the injected depolarizing current steps ( Figure 4C , D ) .", "In addition , the firing thresholds of the different V0v interneurons were graded in a manner that conforms to their order of recruitment during locomotion .", "The slow V0v interneurons display the lowest thresholds , followed by the intermediate and finally the fast and the non-patterned V0v interneurons .", "Thresholds were −46 . 2 ± 0 . 7 mV; −43 . 0 ± 1 . 0 mV; −35 . 2 ± 0 . 6 mV; and −29 . 5 ± 3 . 4 mV for slow , intermediate , fast and non-patterned neurons , respectively ( Figure 4E ) . 10 . 7554/eLife . 18579 . 006Figure 4 . V0v interneuron sub-classes display distinct intrinsic properties .", "( A–D )", "Examples of two different neurons from each group firing in response to an injected subthreshold current step ( A ) The V0v interneurons active already at slow swimming frequencies were either tonically firing or fired in bursts in response to current injections .", "( B ) The V0v recruited at intermediate frequencies also displayed tonic firing or bursting in response to current injections .", "( C ) The fast V0v interneurons always displayed strong adaptation .", "( D ) The V0v interneurons with non-patterned changes in the membrane potential fired only a few action potentials and displayed strong adaptive properties .", "( E ) The different V0v interneurons had different thresholds for firing action potentials in response to injected current .", "( F ) The firing thresholds were significantly different from each other and correlated strongly with the recruitment order as a function of swimming frequency ( one-way ANOVA F ( 3 , 104 ) = 33 . 8 , p<0 . 0001; Tukey’s procedure as post hoc; n = 19 from 17 preparations , 11 from 11 preparations , 74 from 58 preparations , and 4 from 4 preparations for slow , intermediate , fast and non-patterned respectively ) .", "( G ) A principal component analysis revealed that the V0v interneurons with patterned membrane potential oscillations could be separated from each other with those active at slow and intermediate frequencies showing partial overlap .", "The percentage of the total variance explained by PC1 and PC2 were 72 . 48 and 16 . 57 , respectively .", "The percentage contribution of the different variables to PC1 and PC2 , respectively , was: input resistance 20 . 4 and 44 . 9; action potential threshold 26 . 5 and 4 . 1; membrane oscillation amplitude in current clamp 21 . 5 and 48 . 6; minimum recruitment frequency 31 . 6 and 2 . 3 ( n = 13 neurons from 13 preparations , 10 neurons from 9 preparations , and 15 neurons from 13 preparations for slow , intermediate and fast , respectively ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 18579 . 006 In order to confirm the categorization of the rhythmically active V0v interneurons into slow , intermediate and fast based on their intrinsic properties , a principal components analysis was performed .", "This analysis allowed several parameters ( input resistance , action potential threshold , membrane oscillation amplitude in current clamp , and minimum recruitment frequency ) to be taken into account at once , creating a multidimensional view of the data .", "There was a clear separation of the fast V0v interneurons from the slow and intermediate .", "The slow and the intermediate V0v interneurons could also be partly segregated with some overlapping parameters ( Figure 4G ) , concurring with their differential activation during locomotion .", "Thus , the rhythmically active V0v interneurons can be separated into the three different groups previously described for motor neurons and V2a interneurons .", "The morphological identity of V0v interneurons has been previously examined in zebrafish larva .", "By the use of birth date analysis , it was found that the V0v interneurons could be divided into three morphologically distinct groups with ascending , descending and bifurcating axonal projections .", "The latter two groups arise later from common progenitors whereas the former is produced the earliest and from distinct progenitors ( Satou et al . , 2012 ) .", "Additionally , it has been suggested that earlier born spinal neurons in the larval zebrafish form the fast system , whereas those later born are involved in slow swimming ( Liu and Westerfield , 1988; Kimura et al . , 2006; McLean and Fetcho , 2009; Fetcho and McLean , 2010 ) .", "Taken together , these studies suggest that fast V0v interneurons would predominantly be of the ascending morphology , whereas the intermediate and slow V0vs would be predominantly bifurcating and descending .", "The morphologies of V0v interneurons in the adult zebrafish could be analyzed post-experimentally by using neurobiotin in the patch pipette ( Figure 5 ) .", "Contrary to what the studies in larval zebrafish suggest , no correlation was found between the morphology of a neuron and its activity pattern during locomotion ( Figure 5A–D ) .", "All neurobiotin-filled neurons were confirmed to be commissural neurons and a large proportion of them correspond to those that would be recruited at faster swimming speeds ( Figure 5E ) .", "In addition , the majority of V0v interneurons across all four groups had descending axons .", "These axons could be between 2 and 22 segments long and both short and long axons were found in all four groups .", "Apart from descending , we also found ascending , bifurcating and local neurons .", "Bifurcating neurons were defined as having axonal projections stretching over one segment in both ascending and descending directions and local neurons were defined as having projections not extending into the adjacent segment of that where the soma was located .", "Moreover , the descending and bifurcating V0v interneurons could either have axonal projections that were smooth , or extending multiple collaterals , mainly in the dorsal and rostro-caudal directions ( Figure 5D ) .", "Neurons from all four groups displayed extensive dendritic trees in contrast to what has been documented in larvae ( Satou et al . , 2012 ) , suggesting rather large developmental changes in the morphology of neurons between larvae and adults .", "It is worth noting that none of the V0v interneurons had the distinct morphological characteristic of the MCoDs , perhaps suggesting that these V0v interneurons are a larval feature or undergo dramatic morphological remodeling as the network matures into that of the adult . 10 . 7554/eLife . 18579 . 007Figure 5 . Morphological diversity of V0v interneurons .", "( A–D )", "Examples of dye-labeled V0v interneurons .", "( A ) Slow , ( B ) intermediate , ( C ) non-patterned , ( D ) fast .", "The morphologies of the different V0v interneurons show heterogeneity in terms of their axonal and dendritic projections .", "Small , black arrows indicate where the axon crosses to the contralateral side; grey , filled grey arrows indicate that the axon projects further than illustrated ( E ) Graph showing the range of the axonal projections of the different neurobiotin-labeled V0v interneurons . DOI: http://dx . doi . org/10 . 7554/eLife . 18579 . 007" ], [ "This study reveals that the excitatory commissural V0v interneurons represent a functionally heterogeneous class .", "Although they express a defined transcription factor and transmitter phenotype , their activity pattern during locomotion is not uniform .", "This interneuron class could be segregated into two distinct types based on whether they display rhythmic activity or not during locomotion .", "The rhythmically active V0v interneurons could be further subdivided into three main sub-classes engaged sequentially at slow then intermediate and finally fast locomotor speed .", "The order of recruitment of these three sub-classes of V0v interneurons is defined by the integration of their synaptic drive with their intrinsic properties .", "The extent of the rhythmic excitation results from a scaling of the synaptic current with the input resistance of the different V0v interneurons .", "This study thus provides a systematic characterization of the V0v interneurons showing that they represent a functionally heterogeneous population with different sub-classes engaged in a speed-dependent manner during locomotion .", "The molecular mechanisms underlying the development and differentiation of V0 commissural interneurons seem to be conserved between zebrafish and mouse ( reviewed in [Goulding , 2009] ) .", "These interneurons can be segregated into a dorsal inhibitory ( V0d ) , and a ventral excitatory population ( V0v ) .", "The progenitors giving rise to these interneurons express the transcription factor Dbx1 the elimination of which altered the left-right coordination ( Pierani et al . , 2001; Lanuza et al . , 2004; Talpalar et al . , 2013 ) .", "Selective ablation of V0d and V0v interneurons in the immature mouse suggested a differential contribution depending on the speed of locomotion ( Talpalar et al . , 2013; Kiehn , 2016 ) .", "Based on these results , it has been suggested that V0d and V0v interneurons act as two homogenous classes controlling the left-right alternation in a speed-dependent manner with a switch from V0d to V0v as the locomotor activity changes from slow to fast frequencies .", "However , no recordings have thus far been made from identified V0d or V0v interneurons to assess directly whether they do indeed represent a homogenous class and how their activity patterns change as a function of the locomotor frequency .", "A detailed analysis of the development of V0v interneurons in zebrafish has revealed that these commissural excitatory interneurons consist of three sub-classes that differentiate in a specific temporal order correlating with their axonal trajectories ( Satou et al . , 2012 ) .", "Despite the many similarities and conserved developmental mechanisms of V0 interneurons between zebrafish and mice , the available results also point out some apparent discrepancies .", "In addition , the increase in locomotor speed in mice can be associated with change in gait while in fish there is no change in gait and the left-right alternation is maintained at all speeds .", "In mice both V0d and V0v play a role in ensuring the left-right alternation .", "The changes in the locomotor pattern following ablation of V0v interneurons in immature mice suggest that they act via di-synaptic inhibition by recruiting contralateral inhibitory interneurons at relatively faster speeds .", "In contrast , recordings from V0v MCoDs in larval zebrafish show that these neurons are only active during slow swimming ( McLean et al . , 2007; McLean et al . , 2008; Fidelin et al . , 2015 ) .", "Our results now show , in an adult vertebrate , that the V0v interneurons are more functionally diverse .", "Some are engaged at slow swimming while others are active at intermediate and fast swimming .", "The fact that a large proportion of V0v interneurons recorded in our study belong to the fast category , suggests that the V0v interneurons are primarily recruited at faster locomotor speeds .", "Our work indicates some possible developmental changes in the zebrafish V0v interneurons .", "In addition to the prevalence of fast V0v interneurons , we found no evidence for interneurons with morphology resembling the MCoDs described in larval zebrafish , although V0v interneurons active during slow frequency swimming were recorded from in our study .", "The absence of MCoDs in the adult zebrafish could result from the unsuccessful recombination in some V0v interneurons .", "Alternatively , there could be a downregulation of the GFP expression in these neurons .", "However , in larvae , the MCoDs are responsible for driving slow frequency locomotion ( McLean et al . , 2008 ) , whereas in the adult zebrafish this task appears to be carried out largely by slow V2a interneurons ( Ausborn et al . , 2012 ) .", "The results clearly show a heterogeneity in both the V0v interneuron activity pattern and morphology , arguing against the downregulation of GFP or unsuccessful recombination .", "Our results demonstrate that the V0v interneurons that are active during locomotion can be subdivided into slow , intermediate and fast subclasses recruited gradually as swimming speed increases .", "This subdivision matches our previous findings of a similar segregation of motor neurons and the excitatory V2a interneurons in adult zebrafish ( Gabriel et al . , 2011; Ausborn et al . , 2012; Ampatzis et al . , 2013 ) .", "Here , the locomotor circuit is composed of three sub-circuits of connected V2a interneurons and motor neurons engaged sequentially from slow to intermediate and then fast swimming .", "We now show that the same organization also applies to the V0v interneurons , which represent an important population of commissural interneurons in the spinal cord .", "However , in our study , we did not find any clear correlation between the morphology of the V0v interneurons and their order of recruitment .", "It seems that there is no direct relationship between the order of differentiation of these interneurons and their functional engagement during swimming in the adult zebrafish .", "Although slow , intermediate and fast V0v interneurons could be clearly distinguished and identified , there was a larger proportion of fast compared to slow or intermediate V0v interneurons in our random sampling .", "If this is a true representation of the subdivision of the V0v interneurons , it would tend to agree with the assumption put forward in the mouse that they are mostly , albeit in zebrafish not exclusively , active at fast locomotion .", "Moreover , there was an additional type of V0v interneuron not receiving rhythmic inputs during locomotion and whose function in the spinal circuitry is still unclear .", "There is a possibility that these non-patterned neurons are involved in fin movements rather than axial muscle movements .", "In lamprey , a proportion of excitatory commissural neurons have been shown to contact dorsal fin motor neurons ( Mentel et al . , 2008 ) .", "Alternatively , they could be involved in postural control , or could be involved in even more vigorous movement such as struggling .", "In conclusion , our study provides a systematic description of the functional diversity of V0v interneurons in an adult vertebrate .", "These interneurons are organized in a modular fashion and although they seem to contribute at all locomotor frequencies , those active at fast speed seem to be overrepresented in our recordings .", "Thus , we uncover an important organizational principle for an important class of commissural interneurons and the underlying cellular and synaptic mechanisms defining their pattern of recruitment as a function of locomotor speed ." ], [ "Zebrafish ( Danio rerio ) were raised and kept in a core facility at the Karolinska Institute according to established procedures .", "In order to target the excitatory V0 interneurons specifically , two transgenic zebrafish lines were crossed , Tg[dbx1b:Cre] and Tg[vglut2a:loxP-DsRed-loxP-GFP] which have previously been described ( Satou et al . , 2012 , 2013 ) .", "This cross gave offspring in which all dbx1b and vglut2a positive neurons expressed GFP , allowing for specific targeting for patch clamp recordings .", "7–10 weeks old juvenile/adult zebrafish of both sexes were used for all experiments .", "All experimental protocols were approved by the local Animal Research Ethical Committee , Stockholm .", "The preparation was carried out as described previously ( Gabriel et al . , 2011; Kyriakatos et al . , 2011 ) .", "Zebrafish were cold-anesthetized in frozen fish saline containing in mM: 134 NaCl , 2 . 9 KCl , 2 . 1 CaCl2 , 1 . 2 MgCl2 , 10 Hepes and 10 glucose with pH adjusted to 7 . 8 using NaOH and osmolarity adjusted to 290 mOsm .", "The skull was opened and the brain was cut at the level of the midbrain .", "The epaxial musculature was removed up to the caudal end of the dorsal fin and the skin was removed from the remaining tail musculature .", "The vertebral arches at the first two segments of the spinal cord were removed to allow access for extracellular stimulation of descending axons .", "Vertebral arches were also removed over 4–5 segments rostral to the dorsal fin position , to allow access for patch-clamp recording electrodes .", "The preparation consisting of the spinal cord in the vertebral column , the hindbrain and the caudal musculature were cut out and transferred to the recording chamber , placed on the side , and fixed with Vaseline .", "During experiments , the preparation was continuously perfused with oxygenated fish saline at room temperature , containing 10 μM D-tubocurarine ( Sigma-Aldrich , Sweden ) to block the neuromuscular junctions allowing for stability during patch-clamp recordings .", "Fish were anesthetized with 0 . 1% MS-222 ( Sigma-Aldrich ) and fixed in 4% PFA ( vol/vol; Sigma-Aldrich ) overnight at 4°C .", "The spinal cords were dissected out and washed 2 times for 10 min each in phosphate buffered saline ( PBS; 0 . 1 M , pH 7 . 4 , Ambion , Naugatuck , CT ) .", "Spinal cords were then incubated for 40 min at room temperature in blocking solution containing 5% bovine serum albumin ( BSA , Sigma-Aldrich ) , 0 . 15% normal rabbit serum ( NRS , Sigma-Aldrich ) , and 1% PBS-triton ( x-100 ) .", "Spinal cords were then incubated in rabbit anti-GFP antibody ( Life Technologies , Sweden ) , 1:200 in 5% BSA and PBS-triton ( x-100 ) for 48 hr at 4°C .", "The cords were washed 4 times for 10 min each in 1% PBS-triton ( x-100 ) and subsequently incubated in Alexa Fluor-488 anti-rabbit IgG antibody ( Life Technologies ) 1:400 in PBS-triton ( x-100 ) for 4 hr at room temperature .", "Spinal cords were then washed 5 times for 5 min each and 15 min in PBS and subsequently mounted with either lateral or dorsal side up in antifade fluorescent mounting medium ( Vectashield , Vector Labs , Sweden ) .", "Confocal images were obtained with a Zeiss LSM 510 laser-scanning confocal microscope .", "The pictures were put together in CorelDraw and color was added by tracing the neurons in Photoshop for clarification purposes .", "Number of neurons per hemisegment were counted from confocal pictures taken at different rostro-caudal positions along the spinal cord in 6 different preparations stained for GFP .", "Dorso-ventral soma position was calculated from the same confocal pictures .", "Extracellular recording and stimulation electrodes were pulled from borosilicate glass ( 1-mm o . d . , 0 . 87-mm i . d . ; Harvard Apparatus , Holliston , MA ) , broken down to the desired tip diameter ( 15–25 μm ) , and fire-polished .", "Extracellular recordings were made from the motor nerves running through the intermyotomal clefts at the tail , where the musculature was left intact .", "A stimulation electrode was placed dorsally on the rostral spinal cord to elicit locomotor episodes by electrical stimulation ( 1 s at 30 Hz or 10 s at 1 Hz; pulse width , 1 ms; current amplitude , 0 . 3–1 mA ) .", "Extracellular signals were amplified ( gain , 10 , 000 ) with a differential AC amplifier ( A-M Systems ) and filtered with low and high cutoff frequencies of 300 Hz and 1 kHz , respectively .", "Intracellular whole-cell recordings were performed from GFP-labelled V0v interneurons in the mid-body region rostral to the dorsal fin and peripheral nerve recording .", "For intracellular recordings , electrodes were pulled from borosilicate glass ( 1 . 5-mm o . d . , 0 . 87-mm i . d . ; Hilgenberg , Germany ) on a horizontal puller ( Sutter Instruments , Novato , CA ) and filled with intracellular solution containing in mM: 120 K-gluconate , 5 KCl , 10 Hepes , 4 Mg2ATP , 0 . 3 Na4GTP , 10 Na-phosphocreatine with pH adjusted to 7 . 4 with KOH and osmolarity to 275 .", "GFP-labeled V0v interneurons were visualized with a fluorescent microscope ( Axioskop FS Plus; Zeiss , Germany ) equipped with IR-differential interference contrast ( DIC ) optics and a CCD camera with frame grabber ( Hamamatsu , Japan ) .", "Intracellular patch-clamp electrodes were advanced into the exposed portion of the spinal cord through the meninges using a motorized micromanipulator ( Luigs & Neumann , Germany ) while applying constant positive pressure .", "Intracellular signals were amplified with a MultiClamp 700B intracellular amplifier ( Molecular Devices , Sunnyvale , CA ) and low-pass filtered at 10 kHz .", "In current-clamp recordings , no bias current was injected .", "Only V0v interneurons with stable membrane potentials , which fired action potentials in response to suprathreshold depolarizations , and that showed minimal changes in series resistance ( <5% ) were included in this study .", "Images of the outline of the soma as well as the dorsal edge of the spinal cord and the Mauthner axon were captured before patching .", "The soma size and position were analyzed post-experiment using the measurement functions in ImageJ ( http://rsb . info . nih . gov/ij ) .", "For the ventrodorsal soma position , the dorsal edge of the spinal cord was defined as 1 and the dorsal edge of the Mauthner axon as 0 ( Figure 2H ) .", "The mediolateral position of the soma was measured during the experiment by focusing between the soma position , the lateral edge of the Mauthner axon , and the lateral surface of the spinal cord .", "For the mediolateral soma position , the lateral edge of the Mauthner axon was defined as 0 and the lateral surface of the spinal cord as 1 .", "All positions and soma sizes were averaged from at least three individual measurements .", "Neurons were passively filled with neurobiotin tracer ( 0 . 25% vector labs ) during patch clamp experiments to reveal their morphological characteristics post hoc .", "Post-recording , preparations were transferred to 4% PFA solution and left for 48–72 hr at 4°C .", "The spinal cords were dissected out of the vertebral column and washed 4 times for 10 min each in PBS , then incubated in 0 . 5–1% PBS triton for 3–4 hr and subsequently incubated in streptavidin conjugated to Alexa Fluor 555 ( 1:500; Invitrogen , Sweden ) in PBS containing 0 . 5% Triton X-100 ( Sigma-Aldrich ) and 2% normal goat serum ( Sigma-Aldrich ) 24–72 hr at 4°C .", "The tissue was then washed in PBS 3 times for 10 min each and 2 times for 20 min each and mounted in antifade fluorescent mounting medium ( Vectashield , Vector Labs ) .", "Confocal images were obtained with a Zeiss LSM 510 laser-scanning confocal microscope .", "The pictures were put together and colored in Photoshop and CorelDraw .", "Data were digitized at 10 kHz ( extracellular recordings ) or 20 kHz ( patch clamp recordings ) with a Digidata 1322A A/D converter ( Molecular Devices ) and acquired using pClamp software ( version 10; Molecular Devices ) .", "Data analysis was performed in Matlab 2011a , Clampfit software and Axograph version 1 . 5 . 4 .", "The action potential voltage threshold of each V0 interneurons was determined from the measured membrane potential at which the dV/dt exceeded 10 mV/ms .", "The input resistance of neurons was calculated as the slope of a regression line to the linear region of the I–V curve ( membrane potentials of −80 to −60 mV ) , which was obtained by injection of hyperpolarizing current pulses ( duration , 1–2 s ) .", "The minimum recruitment frequency of V0 interneurons was defined as the lowest locomotor frequency of a swimming episode at which the neurons were firing action potentials .", "The minimum locomotor frequency obtained during swimming episodes varied between preparations , therefore , the minimum recruitment frequency of the slow V0v interneurons will be the minimum frequency obtained from those preparations .", "To analyze the postsynaptic currents ( PSCs ) , V0 interneurons were voltage clamped close to the reversal potential of excitation ( 0 mV ) or inhibition ( −65 mV ) .", "The peak-to-trough amplitude of the summed excitatory and inhibitory currents underlying the locomotor-driven membrane potential oscillations were analyzed and not the individual PSCs .", "Graphs and statistical analysis were made with GraphPad Prism 4 .", "Correlation analyzes were carried out using linear regressions .", "For normally distributed data , Student’s t-test ( two groups ) or one-way ANOVAs ( > two groups ) were used with Tukey’s procedure as post-hoc .", "Confidence intervals were 95% .", "Gaussian fits were carried out on all data .", "None of the analyzed data was abnormally distributed .", "Differences were considered to be significant if p<0 . 05 .", "All data are given as mean ± SEM .", "The PCA was carried out using xlStat .", "Factors were included in the PCA if they had a value significantly different from zero using a Pearson correlation matrix .", "Factors included were input resistance , action potential threshold , membrane potential oscillation amplitude and minimum recruitment frequency .", "Figures were put together in CorelDraw ." ] ]

[ "Flexibility in the bilateral coordination of muscle contraction underpins variable locomotor movements or gaits .", "While the locomotor rhythm is generated by ipsilateral excitatory interneurons , less is known about the commissural excitatory interneurons .", "Here we examined how the activity of the V0v interneurons – an important commissural neuronal class – varies with the locomotor speed in adult zebrafish .", "Although V0v interneurons are molecularly homogenous , their activity pattern during locomotion is not uniform .", "They consist of two distinct types dependent on whether they display rhythmicity or not during locomotion .", "The rhythmic V0v interneurons were further subdivided into three sub-classes engaged sequentially , first at slow then intermediate and finally fast locomotor speeds .", "Their order of recruitment is defined by scaling their synaptic current with their input resistance .", "Thus we uncover , in an adult vertebrate , a novel organizational principle for a key class of commissural interneurons and their recruitment pattern as a function of locomotor speed ." ]

[ "During movements such as swimming and walking , the left and right sides of the body are kept coordinated by specific neurons in the spinal cord .", "Some of these neurons – called V0 neurons – can either excite or inhibit neurons on the opposite side of the spinal cord .", "In mice , the inhibitory V0 neurons are responsible for left-right coordination when the mice are moving slowly , while the excitatory neurons operate when the animals are moving more quickly .", "However , in zebrafish larvae a group of excitatory V0 neurons are only active when the larvae are swimming slowly .", "Björnfors and El Manira investigated whether excitatory V0 neurons in adult zebrafish behave like those in the larvae , or whether they act more like those in mice .", "The experiments show that the excitatory V0 neurons in adult zebrafish can be separated into three groups that are activated either at slow , intermediate or fast speeds of movement .", "The activation of the excitatory V0 neurons depends on the properties of the neurons themselves in combination with signals they receive from other neurons in the spinal cord .", "Although the excitatory V0 neurons could be active across all speeds , Björnfors and El Manira found that more neurons were active at faster speeds .", "This suggests that , in the adult zebrafish , there are both similarities and differences in how the V0 neurons are organised compared to larval zebrafish and mice .", "The next step following on from this work would be to find out the specific roles of excitatory and inhibitory V0 neurons during movement ." ]

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

[ "cell biology" ]

[ [ "Targeting of mRNAs to specific locations within the cytoplasm can confer precise spatial control over protein synthesis and function ( Buxbaum et al . , 2015; Holt and Schuman , 2013; Martin and Ephrussi , 2009 ) .", "By compartmentalising protein function , mRNA localisation contributes to diverse processes , including embryonic axis determination , epithelial polarity and neuronal plasticity .", "Trafficking of mRNAs frequently depends on the action of cytoskeletal motors , in particular those that move along the polarised microtubule network ( Mofatteh and Bullock , 2017 ) .", "However , the mechanisms by which specific mRNAs are recruited to , and transported by , microtubule motors remain unclear .", "One of the most tractable systems for microtubule-based mRNA transport operates during early development of Drosophila melanogaster and is responsible for localising spatial determinants of embryonic patterning to microtubule minus ends .", "Transport of these mRNAs is dependent on the Egalitarian ( Egl ) and Bicaudal-D ( BicD ) proteins ( Bullock and Ish-Horowicz , 2001 ) , as well as the minus end-directed motor cytoplasmic dynein-1 ( dynein ) and its accessory complex dynactin ( Wilkie and Davis , 2001 ) .", "Egl is a 1004-amino-acid protein that directly associates with the specialised RNA stem-loops that mediate polarised transport ( so-called RNA localisation signals ) ( Dienstbier et al . , 2009 ) .", "The basis of RNA recognition by Egl is not known , although an exonuclease-like domain between residues 557 and 726 is partly responsible ( Dienstbier et al . , 2009 ) .", "Egl uses a short N-terminal region to bind BicD ( Dienstbier et al . , 2009 ) , and C-terminal features to bind the LC8 dynein light chain ( Navarro et al . , 2004 ) .", "Mammalian BicD orthologues – BICD1 and BICD2 – associate with dynein and dynactin ( Hoogenraad et al . , 2001 ) .", "These observations have led to a model for linkage of localising mRNAs to the dynein transport machinery ( Figure 1A ) .", "It is not known , however , if other factors co-operate with Egl and BicD to bridge mRNAs to the motor complex .", "Another outstanding question is how the assembly of the transport complex , and the activity of the dynein motor within it , is controlled .", "Several lines of evidence indicate that BicD is a key player in these processes .", "By forming an extended coiled-coil homodimer , the isolated N-terminal region of mammalian BICD2 ( BICD2N: containing coiled-coil domain 1 ( CC1 ) and part of CC2 ) can bridge the interaction between dynein and dynactin , forming a mutually dependent triple complex ( Hoogenraad and Akhmanova , 2016; Splinter et al . , 2012; Urnavicius et al . , 2015; Zhang et al . , 2017 ) .", "The binding of dynein to BICD2N and dynactin increases the incidence of processive movement dramatically ( McKenney et al . , 2014; Schlager et al . , 2014 ) , which is associated with repositioning of the dynein motor domains with respect to the microtubule ( Chowdhury et al . , 2015; Zhang et al . , 2017 ) .", "The motor also moves with higher velocity and has increased force output once bound to BICD2N and dynactin ( Belyy et al . , 2016; McKenney et al . , 2014 ) .", "The equivalent N-terminal region of Drosophila BicD stimulates dynein-based transport in vivo ( Dienstbier et al . , 2009 ) , indicating that this mechanism is evolutionarily conserved .", "Full-length BicD proteins interact poorly with dynein and dynactin and are therefore only weak activators of dynein motility ( Dienstbier et al . , 2009; Hoogenraad et al . , 2001 , 2003; Huynh and Vale , 2017; Liu et al . , 2013 ) .", "While mechanistic details are still lacking , BicD appears to be autoinhibited by folding back of the third coiled-coil domain ( CC3 ) onto the dynein-activating sequences in CC1/2 ( Dienstbier et al . , 2009; Hoogenraad et al . , 2001; Stuurman et al . , 1999 ) .", "It has been proposed that the interaction of CC3 with cargo-binding proteins such as Egl or Rab6 ( a G-protein that binds Golgi-derived vesicles ) frees CC1/2 to interact with dynein and dynactin ( Dienstbier et al . , 2009; Hoogenraad and Akhmanova , 2016; Hoogenraad et al . , 2001 , 2003; Matanis et al . , 2002 ) .", "Consistent with this model , mutating an essential residue in the shared Rab6- and Egl-binding site in CC3 prevents Drosophila BicD from associating with dynein in vivo ( Liu et al . , 2013 ) .", "It has recently been shown in vitro that the presence of Rab6 allows full-length BICD2 to associate with dynein and dynactin and thereby activate transport ( Huynh and Vale , 2017 ) .", "This observation provides direct evidence that association of a cargo-binding protein with CC3 stimulates the assembly of an active dynein-dynactin-BicD complex , although the stoichiometry of Rab6 and BICD2 in transport complexes was not investigated .", "Rab6 only associates with mammalian and Drosophila BicD proteins when it is GTP-bound ( Rab6GTP ) ( Huynh and Vale , 2017; Liu et al . , 2013; Matanis et al . , 2002; Short et al . , 2002 ) , a state induced by association with its target membranes ( Hutagalung and Novick , 2011 ) .", "These data suggest a mechanism for linking long-distance movement of dynein with the availability of a vesicular cargo .", "Egl , on the other hand , can associate with BicD CC3 in vitro in the absence of an RNA cargo ( Dienstbier et al . , 2009; Liu et al . , 2013 ) .", "This observation implies that the RNA is not involved in the relief of BicD autoinhibition by Egl , although this hypothesis has not been tested directly .", "We set out to elucidate molecular mechanisms of dynein-based mRNA transport by Egl and BicD by reconstituting this process in vitro with purified components .", "Our results define a minimal set of proteins for RNA translocation on microtubules and show that the RNA strongly activates dynein motility .", "Stimulation of transport by RNA is not dependent on the Egl-LC8 interaction .", "Rather , our data support a model in which the RNA localisation signal overcomes BicD autoinhibition by augmenting the interaction of Egl with BicD CC3 .", "Our study reveals a pivotal role of an RNA localisation signal in gating the activity of a microtubule motor , and give rise to a model in which cargoes stimulate dynein motility by scaffolding active adaptor protein assemblies ." ], [ "We set out to determine if purified dynein , dynactin , Egl and BicD are sufficient to induce mRNA transport in vitro .", "As no method is available for the purification of Drosophila dynein and dynactin , we established a system in which Drosophila Egl and an mRNA target are linked to mammalian dynein and dynactin complexes .", "We took advantage of the strong evolutionary conservation of the Egl/Rab6GTP-binding site of BicD ( Figure 1—figure supplement 1; [Liu et al . , 2013] ) to produce a complex of Drosophila Egl bound to mouse BICD2 .", "This complex was purified from Sf9 insect cells by co-expression of Egl with BICD2 , as soluble Egl could not be recovered in the absence of its binding partner ( Figure 1—figure supplement 2A , B ) .", "The Egl/BICD2 complex , which was captured using an affinity tag on Egl , was not associated with significant amounts of RNA ( Figure 1—figure supplement 2C ) .", "This observation is consistent with previous evidence that RNA is not essential for the interaction of Egl with Drosophila BicD ( Dienstbier et al . , 2009; Liu et al . , 2013 ) .", "The 1 . 4 MDa human dynein complex and 1 . 1 MDa pig dynactin complex were purified from established recombinant and native sources , respectively ( Schlager et al . , 2014 ) .", "The purity of these and other protein preparations used in the study is illustrated in Figure 1—figure supplement 3 .", "RNAs were transcribed in vitro , and body-labelled by stochastic incorporation of fluorescent UTP .", "Interactions of fluorescent RNA molecules with surface-immobilised microtubules were monitored by total internal reflection fluorescence ( TIRF ) microscopy in the presence of dynein , dynactin , and Egl/BICD2 ( Figure 1B ) .", "RNAs and proteins were incubated together for at least 1 hr to promote complex assembly , followed by dilution to concentrations that allow discrimination of single molecules on microtubules .", "We first used the 3’UTR of the hairy mRNA , which mediates transport by a complex containing Egl , BicD , dynein and dynactin in the Drosophila embryo ( Bullock et al . , 2003; Dix et al . , 2013 ) .", "We observed frequent association of hairy RNA with microtubules in the imaging chamber ( Video 1 ) .", "Gratifyingly , 80% of microtubule-associated hairy RNA puncta underwent long-distance transport ( Figure 1C , D and Video 1 ) .", "As observed previously with a Drosophila extract-based system ( Soundararajan and Bullock , 2014 ) , hairy RNAs accumulated at microtubule minus ends following transport ( Figure 1—figure supplement 4A ) and were also capable of diffusive motion on the microtubule lattice ( Figure 1D and Figure 1—figure supplement 4B ) .", "We also performed experiments with the I-factor retrotransposon RNA , which is transported in association with Egl , BicD , dynein and dynactin during Drosophila oogenesis ( Dienstbier et al . , 2009; Dix et al . , 2013; Van De Bor et al . , 2005 ) .", "Like hairy , this RNA exhibited robust minus end-directed transport in our in vitro assay ( Figure 1C ) .", "These experiments reveal that no additional proteins are required for microtubule-based mRNA transport in vitro .", "To test if RNA localisation signals are selectively recognised in our assay conditions , we mixed the well-characterised 59-nucleotide ( nt ) Egl-binding element from the I-factor ( I-factor localisation signal ( ILS ) ) ( Dienstbier et al . , 2009; Van De Bor et al . , 2005 ) , which was labelled with DY647 , with an equimolar amount of a scrambled version of the same sequence labelled with DY547 .", "In the presence of Egl , BICD2 , dynein , and dynactin , the ILS bound to microtubules ~five times more frequently than the mutant RNA and exhibited a similar relative increase in the number of processive movements ( Figure 1E–G ) .", "These data reveal that the transport machinery retains selectivity for RNA localisation signals in our assay .", "Further analysis revealed that ~75% of the processive complexes that contained the scrambled RNA also had a signal from the ILS ( Figure 1E , H ) , raising the possibility that much of the transport of the mutant RNA is an indirect consequence of association with active ILS-bound transport complexes .", "We next investigated the involvement of each of the protein complexes in the RNA transport process .", "We first used SNAP tags to fluorescently label dynein and either Egl or BICD2 in the Egl/BICD2 complex .", "Egl and BICD2 were co-transported with dynein and hairy RNA in the presence of dynactin ( Figure 2A and Figure 2—figure supplement 1; note that dynactin could not be labelled as it is from a native source ) .", "Next , we omitted individual protein complexes from the assembly mix .", "The association of hairy with microtubules was barely detected when Egl/BICD2 , dynactin , or dynein was excluded ( Figure 2B , C and Figure 2—figure supplement 2 ) .", "Thus , the simultaneous presence of all three protein complexes is required to link RNA to microtubules .", "In the absence of Egl/BICD2 or dynactin , dynein rarely exhibited transport but could still associate with microtubules ( Figure 2B and Figure 2—figure supplement 2 ) .", "However , there was an ~two-fold increase in microtubule binding events when both Egl/BICD2 and dynactin were present ( Figure 2D ) .", "Thus , the combination of Egl/BICD2 and dynactin stimulates dynein’s ability to associate with microtubules and move processively in the presence of RNA .", "As described in the Introduction , the prevailing model is that the association of Egl with BicD CC3 is sufficient to free the N-terminal region of BicD to interact with dynein and dynactin .", "Unlike Rab6 , Egl can bind BicD in the absence of associated cargo , leading us to ask whether dynein and dynactin differentiate between RNA-bound and RNA-free Egl/BICD2 .", "To address this question , we performed motility assays with Egl/BICD2 , dynactin and fluorescent dynein in the presence and absence of RNA .", "Strikingly , the number of processive movements of dynein was ~six-fold higher when the RNA was present ( Figure 3A , B ) .", "This reflected an increase in microtubule binding by dynein ( Figure 3C ) , as well as the propensity for processive movement of those complexes associated with the microtubule ( Figure 3D ) .", "The mean velocity and run length of dynein complexes bound to RNA were also significantly higher than those assayed in the absence of RNA ( Figure 3E , F ) .", "We conclude that the RNA is required for robust stimulation of dynein motility and microtubule binding in the presence of Egl/BICD2 and dynactin .", "We next asked if the RNA-directed activation of dynein was a consequence of the combination of Egl with a BicD protein from a different species by performing experiments with a preparation of Drosophila Egl and Drosophila BicD ( DmBicD ) .", "The Egl/DmBicD complex was also produced by co-expression of both proteins in Sf9 insect cells and purification with an affinity tag on Egl .", "The hairy RNA significantly increased the number of processive movements of dynein in the presence of dynactin and Egl/DmBicD ( Figure 3G , H ) .", "This effect was again associated with enhanced microtubule binding of the motor , as well as increased probability of processive movement after engaging with the microtubule ( Figure 3I , J ) .", "As was observed in the experiments with Egl/BICD2 , the RNA also enhanced the mean velocity and length of dynein movements ( Figure 3—figure supplement 1 ) .", "Thus , RNA also gates the activation of dynein motility by a co-evolved Egl/BicD complex .", "We next considered two scenarios for how RNA stimulates dynein motility .", "First , the Egl/BicD/dynein/dynactin complex could be efficiently formed in the absence of RNA , with binding of RNA to Egl triggering a conformational change that activates processive dynein movement .", "Second , the ability of the Egl/BicD complex to interact with dynein and dynactin could be stimulated by the association of Egl with RNA , thus conferring different properties on the motor .", "To distinguish between these possibilities , we fluorescently labelled Egl in the purified Egl/BICD2 complex and monitored how hairy RNA affects its association with microtubule-bound dynein in the presence of dynactin .", "Although there was some association of dynein with Egl in the absence of RNA , the frequency of co-localisation increased by ~six-fold when the RNA was present ( Figure 4A–C ) .", "We confirmed that the RNA also stimulates the association of BICD2 with microtubule-associated dynein by labelling BICD2 within the purified Egl/BICD2 complex ( Figure 4—figure supplement 1 ) .", "Many of the dynein complexes bound to Egl/BICD2 were motile ( regardless of whether RNA was present of absent ) ( Figure 4B , Figure 4—figure supplement 1 and Figure 4—figure supplement 2 ) , indicating that they were also complexed with dynactin ( McKenney et al . , 2014; Schlager et al . , 2014 ) .", "Thus , the ability of RNA to activate processive dynein motion is associated with enhanced assembly of the Egl/BICD2/dynein/dynactin complex .", "We next investigated if the assembly of the endogenous transport complex is stimulated by RNA .", "We immunoprecipitated a transgenically expressed GFP-tagged Egl protein from Drosophila embryo extracts in the presence and absence of exogenous hairy 3’UTR and assayed for co-precipitation of the p150 ( DCTN1/Glued ) subunit of dynactin and the heavy chain of dynein ( Dhc ) by western blotting ( Figure 4D , E ) .", "p150 and Dhc were not detected in the Egl::GFP immunoprecipitate in the absence of exogenous RNA , indicating that the association of Egl with dynein and dynactin is of low affinity or low abundance .", "In contrast , the addition of the hairy RNA led to detectable co-precipitation of the dynein and dynactin components with Egl::GFP .", "Thus , assembly of the transport complex is promoted by the RNA in the context of both purified and endogenously-expressed proteins .", "The results described above raise the question of how RNA binding stimulates the association of Egl and BicD proteins with dynein and dynactin .", "We first asked if this involves the binding of Egl to the LC8 dynein light chain .", "Motility assays were performed with a purified Egl/BICD2 complex in which Egl has two mutations in a consensus LC8-binding site that abolish association with LC8 in vivo and in vitro ( Egldlc2pt; S965K + S969R ) ( Navarro et al . , 2004 ) .", "The Egldlc2pt/BICD2 complex supported robust transport of hairy RNA in the presence of dynein and dynactin ( Figure 5A , B ) .", "Moreover , the mutant Egl/BICD2 complex still supported the RNA-induced increase in processive movement and microtubule binding of dynein in the presence of dynactin ( Figure 5C–E ) , as well as higher mean velocities and run lengths of the motor ( Figure 5—figure supplement 1 ) .", "Thus , the interaction of Egl with LC8 does not play a significant role in activation of dynein by RNA .", "These observations pointed to the other reported interaction of Egl/BICD2 with dynein and dynactin – that is the one mediated by BICD2N – as central to the activation of transport .", "As described in the Introduction , previous studies have indicated that occupancy of the Egl/Rab6GTP-binding site in BICD2 relieves autoinhibition , licensing BICD2N to interact with dynein and dynactin ( Huynh and Vale , 2017; Liu et al . , 2013 ) .", "During handling of the purified Egl/BICD2 complex , we noticed that it had a tendency to dissociate upon dilution .", "This observation suggests dynamic exchange of constituent species .", "We therefore wondered if the RNA relieves BICD2 autoinhibition by stabilising its interaction with Egl .", "To test this hypothesis , we first mixed the 59-nt ILS RNA with purified Egl/BICD2 and performed size exclusion chromatography .", "The RNA localisation signal caused a large change in the elution profile of the protein complex compared to the RNA-free form ( Figure 6—figure supplement 1 ) , indicating a substantial increase in molar mass or a conformational change .", "We next used sedimentation equilibrium analytical ultracentrifugation ( SE-AUC ) to evaluate mean molar masses of complexes in the presence and absence of RNA independently of protein conformation .", "Over a range of protein concentrations , the presence of the ILS caused a large increase in mean molar mass compared to RNA-free samples ( Figure 6A and Figure 6—figure supplement 2 ) .", "An orthogonal method for determining molar masses – size-exclusion chromatography with multi-angle light scattering ( SEC-MALS ) – confirmed that the ILS substantially increases the mean molar mass of the Egl/BICD2 sample ( Figure 6B and Figure 6—figure supplement 3 ) .", "This effect was evident at all salt concentrations examined ( Figure 6B and Figure 6—figure supplement 4 ) .", "Despite being present in a 10-fold molar access to Egl/BICD2 , the scrambled ILS RNA elicited a relatively small increase in mean molar mass ( Figure 6C ) , confirming selectivity of the Egl/BICD2 complex for an active RNA localisation signal .", "Our finding that there is some association of Egl/BICD2 with the mutant RNA is compatible with earlier evidence that Egl is not a highly selective RNA-binding protein ( Bullock et al . , 2006; Dienstbier et al . , 2009; Dix et al . , 2013 ) .", "The ILS induced a broad range of molar masses in the peak fractions , indicating an equilibrating mixture of larger complexes and smaller constituent components ( Figure 6B , C ) .", "Consistent with such dynamics , the mean molar mass of the peak fractions increased with increasing amounts of Egl/BICD2 and RNA ( Figure 6D ) .", "Analysis of peak SEC-MALS fractions by SDS-PAGE revealed that the ILS-induced increases in mass were associated with enhanced interaction of BICD2 and Egl ( Figure 6B and Figure 6—figure supplement 4B ) .", "We also used SEC-MALS to determine the effect of the ILS on the purified complex of Egl and DmBicD .", "The mean molar mass of the peak fractions increased substantially in the presence of the ILS , and this was again associated with increased binding of Egl and the BicD protein ( Figure 6—figure supplement 5 ) .", "Collectively , these experiments reveal that the Egl/BICD2 and Egl/DmBicD complexes readily equilibrate with constituent species and that this is counteracted by the RNA localisation signal .", "In our SE-AUC and SEC-MALS experiments , mean molar masses of the mixtures of ILS , Egl and a BicD protein could reach ~400 kDa .", "The predicted molar masses of the BICD2 , DmBicD and Egl polypeptides are 93 , 89 and 112 kDa , respectively , while the ILS has a molar mass of 19 kDa .", "It was previously shown that DmBicD is a dimer ( Stuurman et al . , 1999 ) , and we confirmed that this is also the case for BICD2 using SEC-MALS ( Figure 6B; observed molar mass 186 . 7 ± 0 . 5 kDa ) .", "The mean molar masses observed in our experiments with the ILS are therefore compatible with a fraction of BicD dimers being occupied by more than one Egl molecule .", "To directly evaluate the stoichiometry of Egl and BicD in mRNA transport complexes , we returned to our in vitro motility assay .", "This system allows investigation of the copy number of these proteins in the fraction of complexes that are able to recruit dynein and dynactin and thus support processive movement on microtubules .", "We first produced Egl/BICD2 complexes with SNAP-tagged BICD2 and labelled them with a mixture of SNAP-reactive dyes such that approximately half of BICD2 polypeptides in the preparation were labelled with TMR , and approximately half were labelled with Alexa647 .", "In an idealised situation , the exclusive presence of BICD2 dimers would result in 50% of complexes with one TMR dye and one Alexa647 dye , 25% with two TMR dyes and 25% with two Alexa647 dyes ( Figure 7A ) .", "However , incomplete labelling of SNAP::BICD2 meant that an obligate BICD2 dimer would result in 40% of complexes labelled with both dyes ( Supplementary file 1 ) .", "When the labelled Egl/SNAP::BICD2 sample was used in motility assays with dynein , dynactin and hairy RNA , 39% of the motile complexes with a BICD2 signal were labelled with both dyes ( Figure 7B , C ) .", "Our co-localisation analysis therefore fits well with there being a single BICD2 dimer in transport complexes ( Supplementary file 2 ) .", "When the procedure was repeated with SNAP-tagged Egl co-expressed with BICD2 , the proportion of fluorescent complexes that was dual-labelled in the presence of RNA was 37% ( Figure 7D , E ) .", "Correcting for the small fraction of Egl::SNAP molecules that are unlabelled , this result indicates that there are two Egl molecules in the vast majority of active RNA transport complexes ( Supplementary files 1 and 2 ) .", "When this experiment was performed in the absence of RNA , the relatively small number of motile Egl complexes observed also had signal from both dyes in 41% of cases ( Figure 7—figure supplement 1 ) .", "These data indicate that even when the assembly of the transport machinery is inefficient , motility is usually associated with the presence of two Egl molecules in a complex .", "The capacity of BicD to bind two Egl molecules is compatible with the symmetrical nature of the Egl-binding region of CC3 ( Liu et al . , 2013 ) .", "Finally , we investigated the copy number of RNA in transport complexes by performing motility assays with equimolar amounts of hairy RNA preparations that were labelled by random incorporation of either Cyanine3 ( Cy3 ) or Cy5 .", "Of the labelled motile RNPs , 14% had signal from both dyes ( Figure 7F , G ) .", "Considering that the same proportion of complexes should have two Cy3 dyes or two Cy5 dyes , and that the labelling efficiency means that a small fraction of RNA molecules will contain neither dye , these data indicate that 30% of RNPs contained two RNAs and 70% contained one RNA ( Supplementary file 3 ) .", "We previously found that transported hairy RNPs assembled in Drosophila extracts exclusively contain a single RNA ( Amrute-Nayak and Bullock , 2012; Soundararajan and Bullock , 2014 ) .", "The subset of complexes containing two hairy RNAs in our current assay presumably reflects a degree of non-specific RNA-RNA interaction or RNA-protein interaction , which is normally blocked in extracts by the binding of other proteins .", "We observed very little co-localisation of Cy3-hairy and Cy5-hairy on a glass surface in the absence of Egl , BICD2 dynein and dynactin ( Figure 7—figure supplement 2 ) , suggesting that the presence of two copies of the RNA in a subset of transport complexes is predominantly due to an interaction of the second RNA molecule with one of the proteins in the transport machinery .", "Motile complexes containing both Cy3 and Cy5 exhibited similar velocity distributions and only a modest increase in run length compared to those containing only a single dye ( Figure 7—figure supplement 3 ) .", "Thus , the presence of a second RNA does not have substantial functional consequences .", "In summary , the results of the dual-labelling experiments are consistent with the vast majority of BicD dimers in active transport complexes associating with two Egl polypeptides , and most associating with a single RNA .", "Together with our earlier results , these data support a model in which the RNA localisation signal licenses BicD to bind dynein and dynactin by facilitating the association of BicD CC3 with two Egl molecules ( see below ) ." ], [ "We have succeeded in reconstituting microtubule-based mRNA transport in vitro using purified proteins and have used this system to define a minimal transport-competent RNP .", "Although genetic experiments indicate that other proteins can modulate the mRNA transport process in vivo ( Dix et al . , 2013; Hain et al . , 2014 ) , no other factors appear to be obligatory for linkage of the RNA to dynein .", "There has recently been considerable focus on the regulation of dynein motility , stemming from the discovery that the isolated N-terminal region of BicD proteins can bridge the interaction of dynein and dynactin and thereby activate transport ( McKenney et al . , 2014; Schlager et al . , 2014; Splinter et al . , 2012 ) .", "Subsequent structural studies have provided important insights into how dynein activity is controlled in this system ( Chowdhury et al . , 2015; Grotjahn et al . , 2018; Urnavicius et al . , 2018 , 2015; Zhang et al . , 2017 ) .", "However , because full-length BicD proteins interact with dynein and dynactin poorly ( Hoogenraad et al . , 2001 , 2003; Huynh and Vale , 2017 ) , it is unclear how dynein activity is controlled within intact cargo-motor complexes .", "Previous observations suggested that binding of a cargo-associated protein such as Egl to the C-terminal region of BicD is sufficient to overcome the autoinhibited state of the full-length protein , and thereby lead to recruitment of dynein and dynactin .", "Our study reveals that a purified Egl/BicD complex does not efficiently associate with dynein and dynactin in the absence of an RNA localisation signal .", "Thus , the availability of the cargo gates robust activation of dynein motility .", "This mechanism presumably limits unproductive long-range movement of the motor complex in the absence of an RNA consignment .", "We show that the previously reported interaction of Egl with LC8 ( Navarro et al . , 2004 ) is not required for RNA-directed activation of dynein motility .", "Characterisation of the features of LC8 that mediate interaction with its binding partners also argue against a role for LC8 as an adaptor between Egl and dynein; the groove that LC8 uses to associate with the consensus binding motif present in Egl is also used for incorporation into the dynein complex , suggesting mutually exclusive interactions ( Benison et al . , 2007; Rapali et al . , 2011 ) .", "However , disrupting the Egl-LC8 interaction in Drosophila does significantly compromise the function of Egl in the maintenance of oocyte fate ( Navarro et al . , 2004 ) .", "There are several examples of LC8 acting as a chaperone for binding partners independently of its association with dynein ( Rapali et al . , 2011 ) , and it may serve the same function for Egl in vivo .", "Our data indicate that a key consequence of RNA binding to Egl is stimulation of the interaction of BicD with dynein and dynactin .", "Thus , RNA-bound Egl must overcome the autoinhibition of full-length BicD that prevents CC1/2 from engaging with dynein and dynactin .", "Negative stain electron microscopy in a contemporary study ( Sladewski et al . , 2018 ) lends further support to this notion; a folded back conformation of full-length DmBicD ( Stuurman et al . , 1999 ) , which is likely to represent the autoinhibited state ( Hoogenraad et al . , 2001 , 2003; Liu et al . , 2013 ) , was retained in the presence of Egl alone , but not detected in the presence of both RNA and Egl .", "Our single-molecule analysis is also consistent with the activation of transport by RNA being mediated by CC1/2 .", "The recruitment of RNA to dynein by Egl/BICD2 is dependent on dynactin ( Figure 2B , C ) , as is also the case for the interaction of BICD2N with the motor complex ( McKenney et al . , 2014 ) .", "Furthermore , the ability of RNA-bound Egl/BICD2 to augment dynein’s binding to microtubules ( Figure 3C ) , as well as its velocity ( Figure 3E ) and run length ( Figure 3F ) , is also shared with BICD2N ( McKenney et al . , 2014 ) .", "Very recently , it has been shown that a single BICD2N dimer and a single dynactin can recruit one or two dynein complexes ( Grotjahn et al . , 2018; Urnavicius et al . , 2018 ) , with the binding of the second motor increasing velocity and run length ( Urnavicius et al . , 2018 ) .", "The two-motor state is associated with a subtle difference in the position of the N-terminal region of BICD2 CC1 ( Urnavicius et al . , 2018 ) .", "Sladewski et al . , 2018 provide evidence that two dynein motors are present in the majority of their transport RNPs , raising the possibility that interaction of RNA-bound Egl with BICD2 modulates the velocity and run length of transport complexes by favouring the two-motor-binding conformation of CC1 .", "An in vitro study of a yeast actin-based mRNA transport complex also reported stimulation of processive movement by the RNA cargo ( Sladewski et al . , 2013 ) ( although an independent investigation of the same complex reported no influence of the RNA [Heym et al . , 2013] ) .", "The mechanism that we and Sladewski et al . , 2018 propose for RNA-mediated activation of dynein – involving relief of autoinhibition of an adaptor – is distinct from the one proposed for the yeast transport complex , which is based on RNA-dependent dimerisation of monomers of the myosin motor ( Sladewski et al . , 2013 ) .", "Thus , multiple strategies may have evolved to co-ordinate the processivity of cytoskeletal motors with the availability of an RNA cargo .", "How could binding of RNA to Egl relieve BicD autoinhibition ?", "Although a complex of Egl bound to BicD can be purified in the absence of RNA following overexpression in insect cells , our data indicate that it readily dissociates into constituent species .", "The interaction between Egl and BicD is mediated by the first 79 amino acids of Egl and a 42-amino-acid region of BicD CC3 ( Dienstbier et al . , 2009; Liu et al . , 2013 ) .", "Although we cannot rule out additional mechanisms of RNA-mediated activation of BicD , the most parsimonious explanation for our data is that the RNA localisation signal promotes the occupancy of CC3 with Egl , thereby freeing CC1/2 to interact with dynein and dynactin ( Figure 8 ) .", "It is not clear how binding of RNA-associated Egl ( or Rab6GTP for that matter ) to BicD CC3 releases CC1/2 .", "One possibility is that binding of Egl/Rab6GTP competes directly with the interaction of CC3 with the N-terminal sequences .", "Alternatively , occupancy of the Rab6GTP- and Egl-binding site in CC3 could induce changes in coiled-coil architecture that are propagated along the molecule to release a discrete autoinhibitory interaction ( Liu et al . , 2013 ) .", "The discovery of crystal forms of CC3 with different coiled-coil registers ( Liu et al . , 2013; Terawaki et al . , 2015 ) lends support to the involvement of coiled-coil dynamics in the activation of BicD .", "It is striking that the vast majority of active transport complexes contain two Egl polypeptides per BicD dimer , even when the transport process is compromised by the omission of RNA .", "This finding suggests that occupancy of both Egl-binding sites of BicD CC3 favours the relief of BicD autoinhibition and recruitment of dynein and dynactin .", "How could the RNA localisation signal stabilise the heterotetrameric Egl/BicD complex ?", "Purified BicD was found not to interact directly with RNA localisation signals ( Dienstbier et al . , 2009 ) , suggesting that the RNA does not act as a bridge between Egl and BicD .", "Our single-molecule experiments are consistent with two Egl proteins being able to associate with a single RNA molecule .", "A structure-function study of an Egl-binding RNA localisation signal revealed two structurally-related helices that must be precisely registered with each other in order to trigger mRNA transport ( Bullock et al . , 2010 ) .", "Our SEC-MALS analysis indicates that free Egl is monomeric ( Figure 6—figure supplement 3 ) .", "It is therefore tempting to speculate that the two helices of the RNA localisation signal are discrete binding sites for Egl monomers , as this offers a simple explanation for how the RNA facilitates the association of two Egl molecules with a BicD dimer .", "Alternatively , binding of the RNA localisation signal could induce a conformational change in Egl that stabilises the protein or increases its affinity for CC3 , thereby favouring full occupancy of BicD .", "High-resolution structures of RNA-protein complexes will be required to discriminate between these possibilities .", "In addition to Rab6GTP-associated vesicles and Egl-associated mRNAs , BicD proteins are implicated in transport of a diverse range of cellular cargoes and pathogens by dynein and dynactin ( Dharan et al . , 2017; Hoogenraad and Akhmanova , 2016; Indran et al . , 2010; Redwine et al . , 2017 ) .", "It is conceivable that the cargo also promotes the activation of dynein in these systems by scaffolding the association of two cargo-associated proteins with CC3 .", "It is easy to envisage how this could occur during membrane trafficking , when the diffusion of CC3-interacting proteins within the membrane would greatly facilitate co-incident association .", "The finding that BicD CC3 can simultaneously bind two Rab6GTP proteins ( Liu et al . , 2013 ) is compatible with this scenario ." ], [ "Sf9 cells ( ThermoFisher Scientific , Waltham , MA ) have not been genetically profiled since purchase but were grown in a tissue culture facility dedicated to insect cell expression .", "The cells were tested for mycoplasma twice a year ( MycoAlert Detection Kit , Lonza ) and the results were always negative .", "Sequences encoding Egalitarian and BicD proteins ( Drosophila melanogaster Egl isoform B:NM_166623 , mouse BICD2:NM_001039179 and Drosophila melanogaster BicD:NM_165220 ) were synthesised commercially ( Epoch Life Sciences , Sugar Land , TX ) with codons optimised for expression in Spodoptera frugiperda Sf9 cells , and cloned for use with the MultiBac expression system .", "Where required , sequences encoding SNAPf tags for fluorescent labelling of protein complexes and ZZ-LTLT tags for IgG-based affinity purification ( Reck-Peterson et al . , 2006 ) were added by Gibson Assembly ( NEB , Ipswich , MA ) of PCR-amplified insert and backbone fragments .", "All constructs were validated by sequencing of the entire open-reading frame .", "Genes encoding Egl::LTLT-ZZ or Egl::SNAP-LTLT-ZZ were cloned downstream of the polh promoter of the pACEBac1 acceptor vector ( Sari et al . , 2016 ) , while genes encoding BICD2 , SNAP::BICD2 , or Drosophila melanogaster BicD ( DmBicD ) were cloned downstream of the polh promoter of the pIDC donor vector ( Sari et al . , 2016 ) .", "The donor and acceptor vectors were recombined at defined Cre loci and incorporated into the baculovirus genome for simultaneous co-expression of Egl and BicD proteins .", "The same strategy was used for assembly of the gene encoding human DHC ( tagged at the N-terminus with ZZ-LTLT-SNAP ) with those encoding other human dynein subunits , as described previously ( Schlager et al . , 2014 ) .", "The isoform composition of the assembled dynein complex is as follows: DHC:NM_001376 . 4; DIC2:AF134477; DLIC2:NM_006141 . 2; Tctex:NM_006519 . 2; LC8:NM_003746 . 2 and Robl:NM_014183 . 3 .", "All recombinant proteins were expressed from the baculovirus genome in Sf9 cells as described previously ( Schlager et al . , 2014 ) .", "Following protein expression , cells were frozen in liquid N2 and stored at −80°C .", "The Egldlc2pt mutations ( S965K + S969R ) ( Navarro et al . , 2004 ) were generated by whole-vector PCR using a single pair of complementary mutagenic primers containing the desired sequence .", "Following amplification , the template DNA was digested with DpnI , and the amplicon ligated and propagated by transformation into α-Select Silver Efficiency chemically competent E . coli ( Bioline , London , UK ) .", "The presence of the desired mutations , and no others , was confirmed by sequencing of the entire open-reading frame .", "All purification steps were performed at 4°C .", "Native dynactin was purified from pig brain as described previously ( Schlager et al . , 2014; Urnavicius et al . , 2015 ) .", "Dynein , BICD2 , Egl/BICD2 and Egl/DmBicD complexes were affinity purified via an N-terminal ZZ-LTLT on DHC ( ZZ-LTLT-SNAP::DHC ) and BICD2 ( ZZ-LTLT-BICD2 ) , or a C-terminal LTLT-ZZ tag on Egl ( Egl::LTLT-ZZ or Egl::SNAP-LTLT-ZZ ) .", "Frozen Sf9 cells were thawed on ice .", "For dynein purification , cells were resuspended in lysis buffer ( 50 mM HEPES pH 7 . 3 , 100 mM NaCl , 10% glycerol , 1 mM DTT , 0 . 1 mM MgATP , 2 mM PMSF , 1 x cOmplete EDTA-free protease inhibitor cocktail ( Sigma-Aldrich , St Louis , MO ) ) .", "For purification of Egl/BICD2 and Egl/DmBicD complexes , lysis buffer was modified to include 500 mM NaCl to disrupt any association of Egl with native RNA species .", "Lysates were generated by repeated passage of resuspended cells through a Wheaton dounce tissue grinder ( Fisher Scientific , Hampton , NH ) and subsequently clarified by ultracentrifugation at 70 , 000 RPM ( 504 , 000 x g ) using a Beckman Coulter Type 70 Ti fixed-angle rotor in a Beckman Coulter Optima L-100 XP preparative ultracentrifuge .", "During centrifugation , IgG Sepharose 6 affinity resin ( GE Healthcare Life Sciences , Little Chalfont , UK ) was applied to a gravity flow Econo-column ( Bio-Rad , Hercules , CA ) and washed twice with five column volumes of lysis buffer ( typically 5 ml of resin slurry was used ) .", "Clarified lysate was added directly to the affinity matrix in the column , which was then sealed and agitated by gentle rolling for 3 hr .", "After incubation , the lysate was allowed to flow through the column by gravity and the retained affinity matrix washed twice with five column volumes of lysis buffer and twice with five column volumes of TEV buffer ( 50 mM Tris-HCl pH 7 . 4 , 150 mM KOAc , 2 mM MgOAc , 1 mM EGTA-KOH pH 7 . 3 , 10% glycerol ) .", "If required , bound SNAP-tagged proteins were fluorescently labelled on-column before proceeding to elution ( see below ) .", "Bound proteins were eluted by overnight TEV cleavage of the ZZ affinity tag using gentle rolling agitation and ~0 . 03 mg ml−1 TEV protease in a final volume of 15 ml TEV buffer .", "Eluted protein was recovered by gravity flow through a fresh Econo-column and concentrated to ~1 . 5 mg ml−1 with a 100 kDa MWCO Amicon Ultra-4 centrifugal filter unit ( Merck , Darmstadt , Germany ) .", "The affinity-purified protein complexes were further purified by FPLC-based gel-filtration chromatography ( AKTA Purifier and AKTA Micro , GE Healthcare Life Sciences ) in GF150 buffer ( 25 mM HEPES pH 7 . 3 , 150 mM KCl , 1 mM MgCl2 , 5 mM DTT , 0 . 1 mM MgATP , 10% glycerol ) to remove large aggregates , TEV protease , and other small contaminants .", "For SEC-MALS and SE-AUC experiments , GF150 was modified to include 5 mM TCEP instead of DTT .", "For the dynein complex , a TSKgel G4000SWxl with guard column ( TOSOH Bioscience Ltd , Reading , UK ) was used , while a Superose 6 Increase 3 . 2/300 column ( GE Healthcare Life Sciences ) was used for BICD2 , Egl/BICD2 and Egl/DmBicD complexes .", "Fractions containing the dynein complex were pooled and concentrated to ~1 mg ml−1 .", "Fractions containing BICD2 , Egl/BICD2 or Egl/DmBicD complexes were pooled without an additional concentration step .", "All purified proteins were dispensed in aliquots for single use , flash frozen in liquid N2 , and stored at −80°C .", "Protein concentrations were determined using a Coomassie Protein Assay Kit ( ThermoFisher Scientific ) .", "To assess purity , proteins were resolved by SDS-PAGE using Novex 4–12% Bis-Tris precast gels ( ThermoFisher Scientific ) and MES-SDS running buffer .", "Protein bands were visualised using Coomassie Instant Blue protein stain ( Expedeon , Over , UK ) and imaged with a ChemiDoc XRS + system ( Bio-Rad ) .", "Protein sizes were evaluated by comparison with Full-Range Rainbow prestained molecular weight markers ( GE Healthcare Life Sciences ) .", "Fluorescent labelling of SNAP-tagged proteins with either SNAP-Cell TMR-Star ( NEB ) or SNAP-Surface Alexa Fluor 647 ( NEB ) was performed on-column during affinity capture according to a previously described method that labels >95% of dynein dimers with at least one dye ( Schlager et al . , 2014 ) .", "For the mixed-labelling of SNAP::BICD2 and Egl::SNAP in Figure 7 , an extended labelling time of 4 hr and a further 10-fold excess of total SNAP-fluorophore reagent was used .", "This method labelled 90% of SNAP-tagged polypeptides ( 81% of complexes containing two protein copies labelled with two dyes ) ( Supplementary file 1 ) .", "Labelling efficiency was determined with spectrophotometry as previously described ( Schlager et al . , 2014 ) .", "The ratio of SNAP-Surface Alexa Fluor 647 to SNAP-Cell TMR-Star that yielded approximately half of labelled polypeptides having one fluorophore and half the other fluorophore was determined empirically for different batches of the dyes .", "Uncapped Cy5-hairy RNA or Cy3-hairy RNA was transcribed in vitro from a gel-purified PCR amplicon template using the MEGAscript T7 Transcription Kit ( Ambion ) .", "The RNA is a 730-nt region of the 3’UTR containing the RNA localisation signal ( Bullock et al . , 2003 ) .", "Cy3-UTP or Cy5-UTP ( PerkinElmer , Waltham , MA ) was added to the transcription reaction together with a 4-fold excess of unlabelled UTP in order to label the RNA at multiple internal sites .", "Alexa488-hairy RNA was synthesised from the same template using a 1:9 ratio of Alexa488-UTP ( ThermoFisher Scientific ) to unlabelled UTP .", "Cy5-I-factor RNA was synthesised from a linearised plasmid template using the MEGAscript SP6 Transcription Kit ( Ambion ) and a 1:3 ratio of Cy5-UTP to unlabelled UTP .", "The RNA is 597-nt long and contains the ILS localisation signal ( Van De Bor et al . , 2005 ) .", "Following digestion of the template DNA with DNase I , proteins were removed using phenol-chloroform-isoamyl alcohol ( ThermoFisher Scientific ) .", "Synthesised RNA was separated from unincorporated nucleotides by two rounds of purification with Sephadex G-50 size-exclusion RNA spin columns ( Sigma-Aldrich ) , precipitated with NH4OAc/ethanol and resuspended in nuclease-free dH2O .", "These procedures typically yield RNA samples with an average of ~3 dyes per molecule .", "Where relevant , the mean number of dyes per RNA molecule was determined by spectrophotometry ( Supplementary file 3 ) .", "ILS wild-type ( Van De Bor et al . , 2005 ) and scrambled mutant RNAs ( with and without a single 5’ DY547 or DY647 dye ) were synthesised , decapped , deprotected , and HPLC purified by GE Dharmacon ( Lafayette , CO ) .", "An additional two A’s were included at the 5’ prime of synthetic RNAs to space the fluorophore from the wild-type or mutant localisation signal .", "Sequences of the RNAs can be found in the Key Resources Table .", "For SE-AUC and SEC-MALS experiments , RNAs were further purified by gel-filtration chromatography in GF150 buffer ( Superose 6 Increase 3 . 2/300 , AKTA Micro ( GE Healthcare ) ) .", "All RNA concentrations were determined by spectrophotometry .", "Glass surfaces were prepared as described previously ( Bieling et al . , 2010 ) .", "Motility chambers with a volume of ~10 μl were assembled by adhering glass cover slips functionalised with PEG/Biotin-PEG ( Rapp Polymere , Tuebingen , Germany ) to glass slides passivated with PLL-g-PEG ( SuSos AG , Duebendorf , Switzerland ) using three segments of double-sided tape distributed along the width of the slide .", "The arrangement of tape yielded two parallel motility chambers per cover slip and allowed side-by-side comparison of two different conditions on the same glass surface .", "For the experiment presented in Figure 7—figure supplement 2 , RNA samples were added to the imaging chambers at this point .", "For all other assays , chamber surfaces were further passivated for 5 min with 1% ( w/v ) Pluronic F-127 ( Sigma-Aldrich ) and washed twice with 20 μl chilled motility buffer ( 30 mM HEPES pH 7 . 3 , 5 mM MgSO4 , 1 mM EGTA pH 7 . 3 , 1 mM DTT , 0 . 5 mg ml−1 BSA ) .", "Chambers were then incubated with 2 mg ml−1 streptavidin ( Sigma-Aldrich ) for 5 min and again washed twice with 20 μl motility buffer .", "To block any unpassivated surface , chambers were incubated with 20 mg ml−1 α-casein ( Sigma-Aldrich ) for 5 min and washed twice with 20 μl motility buffer .", "The prepared chambers were kept in a humidified container until the addition of microtubules and protein/RNA mixtures to prevent desiccation of chamber surfaces .", "Microtubules were polymerised from porcine tubulin ( Cytoskeleton Inc . , Denver , CO ) and labelled with fluorophores and biotin by stochastic incorporation of labelled dimers into the microtubule lattice .", "Mixes of 1 . 66 μM unlabelled tubulin , 0 . 15 μM Hilyte488-tubulin , and 0 . 4 μM biotin-tubulin were incubated in BRB80 ( 80 mM PIPES pH 6 . 85 , 2 mM MgCl2 , 0 . 5 mM EGTA , 1 mM DTT ) with 0 . 5 mM GMPCPP ( Jena Bioscience , Jena , Germany ) for 2–4 hr at 37°C .", "Polymerised microtubules were pelleted in a room temperature table top centrifuge at 18 , 400 x g for 8 . 5 min , and washed once with pre-warmed ( 37°C ) BRB80 .", "After pelleting once more , the microtubules were gently resuspended in pre-warmed ( 37°C ) BRB80 containing 40 μM paclitaxel ( taxol; Sigma-Aldrich ) and used on the same day .", "Constituents of motility assays were incubated together on ice for 1–2 hr by dilution into motility buffer to the following concentrations: 100 nM dynein , 200 nM dynactin , 100 nM Egl/BICD2 or Egl/DmBicD ( using the operational assumption of two Egl molecules and one dimer of the BicD protein per complex ) , and 1 μM RNA .", "To ensure that all complexes assemble at the same ionic strengths , KCl was supplemented to a final concentration of 50 mM during assembly .", "Just prior to imaging , stabilised microtubules were immobilised in a prepared motility chamber for 5 min and subsequently washed once with motility buffer that also contained 50 mM KCl , 1 mg ml−1 α-casein , and 20 μM taxol .", "Assembly mixes were then diluted 40-fold ( with the exception of the complexes in Figure 4A–C , which were diluted 20-fold ) in motility buffer that also contained 50 mM KCl , 1 mg ml−1 α-casein , 20 μM taxol , 2 . 5 mM MgATP , and an oxygen scavenging system ( 1 . 25 μM glucose oxidase , 140 nM catalase , 71 mM 2-mercaptoethanol , 25 mM glucose ) that greatly limits photobleaching ( Yildiz et al . , 2003 ) .", "Diluted assembly mixes were applied to immobilised microtubules in the motility chamber for imaging at room temperature ( 23 ± 1°C ) .", "For the experiment documented in Figure 7—figure supplement 2 , RNA only was added to the chamber and immediately washed with motility buffer containing 50 mM KCl , 1 mg ml−1 α-casein , 20 μM taxol , 2 . 5 mM MgATP , and an oxygen scavenging system .", "For each chamber , a single multicolour acquisition of 500 frames was made at the maximum achievable frame rate ( ~2 frames s−1 ) and 100 ms exposure per frame using a Nikon TIRF microscope system controlled with Micro-Manager open-source acquisition software ( Edelstein et al . , 2010 ) and equipped with a Nikon 100 × oil objective ( APO TIRF , 1 . 49 NA oil ) .", "For the experiment documented in Figure 7—figure supplement 2 , single frames were captured with a 1 s exposure in each channel .", "The following lasers were used: Coherent Sapphire 488 nm ( 150 mW ) , Coherent Sapphire 561 nm ( 150 mW ) , Coherent CUBE 641 nm ( 100 mW ) .", "Images were captured with an iXonEM+ DU-897E EMCCD camera ( Andor , Belfast , UK ) , resulting in pixel dimensions of 105 x 105 nm .", "Multicolour acquisitions used sequential image capture with switching of emission filters ( GFP , Cy3 , and Cy5 ( Chroma Technology Corp . , Bellows Falls , VT ) ) .", "Extracts were generated from embryos of P[tub-Egl::GFP] ( Dienstbier et al . , 2009 ) or Sco/CyO P[actin5C-GFP] flies ( Bloomington Drosophila Stock Center: RRID:BDSC_4533 ) , which contain genomically-integrated transgenes expressing Egl::GFP or GFP from the ubiquitous α-tubulin or β-actin promoters , respectively .", "0–12 hr embryos were dechorionated and flash frozen in liquid N2 .", "300 μl chilled extraction buffer ( 25 mM HEPES pH 7 . 3 , 50 mM KCl , 1 mM MgCl2 , 2 mM DTT , 2x cOmplete EDTA-free protease inhibitor ) was added for each 100 mg of frozen embryos , followed by grinding on ice with a motorised pellet pestle ( ThermoFisher Scientific ) .", "The material was subjected to 25 passes in a Wheaton dounce tissue grinder ( ThermoFisher Scientific ) on ice before the addition of 200 μl chilled extraction buffer containing 0 . 5% Triton-X-100 per 100 mg of embryos .", "Following gentle mixing , samples were incubated on ice for 5 min and passed through a 23G syringe five times before clarification by two centrifugation steps ( each 5 min at 3000 x g ) .", "350 μl aliquots of clarified extract were incubated with 20 units Recombinant RNase Inhibitor ( Promega , Madison , WI ) and either 20 μl of a 6 . 7 μg/μl solution of unlabelled hairy RNA in dH2O or 20 μl dH2O for 30 min at 4°C .", "Magnetic beads coupled to GFP-binding protein ( GFP-Trap MA ( Chromotek , Martinsried , Germany ) ) were washed twice in PBS , followed by blocking of non-specific interaction sites with 1 mg ml−1 casein in PBS for 30 min at 4°C .", "After two washes of the beads in extraction buffer , the equivalent of 30 μl of initial bead slurry was mixed with the embryo extracts with or without hairy RNA .", "Following a 2 hr 30 min incubation at 4°C , beads were washed fives times for 1 min in extraction buffer containing 0 . 05% Triton-X-100 ( three washes in 400 μl of buffer and two washes in 1 ml buffer ) .", "Proteins and RNA-protein complexes were eluted from the beads by the addition of 60 μl 1 x lithium dodecyl sulphate ( LDS ) buffer ( ThermoFisher Scientific ) /50 mM DTT and incubation at 80°C for 10 min .", "Following electrophoresis and blotting onto PVDF membranes , proteins were detected using the following primary antibodies: mouse α-GFP ( mix of clones 7 . 1 and 13 . 1 ( Sigma-Aldrich; RRID:AB_390913 ) ; diluted 1:1000 ) ; mouse α-Dhc ( clone 2C11-C [Sharp et al . , 2000]; RRID:AB_2091523 ) ( provided by the Developmental Studies Hybridoma Bank ( University of Iowa , Iowa , IA ) and diluted 1:1000 ) and rabbit α-p150-C-term ( [Kim et al . , 2007]; provided by V . Gelfand , Northwestern University; diluted 1:10 , 000 ) .", "Secondary antibodies were conjugated to horseradish peroxidase , with signal detected using the ECL Prime system ( GE Healthcare ) and Super RX-N medical X-ray film ( FUJIFILM , Bedford , UK ) .", "Duplicate independent preparations of 1 mg ml−1 Egl/BICD2 ( 2 . 4 μM assuming two Egl molecules and a single BICD2 dimer per complex ) in GF150 buffer ( using 5 mM TCEP instead of 5 mM DTT ) in the presence or absence of a 10-fold molar excess of ILS RNA were pre-incubated on ice for at least 1 hr and subsequently diluted in GF150 ( TCEP ) to yield three samples with volumes of 110 µl and protein concentrations of 1 , 0 . 33 , and 0 . 11 mg ml−1 .", "These samples were loaded in 12 mm six-sector cells and subjected to equilibrium sedimentation in an An50Ti rotor using an Optima XL-I analytical ultracentrifuge ( Beckmann ) at 3200 , 5600 , and 10 , 000 rpm until equilibrium was reached at 4˚C .", "At each speed , comparison of several scans was used to judge whether equilibrium had been reached .", "Data were processed and analysed using SEDPHAT 13b ( [Schuck , 2003]; RRID:SCR_016254 ) and plotted with GUSSI ( [Brautigam , 2015]; RRID:SCR_014962 ) .", "The partial-specific volumes ( v-bar ) , solvent density and viscosity were calculated using Sednterp ( T . Laue , University of New Hampshire; RRID:SCR_016253 ) .", "Samples of BICD2 , Egl/BICD2 and Egl/DmBicD were resolved on a Superdex 200 HR10/300 analytical gel filtration column ( GE Healthcare ) at 0 . 5 ml min−1 in GF150 buffer ( using 5 mM TCEP instead of 5 mM DTT ) , GF75 buffer ( contains 75 mM KCl with 5 mM DTT ) , or GF50 buffer ( contains 50 mM KCl with 5 mM TCEP ) .", "All measurements for BICD2 and Egl/BICD2 were made at room temperature , whereas the relative instability of the Egl/DmBicD complex required measurements be made at 4°C .", "Where indicated , ILS RNA was added at a 10-fold molar excess over Egl/BICD2 or Egl/DmBicD ( based on an operational assumption of two Egl molecules and a dimer of the BicD protein per complex ) and incubated on ice for 1 hr prior to injection on the column .", "Samples lacking RNA were subjected to the same incubation .", "Following SEC fractionation , eluted protein was detected on a Wyatt Heleos II 18 angle light scattering instrument coupled to a Wyatt Optilab rEX online refractive index detector in a standard SEC-MALS format .", "Heleos detector 12 at 99° was replaced with Wyatt’s QELS detector for on-line dynamic light scattering measurements .", "Protein concentration was determined from the excess differential refractive index based on 0 . 186 RI increment for 1 g ml−1 protein solution .", "Concentrations and observed scattered intensities at each point in the chromatograms were used to calculate the absolute molecular mass from the intercept of the Debye plot , using Zimm’s model as implemented in ASTRA software ( Wyatt; RRID:SCR_016255 ) .", "Fractions were analysed by gel electrophoresis and staining with SYPRO Ruby ( Lonza , Cambridge , UK ) or Coomassie Instant Blue according to the manufacturer’s instructions .", "Kymographs were generated and analysed manually using FIJI ( [Schindelin et al . , 2012]; RRID:SCR_002285 ) .", "Typically , three independent chambers were imaged using protein complexes from at least two independent assembly reactions for each experimental condition .", "The positions of microtubules were determined by the fluorescent tubulin signal or a projection of RNA/protein signals over the course of the movie .", "From each of these chambers , 5–10 microtubules were typically selected for analysis with preference given to those that were longer and better isolated from adjacent microtubules .", "No power analysis was used to determine sample size .", "Instead the sample size was chosen to allow the identification of a range of effect sizes .", "To avoid the risk of subconscious bias , microtubules were selected before visualising the motile properties of complexes on them .", "Interactions of fluorescently labelled proteins and RNA with microtubules were scored as binding events if they were ≥1 . 5 s ( three frames ) in duration and as processive events if they achieved predominantly minus end displacement >500 nm ( five pixels ) without significant diffusive behaviour .", "These parameters were chosen in advance of image acquisition following discussion within the team and all particles that fulfilled the criteria were analysed .", "As described previously ( Schlager et al . , 2014 ) , some motile complexes changed velocity during a run , leading us to calculate velocities of individual constant-velocity segments .", "Run lengths were calculated from the total displacement of individual particles regardless of changes in velocity or pauses .", "For both velocity and run length calculations , only particles for which the entire run was observed or those with runs beginning >5 μm from the microtubule minus end were considered .", "When velocities and run lengths were calculated in the presence of RNA , only those complexes clearly associated with RNA were analysed .", "Although plots of 1 - cumulative frequency for run lengths were fitted to a one-phase exponential decay for visualisation purposes , statistical comparison of run lengths were performed on unfitted data .", "For Figures 1D , 2C and 5B , ‘background’ RNA binding was quantified by generating kymographs from random microtubule-free regions of the cover slip of lengths equal to the median microtubule length of those used for analysis .", "For illustrative purposes , the movie and kymographs in the figures had background subtracted in FIJI with a rolling ball radius of 50 pixels .", "All quantitative analysis was performed on the raw data .", "The gel analysis tools of FIJI were used to quantify the SYPRO Ruby signal in background-subtracted images ( rolling ball radius of 50 pixels ) .", "Statistical analyses , curve fitting , and data plotting were performed using Prism 7 . 0b ( GraphPad; RRID:SCR_002798 ) .", "A two-tailed Student’s t-test or a two-tailed Welch’s t-test was used when comparing two groups where a Gaussian data distribution was expected , with the latter test employed in cases of unequal variance .", "A Mann-Whitney test was used to compare two groups with non-Gaussian data distributions .", "An ANOVA test with Dunnett’s correction was used for multiple comparisons ." ] ]

[ "Polarised mRNA transport is a prevalent mechanism for spatial control of protein synthesis .", "However , the composition of transported ribonucleoprotein particles ( RNPs ) and the regulation of their movement are poorly understood .", "We have reconstituted microtubule minus end-directed transport of mRNAs using purified components .", "A Bicaudal-D ( BicD ) adaptor protein and the RNA-binding protein Egalitarian ( Egl ) are sufficient for long-distance mRNA transport by the dynein motor and its accessory complex dynactin , thus defining a minimal transport-competent RNP .", "Unexpectedly , the RNA is required for robust activation of dynein motility .", "We show that a cis-acting RNA localisation signal promotes the interaction of Egl with BicD , which licenses the latter protein to recruit dynein and dynactin .", "Our data support a model for BicD activation based on RNA-induced occupancy of two Egl-binding sites on the BicD dimer .", "Scaffolding of adaptor protein assemblies by cargoes is an attractive mechanism for regulating intracellular transport ." ]

[ "In our cells , tiny molecular motors transport the components necessary for life’s biological processes from one location to another .", "They do so by loading their cargo , and burning up chemical fuel to carry it along pathways made of filaments .", "For example , one such motor , called dynein , can move molecules of messenger RNA ( mRNA ) to specific locations within the cell .", "There , the mRNA will be used as a template to create proteins , which will operate at exactly the right place .", "Transporting mRNA in this way is critical in processes such as embryonic development and the formation of memories; yet , this mechanism is still poorly understood .", "Previous work suggested that the mRNA is simply a passenger of the dynein motor , but McClintock et al . asked if this is really the case .", "Instead , could mRNA regulate its own sorting by controlling the activity of dynein ?", "Studying mRNA trafficking within the complex molecular environment of a cell is challenging , so mRNA transporting machinery was recreated in the laboratory .", "Only the proteins necessary to build a working system were included in the experiments .", "In addition to the filaments , the components included dynein and a complex of proteins known as dynactin , which allows the motor to move together with a protein called BICD2 .", "A protein named Egalitarian was used to link the mRNA to BICD2 .", "By filming fluorescently labelled proteins and mRNAs , McClintock et al . discovered that mRNA strongly promotes the movement of the dynein motor .", "A structured section in the mRNA acts as a docking area for two copies of Egalitarian .", "This activates BICD2 , which then binds to dynein and dynactin , thereby completing the transport machinery .", "According to these results , the mRNA directs the assembly of the system that will carry it within the cell .", "Viruses such as HIV and herpesvirus hijack dynein motors to have their genetic information moved around a cell in order to propagate infection .", "Understanding precisely how mRNA is transported may help to develop new strategies to fight these viruses ." ]

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

[ "cell biology" ]

[ [ "Bone is a dynamic tissue that constantly undergoes remodeling by osteoclast-mediated bone resorption and osteoblast-mediated bone formation ( Eriksen , 2010; Nakahama , 2010; Raggatt and Partridge , 2010 ) .", "In a rapidly growing and mature mammals , bone remodeling is positive or balanced , respectively , allowing for bone mass accrual and later for its maintenance .", "Negatively balanced bone remodeling is a hallmark of pathologies such as osteoporosis and cancerous osteolysis ( Kozlow and Guise , 2005; Novack and Teitelbaum , 2008; Sturge et al . , 2011 ) .", "Skeletal tissues are composed largely of extracellular matrix ( ECM ) .", "Fibrillar ECM proteins , predominately type I collagen in bone and type II collagen in cartilage , provide structural integrity and account for mechanical strength .", "The ECM of bone also contains matricellular proteins that primarily serve as biological modulators .", "Matricellular proteins interact with cell-surface receptors , such as integrins , the structural matrix , and soluble extracellular factors including growth factors and proteases ( Bornstein and Sage , 2002 ) .", "Through these multiple interactions , matricellular proteins modulate cell function and regulate the availability and activity of proteins sequestered in the matrix .", "Therefore , matricellular proteins contribute to skeletal development , homeostasis , and fracture healing ( Alford and Hankenson , 2006 ) .", "Galectins are a family of glycan-binding proteins secreted by a variety of cell types ( Boscher et al . , 2011; Di Lella et al . , 2011 ) .", "As such , they can act as biological cross-linkers for ECM proteins and cell-surface receptors ( Elola et al . , 2007 ) .", "Indeed , certain galectins were reported to function as matricellular proteins ( Troncoso et al . , 2014 ) .", "The minimal structures recognized by these lectins are β-galactosides displayed on the cell surface as part of more complex glycoconjugates ( Di Lella et al . , 2011 ) .", "The prototype galectins ( galectin-1 , -2 , -5 , -7 , -10 , -11 , -13 , -14 , and -15 ) exist as monomers or homodimers of one carbohydrate recognition domain ( CRD ) .", "The tandem-repeat-type galectins ( galectin-4 , -6 , -8 , -9 , and -12 ) harbor two distinct CRDs joined by a peptide linker .", "The chimera-type galectin-3 consists of a CRD connected to a polypeptide that can pentamerize upon binding to glycan ligands ( Rabinovich and Vidal , 2011; Thiemann and Baum , 2011; Ledeen et al . , 2012 ) .", "Being secreted proteins , as well as proteins having intracellular roles , galectins affect a wide range of biological functions including regulation of cell adhesion , migration , cell growth , apoptosis , and autophagy ( Rabinovich and Vidal , 2011; Thiemann and Baum , 2011; Thurston et al . , 2012 ) .", "Still , regulation of bone physiology by galectins has been addressed only to a limited extent .", "Galectin-9 has been shown to induce osteoblast differentiation initiated by coupling of CD44 to bone morphogenetic protein ( BMP ) receptors ( Tanikawa et al . , 2010 ) , whereas GC-1 and GC-8 , the chicken orthologs of galectin-1 and galectin-8 , respectively , were shown to mediate the formation and patterning of pre-cartilage mesenchymal condensations in the developing limb of chicken ( Bhat et al . , 2011 ) .", "To study the possible role of galectins as regulators of bone physiology , we focused upon galectin-8 ( gal-8 ) , initially cloned in our laboratory , which is a tandem-repeat-type galectin having two sugar-binding domains joined by a linker peptide ( Hadari et al . , 1995; Levy et al . , 2006 ) .", "Upon secretion , galectin-8 is equipotent to fibronectin in promoting cell adhesion by ligation and clustering of a selective subset of cell-surface integrins and ECM proteins ( Hadari et al . , 2000; Levy et al . , 2001; Eshkar Sebban et al . , 2007 ) .", "Complex formation between galectin-8 and integrins triggers integrin-mediated signaling cascades ( Levy et al . , 2001 , 2003 ) that affect cell growth , receptor trafficking , and metastatic potential ( Boura-Halfon et al . , 2003; Zick et al . , 2004; Arbel-Goren et al . , 2005; Reticker-Flynn et al . , 2012 ) .", "In this study , we show that galectin-8 regulates bone mass by inducing the secretion of the osteoclastogenic factor , receptor activator of NF-κB ligand ( RANKL ) ( Hanada et al . , 2010 ) , from isolated osteoblasts in a cell autonomous manner .", "As a result , co-culture of galectin-8-treated osteoblasts with bone marrow cells increases their differentiation into active osteoclasts .", "These effects involve the binding of galectin-8 to the osteoblasts' urokinase plasminogen-activated receptor ( uPAR ) ; mannose receptor C , type 2 ( MRC2 ) ; and the low-density lipoprotein receptor-related protein 1 ( LRP1 ) , and seem to be of physiological relevance because galectin-8 transgenic animals exhibit increased expression of RANKL , increased osteoclastogenic activity , and enhanced bone turnover that culminates in reduced bone mass .", "These data identify galectin-8 as a potential drug target for the prevention of diseases associated with excessive bone loss ." ], [ "To study the effects of galectin-8 on osteoblasts in culture , osteoblasts derived from calvaria of newborn CD1 mice were incubated with 50 nM galectin-8 .", "As shown in Figure 1A , such treatment increased by sixfold the expression of RANKL in these cells by 4 hr and resulted in a 2 . 5-fold increase in secretion of soluble RANKL into the medium ( Figure 1B ) by 24 hr .", "Extended incubation with galectin-8 , up to 6 days maintained the high levels of expression of RANKL ( Figure 1C ) .", "Galectin-8 also had a moderate ( 30% ) inhibitory effect on the expression of osteoprotegerin ( OPG ) , a neutralizing decoy receptor of RANKL ( Eriksen , 2010 ) ( Figure 1D ) .", "As a result , there was an overall 10-fold decrease in the ratio of OPG/RANKL transcription in galectin-8-treated osteoblasts ( Figure 1E ) .", "We could therefore conclude that galectin-8 increases the RANKL/OPG ratio in osteoblasts in a cell autonomous manner .", "We have previously shown that galectin-8 is secreted and localizes to the extracellular surface of cells ( Hadari et al . , 2000 ) .", "To determine whether galectin-8 , secreted from osteoblasts , exerts similar effects , the expression of this lectin in osteoblasts was silenced using siRNA .", "As a result , a reduction of 87% in the expression levels of galectin-8 was accompanied by a significant reduction of 33% in the expression levels of RANKL ( Figure 1F ) .", "We could therefore conclude that galectin-8 derived from osteoblasts can mediate RANKL expression , along with other stimuli that induce RANKL . 10 . 7554/eLife . 05914 . 003Figure 1 . Effects of galectin-8 on RANKL and OPG expression in osteoblasts . Osteoblasts derived from calvaria of newborn mice were treated with 50 nM of galectin-8 for 4 hr ( A , D ) ; 24 hr ( B ) ; or for the indicated times ( C ) .", "After treatments , RNA was extracted and qRT-PCR was conducted in order to quantify changes in expression of RANKL ( A , C ) or osteoprotegerin ( OPG ) ( D ) .", "Actin served as a control for normalization purposes .", "The levels of soluble RANKL in the medium were quantified by ELISA ( B ) .", "( E ) OPG/RANKL expression ratio was calculated from the results of A and D . ( F ) Osteoblasts from the calvaria of newborn mice were grown in 12-well plates ( 5 × 104 cells per well ) .", "After 24 hr , cells were transfected with siRNA for galectin-8 .", "Non-targeting siRNA ( siNONT ) served as the control .", "96 hr thereafter , cells were harvested , RNA was extracted , and qRT-PCR was conducted to quantify changes in mRNA levels of galectin-8 and RANKL .", "The content of actin mRNA served as a control for normalization purposes .", "( G ) Bone marrow cells were extracted and analyzed by flow cytometry for the surface expression of galectin-8 .", "Cells were treated with TDG or sucrose ( 10 mM in PBS ) or just PBS before fixation .", "Results shown are of a representative histogram of cell number vs florescence intensity of the secondary antibody ( left ) and quantitation of the averages florescence intensity of the secondary antibody in two experiments carried out in duplicates ( right ) .", "Results are mean values ± SEM of five ( F ) or six independent experiments ( A , D , E ) or of two independent experiments carried out in duplicates ( C , G ) or triplicates ( B ) *p < 0 . 05 , **p < 0 . 01 . DOI: http://dx . doi . org/10 . 7554/eLife . 05914 . 003 To determine whether bone marrow cells can also secrete galectin-8 , they were subjected to analysis by flow cytometry .", "This analysis revealed that indeed primary murine bone marrow cells express and secrete galectin-8 ( Figure 1G ) .", "This surface-bound galectin-8 could be partially displaced by thiodigalactoside ( TDG ) , which blocks lectin–carbohydrate interactions , but not by sucrose , suggesting that surface binding of secreted galectin-8 is mediated , at least in part , through protein–carbohydrate interactions .", "Recent studies have implicated matrix-embedded osteocytes , rather than osteoblasts , in the control of osteoclast formation ( Nakashima et al . , 2011; Xiong et al . , 2011 ) .", "To determine which cell type serves as a target for galectin-8 , calvariae from newborn mice were separated using sequential digestion ( Nakashima et al . , 2011 ) into osteoblast-rich fraction , expressing the osteoblastogenic marker KERA ( keratocan ) ( Nakashima et al . , 2011 ) ( Figure 2A ) , and an osteocyte-enriched fraction , which is almost devoid of KERA-expressing cells ( Paic et al . , 2009 ) , but expresses DMP1 , an osteocyte marker ( Bonewald , 2011 ) .", "As expected , expression of DMP1 was enriched threefold in the kera ( − ) fraction , although a significant number of DMP1 ( + ) cells were also present in the kera ( + ) fraction ( Figure 2B ) .", "Basal RANKL expression was much higher in the Kera ( + ) osteoblasts-enriched fraction than in the osteocyte-enriched fraction ( Figure 2C ) .", "Furthermore , the level of RANKL expression in galectin-8-treated cells was fivefold higher than in basal both in Kera ( + ) and in Kera ( − ) cells ( Figure 2C ) .", "Given that the Kera ( − ) fraction was almost completely devoid of osteoblasts , these results support the conclusion that galectin-8 affects RANKL expression both in cultured osteoblasts and osteocytes , derived from calvaria of newborn mice . 10 . 7554/eLife . 05914 . 004Figure 2 . Effects of galectin-8 on osteoblast fractions isolated from calvaria of newborn mice . Osteoblasts were extracted from calvaria of newborn mice by five sequential incubations with collagenase-dispase solution .", "Osteoblasts derived from the different incubations were seeded for 24 hr .", "KERA ( A ) and DMP1 ( B ) expression , using qRT-PCR , were examined in fractions −2 and −5 that showed the highest and the lowest amount of KERA ( designated kera+ and kera− ) , respectively .", "( C ) Galectin-8 ( 50 nM ) was added to osteoblasts from these cultures for 24 hr; RANKL expression was determined by qRT-PCR .", "Actin served as a control for normalization purposes .", "Results are mean values ± SEM of n = 8 ( A ) , n = 6 ( B ) , n = 7 ( C ) .", "*p < 0 . 05 , **p < 0 . 01 . DOI: http://dx . doi . org/10 . 7554/eLife . 05914 . 004 The increased expression of the osteoclastogenic factor RANKL in osteoblasts treated with galectin-8 prompted us to study the effects of this lectin on osteoclasts differentiation in culture .", "For this purpose , bone marrow cells were co-cultured with osteoblasts derived from calvaria of newborn mice .", "We could demonstrate ( Figure 3A ) that galectin-8 , added to this co-culture , was equipotent to the osteoclastogenic factor PGE2 ( Suda et al . , 2004 ) in the induction of a ∼15-fold increase in osteoclast differentiation , as evident by the appearance of multinucleated TRAP+ cells .", "The effects of PGE2 and galectin-8 were additive to a certain extent , suggesting that they might act by somewhat different mechanisms .", "Very few differentiated osteoclasts appeared in untreated co-cultures .", "Furthermore , galectin-8 had no direct differentiation effect on osteoclasts , as addition of this lectin to naive bone marrow cells in the absence of osteoblasts did not result in osteoclasts differentiation ( Figure 3A ) .", "To verify that RANKL indeed mediates the effects of galectin-8 on osteoclasts differentiation , its expression was silenced using siRNAs .", "As shown in Figure 3B , RANKL-siRNAs reduced its transcription in osteoblasts by 50% , and this was accompanied by a similar 50% reduction in the ability of galectin-8 to induce osteoclastogenesis in co-culture experiments ( Figure 3C ) .", "These results support the conclusion that galectin-8 functions as an osteoclastogenic agent through its action as an inducer of RANKL expression in osteoblasts . 10 . 7554/eLife . 05914 . 005Figure 3 . Effects of galectin-8 on osteoclast differentiation .", "( A ) Osteoblasts ( OBL ) derived from the calvaria of newborn mice were seeded in 24-well plates ( 4 × 104 cells/well ) .", "After reaching 60–70% confluence , murine bone marrow cells ( BM ) extracted from the femur and tibia of 6-week-old mice were added to the culture ( 2 × 106 cells/well ) , together with galectin-8 ( 50 nM ) , PGE2 ( 1 μM ) , or both .", "Galectin-8 and PGE2 were further added on every other day for 10 days .", "TRAP assay was performed , and active osteoclasts ( multinucleated TRAP+ cells ) were counted .", "Results are mean values ± SEM of three independent experiments carried out in duplicates .", "( B ) Osteoblasts were seeded in 12-well plates ( 5 × 104 cells per well ) .", "After 24 hr , cells were transfected with siRNA to RANKL .", "Non-targeting siRNA served as a control .", "72 hr thereafter cells were harvested , RNA was extracted , and qRT-PCR was performed in order to quantify changes in mRNA levels of RANKL .", "The content of actin mRNA served as a control for normalization purposes .", "( C ) Osteoblasts were seeded as in A . After reaching 60–70% confluence , cells were transfected with the indicated siRNAs for 72 hr .", "Thereafter , murine bone marrow cells extracted from the femur and tibia bones of 6-week-old mice were added to the culture ( 2 × 106 cells/well ) .", "Galectin-8 ( 50 nM ) was added on the first , fourth , and sixth days after addition of bone marrow .", "Active osteoclasts were counted as in A . Results are mean values ± SEM of three ( A , B ) and two ( C ) independent experiments each carried out in duplicates ( *p < 0 . 05 , **p < 0 . 01 ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 05914 . 005 The signaling pathways that mediate the effects of galectin-8 on osteoblasts were explored next .", "We could demonstrate that treatment of osteoblasts , derived from calvaria of newborn mice , with soluble galectin-8 ( 50 nM , 4 hr ) , induced the phosphorylation of ERK and Akt , while inhibitors of these signaling pathways—PD98095 and wortmannin , respectively—inhibited these phosphorylations ( Figure 4A ) .", "PD98095 inhibited the ability of galectin-8 to promote transcription of RANKL in osteoblasts , whereas inclusion of wortmannin had no such an effect ( Figure 4B ) , suggesting that the effects of galectin-8 on RANKL gene transcription are mediated by the ERK signaling pathway .", "PD98095 also effectively inhibited the appearance of multinucleated TRAP+-differentiated osteoclasts , when bone marrow cells were co-cultured with osteoblasts in the presence of galectin-8 ( Figure 4C ) , indicating that the ERK signaling pathway is involved in this process as well .", "Wortmannin was capable of eliciting a partial inhibitory effect on osteoclast differentiation ( Figure 4C ) , suggesting that the PI3K/Akt pathway could act at a step downstream or independent of RANKL transcription . 10 . 7554/eLife . 05914 . 006Figure 4 . Signaling pathways activated by galectin-8 . ( A ) Osteoblasts derived from calvaria of newborn mice were treated with 25 μM PD98095 or 1 μM wortmannin for 1 hr before adding galectin-8 ( 50 nM ) .", "After 4 hr , total proteins were extracted and analyzed by Western blotting using antibodies specific for the phosphorylated forms of ERK and Akt .", "Shown is a representative of three experiments .", "( B ) Osteoblasts were treated with PD98095 ( 25 μM ) or wortmannin ( 1 μM ) for 1 hr before being treated with 50 nM galectin-8 .", "After 24 hr , cells were removed from plates , RNA was extracted , and qRT-PCR was performed in order to quantify changes in RANKL transcription .", "Actin served as a control for normalization purposes .", "Results shown are mean values ± SEM of three independent experiments , each done in duplicates .", "( C ) Osteoblasts were seeded in 24-well plates ( 4 × 104 cells/well ) .", "After reaching 60–70% confluence , murine bone marrow cells extracted from the femur and tibia of 6-week-old mice were added to the culture ( 2 × 106 cells/well ) .", "Galectin-8 ( 50 nM ) , PD98095 ( 25 μM ) , and wortmannin ( 1 μM ) were added every other day for 10 days .", "Multinucleated TRAP+ cells were scored as differentiated osteoclasts .", "Results shown in ( C ) are mean values ± SEM of two independent experiments each carried out in duplicate .", "( *p < 0 . 05 , **p < 0 . 01 ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 05914 . 006 To identify osteoblast receptors that could mediate the effects of galectin-8 , proteins extracted from calvaria of newborn rats were affinity purified over columns of immobilized GST-galectin-8 and were analyzed by mass spectrometry .", "Two proteins that specifically bound to the columns were of interest: LRP1 ( low-density lipoprotein receptor-related protein 1 ) ( Grey et al . , 2004 ) and MRC2 ( mannose receptor C , type 2 ) ( Engelholm et al . , 2009 ) .", "Both LRP1 and MRC2 could be detected by staining of proteins that selectively bound to immobilized GST-galectin-8 ( Figure 5A ) .", "Because LRP1 and MRC2 form complexes with the urokinase plasminogen activator receptor ( uPAR ) ( Behrendt , 2004; Gonias et al . , 2011 ) , it was of interest to determine whether uPAR is also part of the proteins complex that binds galectin-8 .", "Indeed , we could show by Western blotting that similar to LRP1 and MRC2 , uPAR also selectively binds to immobilized galectin-8 ( Figure 5B ) . 10 . 7554/eLife . 05914 . 007Figure 5 . Binding of proteins extracted from osteoblasts to GST-galectin-8 . ( A , B ) Calvariae were isolated from newborn rats ( A ) or mice ( B ) ; homogenized , and proteins were extracted and incubated for 16 hr at 4°C with GST- or GST-gal-8-loaded beads .", "Next , the beads were washed in PBS+1% Triton X-100 .", "Elution was performed with 0 . 5M lactose , and the eluted proteins were resolved by SDS-PAGE and were stained with GelCode ( A ) .", "Relevant bands ( marked with a rectangle ) were excised , trypsinized , and subjected to analysis by mass spectrometry .", "Alternatively , the eluted proteins were resolved by SDS-PAGE and were transferred to nitrocellulose membrane for Western blotting with the indicated antibodies ( B ) .", "Blots shown are representatives of four independent experiments with similar results . DOI: http://dx . doi . org/10 . 7554/eLife . 05914 . 007 To evaluate the possible physiological relevance of these galectin-8-binding partners , their siRNAs were introduced into osteoblasts from calvaria of newborn mice .", "Transcription of MRC2 , LRP1 , and uPAR in osteoblasts was reduced >60–80% by their corresponding siRNAs ( Figure 6A–C ) .", "Silencing of MRC2 inhibited ( 65% ) the effects of galectin-8 on RANKL transcription in osteoblasts ( Figure 6D ) and inhibited by 40% the ability of these osteoblasts to promote osteoclastogenesis when co-cultured with bone marrow cells ( Figure 6E ) .", "Similarly , siRNAs to uPAR effectively reduced ( 50% ) the ability of galectin-8 to stimulate expression of RANKL ( Figure 6F ) , suggesting that uPAR , like MRC2 , mediates at least in part , the stimulatory effects of galectin-8 on RANKL transcription and osteoclastogenesis .", "By contrast , silencing of LRP1 significantly increased ∼2 . 5-fold the effects of galectin-8 on RANKL transcription ( Figure 6G ) , suggesting that LRP1 could function as an inhibitory decoy receptor for galectin-8 , impeding its ability to promote expression of RANKL . 10 . 7554/eLife . 05914 . 008Figure 6 . Effects of silencing of MRC2 , LRP1 , and uPAR on the mode of action of galectin-8 . ( A–D , F , G ) Osteoblasts from calvaria of newborn mice were grown in 12-well plates ( 5 × 104 cells per well ) .", "After 24 hr , cells were transfected with the indicated siRNAs .", "Non-targeting siRNA served as control .", "48–72 hr thereafter , galectin-8 ( 50 nM ) was added for another 24 hr .", "Cells were then harvested , RNA was extracted , and qRT-PCR was conducted to quantify changes in mRNA levels of MRC2 ( A ) , LRP1 ( B ) , uPAR ( C ) , and RANKL ( D , F , G ) .", "The content of actin mRNA served as a control for normalization purposes .", "Results shown are mean values ± SEM of ( n = 5 [A–C , F , G]; n = 3 [D] ) **p < 0 . 01 vs control .", "( E ) Osteoblasts were seeded in 24-well plates ( 4 × 104 cells/well ) .", "After reaching 60–70% confluence , cells were transfected with the indicated siRNAs .", "After 72 hr , murine bone marrow cells extracted from the femur and tibia of 6-week-old mice were added to the culture ( 2 × 106 cells/well ) .", "Galectin-8 ( 50 nM ) was added on the first , fourth , and sixth days after addition of the bone marrow .", "TRAP assay was performed , and multinucleated TRAP+ cells were counted .", "Results are mean values ± SEM of triplicate measurements repeated in two independent experiments **p < 0 . 01 vs control . DOI: http://dx . doi . org/10 . 7554/eLife . 05914 . 008 To further assess the physiological significance of the above findings , transgenic mice that overexpress galectin-8 were generated as described under ‘Materials and methods’ .", "These mice express Myc-tagged galectin-8 controlled by the chicken beta-actin promoter that did not include a leader sequence .", "The insert was localized to chromosome 2 , in a region free of genes or other known genomic features .", "Homozygous mice were used in this study .", "Mice were born at normal size and expressed no apparent deformity .", "They were fertile and propagated at a normal Mendelian distribution .", "Age- and sex-matched mice served as the control group .", "Immunohistochemical staining of bone sections and quantitative reverse transcriptase polymerase chain reaction quantitative reverse transcriptase polymerase chain reaction ( qRT-PCR ) analysis revealed that galectin-8 expression was increased ∼sixfold in osteoblasts derived from calvariae of newborn mice , whereas a ∼10-fold increase was observed in osteoblasts derived from long bones of 16-week-old transgenic mice ( Tg ) mice , when compared with the control wild-type ( WT ) mice ( Figure 7A ) .", "Decoration with anti-galectin-8 antibodies of decalcified sections of tibia from 12-week-old WT mice and gal-8 Tg mice confirmed these results .", "A marked increase in anti-galectin-8 antibody binding was observed in sections of tibia derived from gal-8 Tg mice when compared with WT controls ( Figure 7B ) . 10 . 7554/eLife . 05914 . 009Figure 7 . Expression of galectin-8 in femur and tibia of gal-8 Tg mice .", "( A ) RNA was extracted from osteoblasts derived either from calvaria of newborn mice ( n = 7 ) ( left ) or from the femur and tibia of 14-week-old ( n = 5 ) ( right ) wild-type ( WT ) and gal-8 Tg mice .", "qRT-PCR was conducted using primers for galectin-8 or actin ( control ) .", "Result shown are mean ± SEM ( **p < 0 . 01 ) .", "( B ) Tibia was removed from 12-week-old WT ( left ) and gal-8 Tg mice ( right ) .", "Bones were decalcified and fixed in paraffin blocks .", "Sections were cut and stained with anti-galectin-8 antibody ( red ) and DAPI ( blue ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 05914 . 009 To determine whether the osteoclastogenic activity of galectin-8 affects bone morphology , indices of tibial bone mass and architecture of WT and Tg mice were determined by micro-computed tomography ( μCT ) scans both in vivo and in vitro .", "In vitro μCT of the proximal region of the tibia of 16-week-old mice revealed bone osteopenia in the gal-8 Tg mice ( Figure 8 ) .", "This was characterized by a 57% decrease in Tb . N and 62% decrease in BV/TV ratio ( Table 1 ) .", "As a consequence , Tb . Sp was significantly higher ( 2 . 8-fold ) in the Tg animals ( Table 1 ) .", "No change in Tb . Th was observed ( not shown ) .", "A significant reduction ( 32% ) in bone mineral density ( BMD ) of the Tg mice was observed as well ( Table 1 ) .", "Qualitatively , similar changes were also detected by in vivo μCT scans ( Table 1 ) .", "Same changes were also evident upon scanning of the distal region of the tibia ( not shown ) . 10 . 7554/eLife . 05914 . 010Figure 8 . MicroCT scans of tibia proximal regions . In vivo and in vitro μCT scans were performed on 14-week-old WT and Tg mice , or on tibial bones removed from 16-week-old WT and Tg mice , respectively .", "Representative pictures show the proximal region of the tibia ( for in vivo CT ) or a region of interest within the trabecular bone of the tibia metaphysis ( for in vitro CT ) .", "The position of Trabeculae is indicated by arrows . DOI: http://dx . doi . org/10 . 7554/eLife . 05914 . 01010 . 7554/eLife . 05914 . 011Table 1 . Analysis and stereological parameters of tibia proximal region in WT and gal-8 Tg miceDOI: http://dx . doi . org/10 . 7554/eLife . 05914 . 011In vivo CTIn vitro CTWT ( n = 7 ) Tg ( n = 7 ) WT ( n = 5 ) Tg ( n = 5 ) BV/TV0 . 40 ± 0 . 020 . 25 ± 0 . 03**0 . 12 ± 0 . 010 . 04 ± 0 . 01**Tb . N ( 1/mm ) 3 . 19 ± 0 . 021 . 86 ± 0 . 25**3 . 86 ± 0 . 511 . 68 ± 0 . 34**Tb . Sp ( mm ) 0 . 19 ± 0 . 010 . 48 ± 0 . 08**0 . 24 ± 0 . 030 . 68 ± 0 . 18*BMD ( % ) 100% ± 15%52% ± 5%**100% ± 8%68% ± 6%**14-week-old WT and Tg mice ( n = 7 each group ) were scanned using a small animal in vivo μCT scanner .", "Tibial bones were removed from 16-week-old WT and Tg mice ( n = 5 each group ) and scanned using an in vitro CT scanner .", "Analysis was performed on the proximal region of the tibia .", "The parameters calculated are Tb . N ( trabecular number ) , Tb . Sp ( trabecular separation ) , BV/TV ( bone volume/tissue volume ) , and BMD ( bone mineral density ) .", "Results shown are mean values ± SEM .", "BMD is given as relative to the average BMD of WT mice ( **p < 0 . 01 vs WT mice ) .", "To gain further insight into the mechanisms of reduced bone mass in the gal-8 Tg mice , bone formation parameters were determined by dynamic histomorphometry , using calcein double labeling .", "Mineral appositional rate ( MAR ) , a representation of the activity of the average osteoblast , increased by 25% in the Tg animals ( 1 . 12 ± 0 . 05 μm/day vs 1 . 41 ± 0 . 07 μm/day , p < 0 . 005 , Figure 9 , left ) .", "Conversely , bone formation rate ( BFR ) also increased almost twofold in the gal-8 Tg mice compared with that of the control group ( 0 . 30 ± 0 . 03 μm3/mm2/day vs 0 . 55 ± 0 . 06 μm3/mm2/day , p < 0 . 005 , Figure 9 , right ) .", "These results indicate that gal-8 Tg mice experience enhanced bone remodeling that involves enhanced rate of bone formation in spite of overall bone loss in these animals . 10 . 7554/eLife . 05914 . 012Figure 9 . Histomorphometric analysis of femurs from WT and gal-8 Tg mice . 16-week-old WT ( n = 5 ) and Tg ( n = 9 ) mice were injected with calcein .", "MAR ( mineral apposition rate , left ) and BFR ( bone formation rate , right ) were measured and calculated based on sections of the femur bone taken from these mice .", "Results shown are mean values ± SEM ( **p < 0 . 01 vs WT mice ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 05914 . 012 To determine whether the increase in BFR in gal-8 Tg mice could be offset by increased osteoclastogenesis in vivo , RNA was extracted from the femur and tibia of 14- to 16-week-old gal-8 Tg mice and WT control animals .", "As shown in Figure 10A , bones of Tg mice expressed 3 . 3-fold higher amounts of RANKL than those of control WT mice .", "This was accompanied by a fourfold increase in the ratio of osteoclasts surface/bone perimeter in decalcified sections of tibia from the Tg animals ( Figure 10B , C ) and a 40% increase in the abundance of active osteoclasts in culture ( TRAP+ , multinucleated cells ) ( Figure 10D ) .", "Elevated expression of the osteoclast markers TRAP and cathepsin K ( 3 . 5-fold and twofold , respectively ) was also observed in bone marrow cells derived from the Tg mice , when compared with their WT controls ( Figure 10E ) .", "No effects of galectin-8 on the transcription of MCSF , the second key osteoclastogenic factor ( Biskobing et al . , 1995 ) , were observed ( not shown ) .", "Taken together , these results suggest that the increased rate of bone formation in gal-8 -8 Tg mice is apparently offset by the even greater increase in osteoclastogenic activity in the bones of the Tg animals , resulting in a net loss of bone mass . 10 . 7554/eLife . 05914 . 013Figure 10 . Characterization of osteoblasts and osteoclasts derived from WT and gal-8 Tg mice .", "( A ) RNA was extracted from the femur and tibia of 14–16-week-old WT and Tg mice .", "qRT-PCR was conducted in order to quantify changes in expression of RANKL .", "Actin served as a control for normalization purposes .", "Results are mean values ± SEM of five mice per group .", "( B ) Femurs were removed from 16-week-old WT and gal-8 Tg mice .", "Bones were fixed , and TRAP staining was performed .", "Quantification of the ratios of osteoclast number to bone surface was calculated from sections of WT ( n = 5 ) and Tg ( n = 9 ) mice .", "Results are mean values ± SEM ( **p < 0 . 01 ) .", "Representative sections are shown in ( C ) Arrows indicate the position of osteoclasts .", "( D , E )", "Bone marrow cells were extracted from the femur and tibia bone of 14-week-old WT and Tg mice and were seeded in 24-well plates ( 2 × 106 cells/well ) for 24 hr .", "The number of multinucleated TRAP+ cells was determined ( D ) , and qRT-PCR of the indicated genes was performed ( E ) .", "Results are mean values ± SEM of n = 6 and n = 4 mice/group in ( D ) and ( E ) , respectively ( *p < 0 . 05 , **p < 0 . 01 vs WT cells ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 05914 . 013" ], [ "Excessive reduction in bone mass , with osteoporosis as one of its hallmarks , is a major health problem , especially in the elderly population ( Manolagas and Jilka , 1995 ) .", "Still , the mechanisms underlying the development of this disease and some of the critical players in this process remain partially obscure ( Bonucci and Ballanti , 2014 ) .", "In the present work , we provide evidence that an animal lectin of the galectin family regulates osteoclastogenesis and loss of bone mass .", "We show that galectin-8 increases the expression of RANKL , a key osteoclastogenic factor ( Hanada et al . , 2011; Xiong and O'Brien , 2012 ) , in cultured osteoblasts , and promotes their osteoclastogenic potential when co-cultured with bone marrow cells .", "At the same time galectin-8 inhibits the expression of OPG , a neutralizing decoy receptor of RANKL , thus leading to an overall increase in the RANKL/OPG ratio .", "Given that RANKL is both necessary and sufficient for osteoclast differentiation , provided that permissive concentrations of MCSF are present ( Eghbali-Fatourechi et al . , 2003 ) , and given that galectin-8 does not affect the expression of MCSF , our findings strongly suggest that RANKL mediates the potentiating effects of galectin-8 on bone resorption .", "Galectins are secreted by atypical secretory pathway from different cell types ( Boscher et al . , 2011; Di Lella et al . , 2011 ) .", "Therefore , a number of cell types could serve as a source for the secreted galectin-8 that induces RANKL expression by osteoblasts .", "We have shown that silencing of galectin-8 expression in osteoblasts partially inhibits the expression of RANKL , suggesting that galectin-8 , secreted from these cells , could act in an autocrine fashion to induce RANKL expression .", "However , we could also show that galectin-8 is present on the surface of bone marrow cells , turning these cells into additional potential source for galectin-8 that affects osteoblasts .", "Further studies will be required to resolve this complex issue .", "At the molecular level , galectin-8 binds at the cell surface of osteoblasts to the low-density lipoprotein receptor-related protein 1 ( LRP1 ) ( Grey et al . , 2004 ) and the mannose receptor C , type 2 ( MRC2 ) ( Engelholm et al . , 2009 ) that negatively and positively regulate galectin-8 function , respectively .", "LRP1 and MRC2 are part of a multi-protein complex that includes uPAR ( Behrendt et al . , 2000; Smith and Marshall , 2010 ) .", "Indeed , we could demonstrate that uPAR co-precipitates with galectin-8 and that partial silencing the expression of uPAR attenuates the ability of galectin-8 to promote RANKL transcription; findings that conform with the observation that uPAR-deficient mice have increased bone mass ( Furlan et al . , 2007 ) .", "uPAR localizes to integrin-containing adhesion complexes , and co-immunoprecipitates with integrins and integrin-associated signaling molecules such as focal adhesion kinase ( FAK ) and Src family kinases ( reviewed in Smith and Marshall , 2010 ) .", "In particular , it modulates the affinity of β1 , β2 , and β3 integrins ( Wei et al . , 1996 ) for their corresponding matrix ligands ( Smith and Marshall , 2010 ) , and does it , in part , through binding to vitronectin ( Smith and Marshall , 2010 ) .", "Integrins , including α1 , αM , α3β1 , and α6β1 , as well as ECM proteins , also serve as binding partners to galectin-8 that functions as a matricellular protein ( Hadari et al . , 2000; Nishi et al . , 2003; Cárcamo et al . , 2006; Troncoso et al . , 2014 ) .", "Complex formation between galectin-8 and integrins triggers integrin-mediated signaling cascades such as Tyr phosphorylation of FAK and paxillin , and a robust and sustained activation of the ERK and PI3K pathways ( Levy et al . , 2001 , 2003 ) .", "Hence , interaction of galectin-8 with a complex of the uPAR/LRP1/MRC2 that binds integrins could be the mechanism underlying RANKL transcription in osteoblasts treated with this lectin .", "This model is further supported by the fact that integrins promote RANKL transcription through the activation of FAK in osteoblasts ( Nakayamada et al . , 2003 ) .", "Phosphorylated FAK activates several transduction molecules including Src and Grb2 , which activate the ERK and PI3K signaling pathways ( Schwartz and Ginsberg , 2002 ) .", "Indeed , activation of the ERK pathway in osteoblasts is obligatory for the action of galectin-8 as an inducer of osteoclastogenesis , implicating this signaling pathway as being the major pathway activated downstream of galectin-8 .", "In contrast , the PI3K pathway appears not to play a role in the induction of RANKL in response to galectin-8 , while having a partial role in mediating osteoclastogenesis .", "These findings place the PI3K pathway as acting downstream or independent of RANKL transcription in mediating the effects of galectin-8 on osteoclastogenesis .", "The mechanism underlying the inhibitory effects of LRP1 on RANKL transcription in response to galectin-8 is presently unknown .", "LRP1 , which also directly interacts with uPAR ( Gonias et al . , 2011 ) , could , for example , facilitate the endocytosis of uPAR , which was the first cell-signaling receptor identified as a member of the LRP1-regulated plasma membrane proteome ( Gaultier et al . , 2006 ) .", "Because LRP1 down-regulates cell-surface uPAR by facilitating its endocytosis , uPAR-initiated cell signaling may be inhibited by LRP1 .", "Although galectin-8 can potentially bind to receptors that both negatively ( LRP1 ) and positively ( MRC2 and uPAR ) regulate RANKL expression , the net effect is still an increase in RANKL expression and reduction in bone mass , which could be attributed , for example , to different expression levels of these receptors in osteoblasts .", "Alternatively , the duration of the signals emitted by these receptors could differ .", "Osteoblastic cells are considered as the major cell type that expresses RANKL to support osteoclastogenesis; however , recent findings suggest that osteocytes are the main regulators of bone homeostasis through RANKL expression ( Nakashima et al . , 2011; Xiong et al . , 2011 ) .", "Our findings reveal that galectin-8 affects RANKL expression in osteoblasts-enriched fractions of calvariae as well as in osteocyte-enriched fraction .", "These findings support the hypothesis that galectin-8 acts both on osteoblasts and osteocytes , at least in culture , and still , we cannot rule out the possibility that it induces RANKL expression in vivo selectively in osteocytes .", "The increased expression of RANKL in gal-8 transgenic animals enhances their osteoclasts differentiation and results in a reduction in their BMD and bone volume fraction ( BVF ) , thus supporting the notion galectin-8 could induce a physiological bone loss .", "Of interest , dynamic histomorphometry revealed active bone remodeling , associated with increased rate of bone formation in gal-8 Tg mice .", "The increase in bone turnover could be attributed to the enhanced osteoclastogenic activity induced by galectin-8 that subsequently promotes osteoblasts differentiation and increased BFR ( Feng and McDonald , 2011; Bonucci and Ballanti , 2014 ) .", "Still , the net effect is bone loss due to the overall greater activity of the osteoclasts .", "In this respect , our model of Tg mice resembles in certain aspects the changes in bone turnover that take place during postmenopausal osteoporosis ( Raisz , 2005; Feng and McDonald , 2011 ) .", "It is well established that both bone resorption and BFRs are increased during postmenopausal osteoporosis ( Raisz , 2005; Feng and McDonald , 2011 ) ; however , the extent of increased bone resorption exceeds that of augmented bone formation , which causes an imbalance in favor of bone loss ( Arlot et al . , 1990; Ebeling et al . , 1996; Tanizawa et al . , 1999; Raisz , 2005; Eriksen , 2010 ) .", "A number of rodent models for postmenopausal osteoporosis exist ( e . g . , Erlebacher and Derynck , 1996; Bucay et al . , 1998; Mizuno et al . , 2002; Rinotas et al . , 2014 ) .", "One is a model of Tg mice that overexpress human RANKL ( Rinotas et al . , 2014 ) .", "These mice develop bone loss and increased bone turnover rate , which is similar to the phenotype of gal-8 Tg mice , supporting the notion that galectin-8 mainly acts through increased expression of RANKL in vivo .", "In summary , our findings implicate an animal lectin as a novel regulator of osteoclastogenesis and bone remodeling .", "The unique aspect of these observations stems from the fact that galectin-8 , like other secreted animal lectins , binds cell-surface glycoconjugates that enable it to engage in binding to a number of receptors that express the proper repertoire of sugars on their surface .", "This offers a novel mean for spatially controlled regulation of bone remodeling through high-density information coding that involves lectin–sugar interactions .", "Our findings place animal lectins , with galectin-8 as their representative , as novel osteoclastogenic agents and regulators of bone remodeling .", "The phenotype of gal-8 Tg mice , showing enhanced bone turnover and reduced bone mass , supports the conclusion that galectin-8 induces RANKL expression in an in vivo setting .", "The results reveal a potential link between galectins , LRP1 , MRC2 , and uPAR in mediating this process of osteoclastogenesis .", "Therefore , insights from this study might inform efforts to develop novel drug targets for the treatment of diseases associated with bone loss ." ], [ "Commercially available reagents were purchased from the following resources: trypsin-Ethylendiaminetetraacetic acid ( EDTA ) , penicillin , and l-glutamine were purchased from Biological Industries ( Beit Haemek , Israel ) .", "Fetal bovine serum was obtained from Hyclone Laboratories Inc . ( Logan , UT ) .", "Isopropyl-β-d-thiogalactopyranoside ( IPTG ) and PCR Master Mix ( Dreamtaq ) were purchased from MBI Fermentas ( Amherst , NY ) .", "Dispase II ( neutral protease , grade II ) was from Roche Diagnostics ( Mannheim , Germany ) .", "siRNA SMARTpool libraries were provided by Dharmacon ( Lafayette , CO , USA ) .", "GST-coupled resins were purchased from Novagen ( Madison , WI ) .", "Lipofectamine 2000 was from GIBCO-BRL ( Grand Island , NY ) .", "PerfectPure RNA Cell & Tissue for RNA extraction was from 5 PRIME ( Hamburg , Germany ) .", "cDNA reverse transcription kit was purchased from Applied Biosystems ( Carlsbad , CA ) .", "Real-time Polymerase Chain Reaction ( PCR ) kit ( SYBR green PCR master mix ) was purchased from Invitrogen ( Carlsbad , CA ) .", "Thiodigalactoside was purchased from Santa Cruz Biotechnology ( Dallas , TX ) .", "Leukocyte acid phosphatase staining kit , protease inhibitor cocktail , lactosyl-sepharose beads , wortmannin , cycloheximide , proteinase K , lysozyme , collagenase type 1A , prostaglandin E2 , Dulbecco's Modified Eagle Medium ( DMEM ) , sucrose , and diethyl pyrocarbonate ( DEPC ) were purchased from Sigma Chemicals Co . ( St . Louis , MO ) .", "Galectin-8 was a bacterially expressed recombinant protein , encoded by the cDNA of rat galectin-8 ( Hadari et al . , 1995 ) .", "Monoclonal galectin-8 antibodies ( 106 . 1 ) were generated as described ( Levy et al . , 2001 ) .", "CB6F1 mice were used throughout this study .", "All animals were housed under standard light/dark conditions in the animal care unit of the Weizmann Institute of Science .", "Mice were given food and water ad libitum .", "Experiments were approved by the Animal Care and Use Committee of the Weizmann Institute of Science .", "Plasmid-bearing Myc-tagged rat galectin-8 coding sequence under the chicken beta-actin promoter was constructed on the backbone plasmid pQE-TrySystem .", "The plasmid was restricted by NaeI and SphI , and the linear fragment of 3137 bp was microinjected into CB6F1-fertilized oocytes .", "Mice were scanned for the presence of the insert by two PCR reactions using two pairs of primers for amplification of the promoter region ( sense: AAAGGAGATATACCGCGGCGATATCCC , antisense: CTGCAACCTTGAACTCTCGGACATCAC , 630 bp ) and the myc-galectin-8 coding sequence ( sense: CGCCAATAGGGACTTTCCATTGAC , antisense: CTAATTACAGCCCGAAGGAGAAGG , 970 bp ) .", "Isolation and culture of osteoblasts from newborn mice calvariae were carried out as described ( Bakker and Klein-Nulend , 2003; Nakashima et al . , 2011 ) .", "Briefly , calvariae were extracted from up to 1-day-old pups and were subjected to five incubations with 0 . 1% collagenase and 0 . 2% dispase in serum-free medium .", "The medium from the second to the fifth incubations were collected and centrifuged to pellet the osteoblasts .", "Osteoblasts were then grown until sub-confluence and were frozen till further use .", "For each experiment , cells were thawed and grown in α-modified DMEM medium ( Sigma ) supplemented with 10% fetal calf serum .", "The ability of osteoblasts to induce osteoclast differentiation was assayed in a co-culture system , adapted from Takahashi et al . ( 2007 ) .", "Primary osteoblasts ( 1 × 104 cells/well ) extracted from calvariae of newborn mice were co-cultured in 24-well plates with bone marrow cells ( 1 × 106 cells/well ) flushed from the femur and tibia of 6-week-old mice .", "Medium , containing galectin-8 and/or PGE2 , was replaced every other day .", "After 10 days , cells were stained for TRAP ( tartarate-resistant acid phosphatase ) using a commercial staining kit ( Sigma ) following the manufacturer instructions .", "Multinucleated cells stained purple were counted as active osteoclasts .", "Cells were grown in 6-well plates .", "Following treatment , cells were harvested and total RNA was extracted using the PerfectPure RNA kit ( 5 PRIME ) .", "RNA was quantified and cDNA was generated by cDNA Reverse Transcription kit ( Applied Biosystems ) following manufacturer instructions .", "Quantitative detection of mRNA transcripts was carried out by real-time PCR using ABI-Prism 7300 instrument ( Applied Biosystems ) using SYBR Green PCR mix ( Invitrogen ) and specific primers ( 400 nM final concentration ) ( Table 2 ) .", "Results were normalized to mRNA levels of β-actin . 10 . 7554/eLife . 05914 . 014Table 2 . qRT-PCR primer sequences ( 5′–3′ ) DOI: http://dx . doi . org/10 . 7554/eLife . 05914 . 014GeneForward primerReverse primerRANKLATCGGGAAGCGTACCTACAGGTGCTCCCTCCTTTCATCAGTRAPCAGCAGCCAAGGAGGACTACACATAGCCCACACCGTTCTCCTSKCAGCTTCCCCAAGATGTGATAGCACCAACGAGAGGAGAAAMRC2GCCATACGGCTTTGCCCTACGGCCCTGGATTCGGAAACACuPARTGTGCTGGGAAACCGGAGTTGAGGTGGGTCGGGAAGGAGTLRP1TCAGACGAGCCTCCAGACTCTACAGATGAAGGCAGGGTTGGTGal-8 ( mouse ) TGAACACCAATGCCCGAAGCGCGTGGGTTCAAGTGCAGAGGal-8 ( rat ) TGTATGCCCACAGGATCAACATCCGAGCTGAATCTGAACC Femur and tibia bones were removed and placed in RNAlater solution ( Ambion , Foster City , CA ) .", "After flushing of the bone marrow , and two more flushings with RNAlater solution , bones were cut into 1- to 2-mm3 pieces and crushed .", "Total RNA was extracted as detailed above .", "Cells were harvested in lysis buffer ( 25 mM Tris/HCl , 25 mM NaCl , 0 . 5 mM Ethylene Glycol Tetraacetic Acid ( EGTA ) , 2 mM sodium orthovanadate , 10 mM NaF , 10 mM sodium pyrophosphate , 80 mM β-glycerophosphate , 1% Triton X-100 , 0 . 05% Sodium Dodecyl Sulphate ( SDS ) and protease inhibitors 1:1000 , pH 7 . 5 ) and were centrifuged at 12 , 000×g for 20 min at 4°C .", "Supernatants were collected , and samples of 50 μg protein were mixed with 5×Laemmli sample buffer and were resolved by SDS-PAGE under reducing conditions .", "Proteins were transferred to nitrocellulose membrane for Western blotting with the indicated antibodies .", "Media from cells were used for quantification of soluble RANKL using a murine sRANKL ELISA Development Kit ( PeproTech ) according to the manufacturer instructions .", "sRANKL levels were normalized to total cellular protein concentration , quantified by Bradford assay .", "Murine bone marrow cells were extracted and washed with 10 mM TDG or sucrose for 15 min at 4°C .", "Flow cytometry was performed as previously described ( Isaac et al . , 2013 ) .", "Briefly , after 20 min of fixation with 2% p-formaldehyde ( PFA ) , cells were incubated on ice with 0 . 5% BSA for 30 min , followed by incubation with galectin-8 antibodies for 1 hr and Alexa 594-labeled secondary antibodies ( Life Technologies ) for 30 min .", "Cells were washed with cold phosphate-buffered saline ( PBS ) between incubations .", "Flow cytometry analysis was performed by LSR II Flow Cytometer System ( BD Biosciences ) .", "The tibia and femur were removed from 14- to 16-week-old mice and cut open at their distal end .", "Fixation was performed in 2 . 5% PFA for 48 hr , followed by decalcification in 10% EDTA for 3 days .", "Sections were stained with anti-galectin-8 antibodies or with TRAP staining kit ( Sigma ) following manufacturer instructions .", "Measurements were performed on blindly selected regions taken from each slide .", "For in vitro CT analysis , the tibia was removed from 16-week-old mice and scanned using an in vitro μCT scanner ( MicroXCT-400 , Xradia , California , USA ) .", "For each bone , 300 projection images were taken over 180° , with an exposure time of 3 s per projection and geometry set for a voxel size of 5 . 67 μm ( source-to-sample distance 90 mm , sample-to-detector distance 17 mm , linear magnification 4× ) .", "All morphometric parameters were determined by using a direct 3D approach .", "Volume reconstruction was performed with a dedicated software ( Xradia California , USA ) based on the filtered back-projection algorithm .", "Parameters determined in the metaphyseal trabecular bone included bone volume density ( BV/TV ) , trabecular number ( Tb . N ) , and trabecular thickness ( Tb . Th ) and were estimated using a region of interest ( ROI ) size of 150 × 150 × 50 voxels , which was placed in the center of the trabecular region of the tibia , approximately 300 μm below the lowest point of the growth plate .", "For in vivo CT analysis , mice were anesthetized by injection of 2% ketamine and xylasine in PBS .", "Mice were scanned using small animal in vivo μCT scanner ( TomoScope 30S duo , VAMP , Germany ) following instrument-operating instructions .", "Scans were performed using the 65-65-360-90 protocol ( using two micro-focus x-ray tubes of 65 Kv with an integration time of 90 ms ) , with a resolution of 80 μm .", "Image reconstruction was carried out by the Impact View software ( VAMP , Germany ) .", "Files were saved in digital imaging and communications in medicine ( DICOM ) format .", "Conversion of DICOM files to analysis files was carried out using imageJ software ( National Institutes of Health ) .", "BVF , BMD , and stereological bone parameters were calculated using the eXplore MicroView software ( GE Healthcare , UK ) .", "Calculations were performed in a cylinder-shaped ROI , with a size of 10 × 10 × 10 voxels , which was placed inside the trabecular region of the proximal tibia , in similar positions in all mice .", "For BVF analysis , the automated threshold function of MicroView was used for bone segmentation .", "To label bone-forming surfaces , mice were injected subcutaneously with calcein ( Sigma Chemical Co , St . Louis , MO ) at 15 mg/kg , 4 and 1 day before sacrifice .", "After sacrifice , femurs were removed and kept in 70% ethanol until μCT and histomorphometric analyses .", "After μCT image acquisition , femora were embedded undecalcified in polymethylmethacrylate ( Technovit 9100; Heraeus Kulzer , Wehrheim , Germany ) ( Parfitt et al . , 1987 ) .", "Longitudinal 5-μm sections were employed for dynamic histomorphometric measurements based on calcein labeling , using a fluorescent microscope .", "The analysis was carried out in a blinded manner on digital photomicrographs using IMAGE-PRO EXPRESS 4 . 0 image analysis software ( Media Cybernetics , Silver Spring , MD ) .", "The following parameters were determined in a reference area extending 0 . 75–2 . 25 mm proximal of the distal growth plate in a mid-longitudinal plane according to the convention of standardized nomenclature ( Parfitt et al . , 1987 ) : total bone surface ( BS ) , the percentage of single- and double-labeled bone surfaces ( sLS and dLS ) , and interlabel width were measured .", "Mineralizing surface [MS/BS = ( dLS + 1/2sLS ) /BS] , mineral apposition rate [MAR = interlabel width/labeling time interval] , and BFR [= MAR ( MS/BS ) ] were calculated according to convention .", "To determine osteoclast number , consecutive sections were deplasticized , and TRAP ( tartrate-resistant acid phosphatase ) staining was used ( Sigma , St . Louis , MO , USA ) .", "Stained osteoclasts were counted , and their numbers were determined per millimeter of trabecular bone surface ( Oc . N/BS ) in the same reference area as previously described by us ( Dresner-Pollak et al . , 2008 ) .", "Osteoblasts were transfected with siRNA SMARTpool ( Dharmacon ) by using Lipofectamine 2000 transfection reagent ( GIBCO-BRL , Grand Island , NY ) according to manufacturer's instructions .", "Briefly , 50 pmol of the siRNA was diluted in 50 μl of serum-free medium .", "1 μl of Lipofectamine 2000 was also diluted in the same volume of serum-free medium .", "Both solutions were mixed together and incubated at room temperature for 15 min .", "The mix was added at 1:10 ratio to the osteoblasts' culture medium .", "Incubation with the siRNA was carried out for 48 hr .", "Calvariae were extracted from newborn rats and homogenized , and proteins were extracted as described above .", "Glutathione S-transferase ( GST ) and GST-galectin-8 ( 100 μg ) were immobilized on 200 μl glutathione–agarose beads at 4°C .", "After 2 hr , the beads were washed three times with PBS .", "Calvaria proteins were incubated for 16 hr with GST- or GST-gal-8-loaded beads .", "The beads were loaded on a column and washed with 100 ml of PBS in Triton X-100 ( 0 . 1% ) and protease inhibitors .", "Proteins were eluted with 0 . 5M lactose supplemented with 0 . 1% Triton and protease inhibitors cocktail .", "Eluted proteins were resolved by SDS-PAGE and were stained with Gel Code .", "Protein bands that were selectively eluted from GST-gal-8-loaded beads in two independent experiments were excised and trypsinized .", "Resulting peptides were subjected to mass spectrometry analysis and nano-LC-ESI-MS/MS as we previously described ( Isaac et al . , 2013 ) .", "For the analysis of tryptic peptides , survey scans were recorded in the Fault-tolerance ( FT ) -mode followed by data-dependent collision-induced dissociation of the seven most-intense ions in the linear ion trap .", "The data were searched with MASCOT ( Matrix Science , London , UK ) against a Swissprot or National Center for Biotechnology Information ( NCBI ) databases and confirmed by manual inspection of the fragmentation series .", "Relative quantitation was conducted with the Scaffold PTM software ( Proteome Software Inc . , USA ) and the A-score algorithm ( Zhai et al . , 2008 ) .", "Data are presented as mean ± SEM unless otherwise specified .", "Group means were compared using the non-paired t-test .", "Differences of p < 0 . 05 were considered significant ." ] ]

[ "Skeletal integrity is maintained by the co-ordinated activity of osteoblasts , the bone-forming cells , and osteoclasts , the bone-resorbing cells .", "In this study , we show that mice overexpressing galectin-8 , a secreted mammalian lectin of the galectins family , exhibit accelerated osteoclasts activity and bone turnover , which culminates in reduced bone mass , similar to cases of postmenopausal osteoporosis and cancerous osteolysis .", "This phenotype can be attributed to a direct action of galectin-8 on primary cultures of osteoblasts that secrete the osteoclastogenic factor RANKL upon binding of galectin-8 .", "This results in enhanced differentiation into osteoclasts of the bone marrow cells co-cultured with galectin-8-treated osteoblasts .", "Secretion of RANKL by galectin-8-treated osteoblasts can be attributed to binding of galectin-8 to receptor complexes that positively ( uPAR and MRC2 ) and negatively ( LRP1 ) regulate galectin-8 function .", "Our findings identify galectins as new players in osteoclastogenesis and bone remodeling , and highlight a potential regulation of bone mass by animal lectins ." ]

[ "The forces applied to the body during daily activities cause bones to be constantly remodeled , which is essential for keeping them healthy .", "In most adult organisms , new bone is created at the same rate at which old bone is destroyed .", "This means that overall bone mass remains the same .", "But , in diseases such as osteoporosis or bone cancer , bone is destroyed more rapidly than at which new bone is made .", "This leads to brittle bones that are more likely to fracture .", "Understanding how to increase the rate of bone renewal might therefore help scientists develop new treatments for bone diseases .", "Bone is created by cells called osteoblasts and destroyed by other cells called osteoclasts .", "Both of these types of cells develop from stem cells in the bone marrow .", "The activity of these cells is controlled by a number of factors , including the matrix of proteins that holds bone together .", "A group of proteins called galectins are known to act as a bridge between some of the matrix proteins and molecules on the surface of the cells .", "Vinik et al . took osteoblasts from a mouse skull , grew them in the laboratory , and then exposed them to a galectin protein called galectin-8 .", "This made the osteoblasts release a protein called RANKL , which is known to boost osteoclast activity .", "When osteoblasts that had been exposed to galectin-8 were grown alongside bone marrow stem cells , more of the stem cells developed into the bone-destroying osteoclasts .", "Mice that were genetically engineered to produce more galectin-8 than normal mice develop brittle bones , despite also creating new bone at a higher rate than do normal mice .", "This is because osteoclast activity increases at a greater rate , resulting in an overall loss of bone in these animals .", "This is similar to what occurs in some individuals with osteoporosis .", "These experiments therefore suggest that galectin-8 plays an important role in bone remodeling and that it may be a potential target for drugs that treat diseases that weaken bones ." ]

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

[ "cell biology" ]

[ [ "The transcriptional coactivator Yap is an evolutionarily conserved mediator of cell fate decisions such as proliferation , differentiation , and survival ( Yu and Guan , 2013 ) .", "Together with a related protein Taz , Yap is the central target of the Hippo pathway , a growth-suppressive network of kinase complexes that inactivates Yap and Taz ( Hong and Guan , 2012 ) .", "The Hippo pathway has been implicated in organ size control , as overexpression of Yap or inactivation of the Hippo components leads to tissue overgrowth and organomegaly in Drosophila and mouse models ( Camargo et al . , 2007; Dong et al . , 2007; Lin et al . , 2013 ) .", "These effects are largely mediated by Yap-TEAD complexes that activate transcription of genes promoting cell proliferation and inhibiting apoptosis ( Wu et al . , 2008; Zhang et al . , 2008; Zhao et al . , 2008 ) .", "Yap is interconnected with RTK ( Reddy and Irvine , 2013 ) , GPCR ( Yu et al . , 2012 ) , PI3K ( Fan et al . , 2013 ) , Wnt ( Bernascone and Martin-Belmonte , 2013 ) , and TGF-beta ( Ferrigno et al . , 2002; Attisano and Wrana , 2013; Mullen , 2014 ) signaling , and Yap co-regulates transcription by interacting with Smads ( Ferrigno et al . , 2002; Beyer et al . , 2013 ) , TCF/LEF ( Konsavage and Yochum , 2013 ) , Tbx5 ( Beyer et al . , 2013 ) , Runx2 ( Zaidi et al . , 2004 ) , FoxO1 ( Shao et al . , 2014 ) , and p73 ( Strano et al . , 2001 ) , among others ( Barry and Camargo , 2013 ) .", "The intestinal epithelium in both Drosophila ( Karpowicz et al . , 2010; Ren et al . , 2010; Shaw et al . , 2010; Staley and Irvine , 2010 ) and mice does not depend on Yap for homeostatic tissue turnover ( Zhou et al . , 2011 ) , but it does respond very strongly to Yap overexpression ( Barry et al . , 2013 ) and requires Yap for tissue repair ( Cai et al . , 2010 ) .", "Thus , in some tissues Yap may be dispensable for homeostasis but required specifically in response to injury .", "The liver is one of the organs most responsive to excessive Yap activity .", "Transgenic overexpression of Yap or inactivation of its upstream negative regulators causes a dramatic increase in liver size , hepatocyte proliferation , progenitor cell expansion , and tumorigenesis ( Camargo et al . , 2007; Dong et al . , 2007; Lee et al . , 2010; Lu et al . , 2010; Zhang et al . , 2010; Kowalik et al . , 2011; Zheng et al . , 2011 ) .", "In contrast , deletion of Yap in the liver leads to defects in bile duct formation but no apparent defects in hepatocyte number and function ( Bai et al . , 2012 ) , suggesting that Yap may be dispensable for hepatocyte homeostasis .", "However , whether its function is required for hepatocyte homeostasis and response to injury remains to be established ( Yu et al . , 2014 ) .", "Yap activation promotes proliferation , survival , stemness , and tumor development in mouse models ( Camargo et al . , 2007; Dong et al . , 2007; Barry and Camargo , 2013 ) and is commonly observed in human cancers ( Fernandez et al . , 2009; Wang et al . , 2009 ) .", "Collectively , these data suggest that hyperactivation of Yap abrogates organ size control mechanisms and drives tumorigenesis in a seemingly unrestrained fashion .", "However , growth-promoting pathways are normally safeguarded by tumor-suppressive mechanisms ( Hahn and Weinberg , 2002 ) .", "For example , c-myc hyperactivation sensitizes cells to apoptosis ( Evan et al . , 1992 ) , oncogenic Ras induces senescence ( Serrano et al . , 1997 ) , and overexpression of Bcl-2 inhibits cell proliferation ( O'Reilly et al . , 1996 ) .", "Whether or not Yap activity is subject to a similar tumor-suppressive regulation is currently unclear .", "While Yap is known to interact with p73 and promote apoptosis in response to DNA damage in vitro ( Strano et al . , 2001; Lapi et al . , 2008 ) , there is no evidence that Yap can induce apoptosis in vivo .", "Control of cell fate decisions at the tissue level is poorly understood , but it is likely to involve cell contact-dependent regulation .", "Yap activity is regulated by adherens and tight junctions , cell polarity complexes , and the actin cytoskeleton ( Boggiano and Fehon , 2012 ) .", "At high cell densities , Yap is either directly recruited to intercellular junctions or undergoes phosphorylation and cytosolic retention via the Hippo pathway , which itself is also regulated in a cell contact-dependent manner .", "This feature makes Yap competent to direct cell fate decisions depending on cell density and architecture .", "Thus , cell environment may be a major determinant of the outcome of Yap activation .", "Moreover , evidence from Drosophila ( Chen et al . , 2012 ) and mammalian cell culture ( Norman et al . , 2012 ) suggest that the outcome of Yap activation depends on Yap activity in neighboring cells .", "The regulation of Yap activity by such mechanism has not been characterized in mammalian tissues in vivo .", "Here , we describe a mosaic model of Yap activation in the mouse liver , where a high level of Yap expression is induced in a fraction of hepatocytes surrounded by normal tissue .", "We present evidence that a high level of Yap in this context is insufficient to drive clonal expansion , as increased proliferation of Yap-overexpressing hepatocytes is balanced by their increased susceptibility to apoptosis .", "Yap-mediated clonal expansion requires a second signal provided by disruption of tissue homeostasis , such as injury or inflammation .", "Upon returning to homeostasis , excessive Yap-overexpressing cells are eliminated by apoptosis ." ], [ "To study the role of cell environment in the regulation of the mammalian Hippo pathway , we generated a conditional mosaic mouse model of Yap overexpression ( YapKI mice ) .", "We used Yap1 mutant S112A which has enhanced nuclear localization and has been previously shown to cause a several-fold increase in liver size when overexpressed ( Schlegelmilch et al . , 2011; Zhang et al . , 2011 ) in the entire organ .", "A targeting construct driving expression of the Yap1S112A-IRES-GFP downstream of a floxed transcriptional STOP cassete was knocked into the Gt ( ROSA ) 26Sor ( Rosa26 ) locus .", "Upon expression of Cre recombinase , the transcriptional STOP cassette is removed , allowing expression of Yap together with GFP ( YapKI mice; Figure 1—figure supplement 1 ) .", "We used the Rosa26-IRES-GFP targeting construct which is expressed bimodally ( Bondar and Medzhitov , 2010 ) ( likely due to epigenetic regulation of the locus ) , generating GFPlow and GFPhigh populations in all cells of hematopoietic lineages ( Bondar and Medzhitov , 2010 ) and solid tissues tested ( liver , pancreas , muscle , intestine; Figure 1—figure supplement 2 and data not shown ) .", "Both populations have a recombined STOP cassette , but the gene of interest is expressed above physiological level only in GFPhigh cells .", "GFPlow cells serve as an internal control population of the identical genetic background for the GFPhigh cells .", "In the absence of selective pressure , the ratio of GFPlow to GFPhigh cells remains constant throughout life .", "Thus , crossing YapKI mice to Albumin-Cre ( Alb-Cre ) yields mosaic Yap expression in the hepatic lineage , where individual cells overexpressing Yap can be traced by GFP fluorescence ( YapKIAlb-Cre mice ) .", "Similar to the previously reported Rosa26-DNp53 mice ( Bondar and Medzhitov , 2010 ) , the proportion of GFPlow to GFPhigh cells remained stable in the blood of YapKICreER mice upon tamoxifen induction for many months ( Figure 1—figure supplement 2B–D ) .", "In the colons of YapKIVillin-Cre mice , crypts were either entirely GFPhigh or GFPlow ( GFPlow appearing as GFP− due to limited sensitivity of GFP detection by tissue immunostaining ) .", "There were no crypts that have mixed populations , as would be expected if cells randomly switched expression level ( Figure 1—figure supplement 2E ) .", "Collectively , these data illustrate stable inheritance of the Rosa26 allele expression level .", "First , we verified the correlation of Yap and GFP expression by immunofluorescence staining of the YapKIAlb-Cre liver sections .", "Indeed , Yap and GFP in hepatocytes displayed mosaic and overlapping expression pattern , with cytosolic as well as nuclear Yap localization ( Figure 1A ) .", "Utilizing the GFP marker , we tested the recombination efficiency by flow cytometry .", "More than 90% of hepatocytes were GFP-positive , indicating high deletion efficiency of the STOP cassette ( Figure 1B ) .", "Recombination was also confirmed by genomic qPCR ( Figure 1—figure supplement 3 ) .", "The hepatocytes formed two distinct populations differing in GFP brightness .", "To determine the level of Yap activity in these cells , we sorted each of these populations by FACS .", "Since GFP fluorescence varied 10-fold within the GFPlow population , we further subdivided it into GFPlow and GFPmed-low populations ( Figure 1B ) .", "The exogenous transcripts of Yap are highly expressed only in the GFPhigh population , but not in the GFPlow and GFPmed-low populations .", "The small amount of the exogenous Yap mRNA in the GFPlow and GFPmed-low populations is insignificant due to a potential negative feedback mechanism that reduces the endogenous Yap expression , resulting in a similar level of total Yap mRNA compared to that of wild-type ( WT ) ( Figure 1—figure supplement 4 ) .", "Additionally , CTGF , a known Yap downstream target gene ( Zhao et al . , 2008 ) , was significantly upregulated only in the GFPhigh population , whereas its expression remained unchanged in GFPlow and GFPmed-low populations , indicating normal levels of Yap activity in these cells ( Figure 1C ) .", "Thus , GFP and Yap protein levels correlate in YapKIAlb-Cre mice , and Yap is overexpressed and has higher activity specifically in the GFPhigh hepatocytes ( to which we will refer as Yaphigh cells ) . 10 . 7554/eLife . 02948 . 003Figure 1 . YapKIAlb-Cre strain is a hepatocyte-specific mosaic mouse model of Yap activation .", "( A ) Detection of exogenous Yap and GFP by immunofluorescent staining of YapKIAlb-Cre liver sections .", "( B ) Flow cytometric analysis of GFP fluorescence in primary hepatocytes .", "The plots are gated on hepatocytes by forward and side scatter and on live cells by excluding 7-AAD positive events .", "( C ) Expression of Yap and CTGF was determined by qPCR in primary hepatocyte populations sorted based on GFP levels as shown in B . DOI: http://dx . doi . org/10 . 7554/eLife . 02948 . 00310 . 7554/eLife . 02948 . 004Figure 1—figure supplement 1 . The design of the YapKI mice . The targeting construct carrying mouse S112A mutant Yap1 followed by IRES-GFP was integrated into Gt ( ROSA ) 26Sor ( Rosa26 ) locus .", "STOP denotes a strong transcriptional termination signal , removable by loxP recombination . DOI: http://dx . doi . org/10 . 7554/eLife . 02948 . 00410 . 7554/eLife . 02948 . 005Figure 1—figure supplement 2 . Stable mosaic expression of Rosa26 allele in multiple tissues of YapKI mice .", "( A ) Flow cytometry analysis of GFP populations in the peripheral blood of YapKICreER mice 4 weeks after tamoxifen induction .", "CD19 is a marker of B lymphocytes .", "( B ) The proportion of GFP+ B cells ( within CD19+ gate ) ; ( C ) the proportion of GFPhigh cells within GFP+CD19+ gate; ( D ) the proportion of GFPlow cells within GFP+CD19+ gate in the peripheral blood of YapKICreER after a single tamoxifen injection was determined by flow cytometry weekly for 1 year .", "Numbers on the right correspond to mouse eartags .", "( E ) Colons of YapKIVillin-Cre mice and WT littermates were stained with GFP antibody ( green ) and DAPI ( blue ) .", "Green cells in the lamina propria are autofluorescent macrophages . DOI: http://dx . doi . org/10 . 7554/eLife . 02948 . 00510 . 7554/eLife . 02948 . 006Figure 1—figure supplement 3 . Verification of the correct targeting , expression , and recombination of the YapKI allele .", "( A ) Southern blot verifying correct integration of the YapKI targeting construct into the Gt ( ROSA ) 26Sor locus .", "( B ) The level of Yap overexpression in YapKIAlb-Cre mice as determined by Western blot .", "Higher migrating band corresponds to the exogenous Yap ( due to the triple flag tag ) .", "( C ) qPCR on genomic DNA isolated from YapKI ( lox-STOP-lox ) and YapKIAlb-Cre ( lox-STOP-lox + Cre ) hepatocytes with primers that amplify only unrecombined ( STOP-cassette-containing ) region . DOI: http://dx . doi . org/10 . 7554/eLife . 02948 . 00610 . 7554/eLife . 02948 . 007Figure 1—figure supplement 4 . Effect of the YapKI allele on Yap mRNA level . Yap levels were measured by qPCR in YapKIAlb-Cre hepatocytes sorted based on the GFP levels as described in Figure 1B with primers amplifying the total ( A ) , endogenous ( B ) or exogenous ( C ) Yap mRNA . DOI: http://dx . doi . org/10 . 7554/eLife . 02948 . 007 The first question we addressed is whether liver size is affected by mosaic Yap overexpression .", "Previous studies showed that hyperactivation of Yap in the entire liver leads to rapid and progressive hepatomegaly: liver weight increases fivefold in 1 month in Yap transgenic mice ( Camargo et al . , 2007; Dong et al . , 2007 ) and fourfold by 3 months of age in Stk3 , Stk4 ( Mst1/2 ) double null mice ( Lu et al . , 2010; Song et al . , 2010 ) ( where Yap is activated due to an absence of upstream negative regulators ) .", "However , the liver/body weight ratio of the mosaic YapKIAlb-Cre mice was only slightly elevated ( Figure 2A ) .", "Hepatocyte numbers ( Figure 2B ) and liver morphology ( Figure 2C ) also remained normal .", "Moreover , Yaphigh cells did not have a growth advantage over the WT neighbors , since neither the average size of Yaphigh cells clusters in the liver sections ( Figure 2—figure supplement 1 ) nor the average frequency of Yaphigh hepatocytes increased over time ( Figure 2D ) . 10 . 7554/eLife . 02948 . 008Figure 2 . Yap overexpression in hepatocytes does not induce hepatomegaly or clonal expansion at steady state .", "( A ) Liver/body weight ratios of 1- to 3-month-old YapKIAlb-Cre mice ( KI ) and littermate controls ( WT ) , n ≥ 11 .", "****p ≤ 0 . 0001 .", "( B ) Total hepatocyte numbers were determined by quantitative flow cytometry of primary hepatocytes isolated from 1- to 3-month-old mice of the indicated genotypes , n ≥ 14 .", "( C ) A representative image of H&E staining performed on liver sections of 6-week-old control ( WT ) and YapKIAlb-Cre mice ( KI ) .", "( D ) Primary hepatocytes were isolated by collagenase perfusion from YapKIAlb-Cre mice of indicated age groups , and percentage of GFPhigh hepatocytes was determined by flow cytometry; n ≥ 11 .", "( E ) Mice were injected with BrdU for 3 consecutive days and liver sections were stained with BrdU and GFP antibodies .", "Percent of BrdU+ hepatocytes was quantified in liver sections of the controls ( WT ) and within Yaplow ( GFP− ) and Yaphigh ( GFP+ ) populations of the YapKIAlb-Cre livers .", "n ≥ 8 .", "( F ) TUNEL-positive nuclei were quantified on liver sections of control ( WT ) and YapKIAlb-Cre ( KI ) mice .", "3 mice were used for each group and 4 images were taken for each mouse .", "Each dot represents cell count from each image .", "**p ≤ 0 . 01 . DOI: http://dx . doi . org/10 . 7554/eLife . 02948 . 00810 . 7554/eLife . 02948 . 009Figure 2—figure supplement 1 . Yaphigh hepatocyte cluster size does not change over time . Representative images and quantification of GFP cluster size distribution in 1- to 5-month-old YapKIAlb-Cre mouse livers .", "Similar clone size distribution at different ages illustrates lack of clonal expansion of Yaphigh cells . DOI: http://dx . doi . org/10 . 7554/eLife . 02948 . 00910 . 7554/eLife . 02948 . 010Figure 2—figure supplement 2 . Overexpressed Yap induces robust transcription in hepatocytes at steady state .", "( A ) Heatmap showing RNA-sequencing data of genes induced in flow cytometry-sorted Yaphigh hepatocytes compared to the WT ( p ≤ 0 . 005 and ≥twofold ) .", "Two biological replicates were analyzed for WT , Yaplow and Yaphigh hepatocytes .", "The expression fold change was calculated relative to the mean of the WT .", "( B ) Diagram presenting the comparison between Yap-induced genes in RNA-sequencing data on sorted hepatocytes generated in this study and the genes previously reported to be induced in the whole liver of Yap transgenic mice ( Dong et al . , 2007 ) .", "( C ) p-value of gene function analysis for Yap-induced genes shown in A . ( D ) Charts showing the percentages of TEAD binding in different functional categories of genes .", "Gene function groups were identified from Yap induced genes shown in A . p < 0 . 0001 for all categories . DOI: http://dx . doi . org/10 . 7554/eLife . 02948 . 01010 . 7554/eLife . 02948 . 011Figure 2—figure supplement 3 . Comparison of Yap-induced genes identified by the RNA sequencing of Yaphigh hepatocytes ( this study ) and a microarray of Yap-transgenic liver ( Dong et al . , 2007 ) .", "( A ) Heatmap showing the expression fold change of genes induced by Yap in this study as compared to the data reported by Dong et al . ( 2007 ) .", "Genes are sorted based on their fold change in Dong et al . ( 2007 ) .", "( B ) p-value of Pearson correlation between the reported fold change in Dong et al . ( 2007 ) and the fold change calculated with the RNA-sequencing data .", "Mean of the indicated samples and the mean of wild type between duplicates were used . DOI: http://dx . doi . org/10 . 7554/eLife . 02948 . 01110 . 7554/eLife . 02948 . 012Figure 2—figure supplement 4 . Mosaic Mst1/2 loss does not induce hepatocyte expansion or liver size increase . Stk4−/−;Stk3flox/flox ( Mst1/2 ) mice were injected with a low dose adenovirus expressing Cre and GFP .", "( A ) Livers were harvested 5 and 35 days after Ad-Cre-GFP injection; BrdU was injected 1 day before harvesting .", "Liver sections were stained with antibodies to GFP and BrdU .", "( B ) Liver/body weight ratio of Mst1/2 mice injected with Ad-Cre-GFP at the dose shown in A ( 5× ) or fivefold lower dose ( 1× ) .", "( C ) Semi-quantitative analysis of the deleted Stk3 ( ∆Mst2 ) allele in liver genomic DNA of Mst1/2 mice harvested 5 or 35 days after low dose Ad-Cre-GFP infection .", "Rag2 genomic PCR was used as a loading control .", "The triangle denotes serial dilutions of the template DNA ( fourfold ) .", "Representative images are shown ( repeated twice; n > 3/group in each ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 02948 . 012 These results were unexpected , considering the dramatic increase in hepatocyte proliferation and survival driven by Yap in the previously published whole-liver transgenic models ( Camargo et al . , 2007; Dong et al . , 2007 ) .", "We therefore tested whether Yap influenced proliferation and apoptosis in YapKIAlb-Cre liver .", "Indeed , more Yaphigh hepatocytes incorporated BrdU than their GFPlow neighbors or hepatocytes in WT livers ( Figure 2E ) .", "Meanwhile , TUNEL staining indicated more apoptosis in the YapKIAlb-Cre livers ( Figure 2F ) .", "It is noteworthy that only a minor fraction of the Yaphigh cells proliferated , and over 95% remained quiescent throughout the 3-day BrdU labeling period .", "These data suggest that increased Yap levels sensitize hepatocytes to both mitogenic and apoptotic signals but are insufficient to drive cell expansion , resulting in slightly increased cell turnover without net growth advantage .", "This insufficiency was not due to an overall lack of Yap activation , as Yap overexpression induced a significant change in gene expression in our model .", "Sorted WT , GFPlow and GFPhigh hepatocytes were compared by RNA-sequencing analysis .", "Gene expression of GFPlow cells isolated from YapKIAlb-Cre livers was similar to that of the WT hepatocytes , whereas GFPhigh hepatocytes showed robust transcriptional changes: 821 genes were significantly up-regulated ( p ≤ 0 . 005 and fold change ≥2 ) in Yaphigh hepatocytes compared to WT hepatocytes ( Figure 2—figure supplement 2A ) .", "37% of these genes overlapped with genes upregulated in the previously characterized Yap overexpression model ( Dong et al . , 2007 ) ( Figure 2—figure supplements 2B , 3A–B ) , further validating our model .", "Dong et al ( Dong et al . , 2007 ) used an ApoE promoter which is not restricted to hepatocytes , and assayed the whole liver that includes stromal , endothelial and immune cells with microarray , whereas we assayed sorted hepatocytes in a model with hepatocyte-specific Yap induction with RNA-sequencing .", "Thus , a complete overlap in gene profile would not be expected .", "Yap-induced genes showed an enrichment in cell adhesion , extracellular matrix ( ECM ) , and cell–cell junction functional groups ( Figure 2—figure supplement 2C ) , according to DAVID functional cluster analysis ( Huang et al . , 2009a , 2009b ) .", "In contrast , the cell cycle category was not significantly enriched , likely because only a small fraction of all Yaphigh hepatocytes were proliferating , as evidenced by BrdU labeling .", "It is well known that pro-growth effects of Yap overexpression are mediated through TEAD ( Wu et al . , 2008; Zhang et al . , 2008; Zhao et al . , 2008 ) , and the dominant-negative TEAD mutant largely suppresses effects of Yap transgene in the liver ( Liu-Chittenden et al . , 2012 ) .", "To evaluate how many of the Yap-induced genes are potentially regulated directly by Yap-TEAD complexes , we compared these genes to genes with TEAD binding sites identified previously by ChIP-seq ( ENCODE Project Consortium , 2011 ) .", "Indeed , over 25% of all Yap-activated genes are associated with TEAD compared to only 9% of all genes in the genome .", "Moreover , a large fraction of genes induced in Yaphigh hepatocytes and related to cell adhesion ( 33% ) , ECM ( 36% ) , and cell–cell junctions ( 36% ) contain TEAD binding regions ( Figure 2—figure supplement 2D ) .", "The gene expression data shown here suggest that inability of Yap to drive cell expansion in our model is not due to lack of transcriptionally competent Yap-TEAD complexes .", "To test whether mosaic Yap activation can lead to clonal expansion in previously characterized mouse models , we injected Stk4−/−;Stk3flox/flox mice with a low dose of adenovirus expressing Cre and GFP , and compared patterns of BrdU incorporation and GFP+ hepatocytes 5 and 35 days later .", "At both time points , we could only observe single GFP+ hepatocytes , BrdU negative ( Figure 2—figure supplement 4A ) .", "Semi-quantitative PCR of the recombined Stk3 ( ∆Mst2 ) allele also did not show an increase at 35 days , and liver/body mass ratio remained normal ( Figure 2—figure supplement 4B , C ) .", "Taken together , these data suggest that clonal activation of Yap in hepatocytes is not sufficient to overrule growth restricting mechanisms at steady state , but is sufficient to regulate transcription of a large number of genes related to extracellular environment .", "Previous studies demonstrated that Yap is activated during liver injury ( Bai et al . , 2012; Wang et al . , 2012; Anakk et al . , 2013 ) and protects mice from oxidative stress-induced damage ( Wu et al . , 2013 ) .", "We therefore tested whether Yap induction provides selective advantage during liver injury .", "We chose carbon tetrachloride ( CCl4 ) as a damaging agent due to its localized effects around the central vein ( CV ) ( Weber et al . , 2003 ) .", "We first characterized the kinetics of liver damage after a single injection of CCl4 .", "Blood ALT levels in YapKIAlb-Cre and WT mice rose to similar levels , indicating comparable hepatocyte damage ( Figure 3—figure supplement 1 ) .", "By day 4 the acute damage phase ceased completely ( Figure 3—figure supplement 1 ) and very few hepatocytes incorporated BrdU ( Figure 3—figure supplement 2 ) .", "We therefore chose day 4 to study the outcome of the tissue repair response .", "In response to CCl4 damage Yaphigh cells expanded dramatically within 4 days in the pericentral area , comprising over 80% of all hepatocytes in this zone ( Figure 3A , B ) .", "These pericentral Yaphigh cells incorporated significantly more BrdU during days 1–3 after CCl4 administration compared to Yaplow hepatocytes in the same location ( Figure 3C ) .", "In contrast , Yaphigh cells proliferated much less in the CV-distal areas than in the pericentral area ( Figure 3C ) .", "In fact , the BrdU index outside the pericentral zone did not differ between Yaphigh and Yaplow hepatocytes within the same liver .", "Even though Yaphigh cells expanded locally at the injury sites , the effect was strong enough to significantly increase total percentage of Yaphigh cells in the liver ( Figure 3D ) .", "Nevertheless , this expansion of Yaphigh cells did not overrule normal liver size control , as the total number of hepatocytes was similar in YapKIAlb-Cre and WT mice ( Figure 3—figure supplement 3 ) .", "Liver/body weight ratios in both groups also returned to steady-state levels within a month after undergoing a similar increase in response to CCl4 , ( likely due to the infiltration of immune cells during early phase of tissue repair ) ( Figure 3—figure supplements 3–5 ) .", "These results indicate that Yaphigh cells undergo expansion in response to local tissue injury signals . 10 . 7554/eLife . 02948 . 013Figure 3 . Injury induces proliferation and clonal expansion of Yap-overexpressing hepatocytes .", "( A ) Liver sections of control ( WT ) and YapKIAlb-Cre ( KI ) mice isolated at steady state ( SS ) or on days 4 and 14 after CCl4 treatment were stained with antibodies to GFP and BrdU .", "BrdU was injected in all groups for 3 consecutive days before harvesting the livers .", "( B ) The percentage of Yaphigh ( GFP+ ) cells among all hepatocytes was quantified within the pericentral zone ( ‘damage site’ ) and outside the pericentral zone ( ‘outside’ ) and in the liver sections of YapKIAlb-Cre mice on day 4 after CCl4 treatment .", "3–4 mice were used for each group and 3–4 images were taken for each mouse .", "Each dot represents cell count from each image .", "****p ≤ 0 . 0001 .", "( C ) The percentages of BrdU+ hepatocytes among GFP− and GFP+ hepatocytes were quantified outside the CV zone ( ‘outside’ ) and around CV ( ‘damage site’ ) in liver sections of YapKIAlb-Cre mice on day 4 after CCl4 treatment .", "3–4 mice were used for each group and 3–4 images were taken for each mouse .", "Each dot represents a cell count from each image .", "****p ≤ 0 . 0001 .", "( D ) Hepatocytes were isolated by collagenase perfusion from 1- to 2-month-old YapKIAlb-Cre mice at steady state ( SS ) , at day 4 ( D4 ) and day 14 ( D14 ) after CCl4 treatment , or after a 6-day LPS treatment ( LPS ) , and percentage of GFPhigh cells determined by flow cytometry . DOI: http://dx . doi . org/10 . 7554/eLife . 02948 . 01310 . 7554/eLife . 02948 . 014Figure 3—figure supplement 1 . Kinetics of CCl4-induced liver damage in the YapKIAlb-Cre mice . ALT blood level of control ( WT ) and YapKIAlb-Cre ( KI ) mice measured at the indicated days after CCl4 treatment .", "One representative experiment is shown of the two independent repeats , n = 3 per group in each . DOI: http://dx . doi . org/10 . 7554/eLife . 02948 . 01410 . 7554/eLife . 02948 . 015Figure 3—figure supplement 2 . Proliferation rates of WT and YapKIAlb-Cre hepatocytes are similar on day 4 after CCl4 treatment . Mice were injected with BrdU 1 hr before livers were harvested on day 4 after CCl4 treatment .", "Liver sections were stained with antibodies to GFP ( green ) , glutamine synthase ( blue ) , and BrdU ( red ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 02948 . 01510 . 7554/eLife . 02948 . 016Figure 3—figure supplement 3 . Hepatocyte numbers undergo similar changes in response to CCl4 in the WT and YapKIAlb-Cre mice . Hepatocyte numbers were measured by quantitative flow cytometry in the pellet fractions of cells isolated from collagenase-perfused livers at indicated times after CCl4 treatment . DOI: http://dx . doi . org/10 . 7554/eLife . 02948 . 01610 . 7554/eLife . 02948 . 017Figure 3—figure supplement 4 . Liver/body weight ratios undergo similar changes in response to CCl4 in the WT and YapKIAlb-Cre mice . Liver/body weight ratio of YapKIAlb-Cre and littermate control mice was determined at the indicated days after CCl4 injection . DOI: http://dx . doi . org/10 . 7554/eLife . 02948 . 01710 . 7554/eLife . 02948 . 018Figure 3—figure supplement 5 . Hematopoietic cell numbers undergo similar changes in response to CCl4 in the WT and YapKIAlb-Cre mice . CD45+ cell numbers were measured by quantitative flow cytometry in the supernatant fractions of cells isolated from collagenase-perfused livers at indicated times after CCl4 treatment . DOI: http://dx . doi . org/10 . 7554/eLife . 02948 . 018 To investigate whether Yap activity in hepatocytes is required for tissue repair , we generated Yap conditional knockout mice and crossed them to the AlbCre strain to obtain hepatocyte-specific Yap knockout Yap1flox/flox;AlbCre ( YapCKOAlb-Cre ) mice ( Figure 4—figure supplements 1 , 2 ) .", "These mice were treated with CCl4 using the same protocol as described above .", "A similar increase in blood ALT level in YapCKOAlb-Cre and WT mice indicated comparable liver damage in the first 2 days ( Figure 4—figure supplement 3 ) .", "By day 4 , ALT levels returned to baseline ( Figure 4—figure supplement 3 ) , normal pericentral morphology was restored , and expression of the CV-proximal marker glutamine synthase was reestablished in both groups ( Figure 4A ) .", "However , YapCKOAlb-Cre hepatocytes proliferated significantly less than their WT counterparts throughout the repair phase , as shown by BrdU labeling on days 1–3 ( Figure 4B , C ) .", "Consistent with the defective regeneration , YapCKOAlb-Cre livers displayed abnormal collagen deposition around the CV ( Figure 4D ) .", "Interestingly , the steady-state liver/body mass ratio ( Figure 4—figure supplement", "4 ) and BrdU incorporation rate ( Figure 4—figure supplement", "5 ) were higher in the YapCKOAlb-Cre mice than in the WT mice , suggesting that Yap is not required for homeostatic hepatocyte proliferation . 10 . 7554/eLife . 02948 . 019Figure 4 . Yap function in hepatocytes is required for tissue repair .", "( A ) Expression of glutamine synthase detected by immunofluorescent staining of littermate controls ( WT ) and YapCKOAlb-Cre YapCKO livers on day 4 after CCl4 treatment .", "CV , central vein .", "( B ) BrdU staining illustrating lack of hepatocyte proliferation in Yap-deficient hepatocytes in response to CCl4-induced injury .", "The results are quantified in Figure 4C .", "( C ) Total BrdU+ hepatocytes in WT , YapKIAlb-Cre ( KI ) and YapCKOAlb-Cre ( CKO ) mice were quantified outside the CV zone ( ‘outside’ ) and around CV ( ‘damage site’ ) in liver sections on day 4 after CCl4 treatment .", "3–4 mice were used for each group and 3–4 images were taken from each mouse .", "Each dot represents a cell count from each image .", "( D ) Excessive collagen deposition in YapCKOAlb-Cre mice on day 4 after CCl4 treatment revealed by Sirius Red staining . DOI: http://dx . doi . org/10 . 7554/eLife . 02948 . 01910 . 7554/eLife . 02948 . 020Figure 4—figure supplement 1 . The design of the Yap1 conditional knockout targeting construct . Exon 2 is floxed and the neo cassette is removable by frt recombination . DOI: http://dx . doi . org/10 . 7554/eLife . 02948 . 02010 . 7554/eLife . 02948 . 021Figure 4—figure supplement 2 . Verification of the correct targeting , expression , and recombination of the Yap1 conditional knockout allele .", "( A ) Southern blot verifying correct integration of the targeting construct .", "( B ) qPCR on genomic DNA isolated from Yap1flox/flox ( F/F ) and Yap1flox/flox;AlbCre ( F/F + Cre ) hepatocytes with primers that amplify only the unrecombined region .", "( C ) Decreased Yap mRNA levels in YapCKOAlb-Cre mouse liver as measured by qPCR .", "( D ) Decreased Yap protein levels in YapCKOAlb-Cre mouse liver as measured by Western blot . DOI: http://dx . doi . org/10 . 7554/eLife . 02948 . 02110 . 7554/eLife . 02948 . 022Figure 4—figure supplement 3 . Kinetics of CCl4-induced liver damage in the YapCKOAlb-Cre mice . ALT blood level of control ( WT ) and YapCKOAlb-Cre ( KO ) mice measured at indicated days after CCl4 treatment .", "The control group was injected with peanut oil ( the carrier used for CCl4 ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 02948 . 02210 . 7554/eLife . 02948 . 023Figure 4—figure supplement 4 . Increased liver weight of the YapCKOAlb-Cre mice . Liver/body weight ratio of YapCKOAlb-Cre and littermate control mice was determined in the untreated 6- to 8-week-old mice .", "****p ≤ 0 . 0001 . DOI: http://dx . doi . org/10 . 7554/eLife . 02948 . 02310 . 7554/eLife . 02948 . 024Figure 4—figure supplement 5 . No defect in proliferation of the Yap-deficient hepatocytes at steady state . Representative images and quantification of the BrdU+ hepatocytes of the 1 . 5-month-old YapCKOAlb-Cre and littermate control mice at steady state .", "Mice were injected with BrdU for 3 consecutive days and liver sections were stained with BrdU antibody .", "The number of BrdU+ hepatocytes per a 20× field was quantified and normalized to the average values of the WT .", "2–3 mice were used for each group and at least 20 images were taken for each mouse .", "Each dot represents cell count from each image .", "****p ≤ 0 . 0001 . DOI: http://dx . doi . org/10 . 7554/eLife . 02948 . 024 Thus , while Yap is not required for liver development and hepatocyte proliferation under normal conditions , Yap function is required for hepatocyte proliferation during tissue repair .", "Additionally , hepatocytes with a higher level of Yap have growth advantage during the early repair phase .", "Given differential effects of Yap on hepatocyte proliferation during homeostasis vs injury , we wondered how growth control mechanisms might differ under these conditions .", "Tissue injury is accompanied by inflammation , which can promote proliferation by mechanisms distinct from homeostasis .", "To test whether Yap cooperates with inflammatory signals to drive cell cycle progression , we measured hepatocyte BrdU incorporation in YapKIAlb-Cre mice after 6 continuous days of intraperitoneal injections of LPS .", "This chronic LPS treatment induced a massive expansion of Yaphigh hepatocytes , similar to what was seen after CCl4 treatment ( Figure 3D ) .", "Consistently , the BrdU incorporation index indicated that Yaphigh hepatocytes proliferated significantly more than hepatocytes in the WT livers ( Figure 5A , B ) .", "YapKIAlb-Cre livers displayed significant hyperplasia , as visualized by increased hepatocyte density ( Figure 5C ) and measured by total number of hepatocytes per liver ( Figure 5D ) . 10 . 7554/eLife . 02948 . 025Figure 5 . Inflammation induces proliferation of Yap-overexpressing hepatocytes .", "( A ) Control ( WT ) or YapKIAlb-Cre mice were coinjected with BrdU and LPS for 6 days .", "Percentage of BrdU+ hepatocytes was quantified .", "4–6 mice were used for each group and 3–4 images were taken for each mouse .", "Each dot represents cell count from each image .", "( B ) A representative image of chronic ( 6xLPS ) LPS injected livers used for the quantification in A . Arrows indicate BrdU+ hepatocyte nuclei .", "( C ) H&E representative images of the chronic LPS-treated livers .", "( D ) Hepatocyte numbers of the chronic LPS-treated livers was measured by quantitative flow cytometry . DOI: http://dx . doi . org/10 . 7554/eLife . 02948 . 02510 . 7554/eLife . 02948 . 026Figure 5—figure supplement 1 . Role of IL-6 in expansion of Yap-overexpressing hepatocytes .", "( A ) 1- to 2-month-old YapKIAlb-Cre were injected s . c . with LLC cells stably expressing IL-6 ( KI+LLC-IL-6 ) , or empty vector ( KI+LLC ) , and IL-6 levels in the serum were measured 14–20 days later .", "Untreated YapKI and WT mice were bled as controls .", "( B ) Percentage of GFPhigh cells determined by flow cytometry in hepatocytes from 1- to 2-month-old YapKIAlb-Cre mice from ( A ) ( grouped based on serum IL-6 levels ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 02948 . 02610 . 7554/eLife . 02948 . 027Figure 5—figure supplement 2 . Correlation of TEAD and NFkB binding sites in the promoters of genes regulating proliferation . Genes with DNA sequence motifs of NF-kB ( p65 ) and/or TEADs were identified with Motifmap ( NF-kB M00208 , TEADs M01305 ) , using mouse genome MM9 multiz30way .", "NF-kB motifs were found within 1 kb of gene transcription start sites ( TSSs ) and TEAD motifs were found within 10 kb of TSSs .", "Genes related to cell proliferation were identified with David gene ontology GO:0042127 for regulation of cell proliferation .", "Statistical significance was calculated with Chi-square test . DOI: http://dx . doi . org/10 . 7554/eLife . 02948 . 027 We then tested whether IL-6 , an inflammatory cytokine induced by LPS and by liver injury , can cooperate with Yap in promoting hepatocyte growth .", "When IL-6 was produced by LLC cells growing as subcutaneous tumors in YapKIAlb-Cre mice , it caused expansion of Yaphigh hepatocytes in the livers of mice that had the highest levels of systemic IL-6 ( Figure 5—figure supplement 1A , B ) , suggesting that IL-6 may cooperate with Yap to promote hepatocyte proliferation .", "4 days after CCl4 treatment in WT mice , normal liver tissue morphology was restored around the CV .", "In contrast , we observed high cell density , strong cytoplasmic eosinophilia , round to ovoid nuclei , and increased nuclear/cytoplasmic ratio in the pericentral zones of livers from CCl4-treated YapKIAlb-Cre mice ( Figure 6A ) .", "These morphological changes are often seen in hepatocellular carcinomas ( HCC ) and are associated with dedifferentiation ( Chang et al . , 2010; Balani et al . , 2013 ) .", "Immunofluorescent staining indicated that these cells were GFP+ and therefore originated from Yaphigh hepatocytes ( Figure 3A ) .", "To determine whether Yap induction affects differentiation status of hepatocytes , we compared the gene expression of hepatocytes sorted from YapKIAlb-Cre mice at steady state and 4 days after CCl4 damage .", "The majority of liver-specific transcripts ( Pan et al . , 2013 ) ( which roughly represent hepatocyte differentiation state ) were strongly repressed in Yaphigh hepatocytes from CCl4-treated mice , whereas WT and Yaplow hepatocytes showed minimal alterations and no bias towards decreased expression ( Figure 6B ) .", "Immunofluorescent staining and Western blot for hepatocyte-specific gene glutamine synthase also showed a strong downregulation of expression in YapKIAlb-Cre livers after CCl4 damage ( Figure 6C , D ) .", "Downregulation of other hepatocyte-specific genes ( acute phase protein Orm1 and urea cycle component Asl ) was also confirmed by qPCR ( Figure 6—figure supplement 1 ) .", "To further verify RNA-sequencing data and evaluate levels of Yap activation in our model , we performed qPCR analysis of the genes previously shown to be induced in Yap-Tg liver by Pan's group ( Dong et al . , 2007 ) and found comparable effects ( Figure 6—figure supplement 1 ) . 10 . 7554/eLife . 02948 . 028Figure 6 . Yap activation cooperates with tissue injury to repress hepatocyte differentiation and promote progenitor phenotype .", "( A ) H&E staining of liver sections of control ( WT ) and YapKIAlb-Cre ( KI ) mice isolated on day 4 after CCL4 treatment .", "( B ) Heatmap of RNA-sequencing data comparing expression of liver specific genes ( see ‘Materials and methods’ ) .", "Hepatocytes were sorted from wild-type livers ( WT ) or from YapKIAlb-Cre livers based on GFP levels ( Lo and Hi ) , at steady state ( SS ) or on day 4 after CCl4 treatment ( CCl4 ) .", "The fold change is calculated between the indicated samples and WT in steady state .", "Data represent the mean of the duplicates .", "( C ) Liver sections of control ( WT ) and YapKIAlb-Cre ( KI ) mice isolated on days 4 CCL4 treatment were stained with antibodies to glutamine synthase .", "( D ) Glutamine synthase ( GS ) , Yap and beta-actin protein levels in whole-liver protein lysates prepared on day 4 after CCl4 treatment were determined by Western blotting .", "Higher migrating band in the middle panel corresponds to the exogenous Yap ( due to the in-frame triple flag tag ) .", "( E and F )", "Heatmaps showing the RNA-sequencing data for genes enriched in embryonic tissues ( E ) or progenitor markers ( F ) .", "Hepatocytes were sorted from wild type livers ( WT ) or from YapKIAlb-Cre livers based on GFP levels ( Lo and Hi ) , at steady state ( SS ) or on day 4 after CCl4 treatment ( CCl4 ) .", "The fold change is calculated between the indicated samples and wild type in steady state .", "( G and H )", "Flow cytometric analysis of primary hepatocytes isolated by collagenase perfusion from CCl4-treated control ( WT ) or YapKIAlb-Cre ( KI ) mice .", "( G ) Representative flow cytometry plots gated of CD45− CD31− population .", "The numbers on the plots indicate the percentages of the gated population ( EpCam+ progenitors ) .", "( H ) The results from ( G ) were combined with the total hepatocyte numbers to calculate the number of hepatic EpCam+ progenitors per liver . DOI: http://dx . doi . org/10 . 7554/eLife . 02948 . 02810 . 7554/eLife . 02948 . 029Figure 6—figure supplement 1 . Verification of the RNA-sequencing results . Primary hepatocytes of WT and YapKIAlb-Cre ( KI ) mice were sorted by flow cytometry based on GFP levels from steady-state livers ( SS ) or on day 4 after CCl4 treatment ( CCL4 ) and gene expression determined by qPCR .", "Dots represent individual mice . DOI: http://dx . doi . org/10 . 7554/eLife . 02948 . 029 The transcriptional repression was not global: a number of genes expressed predominantly during embryogenesis ( Pan et al . , 2013 ) were induced in Yaphigh hepatocytes , and this effect was strongly enhanced by injury ( Figure 6E ) .", "In addition , several hepatocyte progenitor markers were upregulated in Yaphigh cells after injury , and some of them were already elevated in the steady state ( Figure 6F ) .", "FACS staining of liver cells isolated on day 4 post-CCl4 confirmed expansion of CD45− CD31− EpCam+ hepatic progenitors in YapKI;AlbCre livers ( Figure 6G , H ) .", "These data are in line with the recent report by Yimlamai et al . ( 2014 ) .", "Altogether , these data suggest that Yap cooperates with injury signals to repress hepatocyte differentiation and promote a progenitor phenotype .", "The expansion of Yaphigh cells induced by CCl4 did not persist , and by day 14 after the treatment , the proportion of Yaphigh cells per liver returned to the average value of the untreated group ( Figure 3D ) .", "Yaphigh cells formed a ring of only 1–2 cell layers around CVs , in contrast to multiple layers observed on day 4 ( Figure 3A ) , indicating that the Yaphigh cell pool underwent contraction .", "TUNEL staining suggested that the contraction was mediated by apoptosis: the number of TUNEL positive cells was markedly elevated in the YapKIAlb-Cre livers specifically around the CV on day 4 after CCl4 treatment ( Figure 7A ) .", "Accordingly , RNA sequencing revealed a number of pro-apoptotic genes ( e . g . , Bok , Bax , NGFRAP1 , and Tnfrsf10b ( Dr5 ) ) ( Figure 7B ) upregulated as a consequence of Yap overexpression and further induced after damage .", "We verified by qPCR that Dr5 gene expression was strongly upregulated in the Yaphigh cells after CCl4 ( Figure 7C , D ) .", "Indeed , Yaphigh cells sorted from CCl4-treated livers were more sensitive to killing mediated by TRAIL in vitro , compared to WT or GFPlow counterparts ( Figure 7E ) , suggesting that Yap activation sensitizes hepatocytes to Dr5-mediated cell death .", "This higher sensitivity to cell death was induced by a combination of injury and Yap overexpression , as it was not observed neither in Yaphigh hepatocytes isolated from untreated mice nor in WT hepatocytes from CCl4 livers . 10 . 7554/eLife . 02948 . 030Figure 7 . Yap sensitizes hepatocytes to TRAIL-mediated apoptosis .", "( A ) Cell death as reflected by TUNEL-positive cell numbers quantified outside the CV zone ( ‘outside’ ) and around CV ( ‘damage site’ ) in liver sections of YapKIAlb-Cre mice on day 4 after CCl4 treatment .", "3–4 mice were used for each group and 3–4 images were taken from each mouse .", "Each dot represents cell count from each image .", "( B ) RPKM data from RNA-sequencing illustrating expression of apoptosis-related genes in WT or Yaphigh ( Hi ) hepatocytes sorted from untreated ( SS ) livers or on day 4 after CCl4 treatment ( CCl4 ) .", "( C and D )", "Dr5 mRNA levels in WT and YapKIAlb-Cre livers ( KI ) at indicated time points after CCl4 treatment were determined by qPCR in whole liver extracts ( C ) , or in primary hepatocytes sorted by flow cytometry based on GFP levels from steady-state livers ( SS ) or on day 4 after CCl4 treatment ( CCL4 ) ( D ) .", "2–3 mice were used for each group .", "*p ≤ 0 . 05 .", "( E ) Primary hepatocytes sorted as in ( D ) were cultured on collagen-coated plates with or without TRAIL , and cell viability measured by CellTiter-Blue the next day .", "3–4 mice were used for each group and 3–4 wells were seeded with hepatocytes from each mouse .", "Each dot represents reading from each well . DOI: http://dx . doi . org/10 . 7554/eLife . 02948 . 030 In summary , the expanded population of Yaphigh cells contracts to baseline at later stages of the tissue injury response .", "This process is likely mediated , in part , by sensitization of Yaphigh cells to TRAIL-induced apoptosis via upregulation of Dr5 ." ], [ "Yap has emerged as a central mediator of signals promoting proliferation , survival , and stemness ( Barry and Camargo , 2013 ) .", "Not surprisingly , an expanding number of pathways restricting the pro-growth potential of Yap have been identified , including the Hippo pathway , the actin cytoskeleton and cell junction complexes ( Boggiano and Fehon , 2012 ) , C/EBPa , and Trib2 ( Wang et al . , 2013 ) .", "Despite these multifactorial control mechanisms , mere overexpression of Yap in the whole liver in mouse models leads to hyperplasia , organomegaly , dedifferentiation , and cancer ( Camargo et al . , 2007; Dong et al . , 2007; Schlegelmilch et al . , 2011 ) , raising a question as to whether some layer of homeostatic growth control may be lacking in such models .", "Organ-wide Yap activation in the liver does occur during embryonic development ( Septer et al . , 2012 ) and adaptive hepatomegaly ( Kowalik et al . , 2011 ) .", "Interestingly , these are also examples of physiologically relevant settings of liver size increase .", "Transgenic Yap overexpression in the entire liver may thus be viewed as a model of these physiological scenarios .", "In contrast , during response to a localized injury , only the cells within the damage site need to proliferate , and Yap activation in this case should conform to the physiological organ size limits .", "Our study suggests that under these circumstances , activation of a pro-growth gene program by Yap requires an additional local signal ( e . g . , growth factor , cytokine , or release of contact inhibition ) provided by the injury .", "Our preliminary data implicate IL-6 as at least one of such signals ( Figure 5—figure supplement 1 ) .", "Proto-oncogenes typically require two signals in order to induce proliferation .", "In case of Yap , which acts as a sensor of cell density , its overexpression mimics a condition in which a cell has lost its contacts with the neighbors .", "This in itself should not induce proliferation unless cell-extrinsic signals of tissue damage such as inflammatory cytokines are also present .", "The mechanism of cooperation between activation of Yap and injury/inflammatory signals may occur at the level of gene expression , as many proliferation genes have TEAD as well as NFkB binding sites ( Figure 5—figure supplement 2 ) .", "While it is also possible that cooperation is mediated by increased Yap stability or recruitment of TEAD , this possibility is less likely as we observed induction of high number of TEAD-dependent genes in Yap-overexpressing hepatocytes whereas proliferation genes were not induced .", "Our data suggest that the tissue environment imposes a selective pressure on cells that activate Yap .", "The mosaic model of Yap induction described here reveals selective pressures that determine the fate of Yaphigh hepatocytes: no growth advantage at steady state , positive selection during early stages of tissue injury , and negative selection at later stages when injury induced signals subside .", "The mechanisms opposing clonal expansion of Yaphigh hepatocytes under homeostatic conditions may have many components , but one implicated by our data is apoptosis .", "Pro-apoptotic effects of Yap have been extensively documented in vitro ( Strano et al . , 2001; Lapi et al . , 2008; Tomlinson et al . , 2010 ) , and increased apoptosis has been reported in Mst1/2-deficient mouse livers ( Lu et al . , 2010 ) , but to our knowledge there has been no evidence of Yap promoting cell death in vivo .", "We show that Yap activation in the mouse liver leads to increased apoptosis in vivo and sensitization to TRAIL measured ex vivo .", "The induction of proliferation or apoptosis by Yap is modulated by tissue damage and inflammatory signals .", "This is analogous to the well-characterized functions of c-myc , which is also known induce proliferation or apoptosis , depending on growth factor availability ( Evan et al . , 1992; Harrington et al . , 1994 ) .", "These examples may represent a general principle of mitogenic pathway design , aimed at elimination of aberrant cells .", "YapKIAlb-Cre mice display many features described in other in vivo models of Yap induction , including increased proliferation , dedifferentiation , and activation of TEAD target genes .", "However , the unexpected phenotype unique to YapKIAlb-Cre mice is that the pro-growth effects of Yap are only manifested in a state of altered homeostasis , such as tissue injury or inflammation .", "These differences are unlikely to be due to an insufficient level of Yap expression , as a large number of genes are highly induced by Yap under steady-state conditions in YapKIAlb-Cre mice .", "One relevant difference may be mosaic vs tissue-wide Yap activation .", "We show that mosaic deletion of Mst2 in Mst1−/− background does not lead to clonal expansion or liver size increase .", "Furthermore , in another mouse model recently reported by Camargo et al . ( Yimlamai et al . , 2014 ) while this manuscript was under consideration , mosaic Yap activation in hepatocytes did not induce proliferation of clonal expansion in the first weeks .", "Instead , it promoted hepatocyte dedifferentiation into progenitors , which then expanded .", "Taken together , these results suggest that activation of Yap in hepatic progenitor cells is sufficient for clonal expansion but in mature hepatocytes it requires the second signal to drive proliferation .", "Another possible cause of different phenotypes may lie in different levels of inflammation between our mouse model and the whole liver transgenics .", "Of note , livers with a hepatocyte-specific ablation of Mst1/2 or Sav ( both of which result in Yap activation ) have elevated expression of immune response genes , including TNFa and IL-6 ( Lu et al . , 2010 ) .", "Many variables may contribute to differences in systemic inflammation .", "One likely contributor is the intestinal microbiome composition , which varies greatly across mouse facilities ( Bleich and Hansen , 2012 ) and can induce inflammatory responses in the liver ( Henao-Mejia et al . , 2012 ) .", "Another source of inflammation in case of whole-liver Yap overexpression may be the disruption of systemic metabolism due to Yap-mediated repression of liver-specific genes ( [Yimlamai et al . , 2014] and our RNA-sequencing analysis ) .", "As liver-specific genes are still expressed at normal levels in a large fraction of hepatocytes ( Yaplow cells ) , systemic metabolism is not compromised in YapKIAlb-Cre mice .", "It is therefore possible that the level of systemic inflammation is low in our mice , and this is why they require an exogenous inflammatory signal to enable Yap pro-growth effects .", "Inflammation is an essential component of carcinogenesis ( Ben-Neriah and Karin , 2011 ) and anti-inflammatory treatments can inhibit aberrant proliferation and delay tumor development ( Pribluda et al . , 2013 ) .", "Inflammation can promote tumor development through multiple mechanisms ( Ben-Neriah and Karin , 2011 ) , including provision of a permissive signal for proliferation in the settings of disturbed homeostasis ( Bondar and Medzhitov , 2013; Pribluda et al . , 2013 ) .", "Our RNA-sequencing analysis has revealed two important functions of Yap in hepatocytes: first , the largest category of Yap-regulated genes is related to ECM and cell adhesion .", "These transcriptional targets of Yap appear to be the most common across many cell types and contexts of Yap activation; for example , the gene widely used as a hallmark of Yap activation is a matricellular growth factor CTGF .", "Since loss of cell contacts is known to activate Yap , it makes sense that Yap induces a restorative transcriptional program .", "Second , Yap strongly suppresses liver-specific genes in response to injury .", "Hepatocytes are known to downregulate liver-specific transcripts as they enter the cell cycle during liver regeneration ( Ito et al . , 1991 ) .", "Proliferation and tissue-specific activities are often segregated between progenitor and differentiated cells , especially in tissues with high cell turnover .", "This suggests that extensive proliferation may be generally incompatible with specialized tissue functions .", "In conclusion , tissue remodeling , cell cycle progression , and repression of tissue-specific functions all need to be orchestrated in response to injury , and our findings suggest Yap as a key coordinator of these activities during tissue repair .", "Furthermore , our data highlight the role of tissue microenvironment in the outcome of Yap activation and argue that during homeostasis , growth-promoting function of Yap in differentiated cells requires an additional signal , which may be provided by inflammation or injury ." ], [ "To generate the YapKI and YapCKO lines , targeting vectors were designed and constructed as shown in Figure 1—figure supplement 1A and Figure 4—figure supplement 1 .", "NM-001171147 mouse Yap1 isoform with introduced S112A mutation ( corresponding to S127A of the human protein ) was used to generate YapKI allele .", "After the successful construction of the targeting vectors , the purified plasmid DNA was linearized and transfected into the C57BL/6-derived Bruce4 ES cells .", "Correct recombination and replacement of the DNA were verified by Southern blots .", "Selected clones were expanded and submitted to the Yale Transgene Facility for injections into BALB/c blastocysts to generate chimeric mice .", "In case of the YapCKO line , mice with the germline-transmitted Yap floxed allele were then crossed to Flp-deleter transgenic mice ( ENCODE Project Consortium , 2011 ) to remove the neomycin cassette .", "Animals were maintained at the Yale Animal Resources Center .", "All animal experiments were performed with approval by the Institutional Animal Care and Use Committee of Yale University .", "All mice were on C57BL/6 background .", "AlbCre mice were from Jackson Laboratory .", "For BrdU staining , daily intraperitoneal ( i . p . ) injections of BrdU ( Sigma , St . Louis , MO; 100 μg/mouse ) were performed for 3 days before liver dissection .", "Liver damage was induced by i . p . injection of CCl4 ( Sigma , 1 μl/g , diluted 1:10 with peanut oil ) .", "For the LLC-IL-6 experiment , 1 million LLC cells stably expressing mouse IL-6 or empty vector were injected into each flank of 1- to 1 . 5-month-old YapKIAlb-Cre mice .", "2–3 weeks later when tumors reached approximately 1 cm in diameter , the mice were eye-bled for IL-6 ELISA and sacrificed for hepatocyte cell preparation .", "For the in vivo deletion of Mst2flox/flox allele , AdCMVCre-RSV-GFP adenovirus from KeraFast was amplified in 293A cells , purified by ViraPur kit , dialized against PBS and injected into the tail vein of the 1- to 2-month-old Mst1−/− Mst2flox/flox mice .", "Liver lobes were fixed in 4% PFA overnight and embedded in paraffin .", "Sections were deparaffinized and heated for 20 min at 95°C for antigen retrieval in Tris-EDTA buffer pH 9 .", "TUNEL staining was performed using In Situ Cell Death Detection Kit ( Roche , Germany ) .", "For GFP , Yap , and GS immunofluorescence staining , samples were then blocked in TBS-T with 3% BSA for 2 hr ( staining buffer ) , incubated with the primary antibody overnight and with the secondary antibody for 1 hr .", "For BrdU staining , after the described steps , the samples were fixed in 4% PFA in PBS for 20 min at RT .", "DNA was then denatured in 2 N HCl for 30 min at 37°C .", "After neutralization with 0 . 1 M sodium tetraborate , the sections were stained with mouse anti-BrdU antibodies for 2 hr and visualized with flourophore-conjugated anti-mouse secondary antibodies .", "The slides were mounted in Vectashield anti-fade media containing Hoescht dye to counterstain the nuclei .", "20–30 20× tiled images were obtained using Zeiss Axioplan microscope and AxioVision software .", "Cells were counted manually in AxioVison software .", "Only hepatocytes ( as determined by morphology and GFP expression where applicable ) were scored for BrdU quantification .", "Primary hepatocytes were prepared by collagenase perfusion at Yale Liver Center .", "Hepatocytes were washed twice with FACS buffer ( 2 mM EDTA , 2% FBS in PBS ) , stained with 7-Aminoactinomycin D ( 7AAD ) or propidium iodide , and analyzed on BD Accuri or LSRII , or sorted on MoFlo cytometers .", "Samples were gated on live hepatocytes based on forward and side scatter and live dye exclusion .", "Duplicate hepatocyte samples were prepared in several independent experiments .", "Each of the duplicates contained material pooled from multiple mice .", "Hepatocytes were isolated by flow cytometry , and RNA was extracted using RNeasy kit ( Qiagen , Germany ) .", "1–10 μg of each sample was submitted to the Yale Center of Genome Analysis .", "Sequencing libraries were constructed and were sequenced by Illumina Hiseq 2500 with 76 bp single-end reads , which generated 20 million raw reads per sample on average .", "After removing the low-quality reads ( around 0 . 18% of all reads ) and low-quality portions ( Q value <30 ) of each of the raw reads , the RNA-sequencing data for each sample were mapped to the GRCm38/mm10 mouse reference first .", "The GRCm38 reference was downloaded from the Mouse Genome Informatics ( MGI ) .", "The mapping was performed using TopHat v2 . 0 . 8 , allowing two mismatches .", "The percentages of mapped reads were 71 . 16% on average ( from 70 . 00% to 73 . 79% ) .", "After mapping , Cufflinks v2 . 0 . 2 was applied to assemble and quantify the transcripts and discover the differentially expressed genes , with the annotated gene information from the MGI .", "The gene expression values were calculated for each sample based on the number of fragments per kilobase of exon per million reads mapped ( FPKM ) .", "The significance of differential expression of genes was detected using Cuffdiff for all comparisons of every two samples .", "To select differentially expressed genes , the average of two biological replicates was compared between the conditions examined .", "To select genes with significant activation or repression in any comparison , the p-value of 0 . 005 was used together with a fold-change threshold of twofold .", "A pseudocount of 0 . 01 was added to RPKM value of each gene as a sequencing background , to avoid inflated fold change caused by lowly expressed genes .", "Gene functional analysis was performed with the DAVID functional annotation tools ( Huang et al . , 2009a , 2009b ) ( http://david . abcc . ncifcrf . gov ) .", "The p-value was adjusted with Benjamini methods for multiple hypothesis testing .", "Tissue-specific genes were obtained from Pattern Gene Database ( Pan et al . , 2013 ) ( http://bioinf . xmu . edu . cn/PaGenBase ) .", "Genes expressed specifically ( only in one tissue ) and selectively ( only in a group of samples ) were pooled together as the tissue-specific genes in our analysis .", "Liver-specific genes are identified from mouse liver sample , and mouse embryo specific genes are identified from mouse day 9 . 5 embryo sample .", "Genome-wide binding of Tead4 ( identified by ChIP-sequencing ) was retrieved from the ENCODE Project Consortium ( 2011 ) .", "The uniform peaks determined in all available samples ( HepG2 cell line , K562 cell line , and hESC ) were combined , and genes whose TSS is within 10 kb distance from any Tead4 uniform peak were defined as Tead4-associated genes .", "Hg19 genome annotation was downloaded from the UCSC database ( Karolchik et al . , 2014 ) .", "Primary hepatocytes were plated in collagen-coated wells in Williams' E medium ( WEM ) supplemented with 5% FCS , 10 mM HEPES , 2 mM L-glutamine , 10 mM penicillin/streptomycin , 8 μg/ml gentamicin , 100 μg/ml chloramphenicol , 100 nM dexamethasone , and 1 nM Insulin .", "4 hr after plating , the medium was changed to the supplemented WEM without chloramphenicol and dexamethasone .", "On the second day cells were treated with recombinant TRAIL ( R&D Systems , Minneapolis , MN ) overnight .", "Viable cell number was measured by the CellTiter-Blue assay ( Sigma ) according to the manufacturer protocol .", "Whole liver tissue was snap frozen in liquid nitrogen , homogenized , and resuspended in TNT buffer ( 0 . 1 M Tris-HCL pH 7 . 5 , 150 mM NaCl , 0 . 1% Tween20 ) supplemented with the Complete protease inhibitors ( Roche ) and cleared by centrifugation at 100×g .", "Protein concentration in the supernatants was normalized using Bradford reagent .", "15 μg of the extracts were separated on a gradient SDS-PAGE gel and transferred to PVDF membrane .", "After blocking with 3% BSA in TBST buffer , the membranes were probed with antibodies to Yap ( Cell Signaling , Beverly , MA ) , glutamine synthase ( Genscript , Piscataway , NJ ) , and beta-actin ( Sigma ) .", "ALT tests were performed using ALT activity assay ( Sigma ) according to the manufacturer's protocol .", "RNA was extracted with RNA-bee , and cDNA synthesis was performed using SMART MMLV reverse transcriptase according to the manufacturers' protocols .", "Real time PCR was performed in triplicates using Quanta SYBR reagent on C1000 Thermal Cycler ( BioRad , Hercules , CA ) .", "Primer sequences are listed in the Supplementary file1 .", "Three 1 mm3 pieces of liver tissue per mouse were digested with Pronase overnight , and genomic DNA isolated by NaCl/ethanol precipitation .", "The three DNA samples for each mouse were pooled and used at fourfold dilutions to amplify the Rag2 or recombined Stk3flox/flox alleles using Taqman polymerase .", "Paired antibodies against IL-6 were purchased from BD Biosciences to perform ELISA .", "All statistical analysis was performed by two tailed unpaired Student's t test , unless specified otherwise .", "The RNA sequencing data have been deposited to GEO database under the accession number GSE65207 ." ] ]

[ "The transcriptional coactivator Yes-associated protein ( Yap ) promotes proliferation and inhibits apoptosis , suggesting that Yap functions as an oncogene .", "Most oncogenes , however , require a combination of at least two signals to promote proliferation .", "In this study , we present evidence that Yap activation is insufficient to promote growth in the otherwise normal tissue .", "Using a mosaic mouse model , we demonstrate that Yap overexpression in a fraction of hepatocytes does not lead to their clonal expansion , as proliferation is counterbalanced by increased apoptosis .", "To shift the activity of Yap towards growth , a second signal provided by tissue damage or inflammation is required .", "In response to liver injury , Yap drives clonal expansion , suppresses hepatocyte differentiation , and promotes a progenitor phenotype .", "These results suggest that Yap activation is insufficient to promote growth in the absence of a second signal thus coordinating tissue homeostasis and repair ." ]

[ "As we grow up , the organs in our body tend to stop growing and then remain roughly the same size for the rest of our lives .", "This is possible because of control systems that determine how often the cells within the organ can divide and when they should die .", "If these controls fail , the cells may divide rapidly and not die when they should , which can cause tumors to grow .", "In healthy cells , the proteins that promote cell division and/or prevent cell death are strictly controlled—usually by at least two different ‘on’ signals—so that they are only active at specific times .", "Yap is one such protein that promotes cell division and inhibits programmed cell death .", "Previous studies have reported that artificially increasing the levels of Yap in cells is sufficient to make tumors grow in a seemingly unrestrained fashion .", "This suggests that only one signal is required to over-activate Yap .", "However , it is also possible that these experiments were carried out under conditions where a second unknown growth-promoting signal was also present .", "Here , Su , Bondar et al . genetically altered mice to produce more Yap in some , but not all , cells in the liver .", "This revealed that when surrounded by normal cells , the high levels of Yap in individual cells do not lead to excessive liver growth .", "The cells do divide more , but this is balanced by an increase in the numbers of cells that die .", "However , if the liver is injured , the high levels of Yap can keep the cells in a stem cell-like state and cause them to grow and divide excessively .", "This helps the liver to recover , and once the recovery is complete , the cells that produce more Yap are killed .", "Su , Bondar et al . suggest that the excessive cell growth observed in previous studies may be due to the researchers unintentionally simulating the conditions of inflammation by increasing the levels of Yap in all the cells .", "These findings reveal that Yap needs to be activated by both a signal from within the cell and a second signal from other cells—caused by injury or inflammation—to promote the growth of tumors ." ]

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

[ "genetics and genomics" ]

[ [ "Genome-wide association studies ( GWAS ) have now been conducted for thousands of human complex traits , revealing that the genetic architecture is almost always highly polygenic , that is that the bulk of the heritable variation is due to thousands of genetic variants , each with tiny marginal effects ( Boyle et al . , 2017; Bulik-Sullivan et al . , 2015 ) .", "These findings make it difficult to interpret the molecular basis for variation in a trait , but they lend themselves more immediately to another use: phenotypic prediction .", "Under the assumption that alleles act additively , a 'polygenic score' ( PGS ) can be created by summing the effects of the alleles carried by an individual; this score can then be used to predict that individual’s phenotype ( Henderson , 1984; Meuwissen et al . , 2001; Kathiresan et al . , 2008; Lynch and Walsh , 1998 ) .", "For highly heritable traits , such scores already provide informative predictions in some contexts: for example , prediction accuracies are 24 . 4% for height ( using R2 as a measure ) ( Yengo et al . , 2018 ) and up to 13% for educational attainment ( using incremental R2 ) ( Lee et al . , 2018 ) .", "This genomic approach to phenotypic prediction has been rapidly adopted in three distinct fields .", "In human genetics , PGS have been shown to help identify individuals that are more likely to be at risk of diseases such as breast cancer and cardiovascular disease ( Khera et al . , 2018; Inouye et al . , 2018; Mavaddat et al . , 2019; Khera et al . , 2019 ) .", "Based on these findings , a number of papers have advocated that PGS be adopted in designing clinical studies , and by clinicians as additional risk factors to consider in treating patients ( Torkamani et al . , 2018; Khera et al . , 2018 ) .", "In human evolutionary genetics , several lines of evidence suggest that adaptation may often take the form of shifts in the optimum of a polygenic phenotype and hence act jointly on the many variants that influence the phenotype ( Pritchard and Di Rienzo , 2010; Berg and Coop , 2014; Höllinger et al . , 2019; Sella and Barton , 2019 ) .", "In this context , the goal is to test whether the set of variants that influence a trait are rapidly evolving across populations or over time ( Field et al . , 2016; Berg et al . , 2019; Uricchio et al . , 2019; Edge and Coop , 2019; Racimo et al . , 2018; Berg and Coop , 2014 ) .", "Finally , in various disciplines of the social sciences , PGS are increasingly used to distinguish environmental from genetic sources of variability ( Conley , 2016 ) , as well as to understand how genetic variation among individuals may cause heterogeneous treatment effects when studying how an environmental influence ( e . g . , a schooling reform ) affects an outcome ( such as BMI ) ( Barcellos et al . , 2018; Davies et al . , 2018 ) .", "In all these applications , the premise is that PGS will ‘port’ well across groups—that is that they remain predictive not only in samples very similar to the ones in which the GWAS was conducted , but also in other sets of individuals ( henceforth ‘prediction sets’ ) .", "As recent papers have highlighted , however , PGS are not as predictive in individuals whose genetic ancestry differs substantially from the ancestry of individuals in the original GWAS ( reviewed in Martin et al . , 2019 ) .", "As one illustration , PGS calculated in the UK Biobank predict phenotypes of individuals sampled in the UK Biobank better than those of individuals sampled in the BioBank Japan Project: for instance , the incremental R2 for height is approximately 11% in the UK versus 3% in Japan ( Martin et al . , 2019 ) .", "Similarly , using PGS based on Europeans and European-Americans , the largest educational attainment GWAS to date ( 'EA3' ) reported an incremental R2 of 10 . 6% for European-Americans but only 1 . 6% for African-Americans ( Lee et al . , 2018 ) .", "To date , such observations have been discussed mainly in terms of population genetic factors that reduce portability ( Martin et al . , 2017; Kim et al . , 2018; Duncan et al . , 2018; De La Vega and Bustamante , 2018; Sirugo et al . , 2019; Martin et al . , 2019 ) .", "Notably , GWAS does not pinpoint causal variants , but instead implicates a set of possible causal variants that lie in close physical proximity in the genome .", "The estimated effect of a given SNP depends on the extent of linkage disequilibrium ( LD ) with the causal sites ( Pritchard and Przeworski , 2001; Bulik-Sullivan et al . , 2015 ) .", "LD differences between populations that arose from their distinct demographic and recombination histories will lead to variation in the estimated effect sizes and hence to variable phenotypic prediction accuracies ( Rosenberg et al . , 2019 ) .", "Populations will also differ in the allele frequencies of causal variants .", "This problem is particularly acute for alleles that are rare in the population in which the GWAS was conducted but common in the population in which the trait is being predicted .", "Such variants are likely to have noisy effect size estimates in the estimation sample or may not be included in the PGS at all , and yet they contribute substantially to heritability in the target population .", "Furthermore , causal loci or effect sizes may differ among populations , for instance if the effect of an allele depends on the genetic background on which it arises ( e . g . , Adhikari et al . , 2019 ) .", "For all these reasons , we should expect PGS to be less predictive across ancestries .", "In practice , given that most individuals ( about 80% ) included in current GWAS are of European ancestry ( Popejoy and Fullerton , 2016; Martin et al . , 2019 ) , PGS are systematically more predictive in European-ancestry individuals than among other people .", "As a consequence , the clinical applications and scientific understanding to be gained from PGS will predominantly and unfairly benefit a small subset of humanity .", "A number of papers have therefore highlighted the importance of expanding GWAS efforts to include more diverse ancestries ( Martin et al . , 2018; Bien et al . , 2019; Wojcik et al . , 2019; Martin et al . , 2019; Sirugo et al . , 2019 ) .", "Importantly , factors other than ancestry could also impact the accuracy and portability of PGS .", "For example , the educational attainment of an individual depends not only on their own genotype , but on the genotypes of their parents , due to nurturing effects ( Kong et al . , 2018 ) , and of their peers , due to social genetic effects ( Domingue et al . , 2018 ) , and of course on non-genetic factors .", "Also , traits such as height and educational attainment show strong patterns of assortative mating , which can distort effect size estimates in GWAS ( Domingue et al . , 2014; Robinson et al . , 2017; Ruby et al . , 2018 ) .", "To what extent these effects remain the same across cultures and environments is unknown , but if they differ , so will the prediction accuracy .", "More generally , while we still know little about genotype-environment interactions ( GxE ) in humans , they are well-documented in other species—notably in experimental settings—and would further reduce the portability of PGS across environments ( Gibson , 2008; Tropf et al . , 2017; Mills and Rahal , 2019; Lynch and Walsh , 1998 ) .", "In addition , the extent of environmental variability could differ between GWAS and prediction groups , which would change the proportion of the variance in the trait explained by a PGS ( i . e . , the prediction accuracy ) .", "PGS for some traits may also include a component of environmental or cultural confounding with population structure ( Sohail et al . , 2019; Haworth et al . , 2019; Lawson et al . , 2020; Kerminen et al . , 2018; Berg et al . , 2019 ) ; this source of confounding can increase or decrease prediction accuracy , depending on the structure in the prediction samples .", "Given these considerations , it is important to ask to what extent PGS are portable among groups within the same ancestry .", "To explore this question , we stratified the subset of UK Biobank samples designated as ‘White British’ ( WB ) according to some of the standard sample characteristics of GWAS studies: the ages of the individuals , their sex , and socio-economic status .", "We chose to focus on these particular characteristics because they vary among GWAS samples depending on sample ascertainment procedures .", "Furthermore , these characteristics have been shown to influence heritability for some traits in a study of a subset of the UK Biobank ( Ge et al . , 2017 ) , raising the possibility that these choices also influence prediction accuracy .", "Indeed , for three example traits , we show that there exist major differences in the prediction accuracy of the PGS among these groups , even though they share highly similar genetic ancestries .", "We further demonstrate for a variety of traits that prediction accuracy differs markedly depending on whether the GWAS is conducted in unrelated individuals or in pairs of siblings , even when controlling for the precision of the estimates .", "This finding is again unexpected under standard GWAS assumptions; it underscores the importance of genetic effects that are included in estimates from some study designs and not others and highlights underappreciated challenges with GWAS-based phenotypic prediction .", "At present , it is difficult to determine the reasons why we see such variable prediction accuracy across these strata and study designs .", "Contributing factors probably include indirect genetic effects from relatives , assortative mating , varying levels of genetic and environmental variance , GxE interaction effects and perhaps undetected confounding .", "Nonetheless , our results make clear that the prediction accuracy of PGS can be affected in unpredictable ways by known—and presumably unknown—factors in addition to genetic ancestry ." ], [ "We examined how PGS for a few example traits port across samples that are of similar genetic ancestry but differ in terms of some common study characteristics , such as the male:female ratio ( henceforth ‘sex ratio’ ) , age distribution , or socio-economic status ( SES ) .", "To this end , we limited our analysis to the largest subset of individuals in the UKB with a relatively homogeneous ancestry: 337 , 536 unrelated individuals that were characterized by the UKB , based on self-reported ethnicities as well as genetic analysis , as ‘White British’ ( WB ) ( Bycroft et al . , 2018 ) .", "In all analyses , we further adjusted for the first 20 principal components of the genotype data , to account for population structure within this set of individuals ( Materials and methods ) .", "In all analyses , we randomly selected a subset of individuals to be the prediction set; we then conducted GWAS using the remaining individuals and built a PGS model by LD-based clumping of the associations ( Materials and methods ) .", "To examine the reliability of the prediction , we considered the incremental R2 , that is the R2 increment obtained when adding the PGS to a model with other covariates ( referred to as 'prediction accuracy' henceforth ) .", "Whether this measure is appropriate depends on how PGS are to be used; it is not always the most obvious choice in human genetics , where the goal is often to identify individuals at high risk of developing a particular disease ( i . e . , in the tail of the polygenic score distribution ) .", "Nonetheless , because it has been widely reported in discussions of portability across genetic ancestries ( e . g . , Lee et al . , 2018; Martin et al . , 2019 ) , we also used it here; later , we also present some results on binary traits using incremental area under the receiver operator curve ( AUC ) .", "As a first case , we considered the prediction accuracy of a PGS for diastolic blood pressure in prediction sets stratified by sex , motivated by reports that variation in this trait may arise for somewhat distinct reasons in the two sexes ( Reckelhoff , 2001; Zhou et al . , 2017 ) .", "We randomly selected males and females as prediction sets ( 20K individuals each ) , and used a subset of the rest of the individuals for GWAS , matching the numbers of females and males in the GWAS set ( total sample size 122 , 774 ) ; we refer to this mixed set , somewhat loosely , as the 'diverse GWAS . '", "Adjusting for mean sex effects and medication use ( see Materials and methods ) , the prediction accuracy is about 1 . 15-fold higher for females than for males ( Mann-Whitney p=1 . 1⋅10-5; Figure 1A ) .", "Thus , despite equal representation of males and females in the GWAS set , the prediction accuracy varies depending on the sex ratio of prediction samples .", "To examine this further , we repeated the same analysis but performed the GWAS in only one sex ( which we refer to as 'stratified GWAS' using the same sample size as in the diverse GWAS ) .", "[Note that the diverse GWAS sample is not a merge of the stratified GWAS samples but a mixed-sex sample of equal sample size to that used in the women-only and the men-only GWAS , to allow for direct comparison between GWASs . Results for the merged GWAS ( with a much larger sample size ) are presented in Appendix 1—figure 1A . ] When the GWAS is conducted only in females , the prediction accuracy is about 1 . 35-fold higher for females than for males; in turn , when GWAS was done in only males , the prediction accuracy in both sexes is similar , as well as somewhat decreased ( Figure 1A ) .", "We then considered two other cases , evaluating prediction accuracy in groups stratified by age for BMI—since the UK Biobank participants were enrolled within about a five-year span , differences in age could in principle also be reflective of cohort effects—and by adult SES for years of schooling , using the Townsend deprivation index as a measure; our choices were motivated by prior evidence suggesting that these characteristics of the GWAS influence estimates of SNP-heritability ( Branigan et al . , 2013; Conley et al . , 2015; Belsky et al . , 2018; Elks et al . , 2012; Ge et al . , 2017 ) .", "We withheld a random set of 10K individuals in each quartile of age and SES for prediction and performed GWAS using a subset of the remaining individuals , matching the sample sizes across quartiles in the GWAS set ( total sample sizes of 72 , 328 and 73 , 280 for BMI and years of schooling GWAS , respectively ) .", "Similar to our observation for diastolic blood pressure , the prediction accuracy varies across prediction sets: it is 1 . 4-fold higher for BMI in the youngest quartile compared to the oldest ( Mann-Whitney p=1 . 1⋅10-5; Figure 1B ) , and 2-fold higher for years of schooling in the lowest SES quartile compared to the highest ( Mann-Whitney p=2 . 9⋅10-6; Figure 1C ) .", "Furthermore , the differences across groups are again sensitive to the choice of the GWAS set: the differences are marked when GWAS is restricted to the youngest quartile for BMI and the lowest SES quartile for years of schooling , but diminished when the GWAS is performed in the oldest and the highest SES quartiles for BMI and years of schooling , respectively ( Figure 1B , C ) .", "These results remained qualitatively unchanged when we used R2 instead of incremental R2 to measure prediction accuracy ( Appendix 1—figure 2 ) .", "In these analyses , we used a p-value threshold of 10-4 for inclusion of a SNP in the PGS .", "The choice of how stringent to make the GWAS p-value threshold is important but somewhat arbitrary , with approaches ranging from requiring genome-wide significance to including all SNPs ( Weedon et al . , 2008; Pharoah et al . , 2008; Euesden et al . , 2015; Vilhjálmsson et al . , 2015; Ware et al . , 2017; Mostafavi et al . , 2017; Speidel et al . , 2019 ) .", "Often , this threshold is chosen to maximize prediction accuracy in an independent validation set .", "When the goal is to compare prediction performance across different groups , there is no obvious optimal choice of the p-value threshold .", "[The optimal p-value in this context will differ across studies , as it depends not only on the genetic architecture and heritability of the trait , but also on the GWAS sample size , that is power ( Dudbridge , 2013 ) . ] As we show , however , the qualitative trends reported in Figure 1 do not depend on the p-value threshold choice ( Appendix 1—figure 3 ) ; moreover , the qualitative trends remain when LDpred is used ( with a prior probability of 1 on loci being causal; Vilhjálmsson et al . , 2015 ) instead of pruning approaches ( Appendix 1—figure 3 ) .", "These results pertain to three exemplar traits and do not speak to the prevalence of this phenomenon .", "Nonetheless , they demonstrate that the prediction accuracy of a polygenic score can vary markedly depending on sample characteristics of both the original GWAS and the prediction set , even within a single ancestry , and that this variation in prediction accuracy can be substantial—on the same order as reported for different continental ancestries within the UK Biobank ( Martin et al . , 2019 ) .", "As one example , the prediction accuracy in East Asian samples , averaged across a number of traits , is about half of that in European samples when GWAS was European-based; when the GWAS is done in the lowest SES group for years of schooling , prediction accuracy in the highest SES group is less than half of that in the lowest SES ( Figure 1C ) .", "Moreover , whereas for these traits , we had prior information about which characteristics may be relevant , other aspects that vary across sets of individuals are undoubtedly important as well ( e . g . , smoking behavior and diet may modify genetic effects on lipid traits; Bentley et al . , 2019; Telkar et al . , 2019 ) , and for other traits of interest , much less may be known a priori .", "Our goal in this paper is to highlight that prediction accuracies can vary across groups of highly similar ancestry , rather than to investigate the likely causes for any particular phenotype .", "Nonetheless , we provide some observations that may cast light on these results .", "We first note that in these three examples , the prediction accuracies track SNP heritability differences across strata ( Figure 2A , B , C ) .", "This relationship should be expected , given that the estimation noise decreases with heritability ( Appendix 1 ) , and potentially underlies the observation that prediction accuracies using the diverse GWAS sample are often intermediate between those obtained from stratified GWAS samples of equal sample size ( Figure 1 ) .", "Perhaps the simplest explanation for these findings would be that heritabilities , and hence prediction accuracies , vary only because of differences in the extent of environmental variance across strata , while the genetic variance is the same .", "We can test this hypothesis by examining whether the heritability decreases with increasing phenotypic variance ( more precisely whether it is inversely proportional to it ) , as expected if the genetic variance is fixed across strata .", "What we find instead is that the estimated SNP heritabilities for all three traits increase or remain the same with increasing phenotypic variance ( Figure 2D , E , F ) .", "Thus , for these traits at least , the variable prediction accuracy is not simply the result of differences in the extent of environmental heterogeneity across strata .", "Another possibility is that there is an interaction between genetic effects and sample characteristics , for instance that different sets of genetic variants contribute to blood pressure levels in males and females or to BMI across different stages of life .", "[Although such interactions could in some contexts be thought of as reflecting GxE , we use the term ‘sample characteristic’ rather than ‘environment’ , as environment has different meaning across disciplines , referring in some contexts only to factors that are exogenous to genetics . Viewed in this lens , SES in adulthood cannot be interpreted as exogenous , because it is in part determined by educational achievement , which is itself influenced by genetic factors , and similarly it is questionable whether age or sex are environments . ] This explanation is not supported by bivariate LD score regression , which indicates that the genetic correlations across strata are close to 1 ( Appendix 1—table 2; Materials and methods ) .", "Yet when we re-estimate individual SNP effects in the prediction sets for SNPs ascertained in the original GWAS , the estimated effects of trait-increasing alleles are larger in the groups with higher prediction accuracy ( Appendix 1—figure 4; Materials and methods ) .", "One simple model that could reconcile these findings is if effect sizes are highly correlated across the groups , but systematically larger in those groups with higher prediction accuracy .", "This explanation is reminiscent of the ‘amplification’ model of genetic influences on cognition during development ( Briley and Tucker-Drob , 2013 ) .", "Other factors complicate interpretation , however , and may also contribute to our observations .", "In particular , for the case of years of schooling , conditioning on adult SES induces a form of range restriction , which could contribute to variable prediction accuracy across strata .", "We note , however , that we see highly variable prediction accuracies across SES strata even when the GWAS is conducted in a diverse sample ( i . e . , including individuals from all strata ) ( Figure 1C ) ; in that regard , our approach mimics what happens in practice when polygenic scores are used to predict phenotypes in a sample with a smaller range of SES ( e . g . , Rimfeld et al . , 2018 ) .", "More generally , although this type of range restriction is artificially amplified in our example , SES differences may often be a problem for GWAS in which the sample is not representative of the population; for instance , the most recent major GWAS of educational attainment ( Lee et al . , 2018 ) included numerous medical data sets and the 23andMe data set , which are not representative of the national population .", "Another potentially important factor is that the adjustment for PCs may not be a sufficient control for the different ways in which population structure can confound GWAS results ( Vilhjálmsson and Nordborg , 2013 ) , leading to variable prediction accuracy across strata if they differ in their population structure .", "To examine this possibility , we repeated the analysis in Figure 1 but using a linear mixed model ( LMM ) approach ( including PCs among other covariates; see Materials and methods ) , and obtained qualitatively similar results ( Appendix 1—figure 5 ) .", "Although not a perfect fix ( Listgarten et al . , 2013; Mathieson and McVean , 2013 ) , the fact that we obtain similar results using PCs and LMM suggests that confounding due to population stratification in the UK Biobank alone does not explain the variable prediction accuracies across strata .", "Beyond sample characteristics such as age or sex , a number of other factors may shape the portability of scores across groups of similar ancestry .", "Standard GWAS is done in samples of individuals that deliberately exclude close relatives; as implemented , it detects direct effects of the genetic variants , but also any indirect genetic effects of parents , siblings , or peers , effects of assortative mating among parents , and potentially environmental differences associated with fine-scale population structure ( Young et al . , 2018; Trejo and Benjamin , 2019; Kong et al . , 2018; Lee et al . , 2018; Berg et al . , 2019 ) .", "Given that many of these effects are likely to be culturally mediated ( Stulp et al . , 2017; Selzam et al . , 2019 ) , it seems plausible that they may vary within as well as across groups of individuals with different ancestries .", "If culturally-contingent effects contribute to GWAS estimates ( and hence to PGS ) , they may lead to differences in the prediction accuracy in samples unlike the original GWAS .", "To demonstrate that these considerations are not just hypothetical , we compared the prediction accuracy when the PGS is trained on ‘unrelated’ individuals such as those used in a standard GWAS to one obtained from a sibling-based ( or ‘sib-based’ ) GWAS ( Materials and methods ) .", "In the latter , genotype differences between sibs , a result of random Mendelian segregation in the parents , are tested for association with the phenotypic differences between them .", "Because the tests depend on phenotypic differences between siblings who , of course , have the same parents , these tests are conditioned on the parental genotypes and hence exclude many of the indirect effects signals that may be picked up in standard GWAS ( Appendix 1 ) .", "Differences between standard and sib-based GWAS are thus informative about the presence of factors other than direct genetic effects ( Wood et al . , 2014; Trejo and Benjamin , 2019; Lee et al . , 2018; Berg et al . , 2019; Selzam et al . , 2019 ) .", "A challenge in this comparison is that the UKB contains only ~22K sibling pairs , ~19K of whom are labeled as ‘White British’ ( WB ) .", "The siblings are similar to the unrelated individuals in terms of ages , SES distributions and genetic ancestries ( Appendix 1—figures 6 and 7 ) but include a higher proportion of females; this difference is unlikely to influence our analyses ( see below ) .", "While a large number , 19K pairs is still too few to have adequate power to discover trait-associated SNPs , when compared to a standard GWAS using the much larger sample of unrelated WB individuals ( ~340K ) .", "To increase power and enable a direct comparison between the two designs , we split the SNP ascertainment and effect estimation steps as follows ( Figure 3A ) : we identified SNPs using a standard GWAS with a large sample size ( median ~270K across the traits considered ) ( see Materials and methods ) .", "We then estimated the effect of each significant SNP using", "( i ) a sib-based association test and", "( ii ) a standard association test .", "We chose the size of the estimation set in", "( ii ) such that the median standard error of effect estimates in", "( i ) and", "( ii ) is approximately equal .", "We then compared the prediction accuracy of the two PGS obtained in this way ( ‘standard PGS’ and ‘sib-based PGS’ ) in an independent prediction set of unrelated individuals; as we show in Appendix 1 , our approach leads to highly similar prediction accuracies of the two approaches under a model with direct effects only ( see Materials and methods for details ) .", "A further advantage is that the two scores are compared for the same set of SNPs , such that LD patterns and allele frequency differences do not come into play .", "We applied the approach to 20 traits , focusing on traits with relatively high heritability estimates as well as social and behavioral traits that have been the focus of recent attention in social sciences .", "For the majority of the traits , such as diastolic blood pressure , BMI , and hair color , the prediction accuracies of standard and sib-based PGS were similar ( Figure 3B ) , as expected under standard GWAS assumptions and as observed for traits simulated under these assumptions ( Appendix 1—figure 8 ) .", "However , for height and for a range of social and behavioral traits , such as years of schooling , pack years of smoking and household income , the prediction accuracy of the sib-based PGS was substantially lower than that of the standard PGS ( Figure 3B ) .", "[We caution that , because the first step of our study design is to identify SNPs that are associated with the trait in a large set of unrelated individuals and we subsequently match the sampling variances of sib- and standard GWAS , rather than identify distinct sets of SNPs separately in the two designs , the ratio of prediction accuracies that we obtain cannot be directly compared to those reported in other studies . ] A number of factors could contribute to the differences between prediction accuracies for PGS based on sibs versus unrelated individuals , including confounding effects of population stratification , indirect genetic effects from parents and assortative mating .", "The relative importance of each factor will vary across traits ( Rosenberg et al . , 2019; Kong et al . , 2018; Haworth et al . , 2019; Ruby et al . , 2018; Selzam et al . , 2019 ) .", "For educational attainment , this gap is likely to reflect at least in part the documented contribution of indirect genetic effects to the standard PGS ( Lee et al . , 2018; Kong et al . , 2018; Young et al . , 2018 ) .", "We show in Appendix 1 that in the presence of indirect genetic effects mediated through parents , standard PGS outperforms sib-based PGS unless direct and indirect effects are strongly anticorrelated ( Appendix 1—figure 9 ) , which seems unlikely to be the case for years of schooling .", "The difference in the performance of sib-based and standard PGS observed for other social and behavioral outcomes , such as household income and age at first sexual intercourse ( Figure 3B ) , may reflect a similar phenomenon .", "An additional contribution to divergent prediction accuracies could come from indirect effects among siblings , which would also contribute differentially to standard and sibling-based PGS .", "For height , there may be an important contribution of assortative mating to the difference in prediction accuracies ( Wood et al . , 2014; Robinson et al . , 2017; Lee et al . , 2018 ) .", "In Appendix 1 , we show that under a simple model of positive assortative mating , the prediction accuracy based on a standard PGS is higher than that of a sib-based PGS ( Appendix 1—figure 10 ) .", "We further confirmed that the difference in the sex ratio of the siblings and unrelated individuals , mentioned earlier , has a negligible effect on these differences , though it may underlie the slightly lower prediction accuracy of the standard PGS for pulse rate ( Appendix 1—figure 11 ) .", "The lower prediction accuracies for PGS based on sib-based GWAS indicate that complications such as assortative mating or indirect effects contribute to the standard GWAS estimates .", "In the absence of these complications , we ensure that prediction accuracies are comparable by matching the sampling errors of the two approaches ( Figure 3A ) .", "In the presence of these complications , the magnitude of the ratio of prediction accuracies should reflect the strength of assortative mating , the relative contribution of indirect genetic effects compared to direct effects , and so forth .", "However , interpreting the magnitude of the deviation from 1 is far from straightforward: as we show in Appendix 1 , the relative difference in prediction accuracies between the two approaches stems in part from the noise-to-signal ratio for the effect estimates in sib-based versus standard GWAS ( Appendix 1 , Appendix 1—figures 9 and 10 ) , and as a result also depends on features of the comparison like the sample sizes used and the PGS model .", "Motivated by these considerations , we examined how the prediction accuracy varies when progressively relaxing the GWAS p-value threshold for inclusion of SNPs , that is when including more weakly associated SNPs in the PGS .", "[In Figure 3B , results are shown for the p-value threshold that maximizes the prediction accuracy of the standard PGS , replicating the practice when comparing populations of different ancestry; Martin et al . , 2019 . ] For hair color and diastolic blood pressure , there is little to no difference in prediction accuracy between the two estimation methods , regardless of the number of SNPs included in the score ( Figure 3C , D ) .", "In contrast , for height , standard and sib-based PGS perform similarly when based on the most significantly associated SNPs , but standard PGS progressively outperforms sib-based PGS when more SNPs are included ( Figure 3E ) .", "Similarly , the difference in prediction accuracy between sib-based and standard PGS changes markedly for years of schooling , household income and other social and behavioral traits ( Figure 3F and Appendix 1—figure 12 ) .", "The growing gap in performance with increasing p-value threshold likely reflects a combination of an increasing noise-to-signal ratio for the effect estimates in sib-based versus standard GWAS ( see Appendix 1 ) and changes in the relative importance of direct effects versus other factors such as indirect parental effects and assortative mating .", "In summary , the differences between the prediction accuracies of standard and sib-based PGS seen for a number of traits ( Figure 3B ) , notably social and behavioral ones , demonstrate that standard GWAS estimates often include a substantial contribution of factors other than direct effects .", "In these cases , even if the power to detect direct effects were comparable , standard GWAS would lead to higher prediction accuracy than sib-GWAS .", "In some contexts that may be a sufficient reason to rely on PGS derived from standard GWAS .", "However , that gain stems from the inclusion of factors such as indirect effects and assortative mating that are likely to be modulated by SES , environment and culture ( e . g . , Selzam et al . , 2019; Stulp et al . , 2017 ) .", "Thus , the increased prediction accuracy likely comes at a cost of not always porting well across groups , even of the same ancestry , in ways that may be difficult to anticipate ." ], [ "Although the conversation around the portability of PGS has largely focused on genetic ancestries , our results show that prediction accuracy can also differ , in some cases substantially , across groups of similar ancestry—even due to basic study design differences such as age , sex or SES composition .", "When due only to increased environmental variance , such decreased accuracy may not pose a problem , at least for certain applications .", "But as we have shown , differences in the degree of environmental variance are not the primary explanation for the patterns we report ( Figure 2 ) , and other factors , including differences in the magnitude of genetic effects among groups , indirect effects and assortative mating , also lead to differences in the prediction accuracy of PGS , in ways that may make applications of phenotypic prediction less reliable , even within a single ancestry group .", "For some traits , there is prior information about which factors are likely to be important , but not always , and even for well-studied traits , it may be difficult to enumerate all the influential factors .", "As an example , we considered the accuracy of the polygenic score for years of schooling and found that it also varies somewhat depending on whether individuals have no sibling or one sibling in the prediction sets ( Materials and methods; Appendix 1—figure 13 ) .", "Following the discussion of portability across ancestries , we have focused on incremental R2 as a measure of portability .", "This measure is less directly informative when the goal is to use PGS to reliably identify individuals in the tails of the distribution , that is those at elevated risk of developing a disease—the main application of PGS in human genetics , as distinct from social science or evolutionary biology .", "Nonetheless , the same concerns raised here are likely to apply .", "To illustrate that point , we considered binary outcomes of the traits considered in Figure 1 , 'hypertension' ( defined as diastolic blood pressure > 110 mmHG ) , 'obesity' ( defined as BMI > 35 kg/m2 ) , and 'college completion' , and evaluated the prediction accuracy as measured by incremental AUC ( Appendix 1—figure 14 ) .", "The qualitative results are the same as in Figure 1 .", "We also examined how incremental AUC varies by sex for five binary disease traits that we chose because they have relatively high heritability .", "For three of them , hypothyroidism and two cardiovascular outcomes , prediction accuracy varies depending on both the GWAS and prediction sets ( Appendix 1—figure 15 ) .", "Thus , for both quantitative and binary traits , the question of the domain over which a PGS applies is not just about LD patterns , allele frequencies or GxG effects but also about the extent of environmental and genetic variance , GxE , as well as the contribution of direct effects versus indirect effects , assortative mating and environmental confounding .", "An important implication is that differences in prediction accuracies among groups with distinct ancestries cannot be interpreted exclusively or even primarily in terms of population genetic parameters when these groups differ dramatically in their SES ( Chetty and Hendren , 2018; Conley , 2010; Nuru-Jeter et al . , 2018; Reich , 2017 ) and other factors that may affect portability—especially when the relative contribution of these factors to GWAS signals remains unknown ( Young et al . , 2019; Mills and Rahal , 2019 ) .", "Thus , efforts to conduct GWAS in groups that vary in ancestry and geographic locations will need to be accompanied by a careful examination of variation in portability along other dimensions .", "While these results raise the question of how to best construct a PGS , the answer is not obvious , and likely depends on the specific trait and samples .", "For example , for the three cases shown in Figure 1 , considering a fixed GWAS sample size , the highest prediction accuracy is attained with a GWAS sample limited to some stratum ( e . g . , women for diastolic blood pressure ) .", "Yet a much larger merged data set containing the union of strata generates the most predictive PGS ( Appendix 1—figure 1 ) .", "Together , these observations suggest a trade-off between the factors that are shared among strata and lead to increased power with sample size and those that differ across strata and underlie the variable prediction accuracy .", "In principle then , if influential factors were known , the composition of the GWAS sample could be optimized to yield the highest accuracy in a given prediction set , but how much each stratum should be weighted will depend on a number of factors such as the genetic and environmental variance in each stratum , genetic correlation across strata , and sample sizes .", "Moreover , factors such as assortative mating and indirect effects are soaked up into the GWAS estimates—and critically also into the SNP heritability estimates .", "Thus , the choice of a GWAS sample is about more than power; it is implicitly making a choice about all sorts of sample characteristics that may or may not hold true of the prediction set .", "In that regard , it is worth noting that while classical twin studies were often constituted to be representative of a reference population ( often national in nature ) ( Polderman et al . , 2015; Branigan et al . , 2013 ) , the same is not true of most contemporary human genetic datasets , which are skewed towards medical case-control studies , biobanks that are opt-in ( and thus tend to include individuals who are wealthier and better educated than the population average ) or direct-to-consumer proprietary genetic databases ( which are even more skewed along these dimensions ) ( Lee et al . , 2018 ) .", "For instance , individuals in UK Biobank have higher SES than the rest of the British population ( Fry et al . , 2017 ) and are presumably self-selected for a certain level of interest in biomedical research .", "These factors alone raise challenges as to the broad portability of PGS derived from them .", "More generally , it seems plausible that individuals included in a GWAS differ from those that , for myriad reasons , do not end up participating ( Taylor et al . , 2018 ) , in ways that make it difficult to predict the domain over which GWAS-based estimates can be reliably generalized .", "One fruitful way forward may be to study data from related individuals , in which it should be possible to decompose the components of the signals identified in GWAS into direct and indirect effects , the degree of assortative mating and the contribution of residual stratification ( Zhang et al . , 2015; Young et al . , 2018; Kong et al . , 2018 ) .", "Not only will this decomposition help us to better interpret the results of GWAS and the resulting PGS , it will make it possible to examine under which circumstances , and for which phenotypes , components port more reliably to other sets of individuals , both unrelated and related .", "Ultimately , we envisage that in order to be broadly applicable , GWAS-based phenotypic prediction models will need to include not only a PGS but some study characteristics , other social and environmental measures and , perhaps crucially , their interactions ." ], [ "The UK Biobank ( UKB ) is a large study of about half a million United Kingdom residents , recruited between years 2006 to 2010 ( Bycroft et al . , 2018 ) .", "In addition to genetic data , hundreds of phenotypes were collected through measurements and questionnaires at assessment centers , and by accessing medical records of the participants .", "We focused on 25 traits , including traits with relatively high heritability estimates as well as social and behavioral traits that have been the focus of recent attention in social sciences ( see Appendix 1—table 1 for a complete list of phenotype data used in this work , and their corresponding numeric field codes in the UKB data showcase ) .", "We calculated the phenotype ‘years of schooling’ by converting the maximal educational qualification of the participants to years following Okbay et al . ( 2016 ) ( Appendix 1—table 4 ) .", "For diastolic blood pressure , pulse rate , and forced vital capacity , we took the average of the first two rounds of measurement taken during the same examination at UKB assessment centers .", "We adjusted the diastolic blood pressure levels for blood pressure lowering medication following Evangelou et al . ( 2018 ) by shifting the values upward by 10 mmHg for individuals taking medication .", "For hand grip strength , we took the average of the measurements for the two hands .", "For categorical phenotypes , we assigned integer values to each category ( Appendix 1—table 1 ) .", "For hair color , individuals who reported hair color variable ‘Other’ were excluded from the analyses .", "We considered binary traits , ‘hypertension’ defined as diastolic blood pressure >110 mmHG , ‘obesity’ defined as BMI >35 kg/m2 , and ‘college completion’ defined based on attainment of a college or a university degree .", "Disease outcomes were ascertained using self-reported information and/or using the hospital inpatient main and secondary diagnoses coded according to the International Classification of Diseases ( ICD-9 and ICD-10 ) .", "Hypothyroidism , type 2 diabetes , and rheumatoid arthritis were ascertained based on ICD-10 codes of E03 . X , E11 . X and M06 . X , respectively .", "Myocardial infarction was ascertained based on ICD-9 codes of 410 . 9 , 411 . 9 , 412 . 9 , or ICD-10 codes of I21 . X , I22 . X , I23 . X , I24 . 1 , I25 . 2 following Khera et al . ( 2018 ) , or participants with myocardial infarction outcome data among the UK Biobank’s algorithmically-defined outcomes .", "We also considered the binary outcome of ever being diagnosed to have had a heart attack , angina or stroke .", "For a subset of individuals , multiple measurements of a phenotype were provided , corresponding to multiple visits to UKB assessment centers; in those cases , we used the measurements during the first visit .", "UKB participants were genotyped on either of two similar genotyping arrays , UK Biobank Axiom and UK BiLEVE arrays , at a total of ~850K markers .", "We focused on autosomal bi-allelic SNPs shared between both arrays , and used plink v . 1 . 90b5 ( Chang et al . , 2015 ) to filter SNPs with calling rate >0 . 95 , minor allele frequency >10−3 , and Hardy-Weinberg equilibrium test p-val >10−10 among the WB samples , resulting in 616 , 323 SNPs .", "We calculated SNP heritability across sex , age and SES groups for diastolic blood pressure , BMI and years of schooling , respectively ( as described in the section ‘GWAS by sample characteristics’ ) as well as genetic correlations across pairs of groups: we first performed GWAS using all unrelated WB individuals in each group .", "We then used the GWAS summary statistics to perform LD score regression with LD scores computed from the 1000 Genomes European-ancestry samples ( Bulik-Sullivan et al . , 2015 ) ." ] ]

[ "Fields as diverse as human genetics and sociology are increasingly using polygenic scores based on genome-wide association studies ( GWAS ) for phenotypic prediction .", "However , recent work has shown that polygenic scores have limited portability across groups of different genetic ancestries , restricting the contexts in which they can be used reliably and potentially creating serious inequities in future clinical applications .", "Using the UK Biobank data , we demonstrate that even within a single ancestry group ( i . e . , when there are negligible differences in linkage disequilibrium or in causal alleles frequencies ) , the prediction accuracy of polygenic scores can depend on characteristics such as the socio-economic status , age or sex of the individuals in which the GWAS and the prediction were conducted , as well as on the GWAS design .", "Our findings highlight both the complexities of interpreting polygenic scores and underappreciated obstacles to their broad use ." ]

[ "Complex diseases like cancer and heart disease are caused by the interplay of many factors: the variants of genes we inherit , the lifestyles we lead and the environments we inhabit , plus the interaction of all these factors .", "In fact , almost every trait , even how many years we will spend studying , is influenced both by our environment and our genes .", "To identify some of the genetic factors at play , scientists perform analyses known as genome-wide association studies , or GWAS for short .", "In these studies , the genomes from many different people are scanned to look for genetic differences associated with differences in traits .", "By summing up all the small genetic differences , so-called “polygenic scores” can be calculated .", "When there is a large genetic component to a trait , polygenic scores can be useful predictive tools .", "But there is a catch: polygenic scores make less accurate predictions for individuals of a different ancestry than those involved in the GWAS , which limits the use of these tools around the world .", "Mostafavi , Harpak et al . set out to understand if there are other factors in addition to ancestry that could influence the performance of polygenic scores .", "Using data from the UK Biobank , an international health resource that pairs genomic data and clinical information , Mostafavi , Harpak et al . examined polygenic scores among individuals that share a single , common ancestry .", "These polygenic scores were used to predict three traits ( blood pressure , body mass index and educational attainment ) in individuals and the predictions were then compared to the actual trait values to see how accurate they were .", "The analysis revealed that even within a group of people with similar ancestry , the accuracy of polygenic scores can vary , depending on characteristics such as the sex , age or socioeconomic status of the individuals .", "This analysis emphasises how variable GWAS and their predictive value can be even within seemingly similar population groups .", "It further highlights both the complexities of interpreting polygenic scores and underappreciated obstacles to their broad use in medical and social sciences ." ]

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

[ "neuroscience" ]

[ [ "The function of dendritic spines , sites of synaptic input for neurons in the central nervous system , is linked intimately to their unique geometry .", "For example , the thin spine neck limits diffusion of signaling molecules from the spine head ( Svoboda et al . , 1996; Tonnesen et al . , 2014 ) , a feature critical for synapse specificity during plasticity ( Harvey and Svoboda , 2007; Matsuzaki et al . , 2004; Yuste and Denk , 1995 ) .", "Furthermore , theoretical studies hypothesize that the resistance of the spine neck , if high relative to the impedance of the dendrite , may electrically compartmentalize synaptic potentials in the spine head ( Jack et al . , 1975 ) .", "As a result , the high neck resistance of spines can lead to passive amplification the voltage in the spine head , but also attenuation of signals as they travel across the spine neck ( Araya , 2014; Sala and Segal , 2014; Yuste , 2013 ) .", "Accordingly , long-necked spines may be expected to have higher neck resistances , producing strong attenuation of synaptic potentials .", "However , experimental evidence for this hypothesis has been less conclusive .", "Studies of cortical pyramidal cells show a clear negative correlation between neck length and EPSP amplitude ( Araya et al . , 2006; 2014 ) , while studies in hippocampal pyramidal cells ( Takasaki and Sabatini , 2014 ) and olfactory granule neurons ( Bywalez et al . , 2015 ) observe either a weak relationship or none at all .", "Therefore , better knowledge of the relationship between spine geometry and synaptic function will forward our understanding of how synaptic input shapes neuronal excitability .", "Dopaminergic neurons of the substantia nigra ( SNc ) are commonly categorized as aspiny neurons , however early anatomical studies are less definitive regarding this view .", "Spine-like appendages have been reported on dopamine neurons in a variety of species including humans ( Grace and Onn , 1989; Kline and Felten , 1985; Phelps et al . , 1983; Preston et al . , 1981; Rinvik and Grofova , 1970; Sarti et al . , 2007; Schwyn and Fox , 1974; Yung et al . , 1991; Cruz-Sanchez et al . , 1995; Patt et al . , 1991 ) .", "In other studies , spines were encountered only rarely ( Juraska et al . , 1977; Tepper et al . , 1987 ) .", "While many of these studies provide important descriptions of spine density , quantitative analyses of the density of spines in SNc dopamine neurons have seldom been performed .", "Likewise , the function of spines on dopamine neurons is unclear .", "For example , high densities of vesicular monoamine transporters , dopamine transporters and dopamine D2-autoreceptors have been localized to spines ( Gantz et al . , 2015; Nirenberg et al . , 1996a; 1996b ) , which are associated with the reuptake and storage of dopamine .", "However , these observations raise the question of whether spines on dopamine neurons also function as typical sites of excitatory synaptic input .", "Using two-photon laser scanning microscopy , we examined the density and morphology of dendritic spines on dopamine neurons in live and fixed tissue preparations from juvenile ( P6–P25 ) and adult mice ( up to 7 months old ) .", "During preparation of this study , a separate study was published that reports a mixture of spine and shaft synapses on dopamine neurons and tests glutamate receptors on spines ( Jang et al . , 2015 ) .", "Here , we perform a comprehensive analysis of the functionality , chemical and electrical compartmentalization as well as Ca2+ signaling in short spines ( <2 µm ) , long spines ( 2–5 µm ) and shaft synapses .", "In addition , we tested the influence of subthreshold voltage changes on spine Ca2+ during slow pacemaking , a characteristic firing pattern of dopamine neurons .", "We demonstrate the presence of functional dendritic spines on SNc dopaminergic neurons and provide evidence that activation of spines during pacemaking leads to novel enhancement of spine Ca2+ that occurs periodically in a window starting at the middle phase and lasts throughout remainder of the spike cycle ." ], [ "We analyzed the density and morphology of dendritic spines on dopaminergic neurons in live brain slices obtained from juvenile ( P6–25 ) mice .", "Figure 1 shows a typical example of an Alexa-594-filled dopamine neuron .", "Dendritic spines were clearly visible on all dendrites that were imaged , including on proximal and distal dendrites ( Figure 1A ) .", "However , the density of spines varied widely across the population of dendritic segments from 0 . 5 spines/10 µm up to 4 . 4 spines/10 µm , with the average density on segments at 2 . 08 ± 0 . 10 spines/10 µm ( n = 76; Figure 1B ) .", "Plotting the spine density versus age ( Figure 1B ) , we observed a weak but statistically significant correlation indicating a reduction in density with age ( Pearson’s R = -0 . 23569 , p=0 . 040 , n = 76 dendritic segments ) . 10 . 7554/eLife . 13905 . 003Figure 1 . Dendritic spines on SNc dopamine neurons visualized in live slices .", "( A ) SNc dopamine neuron filled with Alexa-594 via patch pipette , visualized on a two-photon microscope .", "Higher magnification of selected dendritic segments are shown in green , blue and red boxes .", "( B ) Plot of spine density versus age for dendritic segments visualized in live slices .", "( C ) Cumulative histogram showing distribution of spine lengths for P6 – P11 ( green ) and P14 – P25 ( purple ) mice .", "( D ) Distribution of spines ( indicated by vertical lines ) along continuous stretches of dendrite from 20 different cells .", "( E ) Plot of spine density versus distance from the 20 dendrites in previous panel ( gray dashed lines ) and averages with s . e . m . ( white boxes ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 13905 . 003 Analysis of spine morphology showed many of the typical spine shapes ( e . g . stubby and mushroom spines ) similar to those reported on more traditional spiny neurons like pyramidal cells .", "In addition , we observed many spines that exhibited strikingly long necks , characteristic of dendritic filopodia ( Figure 1C ) .", "In young mice ( P6–P11 ) , the average spine length was 1 . 97 ± 0 . 06 µm ( range , 0 . 18–4 . 85 μm ) with 142 of 355 spines ( 40% ) measuring >2 µm in length ( Figure 1C ) .", "In older juveniles ( P14–P25 ) , spines were somewhat shorter at an average length of 1 . 74 ± 0 . 05 µm ( range , 0 . 06–4 . 95 µm ) with 166 of 518 spines ( 32% ) measuring >2 µm .", "Therefore , we observe a significant shift in the density of spines during development away from long spines toward shorter spines , consistent with the notion that longer , filopodial-like spines may be immature structures .", "In separate experiments , we analyzed the density of spines across the entire visible dendritic tree in individual cells ( age P14–P18 ) .", "In the ‘whole cell’ images , the average spine density was 1 . 86 ± 0 . 02 spines/10 µm ( n = 20; Figure 1D , E ) .", "Figure 1D shows the positions of individual spines within a continuous stretch of dendrite for 20 neurons .", "In some cells , spines were evenly distributed throughout the dendrites while in others , we observed stretches of dendrites that were only sparsely populated with spines .", "This fits with the large variability in spine density that we observe between individual dendritic segments ( Figure 1B ) , as well as observations in primates that spines can occur in patches ( Schwyn and Fox , 1974 ) .", "On average , the density of spines decreased modestly with distance from 2 . 33 ± 0 . 30 spines/10 µm ( n = 20 ) in the proximal dendrite ( 0–100 µm ) to 1 . 52 ± 0 . 22 spines/10 µm ( n = 12 ) in the most distal dendrites ( 300–545 µm ) ( p=0 . 037 , student’s unpaired ) .", "This is also consistent with results in guinea pig ( Yung et al . , 1991 ) and humans ( Cruz-Sanchez et al . , 1995 ) that describe a higher density of spines in proximal versus distal dendrites .", "To test the possibility that the dendritic spines identified in live SNc dopamine neurons may be an artifact of the slicing procedure ( Kirov et al . , 1999 ) , we imaged dopamine neurons in both Golgi-stained tissue and fast-perfusion fixed tissue .", "In Golgi-stained tissue ( Figure 2A ) , SNc dopamine neurons exhibited dendritic spines consistent with past experiments analyzing Golgi-stained SNc cells ( Juraska et al . , 1977; Phelps and Adinolfi , 1982; Rinvik and Grofova , 1970; Schwyn and Fox , 1974 ) .", "To visualize dopamine neurons in tissue from fast transcardially-perfused mice , we used transgenic mice in which GFP is driven by the tyrosine hydroxylase promoter and visualized their morphology using juxtacellular labeling with Alexa-594 .", "Spines in fixed tissue were observed along the dendrites of SNc dopamine neurons , though at somewhat lower densities than in live tissue ( Figure 2B ) .", "This raises the possibility that some spine growth in dopamine neurons may result from acute slicing .", "Alternatively , it has been suggested that tissue fixation may limit diffusion of fluorescent dye and visualization of spines ( Kim et al . , 2007 ) .", "Comparing cells from young ( P15–P25 ) and mature ( P75–P119 ) mice ( Figure 2C ) , we found no significant difference in the density of spines ( young , 0 . 90 ± 0 . 15 spines/10 µm , n = 16; mature , 0 . 75 ± 0 . 13 spines/10 µm , n = 13; p=0 . 47 ) .", "Therefore , we demonstrate that dendritic spines are present in live and fixed tissue preparations from both juvenile and adult mice , suggesting that dendritic spines are a common feature of mammalian SNc dopamine neurons . 10 . 7554/eLife . 13905 . 004Figure 2 . Dendritic spines on SNc dopamine neurons visualized in perfusion-fixed slices .", "( A ) Golgi stained sagittal slice from P24 mouse .", "SNc indicated by black outline .", "Red box shows a putative dopamine neuron at higher magnification .", "Blue box shows selected dendritic segment with dendritic spines .", "( B ) SNc dopamine neuron from brain of transcardially-perfused , P75 mouse visualized by juxtacellular labeling with Alexa-594 .", "Red box shows spiny dendritic segment at higher magnification .", "( C ) Bar plot of average SNc dopamine neuron spine densities measured in perfusion-fixed brain slices . DOI: http://dx . doi . org/10 . 7554/eLife . 13905 . 004 We next tested whether dendritic spines on SNc dopamine neurons are sites of glutamatergic synaptic input .", "Using locally-positioned theta-glass electrodes ( diameter , 5–10 µm ) , we tested whether electrical stimulation would generate localized Ca2+ influx into the spine head indicating the presence of active presynaptic inputs ( Figure 3A ) ( Chalifoux and Carter , 2010; Oertner et al . , 2002; Sabatini et al . , 2002 ) .", "Dopamine neurons were filled via patch pipette with Alexa-594 and Fluo5F to visualize cell morphology and intracellular Ca2+ .", "To eliminate spontaneous firing and Ca2+-dependent oscillations ( Nedergaard et al . , 1993; Puopolo et al . , 2007; Wilson and Callaway , 2000 ) , we added QX-314 ( 1 mM ) to the pipette solution and nifedipine ( 10 µM ) to bath solutions to block voltage-gated sodium and calcium channels . 10 . 7554/eLife . 13905 . 005Figure 3 . Dendritic spines on SNc dopamine neurons are sites of glutamatergic synaptic inputs .", "( A ) Dendritic segment visualized with Alexa-594 and Dodt contrast image to visualize the stimulation electrode .", "White dashed line indicates path of linescan .", "( B ) Linescan images of Alexa-594 ( red ) and Fluo5F ( green ) and quantified Ca2+ signals in spine 1 ( green ) , spine 2 ( black ) and the dendrite ( blue ) .", "Simultaneously recorded somatic EPSP shown below .", "( C ) Peak amplitudes and rise times of synaptically-evoked Ca2+ signals into a spine and parent dendrite for all spines tested ( black lines ) .", "Average values and s . e . m . for spine ( green ) and dendrite ( blue ) shown in outer circles .", "( D ) top: Ca2+ influx into the spine head in response to synaptic stimulation in control conditions ( black ) and after wash on of 50 µM D-AP5 ( red ) .", "bottom: Corresponding somatically recorded EPSPs .", "( E ) Peak amplitude of spine Ca2+ signal before and after application of D-AP5 . DOI: http://dx . doi . org/10 . 7554/eLife . 13905 . 005 Figure 3B provides an example of Ca2+ signals recorded in two adjacent spines and the neighboring dendrite in response to electrical stimulation .", "Following stimulation , we observed a large , rapid increase in the Ca2+ signal in spine 1 ( green trace ) , but not in spine 2 ( black trace ) or the nearby dendrite ( blue trace ) .", "Comparing the amplitude and rise times of Ca2+ signals in spines versus dendrites , Ca2+ signals in individual spine heads were larger ( p=3 . 4e-7 ) and faster ( p=6 . 3e-7 , student’s paired t-test; n = 19 ) than in the nearby dendrite ( Figure 3C ) .", "Finally , we tested the effect of NMDA receptor blockade on synaptically-evoked Ca2+ signals .", "Across all spines tested , application D ( - ) -2-amino-5-phosphonopentanoic acid ( D-AP5; 50 µM ) produced a dramatic reduction in Ca2+ influx into the spine ( Figure 3D , E; control: 10 . 1 ± 1 . 9% ∆G/GS; D-AP5: 2 . 0 ± 0 . 6% ∆G/GS; p=0 . 9e-4 , students paired t-test , n = 12 ) , suggesting that NMDA receptors are the main source of Ca2+ entering spines .", "This observation is consistent with the spine as a site of glutamatergic synaptic input innervated by active presynaptic terminals .", "The presence of glutamatergic synapses on both dendritic shafts and spines in dopamine neurons raises the possibility that these two structural classes of synapses could differ in chemical and electrical signaling .", "Chemical compartmentalization by the spine , due to slower diffusion through the narrow spine neck , could result in greater spatial-specificity of cell signaling pathways in the spine head .", "Similarly , spine and shaft synapses could differ in synaptic strength due to attenuation of the EPSP by the resistance of the spine neck .", "Finally , the spine neck could produce local boosting of the EPSP within the spine head leading to activation of voltage-dependent ion channels that would not take place at shaft synapses .", "Differences in the geometry between individual spines are thought to shape chemical and electrical signaling ( Araya et al . , 2006; Bloodgood and Sabatini , 2005; Noguchi et al . , 2005; Takasaki and Sabatini , 2014 ) .", "For example , work in cortical pyramidal neurons has shown a clear inverse correlation between synaptic potential and neck length ( Araya et al . , 2014 ) while similar work in hippocampal pyramidal neurons show only a weak relationship ( Takasaki and Sabatini , 2014 ) .", "Therefore , we tested electrical signaling in single spines of different lengths using glutamate uncaging .", "Much like the experiments examining synaptically-evoked responses , glutamate uncaging resulted in uncaging-evoked EPSPs ( uEPSPs ) , clear Ca2+ influx into the spine head , and a smaller , slowly rising Ca2+ signal in the parent dendrite ( Figure 4— figure supplement 1 ) .", "Figure 4A shows an example of two neighboring spines of different lengths and their uncaging-evoked responses .", "Using the same uncaging power to activate each spine , we found that the two spines display comparable uncaging-evoked Ca2+ signals ( Figure 4B , top ) .", "However , we found clear differences in the amplitudes of uEPSPs .", "Glutamate uncaging generated a uEPSP of 0 . 91 mV in the shorter spine ( 1 . 55 µm ) versus a uEPSP of 0 . 32 mV in the longer spine ( 4 . 15 µm ) .", "In collected experiments , we observed no correlation between the amplitude of the spine Ca2+ signal and spine length ( Figure 4C; Pearson’s R = 0 . 074; p=0 . 54 , n = 71 ) .", "However , comparing uEPSP amplitudes for neighboring spines , uEPSPs from short spines ( <2 µm ) were significantly larger than uEPSPs generated in nearby longer spines ( >2 µm ) ( short: 1 . 46 ± 0 . 16 mV versus long: 0 . 98 ± 0 . 19 mV; p=9 . 8e-3 , paired , n = 14 ) ( Figure 4D ) .", "Furthermore , plotting the amplitude of uEPSPs against spine length for all spines assayed ( Figure 4E ) , we observed a strong correlation between the size of the somatically-recorded uEPSP and spine length ( slope = -0 . 39 mV/µm; Pearson’s R = -0 . 50; p=1 . 5e-5 , n = 71 ) .", "In agreement with Araya et al . ( 2006 ) , our data demonstrate that synapses formed onto long spines result in smaller amplitude uEPSPs , and likely exert a weaker influence on the membrane potential in SNc dopamine neurons . 10 . 7554/eLife . 13905 . 006Figure 4 . Comparison of uEPSPs for short and long spines .", "( A ) Dendritic segment with neighboring spines of different lengths visualized by Alexa-594 ( bottom spine = 4 . 2 µm , top spine = 1 . 6 µm ) .", "Sites of glutamate uncaging are indicated by blue and orange circles .", "( B ) Glutamate uncaging-evoked responses of spines in A . Blue traces correspond to the long spine , orange traces correspond to the short spine .", "Top: glutamate-evoked spine Ca2+ signals .", "Bottom: uEPSPs .", "( C ) Amplitude of spine Ca2+ signal plotted against spine length for all spines tested ( dots ) and linear regression .", "( D ) uEPSP amplitudes of neighboring short ( <2 µm ) and long ( >2 µm ) spines .", "Circles represent mean and s . e . m . ( E ) Amplitudes of uncaging-evoked EPSPs plotted against spine length for all spines tested ( dots ) and linear regression . DOI: http://dx . doi . org/10 . 7554/eLife . 13905 . 00610 . 7554/eLife . 13905 . 007Figure 4—figure supplement 1 . Glutamate uncaging-evoked responses of dendritic spines .", "( A ) Dendritic segment visualized by Alexa-594 .", "Site of glutamate uncaging indicated by yellow circle .", "( B ) Glutamate uncaging-evoked responses .", "Green trace: spine Ca2+ signal; blue trace: dendrite Ca2+ signal; red trace: somatic EPSP .", "( C ) Peak amplitudes of uncaging-evoked Ca2+ signals into a spine and parent dendrite for all spines tested ( black lines ) .", "Average values and s . e . m . for spine ( green ) and dendrite ( blue ) shown in outer circles .", "Amplitudes were significantly larger in the spines than in nearby dendrites ( p=4 . 7e-15 ) .", "( D ) Rise times of uncaging-evoked Ca2+ signals into a spine and parent dendrite for all spines tested ( black lines ) .", "Average values and s . e . m . for spine ( green ) and dendrite ( blue ) shown in outer circles .", "Rise times were significantly faster in the spines than in nearby dendrites ( p=3 . 4e-10 ) DOI: http://dx . doi . org/10 . 7554/eLife . 13905 . 007 Because spines can compartmentalize chemical signals due to their thin spine neck which acts as diffusion barrier , we next analyzed the influence of spine length on chemical compartmentalization .", "We measured the rate of fluorescence recovery after photobleaching ( FRAP ) in spines of varying lengths ( 0 . 88–4 . 54 µm ) .", "As seen in Figure 5A and B , fluorescence intensity of Alexa-594 in the spine head was measured by linescans across the spine , while a second two-photon laser was used to partially bleach the dye within the spine ( 0 . 5 ms pulse , 725 nm ) .", "Traces were corrected for a small amount of photobleaching ( ~3 . 5% ) which occurred as a result of scanning by the imaging laser alone ( see Materials and methods ) .", "We then fit the recovery of spine fluorescence intensity with a double-exponential function ( Figure 5B , purple line , weighted tau = 66 . 5 ms ) and plotted the weighted time constant of FRAP against spine length for 33 spines ( Figure 5C ) .", "We observed a significant correlation in which the time course of FRAP slowed with increased spine lengths ( slope = 46 . 1 ms/µm; Pearson’s R = 0 . 52; p=0 . 0020 ) .", "The range of FRAP time courses observed ( 8 . 1–299 ms , mean = 115 ms , median = 103 ms ) were similar to analogous experiments conducted in hippocampal pyramidal neurons ( Grunditz et al . , 2008; Takasaki and Sabatini , 2014; Tonnesen et al . , 2014 ) .", "These FRAP experiments demonstrate that spine length significantly influences the compartmentalization of chemical signals in SNc dopamine neurons . 10 . 7554/eLife . 13905 . 008Figure 5 . Spine length correlates with chemical compartmentalization .", "( A ) Frame scan of assayed spine , path of linescan ( red line ) , and site of photobleaching ( yellow ) .", "( B ) Spine fluorescence intensity following selective photobleaching of spine head .", "Timing of photobleaching pulse indicated by vertical yellow line .", "Subsequent recovery of fluorescence was fit with a double-exponential function ( purple ) .", "( C ) Time constant of FRAP plotted against spine length with linear regression ( purple line ) .", "( D ) Time constant of FRAP plotted against spine head volume with linear regression ( purple line ) .", "( E ) uEPSP evoked from spine shown in A . ( F ) uEPSP amplitude plotted against estimated spine neck resistance with linear regression for all data ( solid red line ) .", "Linear regression for all data except the 2 spines with the highest estimated neck resistance indicated by dashed line . DOI: http://dx . doi . org/10 . 7554/eLife . 13905 . 008 Previous work has shown that the dimensions of the spine head can influence the time course of the FRAP measurements ( Svoboda et al . , 1996; Tonnesen et al . , 2014 ) .", "Therefore , we estimated the volume of the spine head based on the full width at half maximum ( FWHM ) of Alexa-594 signal intensity across the spine head .", "Assuming that the spine head is a sphere with the FWHM as the diameter , we estimated that spine head volumes cover a range from 0 . 016 to 0 . 64 μm3 .", "We found no correlation between the FRAP time constants and spine head volume ( Pearson’s R = 0 . 193 , p=0 . 28 ) .", "We next used estimates of the spine volume and FRAP measurements to calculate the resistance of the spine neck with Fick’s law: Rneck=tauFRAPDAlexaRaxial/Vhead .", "In this equation , Rneck is the neck resistance , tauFRAP is the time constant from FRAP measurements , DAlexa is the free diffusion time constant for Alexa-594 ( 120 μm2/s ) , Raxial is the axial resistance ( 150 Ω*cm ) , and Vhead is the volume of the spine head .", "Using these values , the estimated spine neck resistance ranged from 8 . 4 MΩ–1 . 3 GΩ , with a median of 72 MΩ .", "These estimates assume a spherical spine head as well as an intermediate value of cytoplasmic Raxial of 150 Ω*cm , while values can range from 50–250 Ω*cm .", "Despite the assumptions , however , our neck resistance values fall roughly in line with estimates from studies in other cell types ( Tonnesen et al . , 2014 ) .", "Given the estimates of Rneck that we obtained using the spine morphology and FRAP measurements , we compared these measurements with the size of evoked synaptic potentials .", "Therefore , we uncaged glutamate onto the same spines from which FRAP analysis was performed and measured the uEPSPs ( Figure 5E ) .", "Plotting the uEPSP amplitude against the Rneck , we found a moderate inverse correlation between uEPSP amplitude and Rneck ( Pearson’s R = -0 . 503 , p=0 . 0046 ) ( Figure 5F ) .", "Intriguingly , the 2 spines which produced the smallest amplitude uEPSPs also had estimated neck resistances that were dramatically greater than the other spines assayed .", "A significant correlation is still observed if these data points are excluded from analysis ( Figure 5F dashed line; R = -0 . 488 , p=0 . 0085 ) .", "In summary , these findings are consistent with the idea that the higher resistance of longer spines may result in stronger attenuation of the EPSP across the spine neck ( Araya et al . , 2006; 2014 ) .", "In addition to spine geometry , synaptic receptor composition such as the density of spine AMPA receptors can be an effective determinant of synaptic strength ( Matsuzaki et al . , 2001 ) .", "Comparisons of uEPSPs kinetics in long and short spines indirectly support this idea .", "We found significantly faster rise times in short spines than in longer spines ( 10–90% uEPSP rise time; short <2 µm spines: 42 . 8 ± 6 . 5 ms , long ≥ 2 µm spines: 71 . 9 ± 10 . 4 ms; n = 45 and 26; p=0 . 023 student’s unpaired t-test ) .", "Therefore , we hypothesized that the variability in the uEPSP amplitudes may result from distinct compositions of synaptic glutamate receptors in the short and long spines .", "To test this , we recorded glutamate uncaging-evoked AMPA or NMDA receptor-mediated currents from short and long-necked spines by voltage clamping neurons to either −70 mV or +40 mV ( Figure 6A , B ) .", "To maximize space clamp , we analyzed spines located within 50 µm of the soma and recorded using Cs+ -based internal solutions and bath applied TTX ( 500 nM ) .", "NMDAR-mediated currents were quantified 100 ms following onset of the uncaging pulse when AMPA receptors were likely desensitized . 10 . 7554/eLife . 13905 . 009Figure 6 . Comparison of AMPA/NMDA ratio for short and long spines .", "( A ) Uncaging-evoked currents recorded at -70 mV ( red ) and +40 mV ( black ) from targeting a short spine ( 0 . 9 µm ) .", "( B ) Uncaging-evoked currents as in A from targeting a nearby long spine ( 3 . 4 µm ) .", "( C ) Plot of AMPAR ( red ) and NMDAR ( black ) – mediated current amplitudes versus spine length for all spines tested and corresponding linear regressions .", "( D ) Plot of AMPA/NMDA ratio versus spine length for all spines tested and linear regression .", "Circles indicate mean and s . e . m . for short and long spines .", "( E ) Plot of AMPAR charge transfer versus spine length for all spines test and linear regression .", "( F ) Uncaging-evoked currents as in A in the presence of CTZ and D-AP5 while targeting a short spine ( 1 . 4 µm ) .", "( G ) Uncaging-evoked currents as in F from targeting a nearby long spine ( 4 . 2 µm ) .", "( H ) Plot of AMPAR– mediated current amplitudes measured at −70 mV and +40 mV in the presence of CTZ and corresponding linear regressions .", "( I ) Plot of AMPAR charge transfer versus spine length and linear regression .", "( J ) Plot of the ratio of AMPAR current amplitudes measured at -70 and +40 mV versus spine length and linear regression . DOI: http://dx . doi . org/10 . 7554/eLife . 13905 . 009 The uncaging-evoked AMPAR-mediated current measured at −70 mV was dramatically larger in short spines versus longer spines ( slope = 4 . 24 pA/µm; Pearson’s R = -0 . 60 , p=2 . 9e-6 , n = 52 spines ) ( Figure 6C ) .", "Holding at +40 mV to measure NMDAR-mediated currents , again we found a significant , yet somewhat weaker negative relationship between the uncaging-evoked NMDAR current and spine length ( slope = -2 . 3 pA/µm; Pearson’s R = -0 . 49; p=2 . 3e-4 , n = 52 spines ) ( Figure 6C ) .", "Lastly , we found that the AMPA to NMDA ratio was significantly lower in long spines ( >2 µm ) as compared to short spines ( spine length <2 µm , AMPA/NMDA ratio = 1 . 54 ± 0 . 17; spine length >2 µm , AMPA/NMDA ratio = 0 . 71 ± 0 . 17; p=0 . 0017 , n = 52 spines ) ( Figure 6D ) .", "In addition to spine neck resistance , these findings suggest that the negative correlation between uEPSP amplitude and spine length ( Figure 4E ) also involves a differential glutamate receptor expression and composition , with a weaker AMPA-receptor component present in long-necked spines .", "The spine neck imparts an electrical resistance , and therefore may attenuate synaptic current entering the dendrite .", "The extent of this attenuation will depend on the relative values of the synaptic conductance and spine neck resistance .", "Faster currents ( such as those mediated by AMPARs ) will likely display greater attenuation than slower NMDAR-mediated currents ( Johnston and Wu , 1995 ) .", "Therefore , it is possible that the observed difference in AMPA to NMDA ratio is due to greater filtering of AMPAR currents by the spine neck resistance of long spines .", "Measurements of synaptic charge transfer are less distorted by the cable properties of the neuron and spine neck than are measurements of current amplitude ( Johnston and Wu , 1995 ) .", "Therefore , we examined the relationship between the AMPAR-mediated charge transfer ( measured as the integral of the uncaging-evoked current at −70 mV ) and the length of the spines ( Figure 6E ) .", "We observed a moderate and highly significant correlation between these values ( R = −0 . 56 , p=1 . 8e-5 , n = 52 spines ) , further supporting the hypothesis that long spines are of weaker strength due in part to a smaller contribution of AMPARs .", "To further disambiguate whether the relationship between AMPAR current amplitude and spine length was due to differences in receptor expression or differences in spine neck resistance , we examined the relationship between isolated AMPAR-currents ( NMDARs were blocked with 50 µM D-AP5 ) and spine length in the presence of cyclothiazide ( CTZ , 100 µM ) to inhibit AMPAR desensitization .", "CTZ increased the maximum synaptic conductance and slowed the time course of the AMPAR-mediated conductance changes .", "Both effects will minimize the impact of the spine neck resistance on measurement of the synaptic current .", "Examples of uncaging-evoked AMPAR currents at positive and negative potentials in the presence of CTZ are shown in Figure 6F , G .", "We observed a significant correlation between AMPAR current amplitude and spine length in the presence of CTZ at both positive and negative voltages ( at -70 mV R = 0 . 92 , p=2 . 5e-5; at +40 mV R = 0 . 95 , p<1e-5; n = 6 spines , 3 shafts ) ( Figure 6H ) .", "We also observed a significant correlation between AMPAR-mediated charge transfer and spine length in the presence of CTZ ( R = 0 . 66 , p=0 . 02 , n = 6 spines , 3 shafts ) ( Figure 6I ) further supporting the hypothesis that long spines display lower AMPAR density .", "Finally , we confirmed that the spine neck acts as an ohmic resistor .", "If the spine neck was non-ohmic ( i . e . voltage-dependent ) it could differentially affect measurement of the AMPA/NMDA ratio in spines with different neck resistances .", "Measuring isolated AMPAR currents at different voltages allowed us to test the ohmic nature of the spine neck resistance because changes in synaptic conductance will be due the presence of the ligand alone .", "To measure the synaptic conductance in the absence of any spine neck resistance , we uncaged glutamate directly near the dendritic shaft .", "When holding the cell at −70 mV and targeting the dendritic shaft , uncaging-evoked synaptic currents were 1 . 60 ± 0 . 05 times larger than the current measured at +40 mV ( Figure 6J ) .", "If the spine neck resistance changed with membrane potential , one would predict that the ratio between AMPAR currents measured at −70 mV and +40 mV would differ according to spine neck resistance .", "However , when uncaging onto spines the ratio of AMPAR current amplitudes measured at −70 and +40 mV was 1 . 56 ± 0 . 05 ( Figure 6J ) - similar to trials targeting dendritic shafts .", "Furthermore , we observe no correlation between spine length and the ratio of AMPAR currents measured at −70 and +40 mV ( R = -0 . 010 , p=0 . 97 , n = 3 shafts , 6 spines ) ( Figure 6J ) .", "Therefore , it appears that the resistance of the spine neck does not change with membrane potential under our experimental conditions .", "Glutamatergic excitation of SNc dopamine neurons has long been recognized to occur via input to synapses located on the dendritic shaft ( Chatha et al . , 2000; Henny et al . , 2012; Paquet et al . , 1997; Rinvik and Grofova , 1970 ) .", "However , it remains unclear how spine and shaft synapses compare in both density and function .", "The difficulty in identifying shaft synapses in live slices has limited functional investigation of shaft synapses .", "To address this limitation , we utilized a recently developed transgenic mouse in which postsynaptic protein PSD-95 is tagged with mVenus ( ENABLED ) to allow unambiguous identification of shaft synapses ( Fortin et al . , 2014 ) .", "Crossing the ENABLED mouse with a dopamine transporter driven Cre mouse line ( DAT-Cre ) , resulted in dopamine neuron-specific expression of the postsynaptic protein PSD-95 tagged with mVenus .", "Figure 7A shows an example dendritic segment filled with Alexa-594 , in which postsynaptic densities are identified as mVenus-positive puncta .", "We compared activation of spine synapses with puncta-labeled synapses on the neighboring dendritic shaft ( Figure 7B ) .", "Uncaging onto shaft synapses resulted in uEPSPs that were 100% larger than uEPSPs from nearby spine synapses ( shaft: 1 . 16 ± 0 . 11 mV; spine: 0 . 58 ± 0 . 07 mV; n = 24 , p=2 . 7e-5 , student’s paired t-test ) ( Figure 7C , D ) .", "By contrast , uncaging onto mVenus-negative dendritic regions resulted in uEPSPs that were minimal in amplitude ( puncta: 1 . 40 ± 0 . 15 mV , no puncta: 0 . 14 ± 0 . 05 mV; n = 6 , p=4 . 9e-4 , student’s paired t-test ) .", "These results confirm that mVenus puncta clearly identify shaft synapses . 10 . 7554/eLife . 13905 . 010Figure 7 . Comparison of spine and shaft synapses .", "( A ) Maximum intensity projections of two photon images from a recorded neuron in DAT-Cre x PSD-95-ENABLED mouse . Left: Alexa-594-filled dendrite .", "Right: PSD-95mVenus puncta .", "Red boxes indicate region shown in B . ( B ) Overlay of a single slice of the z-stack from images in A , asterisks indicate sites of glutamate uncaging targeting a spine ( orange ) and a shaft synapse ( blue ) .", "( C ) uEPSPs evoked in response to targeting of a shaft puncta ( blue ) and spine ( orange ) located on the same dendrite .", "( D ) Comparison of uEPSP amplitudes when targeting shaft synapses or nearby spines .", "Circles represent averages and s . e . m . ( E ) Plot of uEPSP amplitude versus mVenus pixel intensity for shafts ( blue ) and spines ( orange ) .", "( F ) Plot of uEPSP amplitude versus spine length with shaft uncaging sites indicated by grey box .", "Green circles indicate that mVenus pixel intensity was >2 standard deviations above background green pixel intensity .", "Magenta circles indicate that the spine or shaft did not display significant mVenus signal .", "( G ) Plot of mVenus pixel intensity and the fraction of mVenus-positive spines against spine length .", "Grey box indicates average background mVenus signal ± 2 standard deviations .", "Green and magenta circles indicate spines with or without significant mVenus signal . DOI: http://dx . doi . org/10 . 7554/eLife . 13905 . 01010 . 7554/eLife . 13905 . 011Figure 7—figure supplement 1 . Plot of the measured spine density versus the measured shaft synapse density for individual dendritic segments ( filled circles ) and average values ( empty circle ) .", "Dashed line indicates equal densities . DOI: http://dx . doi . org/10 . 7554/eLife . 13905 . 011 The presence of PSD-95 has been shown to stabilize AMPARs within the synapse ( Béïque et al . , 2006; El-HusseinI et al . , 2000; Taft and Turrigiano , 2014 ) , which is expected to correlate with stronger postsynaptic potentials .", "We next compared the pixel intensity of mVenus at the position of uncaging with uEPSP amplitudes ( Figure 7E ) .", "We observed a significant correlation between uEPSP amplitude and mVenus pixel intensity ( Pearson’s R = 0 . 57 , n = 67 , p=4 . 8e-7 ) .", "Regions of the dendrites , including the dendritic shaft and spines , were considered mVenus-positive when the green pixel intensity exceeded 2 standard deviations of the average green background intensity measured in the dendrite shaft .", "It is possible that small mVenus puncta could escape our detection , biasing our results to synapses with large postsynaptic densities .", "Using this criteria , we compared spines of similar lengths ( <2 μm ) and found that uEPSPs were substantially larger for PSD-95 positive spines than for PSD-95 negative spines ( Figure 7F ) ( PSD-95 positive uEPSP = 0 . 85 ± 0 . 13 mV , n = 12; PSD-95 negative uEPSP=0 . 42 ± 0 . 12 mV , n = 5; p=0 . 030 ) .", "These results show that in both shaft and spine synapses , synaptic strength correlates with PSD-95 expression .", "Given our observations that synaptic strength is correlated with both spine length and PSD-95 expression , we next asked whether PSD-95 expression differs in spines of various lengths .", "We measured mVenus pixel intensity in 327 spines from 11 dopamine neurons and plotted mVenus pixel intensity against spine length ( Figure 7G ) .", "We observed a weak , but significant correlation between mVenus expression and spine length ( R = −0 . 23 , p=2 . 5e-5 ) .", "We again categorized spines as mVenus positive if pixel intensity was more than 2 standard deviations greater than the background signal .", "75% of spines with lengths <2 µm ( 179 of 240 ) were scored mVenus positive whereas only 40% of spines longer than 2 µm ( 33 of 83 ) displayed detectable mVenus expression .", "This observation is consistent with the conclusion from our voltage-clamp analyses , that longer spines display smaller AMPAR currents ( Figure 6 ) , likely contributing to the smaller uEPSPs observed in current clamp ( Figure 4 ) .", "In total , our data support the hypothesis that the long spines are immature synaptic structures that have few AMPARs .", "Lastly , we compared the relative densities of spines and shaft synapses on individual dendritic segments ( Figure 7—figure supplement 1 ) .", "In most cases , mVenus-positive shaft synapses outnumbered spines ( average density shaft synapses: 2 . 53 ± 0 . 14 puncta/10 µm; average density spine synapses: 2 . 01 ± 0 . 19 spines/10 µm ) .", "However , there were several instances ( 9/23 dendritic segments ) in which shaft synapses and spines were observed in equal numbers , or spines outnumbered shaft synapses ( data were measured from 23 dendritic segments consisting of total of 1525 µm of dendrite , 317 spines and 392 shaft synapses ) .", "Altogether , these data reveal that excitatory synaptic inputs of both structural classes , spines and shaft synapses , coexist and must act together in the integration of synaptic inputs on SNc dopamine neurons .", "Dopamine neurons are spontaneously active and therefore , synaptic inputs will be received on an ever changing membrane potential ( Hage and Khaliq , 2015; Puopolo et al . , 2007 ) .", "Therefore , we first examined how steady-state changes in subthreshold potential ( −75 to −45 mV ) influence glutamate-evoked signals from spines as compared to shaft synapses ( Figure 8 ) .", "Recordings were made in bath applied TTX ( 500 nM ) and nifedipine ( 10 µM ) to limit spontaneous membrane oscillations at depolarized potentials .", "In Purkinje neurons , T-type Ca2+ channels display greater expression within the spines than the dendrites ( Isope et al . , 2012 ) .", "While the sub-cellular distribution T-type channels has not been examined in dopamine neurons , differential expression in the spines and dendrites could influence the relationship between glutamate-evoked Ca2+ influx and membrane potential .", "Therefore , in a subset of experiments , T-type Ca2+ channels were blocked with TTA-P2 ( 1 µM ) .", "Results in the two conditions were similar , and therefore data was pooled ( 16 spines and 16 shaft synapses recorded with TTA-P2 , 15 spines and 10 shaft synapses recorded without TTA-P2 ) .", "Because of the large spine-to-spine variability in absolute Ca2+ signals observed ( Figure 4C ) , we normalized the amplitude of glutamate-evoked Ca2+ signals to those recorded at −45 mV within the same spine or shaft .", "This normalization will also account for possible differences in receptor expression between shafts and spines . 10 . 7554/eLife . 13905 . 012Figure 8 . Voltage-dependence of shaft and spine synaptic responses .", "( A ) Examples of glutamate-evoked Ca2+ signals and uEPSPs evoked from 4 different stable membrane potentials for a single shaft synapse .", "( B ) Examples of glutamate-evoked Ca2+ signals and uEPSPs evoked from 4 different stable membrane potentials for a single spine .", "( C ) Plot of normalized Ca2+ signal amplitudes against starting membrane potential for spines ( green dots and lines ) and shaft synapses ( purple dots and lines ) .", "( D ) Plot of normalized uEPSP amplitudes against starting membrane potential for spines ( green dots and lines ) and shaft synapses ( purple dots and lines ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 13905 . 012 In both spines and shaft synapses , glutamate-evoked Ca2+ signals increased in amplitude with depolarization , likely due to Mg2+ unblock of NMDA receptors ( Figure 8A , B ) .", "Interestingly at hyperpolarized potentials ( −60 to −65 mV ) , the normalized Ca2+ signals in spines were significantly larger in amplitude than analogous shaft Ca2+ signals ( normalized Ca2+ signals in spines vs shafts: 0 . 61 ± 0 . 03 vs 0 . 50 ± 0 . 03; spine , n = 52 measurements , 31 spines; shafts , n = 46 measurements , 21 shafts; p=3 . 3e-3 ) ( Figure 8C ) .", "Therefore , spine Ca2+ signals were less sensitive to hyperpolarization than shafts signals .", "We also measured the impact of steady-state voltage changes on the amplitudes of uEPSPs for shaft versus spine synapses ( Figure 8D ) .", "Here , we normalized the amplitudes of all uEPSPs to the amplitude measured at −75 mV , where the driving force of the synaptic current is largest .", "Between −55 to −50 mV , the average normalized amplitude of shaft uEPSPs was 0 . 71 ± 0 . 03 ( n = 49 measurements from 26 shafts ) .", "By contrast , the average normalized amplitude of spine uEPSPs was 1 . 06 ± 0 . 11 ( significantly larger than normalized shaft uEPSPs , p=0 . 004 , n = 57 measurements from 31 spines ) , indicating that there was strikingly little effect of depolarization on the amplitude of spine uEPSPs .", "This suggests that at hyperpolarized membrane potentials , NMDA receptors are more effectively recruited in spines relative to shaft synapses which we reason may occur through boosting of the voltage in the spine head .", "Firing in dopamine neurons is shaped by an interaction between synaptic inputs and active subthreshold conductances that drive pacemaking .", "To better understand the functional contribution of spiny synapses to dopamine neuron excitability , we imaged glutamate-evoked Ca2+ responses in spines during slow tonic firing .", "Figure 9A shows AP-evoked Ca2+ signals measured during tonic firing for a spine and nearby dendrite .", "Figure 9B shows glutamate uncaging-evoked Ca2+ signals and the uEPSP measured while injecting constant negative current to hold the membrane potential near −63 mV .", "We then uncaged glutamate on a background of tonic firing .", "We found that when glutamate uncaging shortly preceded an action potential , Ca2+ influx into the spine was dramatically larger than the linear sum of the isolated AP- and glutamate-evoked signals ( Figure 9C ) .", "In contrast , when glutamate uncaging occurred shortly after an AP ( Figure 9D left ) , spine Ca2+ signals were smaller and resembled the predicted linear sum .", "We measured glutamate-evoked Ca2+ signals throughout the entire pacemaker cycle and found that there was a dramatic enhancement of the spine Ca2+ signal midway through the firing cycle , much earlier than we had expected ( Figure 9D right ) .", "Notably , the Ca2+ signal was enhanced before the onset of the following AP ( open triangle in Figure 9D ) , suggesting small changes in subthreshold voltage during tonic firing can dramatically influence synaptic Ca2+ influx . 10 . 7554/eLife . 13905 . 013Figure 9 . Phase-dependent enhancement of glutamate-evoked Ca2+ signals during tonic firing .", "( A ) AP-evoked Ca2+ signals measured in a spine ( green ) and nearby dendrite ( blue ) during tonic firing .", "Signals are averages of 3 individual AP-evoked Ca2+ transients .", "( B ) Ca2+ signals as in A , for glutamate uncaging near the spine head while holding at −63 mV .", "Signals are averages of 6 uncaging trials .", "( C ) Spine and dendrite Ca2+ signals evoked by uncaging glutamate during tonic firing .", "Dotted lines represent the ‘linear sum’ , defined as ‘AP-alone’ plus ‘uncage- alone’ signals from panels A and B . ( D ) Comparison of spine Ca2+ signals and corresponding linear sums for trials in which glutamate uncaging occurred at either an early phase ( left ) or intermediate phase ( right ) of the firing cycle .", "Data are from the same spine as panels A–C .", "Dashed lines indicate baseline signals before the uncaging pulse .", "In the early phase example , the Ca2+ signal displayed an AP-evoked increase before the uncaging pulse that was not measured as part of the glutamate-evoked response ( arrow ) .", "Inverted triangles in the mid-phase example indicate the peaks of the glutamate-evoked Ca2+ signal measured before ( open symbol ) and after the subsequent AP ( closed symbol ) .", "( E ) Top: Normalized spine Ca2+ amplitude plotted against the phase at which glutamate uncaging occurred for measured data ( green ) and linear sums ( black ) .", "Bottom: Plot of membrane potential immediately prior to glutamate uncaging versus phase .", "The black trace shows a typical interspike interval .", "( F ) As in E , except glutamate-evoked Ca2+ signals were only measured before the onset of the subsequent AP . DOI: http://dx . doi . org/10 . 7554/eLife . 13905 . 013 To enable comparison of Ca2+ signals across multiple spines , we normalized all uncaging-evoked responses to those recorded from a steady holding potential of -63 mV ( Figure 9E ) .", "Performing this analysis on the linear summations of the isolated AP- and uncaging-evoked Ca2+ signals predicts a small phase-dependence of spine Ca2+ influx due to additional AP-evoked Ca2+ in late phases of the firing cycle ( Figure 9E ) .", "Values slightly <1 are observed early in the firing cycle due to the decay of the AP-evoked Ca2+ transient .", "We observe a much more dramatic influence of phase on glutamate-evoked spine Ca2+ signals within our measured data ( Figure 9E ) .", "When glutamate uncaging occurred in the last third of the spike cycle , spine Ca2+ influx was 1 . 90 ± 0 . 07 fold larger than Ca2+ influx produced by uncaging alone .", "By contrast , linear summation of the AP-evoked and uncaging evoked signals during this same range of the spike cycle predicts signals just 1 . 28 ± 0 . 04 fold larger than uncaging alone ( measured data was significantly greater than linear sums , p=1 . 4e-9 , n = 58 measurements from 20 spines ) .", "Action potentials immediately following synaptic activation are known to enhance Ca2+ signals ( Nevian and Sakmann , 2004 ) .", "However , we wanted to determine if the subthreshold voltage alone was sufficient to enhance glutamate-evoked Ca2+ influx .", "To isolate the effect of subthreshold voltage on spine Ca2+ signals , we measured the peak Ca2+ signals before the onset of spikes and excluded Ca2+ signals if spikes occurred <100 ms after the uncaging pulse ( Figure 9F ) .", "Using this alternative measure , we observed dramatic enhancement of spine Ca2+ influx in the middle and late phases of the spike cycle ( middle phase: measured Ca2+ = 1 . 67 ± 0 . 07 , linear sum = 0 . 95 ± 0 . 03 , p=5 . 5e-12 , n = 41 measurements from 20 spines; late phase: measured Ca2+ = 1 . 64 ± 0 . 07 , linear sum = 0 . 85 ± 0 . 04 , p=2 . 7e-13 , n = 36 measurements from 20 spines ) .", "Therefore , we find that glutamate-evoked spine Ca2+ influx in dopamine neurons is enhanced in a time window that occurs periodically during the natural pacemaker cycle .", "Finally , if the spine neck resistance results in amplification of EPSPs within the spine head ( Figure 8 ) , then voltage-gated channels may be more likely to be activated and shape the synaptic response .", "To determine whether voltage-gated sodiumchannels contribute to synaptic responses of spines , we measured the effect of TTX ( 500 nM ) on glutamate-evoked spine Ca2+ signals from a hyperpolarized membrane potential ( -63 mV ) .", "In the example spine show in Figure 10A , application of TTX reduced the amplitude of the spine Ca2+ signal from 30% ∆G/GS to 17% ∆G/GS .", "On average , TTX reduced the spine Ca2+ signal by 25 ± 10 . 0% ( p=0 . 039 , student’s paired ) ( Figure 10B ) but had little effect on dendritic Ca2+ signals ( reduced by 9 ± 10% , p=0 . 45 ) ( Figure 10C ) .", "Therefore , in addition to the known involvement of sodium channels in enabling robust backpropagation of action potentials in the dendrites of dopamine neurons ( Gentet and Williams , 2007; Hausser et al . , 1995 ) , we find a likely role of sodium channels in boosting synaptic potentials in spines of substantia nigra dopamine neurons . 10 . 7554/eLife . 13905 . 014Figure 10 . Voltage-gated sodium channels and NMDAR shape spine Ca2+ signals .", "( A ) Example of uncaging evoked Ca2+ signals in control conditions and following application of TTX ( 500 nM ) .", "( B ) Plot of the effect of TTX on spine Ca2+ signals .", "( C ) Plot of the effect of TTX on dendrite Ca2+ signals .", "( D ) Example of uncaging evoked Ca2+ signals in control conditions and following application of D-AP5 ( 50 µM ) .", "( E ) Plot of the effect of D-AP5 on spine Ca2+ signals .", "( F ) Plot of the effect of D-AP5 on dendrite Ca2+ signals . DOI: http://dx . doi . org/10 . 7554/eLife . 13905 . 014 In separate experiments , we tested the effect of blockade of NMDA receptors with D-AP5 ( 50 µM ) .", "In the example shown in Figure 10D , D-AP5 reduced the glutamate-evoked spine Ca2+ signal from 12% ∆G/GS to 6% ∆G/GS .", "On average , blockade of NMDA receptors reduced spine Ca2+ influx by 49 ± 6% ( p=2 . 2e-3 ) ( Figure 10E ) .", "The effect of D-AP5 on the dendrite Ca2+ signal was smaller , on average reduced by 36 ± 10% ( p=0 . 02 ) ( Figure 10F ) .", "The finding that TTX and D-AP5 produced greater attenuation of the spine Ca2+ signal than the dendritic Ca2+ signal suggests that there is greater activation of NMDA receptors and voltage-dependent channels in the spine head than the dendrite in response to glutamate uncaging .", "The contribution of voltage-gated channels to glutamate-evoked responses provides a mechanism by which subtle changes in the subthreshold voltage—like those observed during tonic firing—can shape the synaptic responses of dendritic spines ." ], [ "Our results are in accord with a number of past studies that have observed dendritic spines on midbrain dopamine neurons .", "To date , spines have been identified on dopamine neurons in rat ( Grace and Onn , 1989; Nirenberg et al . , 1996b; Phillipson , 1979; Sarti et al . , 2007 ) , guinea pig ( Yung et al . , 1991 ) , cat ( Phelps et al . , 1983; Preston et al . , 1981; Rinvik and Grofova , 1970 ) , rabbit ( Kline and Felten , 1985 ) , primates ( Schwyn and Fox , 1974 ) and humans ( Cruz-Sanchez et al . , 1995; Patt et al . , 1991 ) .", "We estimated spine densities from dendritic segments and whole-cell reconstructions and found that dendritic spines are present at an average density of 2 . 08 spines/10 μm .", "These values are also in close agreement with a recent study of dopamine neurons spines in juvenile mice ( Jang et al . , 2015 ) .", "Along with past work , therefore , our data provide clear evidence that synaptic excitation of dopamine neurons likely involves shaft synapses as well as input onto dendritic spines .", "We examined dopamine neurons in both live slices as well as fast-perfusion fixed tissue and found a modest but consistent reduction in spine density with age .", "This finding is consistent with developmental studies that report fewer spines on dopamine neurons in adult animals ( Phelps and Adinolfi , 1982; Phelps et al . , 1983 ) .", "Humans , however , may be a notable exception to this rule according to two studies that characterized the morphology of dopamine neurons in substantia nigra from adult deceased patients ( Cruz-Sanchez et al . , 1995; Patt et al . , 1991 ) .", "Both studies report significant numbers of dendritic spines in normal , nondiseased adult patients ( age range , 20–93 years old ) .", "Interestingly , one of these studies reported that the density of spines on putative SNc dopamine neurons in normal adults was 1–2 spines/10 μm ( Cruz-Sanchez et al . , 1995 ) , in striking agreement with the values that we report here in mice .", "In another study , the authors observed prominent pathological changes to dopamine neurons in Parkinson’s patients including reduction in dendritic branching and a loss in the number of dendritic spines ( Patt et al . , 1991 ) .", "In future work , it will be important to determine whether a causative relationship exists between the observed changes in dendritic morphology/spine density and selective death of substantia nigra dopamine neurons which occurs in Parkinson’s patients .", "Comparing dopamine neurons to other well-studied spiny neurons reveals similarities but also important differences .", "For example , pyramidal neurons receive the vast majority ( >85% ) of excitatory inputs onto spines ( Kasthuri et al . , 2015 ) while dopamine neurons are unusual in that they receive inputs onto a mixture of shaft and spine synapses .", "The density of spines on dopamine neurons is considerably lower than pyramidal neurons ( typically >10 spines/10 μm ) but similar to some GABAergic interneurons neurons of the hippocampus and cortex that have spine densities of 2–4 spines/10 μm ( Kawaguchi et al . , 2006; Scheuss and Bonhoeffer , 2014 ) .", "It also must be noted that unlike cortical and hippocampal neurons that receive mostly excitatory input , substantia nigra dopamine neurons function within a largely inhibitory network .", "Up to 70% of synaptic inputs that are received by the SNc dopamine neurons are inhibitory while a much smaller fraction of the synaptic inputs arrive from excitatory sources ( Bolam and Smith , 1991; Henny et al . , 2012; Smith et al . , 1996 ) .", "This raises the possibility that the low density of dendritic spines on SNc dopamine neurons may simply reflect the low density of excitatory inputs overall .", "Consistent with this idea , we find that the ratio of spines to PSD95-mVenus positive shaft puncta is 4 to 5 .", "Therefore , despite a low absolute density of spines on dopamine neurons , the relative density of shaft and spine synapses is comparable .", "Whether long-necked spines more strongly attenuate synaptic potentials is a matter of current debate .", "Studies of cortical pyramidal cells show a negative correlation between neck length and EPSP amplitude ( Araya et al . , 2006; 2014 ) , while studies in hippocampal pyramidal cells ( Takasaki and Sabatini , 2014 ) show only a weak correlation .", "It is important to note that Takasaki and Sabatini tested spines that were below 1 . 2 μm in length whereas Araya et al . tested substantially longer spines ( ≥2 μm ) .", "In the dopamine neurons , we found a wide range of spine lengths up to 5 μm .", "However if we only consider spines shorter than 1 . 2 μm , we also observe no significant correlation between spine length and uEPSP amplitude ( Pearson’s R = −0 . 054; n = 21 spines; p=0 . 81 ) .", "In a different study , Bywalez et al . ( 2015 ) found no correlation between spine length and EPSP amplitude in olfactory bulb granule neurons , which have spines up to 15 µm in length .", "However , spines on the granule cells are excitable and possess voltage-gated sodium and calcium channels which shape the release of neurotransmitter from the spines ( Egger et al . , 2005 ) .", "As Bywalez et al . proposed , the spine sodium channels may function to reduce variability in spine potentials , Ca2+ influx and subsequent neurotransmitter release from spines .", "The dendrites of many neurons are excitable and express a wide variety of voltage-gated ion channels that shape action potential backpropagation and synaptic integration ( Stuart and Spruston , 2015 ) .", "However , there is not a consensus regarding whether or not stimulation of a single spine leads to downstream activation of voltage-gated ion channels .", "Studies using voltage-sensitive fluorescent indicators to assay spine voltage report that the depolarization of the spine head following single spine stimulation is insufficient for activation of voltage-gated channels ( Palmer and Stuart , 2009; Popovic et al . , 2015 ) .", "By contrast , Ca2+ imaging studies examining single spine responses have described a clear contribution of voltage-gated ion channels in the spine head with many observing activation of high-voltage activated calcium channels ( Bloodgood et al . , 2009; Bywalez et al . , 2015; Carter and Sabatini , 2004; Grunditz et al . , 2008; Harnett et al . , 2012; Seong et al . , 2014 ) .", "These observations predict that the electrical resistance of the spine neck amplifies the EPSP in the spine head to potentials considerably greater than those recorded in the dendrite or soma .", "We observed a significant contribution of voltage-gated sodium channels to the glutamate-evoked responses of individual spines ( Figure 10 ) , suggesting the depolarization of the spine head is sufficient for sodium channel activation in dopamine neurons .", "Most studies of spine function have examined pyramidal neurons that rest at hyperpolarized membrane potentials near -70 mV .", "However , during pacemaking in dopamine neurons , the interspike membrane potential covers a narrow but relatively depolarized subthreshold voltage range with an average non-spike voltage of ~-55 mV ( Hage and Khaliq , 2015 ) .", "At these depolarized voltages , the membrane potential is within the steepest region of the voltage-activation curve of sodium channels , where even a small amplification of the spine head EPSP may lead to significant sodium channel activation .", "In a clear demonstration of the relationship of EPSPs with voltage-gated sodium channels , Carter et al . ( 2012 ) showed that EPSPs of 5 mV can lead to activation of both persistent and transient sodium current .", "Therefore , even if the effects of the spine neck resistance on electrical signaling are small , the intrinsic properties of dopamine neurons may allow minor amplification to produce major effects on the synaptic voltage response ." ], [ "For experiments utilizing wildtype animals , sagittal brain slices containing SNc were prepared from postnatal day 14–25 Swiss Webster mice of either sex according to the institutional guidelines at the National Institutes of Health .", "Experiments utilizing the PSD-95-ENABLED mouse line performed on juvenile mouse progeny generated by crossing heterozygous PSD-95-ENABLED mouse with a heterozygous DAT-Cre mouse .", "Mice were genotyped using previously published primers ( Fortin et al . , 2014 ) .", "Animals were anesthetized with isoflurane and swiftly decapitated .", "Brains were quickly removed and placed in ice-cold slicing solution containing ( in mM ) 250 glycerol , 2 . 5 KCl , 2 MgCl2 , 2 CaCl2 , 1 . 2 NaH2PO4 , 10 HEPES , 21 NaHCO3 , 5 glucose , bubbled with 95/5% O2/CO2 .", "Slices were cut at 300 µm thickness using a vibrotome ( DTK-1000; DSK , Dosaka ) and incubated for 30 min at 34° C in artificial cerebrospinal fluid ( ACSF ) containing ( in mM ) 125 NaCl , 25 NaHCO3 , 1 . 25 NaH2PO4 , 3 . 5 KCl , 1 MgCl2 , 2 CaCl2 , and 10 glucose , bubbled with 95/5% O2/CO2 .", "Slices were then stored at room temperature until time of use .", "Slices were placed into a recording chamber and perfused continuously with warm ACSF ( 32–34°C ) .", "Dopamine neurons were targeted primarily by their location within the SNc .", "Other criteria included the presence of slow pacemaking ( <5 Hz ) during cell-attached or whole-cell recordings , broad APs ( halfwidth >1 . 35 ms ) and prominent voltage sag in response to negative current injection—associated with hyperpolarization-activated cation current ( IH ) .", "Current-clamp and voltage-clamp recordings were made with a Multiclamp 700B amplifier and digitized using a Digidata 1440A ( Molecular Devices ) .", "Low-resistance patch electrodes ( 2–4 MΩ ) were pulled from filamented borosilicate glass .", "In voltage-clamp recordings , pipette series resistance was compensated by 70–80% and was monitored carefully throughout the experiment .", "Recordings were terminated if series resistance changed by >20% .", "Current-clamp recordings were bridge balanced and monitored frequently throughout the experiment .", "For current-clamp recordings , internal recording solution contained ( in mM ) 122 K methanesulfonate , 9 NaCl , 1 . 8 MgCl2 , 4 Mg-ATP , 0 . 3 Na-GTP , 14 phosphocreatine , 10 HEPES , 0 . 5 EGTA , 0 . 1 CaCl2 and 0 . 05 Alexa Fluor 594 hydrazide ( Molecular Probes , Eugene , OR ) adjusted to 7 . 35 with NaOH .", "For Ca2+ imaging experiments , EGTA and CaCl2 were excluded from the internal solution and 300 µM Fluo-5F ( KD = ~ 2 . 3 µM ) was added .", "For voltage-clamp recordings internal solution contained ( in mM ) 135 CsCl , 10 NaCl , 10 HEPES , 2 MgCl2 , 0 . 5 EGTA and 0 . 1 CaCl2 , except where specifically indicated .", "In some cases , recordings were made using QX-314 ( 1 mM ) added to the internal solution as well as bath applied nifedipine ( 10 μM ) to reduce spontaneous membrane oscillations when holding at depolarized potentials ( Puopolo et al . , 2007 ) .", "Imaging experiments were performed using a custom two-photon microscope from Prairie Technologies Ultima ( Middleton WI ) along with a Mai Tai ultrafast Ti:Sapphire laser ( Spectra-Physics , Mountain View CA ) tuned to 810 nm for Ca2+ imaging .", "For experiments using the PSD-95-ENABLED mice the excitation laser was tuned to 960 nm to visualize the mVenus signal .", "Cells were imaged using a 40x , 0 . 8 NA objective ( Olympus , Melville NY ) .", "Fluorescence was split into red and green channels using a 575 nm dichroic longpass mirror and passed through 607/45 nm and 525/70 nm barrier filters before being detected by multi-alkali photomultiplier tubes ( Hamamatsu ) .", "Ca2+ imaging was initiated 15–20 min after whole-cell break in to allow diffusion of fluorescent indicators .", "Linescan imaging of dendritic Ca2+ was performed at 30 s intervals .", "Ca2+ imaging data are presented as ΔG/GS and were quantified as changes in green fluorescence divided by red fluorescence ( ΔG/R ) , normalized to GS/R * 100% .", "GS/R was measured by imaging a pipette filled with internal recording solution plus saturating Ca2+ ( 2 mM CaCl2 ) placed directly above the slice at the end of experiment ( Yasuda et al . , 2004 ) .", "Simultaneous glutamate uncaging experiments were performed using a second Mai Tai laser tuned to 725 nm with an uncaging pulse width of 500 µs .", "Uncaging laser power measured at the back of the objective was between 35 and 45 mW .", "Synapses assayed by glutamate uncaging were 30–40 µm below the surface of the slice .", "The same parameters were used to bleach the spine head in FRAP recordings .", "The extent of bleaching by the uncaging laser was consistent from spine to spine ( fluorescence reduced by 34 ± 2% ) , suggesting effective uncaging power was similar across experiments .", "External solutions for glutamate uncaging experiments contained 3 mM 4-Methoxy-7-nitroindolinyl-caged-L-glutamate ( MNI-glutamate ) ( Tocris bioscience ) and 10 µM D-serine to prevent NMDAR desensitization .", "Solutions were recirculated to conserve MNI-glutamate ( volume = 10 mL ) .", "Local stimulation of dendritically-located synapses was performed using bipolar electrodes placed in theta glass pipettes filled with ACSF ( tip diameter ~ 5 µm ) .", "Electrodes were placed within 10 µm of an Alexa-594 labeled dendrite .", "Stimulus intensity was set to 15–45 V with 0 . 5 ms pulse duration using an Iso-Flex stimulus isolator ( A . M . P . I . ) .", "Picrotoxin ( 50 µM ) , CGP 55 , 845 hydrochloride ( 1 µM ) , sulpiride ( 1 µM ) , SCH 39 , 166 hydrobromide ( 1 µM ) and LY 341495 ( 1 µM ) were added to ACSF to block activity of GABAA , GABAB , D2 dopamine , D1 dopamine and group II metabotropic glutamate receptors .", "Golgi-Cox staining of Swiss Webster mouse brain tissue was performed using FD Rapid GolgiStain Kit ( FD NeuroTechnologies , Inc . , Columbia MD ) according to manufacturer instructions .", "Juxtacellular labelling of SNc dopamine neurons was performed using Tg ( TH-EGFP ) 1Gsat GENSAT line mice which express enhanced GFP under the control of the tyrosine hydroxylase promoter .", "Mice were transcardially perfused with cold 1 . 5% PFA in phosphate buffered saline ( PBS ) after which brains were dissected and postfixed for 1 hr in 1 . 5% PFA .", "Brains were sectioned at 300 µm .", "Fixed brain slices containing the SNc were transferred to a microscope stage and dopaminergic neurons were identified by green fluorescence .", "A high resistance sharp electrode containing 0 . 5 mM Alexa-594 was then placed against the soma of an identified dopamine neuron and large , brief ( 500 µs ) current pulses were applied once every 30 s for 5–10 min until the soma was clearly filled with Alexa-594 .", "Afterwards , the slice was mounted on a glass slide with VectaShield mounting medium ( Vecta ) and imaged on a two photon microscope ( described above ) .", "Images used for measurements of spine length and density along dendritic segments were performed using z-stacks of 64 nm2xy-resolution .", "Images used to measure spine distribution along entire dendrites had a resolution of 146 nm2 .", "Planes of z-stacks were separated by either 0 . 8 or 1 . 0 µm .", "When dendritic regions examined extended beyond the borders of a single z-stack , images were integrated using the Volume Integration and Alignment System ( VIAS ) software package ( Rodriguez et al . , 2003 ) .", "The combined data set was then loaded into Neuron Studio ( Wearne et al . , 2005 ) where dendrites were semi-automatically reconstructed and dendritic spines were manually identified .", "Spine morphology was measured in ImageJ .", "The spine length was measured from the base of the dendrite to the tip of the spine head .", "Spine head diameter was estimated by measuring full-width at half maximum of the pixel intensity across a 3 pixel wide line drawn orthogonal to the spine neck .", "The spine head diameter was then used to estimate the spine head volume by Vhead = 4/3*π*r3 , where r is the radius of the spine head .", "The pixel intensity of mVenus signal in PSD-95-ENABLED mice was measured by drawing an ROI around the dendritic shaft or spine of interest and measuring the pixel intensity throughout the z-stack .", "After background subtraction , green and red pixel intensities were measured as the integral of 3 consecutive z-slices , with the middle slice corresponding to the highest red intensity .", "Bleedthrough of Alexa-594 signal into the green channel was measured in regions of the dendritic shaft with no apparent mVenus signal ( average bleedthrough = 3 . 3% ) .", "mVenus pixel intensities for dendritic shafts and spines were therefore corrected by subtracting the product of the corresponding red pixel intensity and the bleedthrough measured for that dendrite .", "Electrophysiological data were analyzed using custom routines written in Igor Pro ( Wavemetrics ) .", "Ca2+ imaging and FRAP data were quantified using ImageJ to measure fluorescence intensities and further analyzed using Igor .", "FRAP data were corrected for slight and gradual acquisition- related bleaching due to scanning by the imaging laser ( Ishikawa-Ankerhold et al . , 2012 ) .", "To isolate the extent of acquisition-related bleaching , we measured the Alexa-594 fluorescence intensity during linescans in which the uncaging laser was not targeted to the spine head .", "On average , the baseline fluorescence was reduced by 3 . 5 ± 0 . 7% ( n=33 ) over the course of imaging .", "These acquisition-only signals were fit with a straight line and FRAP traces were normalized to these functions .", "Corrected FRAP traces were then fit with a double-exponential function constrained to return to baseline fluorescence levels .", "The weighted time constant was calculated as:Afast× τfast+ Aslow× τslowAfast +Aslow ." ] ]

[ "Little is known about the density and function of dendritic spines on midbrain dopamine neurons , or the relative contribution of spine and shaft synapses to excitability .", "Using Ca2+ imaging , glutamate uncaging , fluorescence recovery after photobleaching and transgenic mice expressing labeled PSD-95 , we comparatively analyzed electrical and Ca2+ signaling in spines and shaft synapses of dopamine neurons .", "Dendritic spines were present on dopaminergic neurons at low densities in live and fixed tissue .", "Uncaging-evoked potential amplitudes correlated inversely with spine length but positively with the presence of PSD-95 .", "Spine Ca2+ signals were less sensitive to hyperpolarization than shaft synapses , suggesting amplification of spine head voltages .", "Lastly , activating spines during pacemaking , we observed an unexpected enhancement of spine Ca2+ midway throughout the spike cycle , likely involving recruitment of NMDA receptors and voltage-gated conductances .", "These results demonstrate functionality of spines in dopamine neurons and reveal a novel modulation of spine Ca2+ signaling during pacemaking ." ]

[ "When a nerve cell is viewed under the microscope , its structure looks a little like that of a tree .", "Each nerve cell , or neuron , has an array of ‘branches’ known as dendrites , which receive chemical messages from other cells .", "These messages are converted into electrical signals in the cell body and then travel down the main nerve fiber , which is the output of the cell .", "At the end of the nerve fiber , the signals are converted into chemical messages again and passed on to the dendrites of the next neuron .", "The dendrites of most neurons are covered with spines that resemble thorns on a stem .", "These are the sites that typically connect neurons with other cells .", "However , neurons that release a chemical messenger called dopamine have largely smooth dendrites .", "These neurons still belong to extensive neural circuits that are involved in movement and reward processing .", "As such , it is not clear whether dopamine neurons receive connections that form onto dendritic spines , or whether all of their connections are formed directly onto dendrites .", "By studying slices of mouse brain , Hage et al . now show that inputs onto dopamine neurons in fact establish both types of connections .", "The connections formed directly onto dendrites usually have more influence on the neuron than those on spines .", "However , the size and shape of spines determines their properties .", "Most spines are attached to dendrites via a narrow neck , and spines with long necks have less influence on the electrical signaling of the cell than short-necked spines .", "This is because the neck limits the movement of electrical charges .", "Long-necked spines also possess fewer receptors for the chemical messenger glutamate , which reduces their ability to transmit signals arriving from other cells .", "Dopamine neurons receive input from many different areas of the brain .", "A key next step is to determine whether neurons from specific brain regions are more likely to form connections with spines as opposed to directly on dendrites .", "Given that these inputs often arrive at the same time , another question is whether there is crosstalk between these two types of connections ." ]

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

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

[ [ "Gaining insight into the dynamics of viral gene delivery into a host cell during infection is critical for understanding the virus replication dynamics ( Ellis and Delbrück , 1939; You and Yin , 1999; Gallet et al . , 2011; Lee et al . , 1997 ) and viral fitness ( Burch and Chao , 2000; Abedon and Culler , 2007 ) .", "A central method for studying virus replication dynamics is the ‘one-step growth cycle’ method , which was developed for an ex vivo model of phage growth in E . coli by Ellis and Delbruck ( Ellis and Delbrück , 1939 ) .", "This quantitative method measures the rate of one cycle of phage replication .", "For accurate quantification of the infectious cycle rate , it is critical that the infection is synchronized so that cells are infected at the same time .", "This is achieved by incubating bacterial culture with phage , diluting the solution after phage has adsorbed to cells so that no more adsorption occurs , and then measuring virus growth at different time intervals using a plaque assay .", "Modified versions of the one-step and multi-step replication assays remain central today for monitoring the growth and evolution of viruses as they mutate to evade antiviral drugs , the immune system or environmental factors inhibiting viral spread ( Lee et al . , 1997; Coffin , 1995; Perelson et al . , 1996; Nowak , 1992 ) .", "The one-step growth cycle method builds on an assumption that initiation of infection through phage DNA injection into cells occurs rapidly and simultaneously after phage adsorbs to cells ( Ellis and Delbrück , 1939 ) .", "Although the dynamics of the phage adsorption step has been investigated ( Moldovan et al . , 2007; Mackay and Bode , 1976 ) , the dynamics of the initiation of phage injection is poorly understood even though it has critical importance for virus growth rate studies .", "The one-step growth cycle method focuses on the lytic infection pathway ( where a virus replicates immediately , leading to cell lysis ) .", "However , the cell decision between a lytic or lysogenic infection ( latent state where phage DNA is integrated into a cell chromosome without killing the cell ) may depend on the dynamics of DNA injection from multiple phages adsorbed to a single bacterial cell ( Zeng et al . , 2010; Trinh et al . , 2017; Kourilsky , 1974; Kourilsky , 1973 ) .", "Some studies claimed that DNA ejection from such a phage population occurs in a stochastic manner and is solely attributed to the opening dynamics of the portal-tail connector channel through which DNA is being ejected ( Raspaud et al . , 2007 ) .", "This description fits the generalized picture of biochemical stochasticity proposed in the past for many cellular processes ( Arkin et al . , 1998; Singh and Weinberger , 2009 ) .", "However , as shown below , our data does not support these claims .", "Furthermore , biochemical stochasticity may not play as much of a role in virus-cell interactions as previously thought ( Zeng et al . , 2010; St-Pierre and Endy , 2008 ) , specifically concerning the process of cell-fate decision making between lysogenic and lytic pathways in E . coli following phage λ infection ( Zeng et al . , 2010; Trinh et al . , 2017 ) .", "While host functions can influence this decision process ( Casjens and Hendrix , 2015 ) , the initial choice is made at the individual phage level ( Trinh et al . , 2017 ) .", "This , in turn , determines how phages interact with one another during cell infection , where the central interaction parameter is replication dynamics of multiple viral genomes being injected into a cell .", "Interestingly , replication dynamics occurs on the timescale of the genome injection process , varying from seconds to minutes ( Mackay and Bode , 1976; Van Valen et al . , 2012 ) .", "This is also evidenced by an important role that the order of phage genome injection is playing in CRISPR sequence acquisition ( Modell et al . , 2017 ) .", "Therefore , timing of phage DNA injections into a cell has been shown to affect the decision between lysogeny and cell lysis ( Trinh et al . , 2017; Cortes et al . , 2017 ) .", "Specifically , it was proposed that simultaneous , instant injection events from all phages infecting one cell lead to a lack of competition for replication of phage genomes , which results in higher lysogenizaton frequency ( Trinh et al . , 2017; Kourilsky , 1973 ) .", "It was indeed observed that probability of lysogeny increases with the number of simultaneously infecting phages ( Trinh et al . , 2017; Kourilsky , 1973 ) .", "On the contrary , delayed asynchronous injections lead to competitive interactions between phage genomes observed during lytic infection .", "Consistently , infection delays were shown to decrease the chance of lysogeny by also lowering CII levels in the cell , where CII is a viral DNA-binding transcription factor governing postinfection decision-making ( Cortes et al . , 2017 ) .", "Lytic phage that delivers its genome first overrides cell’s lysogeny decision and uses cell’s limited resources to replicate its own progeny ( Trinh et al . , 2017; Kourilsky , 1973 ) .", "A pioneering study demonstrated that DNA injection from phage λ , after preadsorption to E . coli , occurred instantaneously at 37°C , but at lower temperatures the heterogeneous injection dynamics with minutes-long lag times was observed , with injections completely inhibited at 4°C ( Mackay and Bode , 1976 ) .", "The mechanism behind this injection behavior remained unknown .", "Thus , understanding the mechanism and environmental factors determining the switch between synchronized and desynchronized injection dynamics from a phage population is of key importance for studies of the infectious cycle where the cell decision between lytic and lysogenic infection plays a crucial role .", "As shown here , the main factor influencing injection dynamics is the structural state of DNA inside the viral capsid .", "DNA is tightly packaged inside phage λ capsid with DNA-DNA surface separation of only 7 Å ( Lander et al . , 2013; Earnshaw and Harrison , 1977 ) .", "This leads to high interstrand repulsive forces that , along with DNA bending stress , result in internal genome pressure of tens of atmospheres ( Evilevitch et al . , 2003; Tzlil et al . , 2003; Purohit et al . , 2003 ) .", "This pressure powers DNA ejection into a bacterial cell .", "However , the small interstrand distance also generates electrostatic and hydration sliding friction between neighboring DNA strands ( Gabashvili and Grosberg , 1992; Berndsen et al . , 2014; Liu et al . , 2014 ) , which limits the packaged genome’s mobility ( or fluidity ) .", "Using atomic force microscopy ( AFM ) combined with isothermal titration calorimetry ( ITC ) , we have recently shown that restricted intra-capsid DNA mobility can be characterized by a solid-like mechanical response to nano-indentation of a DNA-filled capsid ( Liu et al . , 2014; Sae-Ueng et al . , 2014 ) .", "At the same time , DNA packaged in the capsid can undergo a mechano-structural transition from a solid- to a fluid-like state .", "This transition is induced by increasing the temperature ( which affects DNA bending stress and packing defects ) or by varying external ionic conditions ( which affects DNA-DNA repulsive interactions and overall genome stress in the capsid ) ( Li et al . , 2015 ) .", "In the fluid-like state , the interstrand DNA-DNA friction of the encapsidated genome is significantly reduced due to an increase in distance between packaged DNA strands occurring with local DNA disordering .", "These observations suggest that dynamics of DNA ejection can be affected by a transition in mobility of the encapsidated genome .", "To investigate the effect of this mechano-structural DNA transition on DNA ejection dynamics from phage λ , we designed a new ITC assay that provides ultra-sensitive detection of ejection dynamics from a phage population triggered by instant mixing with LamB λ-receptor in vitro .", "We also used time-resolved small angle X-ray scattering ( SAXS ) to further support our findings .", "We discovered striking bimodal dynamics of coexisting phage populations displaying fast synchronized and slow desynchronized ejection events .", "Here , we show that this complex ejection dynamics is directly linked to the two mechano-structural states of DNA—solid- and fluid-like—inside the λ capsid .", "This remarkable observation of mechano-regulation of phage genome ejection dynamics can be compared to gene expression regulation in meters-long , tightly packaged chromosomal DNA in eukaryotic cells ( Eslami-Mossallam et al . , 2016 ) , where gene expression is controlled not only by genetic regulation but also by mechanical properties of DNA in nucleosomes .", "Environmental stress factors , such as an abrupt change in temperature or in ionic conditions , were demonstrated to play a major role in the mechano-regulated gene activation process , turning certain genes on and off , in a process termed epigenetics .", "Similarly , we show that temperature and ionic conditions control the mechanical properties of virally encapsidated DNA and act as an on-off switch between synchronized and desynchronized ejection dynamics .", "This can influence timing of viral replication and the lytic-lysogenic cell decision process ( Trinh et al . , 2017 ) .", "These in vitro ejection dyamics results are in agreement with earlier in vivo observations of temperature’s effect on DNA injection dynamics from phage λ preadsorbed to E . coli ( Mackay and Bode , 1976 ) ." ], [ "Dynamics of DNA ejection from phage has been previously investigated using single particle fluorescence ( Grayson et al . , 2007 ) as well as bulk light scattering analysis ( LS ) ( Raspaud et al . , 2007; Freeman et al . , 2016 ) .", "Single particle fluorescence studies have been mainly focused on determining DNA translocation rates from a phage capsid , rather than population ejection dynamics , due to limited statistics as well as uneven timing for delivery of phage receptors to trigger ejection ( because of flow cell geometry ) .", "LS measurements , on the other hand , record a decrease in total scattering intensity , I ( 0 ) , from a phage population .", "This decrease is associated with a change in DNA scattering density inside capsids , reflecting the number of DNA-filled phage particles remaining as ejection events occur .", "When DNA is ejected and relaxed in solution to a micron-size coil , it no longer contributes to the scattering intensity ( Freeman et al . , 2016 ) .", "Figure 1 shows our LS-measured decay of normalized scattering intensity versus time , ΔI", "( t ) = I", "( t ) −I ( final ) I ( initial ) −I ( final ) , for DNA ejection from wild-type ( wt ) DNA length phage λ ( corresponding to 48 , 500 bp ) when it is mixed with LamB solution at 22°C in TM-buffer ( 10 mM MgCl2 , 50 mM TrisHCl , pH 7 . 5 ) .", "As observed in the figure , the overall time for the ejection process is much slower ( here > 1000", "s ) than the ejection time for DNA translocation from a single phage ( ≤10", "s ) ( Grayson et al . , 2007 ) because ejection events at these conditions occur in a stochastic manner ( Freeman et al . , 2016 ) ( if ejections were synchronized , the overall ejection time would be equivalent to DNA translocation time from a capsid ) .", "Thus , LS-measured scattering intensity decay reflects dynamics of ejection events versus time , discerning only between DNA-filled and empty capsids ( Freeman et al . , 2016 ) .", "However , this observation does not exclude that a fraction of phages can eject DNA synchronously .", "As DNA ejection is progressing , each ejecting particle is given the same weight in the total scattering intensity signal change .", "Therefore , the signal from a phage population with fast synchronized ejection events occurring within seconds will be dominated by much slower desynchronized ejections occurring in parallel but on a much longer timescale of minutes .", "Something similar occurs in reaction kinetics measurements where resolution is determined by the slowest rate-limiting step .", "In our case , this will prevent differentiation between phage populations with synchronized and desynchronized ejection dynamics .", "Therefore , in order to be able to distinguish between synchronized and desynchronized ejection populations , a heavier weight must be assigned to the readout signal from the phage population with synchronized ejection dynamics .", "Such a measurement is possible with ITC-measured heat flow ( i . e . rate of heat production measured in µJ/s or µcal/s or W ) for the DNA ejection process when phage is titrated into LamB solution ( Liu et al . , 2014; Jeembaeva et al . , 2010 ) .", "ITC is mainly used for thermodynamic analysis .", "However , it was recently demonstrated that it can be successfully used to measure reaction kinetics reflected by heat flow ( so-called kinITC ) due to the very short response time of modern ITC instruments ( Burnouf et al . , 2012 ) .", "We designed a new assay using ultra-sensitive micro-ITC to determine ejection dynamics from phage λ with the aim to differentiate between populations with synchronized and desynchronized ejection events .", "We have previously shown that DNA ejection from phage λ is associated with an exothermic heat release , which can be accurately measured by ITC ( Liu et al . , 2014; Jeembaeva et al . , 2010 ) .", "Synchronized genome ejections will result in a significantly higher heat flow due to additive energy contributions from multiple ejecting λ-particles in one time instant , compared to that of desynchronized ejections where heat flow from single ejection events is spread over a longer period of time .", "Here , phage λsolution is titrated in LamB receptor solution with LamB present in a large excess ( 104:1 LamB:phage which ensures that ejection dynamics is not limited by receptor concentration ) .", "The instrument records differential power ( DP ) versus time ( DP is equivalent to heat flow , measured in μcal/s ) between the reference cell ( containing buffer ) and the sample cell ( containing phage-LamB reaction mixture ) .", "Figure 1 inset shows heat flow ( µcal/s ) versus time for the DNA ejection process occurring when wt-DNA length phage λ particles are titrated into LamB receptor solution at 22°C ( all conditions remained identical to LS measurement for direct comparison ) .", "Heat flow contributions associated with enthalpy of mixing and pressure-volume work arising from titrating phage into buffer , buffer into LamB solution , and buffer into buffer are measured separately for each set of parameters and subtracted from the raw DP curve ( Liu et al . , 2014; Jeembaeva et al . , 2010 ) .", "All titration curves shown in this work ( including Figure 1 inset ) are shown with background DP contributions subtracted .", "The response time of our ITC instrument ( Malvern iTC200 , see Materials and methods Section ) is only ∼3 . 5 s , which is significantly shorter than equilibration time for the DNA ejection process ( e . g . it took ∼250 s for the DP curve in Figure 1 inset to return to baseline after titrating phage into LamB solution ) .", "This allows analysis of DNA ejection dynamics by directly evaluating the equilibration times of exothermic DP-peaks from phage-into-LamB titrations .", "( All separately-measured background DP contributions subtracted from the raw DP signal occur on a timescale of <10 s [Liu et al . , 2014; Jeembaeva et al . , 2010] )", "Here , we demonstrate how ITC provides information on population dynamics of synchronized and desynchronized ejection events not distinguishable by LS .", "As explained above , LS-measured scattering intensity in Figure 1 reflects the total number of DNA-filled phage particles remaining over time as DNA ejections are progressing .", "On the contrary , ITC-measured heat flow in Figure 1 inset corresponds to the instantaneous change in the heat released from a population of ejecting phage particles versus time .", "Thus , in order to compare the ejection dynamics from LS and ITC measurements , we need to take the derivative with respect to time of the LS-measured normalized forward scattering curve in Figure 1 .", "This yields the instantaneous change in the number of ejecting phages versus time ( dΔI/dt ) .", "The resulting dΔI/dt", "( t ) curve is scaled to correspond to the phage concentration used in an ITC measurement and multiplied by the heat amount ( enthalpy ) released by one phage during ejection at a given temperature ( Liu et al . , 2014; Li et al . , 2015 ) .", "Figure 1 inset shows the resulting LS scattering intensity derivative ( blue curve ) compared to the ITC titration curve ( green curve ) .", "The derivate of LS data displays a slower decay than ITC data .", "As we have previously shown , LS overestimates the total ejection time since it also records the scattering intensity contributions from DNA condensates formed immediately after rapid DNA ejection ( due to DNA ejection occurring faster than DNA diffusion in the bulk solution once DNA is ejected from the capsid ) ( Freeman et al . , 2016; Löf et al . , 2007 ) .", "Ejected DNA condensates will relax in solution through diffusion , forming larger random coil aggregates with negligible contribution to the LS signal .", "This diffusive DNA coil relaxation process is masking the DNA ejection dynamics , making it appear slower ( Freeman et al . , 2016; Löf et al . , 2007 ) .", "The most marked difference , however , between LS derivative and ITC data in Figure 1 inset is in the two exothermic peaks on the DP titration curve , which correspond to bimodal phage ejection dynamics from synchronized and desynchronized ejection events ( in order to accommodate both slower LS data and ITC data on the time scale in Figure 1 inset , the two ITC peaks are not clearly visible but they are well resolved when plotted on a shorter time scale as shown in the next section ) .", "This is not observed in the LS derivate displaying only averaged unimodal ejection dynamics .", "Thus , unlike LS scattering intensity , ITC heat flow data does not obscure the contribution from rapidly occurring synchronized ejections with slower decay from desynchronized ejections .", "This makes ITC a suitable method to investigate the effect of solid-to-fluid-like intra-capsid DNA transition on phage ejection population dynamics .", "As described above , we have recently found , using ITC ( and supported by other methods ) , that DNA packaged in phage λ capsid undergoes a mechano-structural transition as a function of temperature ( Liu et al . , 2014 ) , as illustrated in Figure 2A .", "By integrating the area under the exothermic DP titration curve for phage-in-LamB titration ( with all mixing enthalpies and pressure-volume work subtracted ) , the enthalpy change associated with DNA ejection from phage can be derived and corresponds to ΔHej ( T ) = ΔH ( T ) DNA ejected – ΔH ( T ) DNA inside phage .", "Since the total volume of the system does not change during DNA ejection , and the pressure is constant , enthalpy and internal energy are approximately equal ( Liu et al . , 2014; Jeembaeva et al . , 2010 ) .", "Figure 2B shows ITC-measured enthalpy ΔHej ( T ) for DNA ejection from wt phage λ when it is titrated into a solution with LamB receptor in the temperature range between 22°C and 42°C in 10 mM MgCl2 Tris-buffer .", "Figure 2B shows a discontinuity in the approximately linear dependence of ΔHej on temperature occurring at T* ∼33°C for wt λ-DNA length ejection from phage λ .", "The discontinuity is attributed to DNA structure disordering inside the capsid , which leads to a transition from a solid-like to a fluid-like state ( Liu et al . , 2014 ) .", "The slopes in the linear regions of ΔHej ( T ) in Figure 2B , correspond to specific heat capacity ΔCp ( T ) for DNA ejection process [ΔCp ( T ) =Cp ( ejected DNA ) – Cp ( packaged DNA ) ] .", "Since change in Cp ( ejected DNA ) ( i . e . free DNA in solution ) is relatively small in the measured temperature interval ( 22° - 43°C ) ( Tsereteli et al . , 2000 ) , the inversion of ΔHej ( T ) slope at transition temperature ( T* ∼33°C ) suggests a change in Cp ( packaged DNA ) value for DNA inside the capsid .", "This Cp change is in turn associated with transition in ordering and hydration of the packaged genome , which we have previously analyzed with ITC in ref . ( Jeembaeva et al . , 2010 ) .", "To investigate the effect of the DNA transition in λ-capsid on dynamics of the DNA ejection process , we analyze the equilibration time and median time of exothermic DP peaks on titration curves .", "Figure 3A shows ITC titration curves ( shown as heat flow versus time ) recorded for DNA ejection from wt phage λ at 22°C and 32°C ( below the DNA transition temperature ) , and at 37°C and 42°C ( above the DNA transition temperature ) in 10 mM MgCl2 Tris-buffer .", "Figure 3A shows that , above the DNA transition temperature ( ∼33°C ) , only one enthalpy peak is observed , starting immediately after titration of phage in LamB ( at time zero ) .", "However , the DP curve is not symmetrical and shows a right shoulder suggesting an unresolved second peak .", "Indeed , when the temperature was decreased below the transition temperature , the DP curve splits into two modes corresponding to two phage populations with different heat flow dynamics .", "At both 22° and 32°C , the first peak returns to baseline after ∼20 s when phage is titrated into LamB , corresponding to a phage population with fast ejection dynamics .", "The second DP peak lasts for ∼250 s at 22°C before returning to baseline value , corresponding to a phage population with slow ejection dynamics ( the blue titration curve in Figure 3A is truncated at 150 s for clarity of presentation , however full ITC titration curve at 22°C returning to baseline after ∼250 is shown in the Figure 1 inset ) .", "Since these two exothermic peaks on a DP curve are not completely resolved , we have deconvoluted ( Goodman and Brenna , 1994 ) the DP curves into two asymmetric Gaussian functions with an exponential damping term , using a precise deconvolution method ( Goodman and Brenna , 1994 ) ( see details in Supplemental Material Section ) .", "Figure 3B shows fitting of two exothermic DP peaks ( dashed curves ) for the DNA ejection process at 32°C .", "The same deconvolution fitting procedure was applied to the single asymmetric exothermic peaks with right skewed shoulder on the DP curves above the DNA transition temperature in order to make a consistent comparison of DNA ejection dynamics at all measured temperatures .", "However , above the DNA transition temperature , two deconvoluted peaks derived from the fitting essentially converge into a single Gaussian , suggesting that there is mainly one dominant population of phage λ particles with faster ejection dynamics .", "This is illustrated in Figure 3C , which shows a DP curve for DNA ejection process at 37°C with deconvoluted bimodal and unimodal Gaussian fittings .", "Since most fitted Gaussian peaks on the DP curve are right skewed , we use the median value ( instead of mode value ) to estimate the central tendency time corresponding to average time for the overall DNA ejection process from a phage population .", "( In Supplemental Material Section we explain why the DP exothermic peaks are right skewed for DNA ejection process ) .", "Figure 3D summarizes median time values obtained from the Gaussian fits for DNA ejection from phage populations with fast and slow ejection dynamics at temperatures 22–42°C .", "The error bars are derived from the width of the Gaussian fits at the median value .", "Figure 3D shows that a phage population with fast ejection dynamics ( corresponding to the first exothermic peak and referred to as ‘fast population’ in the plots ) has an average ejection time of ∼10 s at all temperatures .", "This ejection time corresponds to the time of DNA translocation from a single phage λ capsid under similar buffer conditions previously measured by single particle fluorescence ( Grayson et al . , 2007 ) .", "This observation suggests that phage particles in the fast population eject their genomes immediately after receptor addition in a synchronized manner , which results in high heat flow ( additive from many phage particles ) and a sharp exothermic peak .", "On the other hand , the phage population with slow ejection dynamics ( corresponding to the second exothermic peak and referred to as ‘slow population’ in all plots ) is present only below the DNA transition temperature , where the slow ejection population coexists with the fast ejection population .", "Below the transition temperature , the average ejection time for the slow population is ∼30 s at 22°C and ∼45 s at 32°C ( while DP peak equilibration to the baseline takes ∼100 and ∼250 s , respectively ) ; see Figure 3D .", "These ejection times are significantly longer than DNA translocation time from a single phage ( ∼10", "s ) ( Grayson et al . , 2007 ) , suggesting that ejection events occur with delays in a desynchronized manner , as we had previously observed with LS ( Freeman et al . , 2016 ) .", "These observations demonstrate that a temperature-induced DNA transition from a solid- to a fluid-like state in λ capsid strongly affects the dynamics of genome ejection from phage .", "Below ∼33°C , the temperature of intracapsid DNA transition , two exothermic peaks on the DP curve indicate ejection dynamics corresponding to two virus populations with synchronized and desynchronized ejections .", "The presence of synchronized and desynchronized populations suggests the co-existence of phage particles with packaged DNA in both fluid-like and solid-like states .", "This is supported by the fact that DNA is trapped in a metastable state while it is being packaged into the capsid due to high DNA-DNA electrostatic sliding friction , which leads to different non-equilibrium DNA conformations in the capsids ( Berndsen et al . , 2014; Keller et al . , 2016 ) .", "When temperature is increased above that of DNA transition ( at T > 33°C ) , most of the phage population with slow desynchronized ejection dynamics is converted into a population with fast synchronized dynamics; the DP curve displays only one exothermic peak .", "The fast population corresponds to phage with DNA in a fluid-like state , where DNA-DNA electrostatic sliding friction is low ( Liu et al . , 2014 ) and genome ejection occurs immediately after phage is titrated into LamB solution .", "In many dsDNA phages , there is a portal ‘plug’ protein at the portal-tail vertex that seals the portal opening through which DNA is ejected , facilitating retention of the packaged genome .", "For example , in phage P22 , a homotrimer of the tail needle protein ( gp26 ) acts as a portal plug ( Bauer et al . , 2015 ) .", "Similarly in phage λ , protein gpW ( the gp26 homolog ) has been suggested to have a similar portal sealing function ( Bauer et al . , 2015; Gaussier et al . , 2006 ) .", "Adsorption of phage particles to cell receptors ( LamB for phage λ ) leads to portal plug opening , resulting in DNA ejection .", "As we have shown above , when DNA in the capsid is in a fluid-like pressurized state , phage-to-receptor adsorption results in immediate synchronized DNA ejections from all virions .", "By using time-resolved SAXS , we estimate the energy of activation for synchronized and desynchronized ejection processes by analyzing Arrhenius' dependence of DNA ejection rates on temperature ( see data and method description in Figure 3—figure supplement 1 and Materials and methods Section ) .", "The ejection rates are determined by measuring encapsidated DNA diffraction peak area versus time when phage λ is instantly mixed with LamB in a stopped-flow SAXS chamber .", "This reflects the number of remaining DNA-filled phage particles over time .", "We found that the energy of activation required to initiate the synchronized DNA ejection process from phage population with DNA in a fluid-like state is small , with ejection rates essentially not influenced by temperature ( Figure 3—figure supplement 1 ) .", "On the contrary , the rates of DNA ejection events from phage with intracapsid DNA in a solid-like state show a strong dependence on temperature with relatively high activation energy ( ∼20 times higher than molecular thermal energy ) required to initiate ejection by overcoming a significant DNA-DNA sliding friction ( Berndsen et al . , 2014; Liu et al . , 2014 ) .", "This leads to stochastic delays in ejection events or even complete arrest of the ejection process at lower temperatures , as has been previously observed in vivo ( Mackay and Bode , 1976 ) ( e . g . no ejection occurs when phage λ is preadsorbed to E . coli cells at 4°C ) .", "These observations suggest that measured activation energy for phage DNA ejection is associated with interstrand DNA sliding friction and mechano-strucutral transition of encapsidated genome , which further supports the ITC data above .", "As we previously found ( Li et al . , 2015 ) , the temperature at which the solid-to-fluid like DNA transition in phage λ occurs is strongly influenced by ionic conditions , since small polyvalent ions freely diffuse through the capsid pores and directly affect the DNA-DNA sliding friction and genome stress .", "Specifically , we had shown ( Li et al . , 2015 ) that , at physiologic temperature for phage infection ( 37°C ) , varying ionic conditions will affect mobility of packaged DNA .", "As shown above , the transition from solid- and fluid-like DNA state in phage capsids results in desynchronized or synchronized DNA ejection dynamics , which in turn has been proposed to affect the cell decision between lysogenic and lytic infection ( Zeng et al . , 2010; Trinh et al . , 2017 ) .", "This raises an important biological question of how the extra- and intra-cellular ionic environment of a bacterial host affects ejection dynamics and consequentially the viral replication pathway .", "Therefore , in the next Section we investigate how deviations from ionic conditions mimicking those for optimum phage infectivity influence DNA ejection dynamics by switching between desynchronized and synchronized ejection behavior .", "Polyvalent cations present in the bacterial cytoplasm , such as polyamines and Mg2+ , have been shown to have a strong effect on the repulsive interactions between packaged DNA helices in a viral capsid through screening of negative DNA charges and/or introduction of attractive interaction ( Li et al . , 2015; Evilevitch et al . , 2008; Qiu et al . , 2011 ) .", "Since free polyamine concentration in cells is very low as most polyamines are bound to cellular DNA and RNA ( Davis et al . , 1992; Gibson and Roizman , 1971; Igarashi and Kashiwagi , 2000 ) , free Mg-ions will have a large effect on DNA interstrand interactions inside the capsid .", "Therefore , we are specifically interested in the effect of Mg-ion concentration on the dynamics of DNA ejection from phage λ at concentrations similar to those of extra- and intracellular Mg2+ in vivo .", "Mg-ions are essential for both cellular metabolism ( e . g . enzyme activity , protein synthesis , preservation of ribosome and nucleic acid structures ) ( Coates , 2010; Rodgers , 1964; Morgan et al . , 1966; Lusk et al . , 1968 ) and the phage λ infectious cycle ( Fry , 1959; Gaussier et al . , 2006 ) .", "It has been shown that a concentration of ∼5–10 mM of Mg2+ ions in the extracellular solution is critically important for both optimum adsorption of phage λ ( Fry , 1959 ) to E . coli , as well as phage replication rate ( Mackay and Bode , 1976; Harrison and Bode , 1975 ) .", "A similar Mg-concentration has also been shown to provide optimum conditions for DNA packaging in phage λ ( Gaussier et al . , 2006; Nurmemmedov et al . , 2012 ) and corresponds to the free Mg-concentration in E . coli cytoplasm ( Lusk et al . , 1968; Kung et al . , 1976; Hurwitz and Rosano , 1967 ) .", "As mentioned above , Mg-concentration affects DNA stress in the capsid and therefore strongly influences the temperature of transition between solid-like and fluid-like states ( Li et al . , 2015 ) .", "At optimum temperature for infection ( 37°C ) , this Mg-concentration should affect phage ejection dynamics leading to synchronized or desynchronized ejections depending on the intra-capsid DNA state .", "A correlation between physiologic Mg-concentration and phage λ genome ejection dynamics would suggest that mechanical intracapsid DNA transition is an important regulatory mechanism for viral replication .", "This will be discussed in Conclusions Section .", "In the first set of measurements using ITC assay ( Figure 4A ) , we determined DNA transition temperatures for wt λ-DNA length phage in MgCl2 Tris-buffer with MgCl2 concentrations varied between 5 and 50 mM .", "Figure 4A shows enthalpy ΔHej ( T ) versus temperature for the DNA ejection process triggered by phage λ titration into LamB solution .", "With increasing temperature , an abrupt discontinuity and inversion in an essentially linear enthalpy change ΔHej ( T ) indicates the temperature T* at which intracapsid DNA transition occurs .", "Increasing Mg-concentration will initially significantly reduce the strength of the interstrand repulsive interactions in the capsid due to counterion screening of the negative charges between packaged DNA helices ( Lander et al . , 2013; Evilevitch et al . , 2008 ) .", "However , we have previously shown that the screening effect of Mg-ions will become progressively smaller due to the counter-ion saturation ( Evilevitch et al . , 2008 ) .", "As described above , DNA packaged in the capsid has to reach the critical stress level which increases with increasing temperature until the structural transition relieving the genome stress occurs at T* .", "Thus , reducing the intra-capsid DNA stress by increasing Mg-ion concentration leads to an increase in T* .", "That is , a higher temperature is required to reach the critical DNA stress limit in order to overcome the energetic barrier triggering the structural genome transition .", "Indeed , Figure 4A and B shows that the DNA transition temperature T* strongly rises with the initial increase in Mg-concentration , from T*∼23°C at 5 mM MgCl2 Tris-buffer to T*∼37°C at 20 mM MgCl2 Tris-buffer .", "However , between 20 and 50 mM MgCl2 , T* remains essentially unchanged due to Mg-ion saturation at the DNA helices ( Evilevitch et al . , 2008 ) .", "As we have previously shown , at 5 mM MgCl2 at 37°C ( well above the DNA transition temperature ) , DNA in the λcapsid is in a fluid-like state ( Li et al . , 2015 ) .", "However , at [Mg2+] ≥ 10 mM at 37°C , where the solid-to-fluid like DNA transition occurs at or close to this temperature , there is likely a coexistence between phage populations with DNA in fluid- and solid-like states inside the capsid .", "Therefore , using ITC dynamics measurements , we investigate how a Mg-ion regulated switch between solid-like and fluid-like DNA states in λ-capsid affects DNA ejection dynamics at physiologic temperature of infection ( 37°C ) .", "Heat flow ( μcal/s ) versus time of exothermic peaks on DP titration curves was analyzed for phage λ into LamB titrations at 37°C and Mg2+ concentrations between 5 and 50 mM .", "For clarity , Figure 5A shows selectively DP titration curves at 5 mM Mg2+ Tris-buffer ( above DNA transition ) and 20 mM Mg2+ Tris-buffer ( below DNA transition ) .", "At 5 MgCl2 , where packaged DNA is in a predominantly fluid-like state ( since T* ∼23°C ) , only one exothermic DP peak corresponding to fast ejection dynamics is observed on the titration curve , with median ejection time of ∼10 s , see Figure 5A .", "As discussed above , this average ejection time corresponds to DNA translocation time from a single phage capsid and reflects synchronized ejection events from a majority of phage population .", "On the contrary , at 20 mM MgCl2 , the intracapsid DNA is at its transition temperature ( T* ∼37°C ) .", "Now , the DP titration curve shows two distinct exothermic peaks attributed to fast and slow ejection dynamics .", "The first peak has median ejection time of ∼10 s corresponding to a phage population with DNA in a fluid-like state and synchronized ejection dynamics .", "The second peak ( corresponding to a phage population with DNA in a solid-like state ) has median ejection time of ∼55 s suggesting stochastic delays in initiation of DNA ejection resulting in slower ejection dynamics .", "In Figure 5B , we summarize these data by plotting median ejection times ( corresponding to average ejection times ) derived from deconvoluted skewed Gaussian fits to DNA ejection DP peaks in 5–50 mM MgCl2 Tris-buffers at 37°C .", "To further quantify these results , in Figure 5C we show the ratio between fast population peak area ( for synchronized ejection events ) and total area of both fast and slow exothermic peaks .", "Assuming that ejection enthalpy per virion is approximately the same for phage with DNA in a fluid- or in a solid-like state , this plot reflects the fraction of phages ( from the whole phage population ) with DNA in a fluid-like state and synchronized ejection dynamics .", "The observed fraction of fast phage population with synchronized ejections varies between 100% ( at 5 mM Mg2+ ) and only ∼10% ( at [Mg2+] ≥ 20 mM ) .", "Thus , Figure 5C shows how variation in Mg-concentration at 37°C has a dramatic effect on ejection dynamics from phage λ by switching from all synchronized to nearly all desynchronized ejection events .", "Consequentially , at Mg-concentrations above that of physiologic optimum for infection ( ∼5–10 mM ) , DNA ejection dynamics is significantly reduced with an order of magnitude slower average ejection time compared to that of a fast population with synchronized ejections .", "Different bacteriophage traits determine the dynamics and heterogeneity of viral populations .", "Investigating these traits is of fundamental importance for understanding viral replication pathways and for improving the potency of treatments and viral vaccines ( Lee et al . , 1997 ) , and studying viral fitness ( Goldhill and Turner , 2014 ) .", "Structural virology has been instrumental in providing a detailed morphological description and classification of viruses and their receptors required for studies of traits influencing virus-host interaction dynamics ( Gallet et al . , 2011 ) .", "However , most of the structural studies have been focused on viral capsids due to their symmetry , rather than on the encapsidated genome where the lack of symmetry prevents high-resolution analysis .", "Recent findings of pressure-driven viral DNA ejection into cells from phage ( Evilevitch et al . , 2003; Castelnovo and Evilevitch , 2007 ) , Archaeal viruses ( Hanhijärvi et al . , 2013 ) and Herpesviruses ( Bauer et al . , 2013 ) , have raised strong interest in understanding the role that packaged dsDNA structure plays in dynamics of genome ejection and viral replication .", "However , the structure of the encapsidated genome and its mechanical properties have until now not been sufficiently investigated nor considered as a trait influencing viral population dynamics .", "While this work is an in vitro study , it suggests a novel mechanism affecting viral replication dynamics that can influence the cell’s decision between lytic and lysogenic infection , prompting further investigations in vivo .", "In this work , we discovered , for the first time , a direct link between the structure ( and its associated mobility ) of the encapsidated dsDNA in phage λ and dynamics of initiation of its release from the capsid .", "We demonstrate that the solid-to-fluid-like intracapsid DNA transition ( Liu et al . , 2014 ) , is a determining factor for either rapid synchronized or slow desynchronized ejection dynamics from a phage population .", "We show that at optimum temperature for infection ( i . e . ∼37°C ) and Mg-concentration ≤ 10 mM , the encapsidated DNA is in a fluid-like state and ejection events from the majority of the phage population are synchronized with DNA ejections occurring within ∼10 s directly after phage adsorption to E . coli receptors ( this time corresponds to DNA translocation time from a capsid ) .", "However , deviations from these conditions for infection ( e . g . low temperature , high Mg-concentration ) result in intracapsid DNA being in a solid-like state where DNA is arrested in different conformations due to high interstrand sliding friction ( Berndsen et al . , 2014; Liu et al . , 2014 ) .", "This leads to a striking heterogeneity in phage ejection dynamics , displaying coexistence of populations with synchronized ejection events ( where DNA is in a fluid-like state ) and desynchronized stochastically occurring ejection events ( where DNA is in a solid-like state ) .", "The latter presents one to two orders-of-magnitude slower ejection dynamics , ranging from minutes to tens of minutes , depending on salt conditions and temperature , compared to DNA translocation time from a capsid .", "This ejection dynamics behavior is in good agreement with earlier observations of temperature’s effect on DNA injection dynamics from phage λ after preadsorption to E . coli culture ( Mackay and Bode , 1976 ) .", "Our findings also dismiss an earlier claim that stochasticity of DNA ejections from phage is solely controlled by capsid portal opening dynamics ( Raspaud et al . , 2007; Frutos et al . , 2016 ) .", "These assumptions were based on earlier LS measurements of phage ejection dynamics that could not distinguish between synchronized and desynchronized ejection events , as we have demonstrated above .", "( The same reference [Frutos et al . , 2016] ) also incorrectly concludes that there is no intracapsid DNA transition in phageλ with increasing temperature .", "As described above , we have clearly demonstrated that solid-to-fluid like DNA transition occurs as a function of salt and temperature , signified by an abrupt change in mobility ( measured with AFM nanoindentation ) and internal energy ( measured with ITC ) of the packaged λ-genome ( Li et al . , 2015; Liu et al . , 2014 ) .", "As discussed above , it was recently proposed that the lysogeny-lytic cell decision initially occurs at the phage level through the timing of DNA delivery from multiple phages into a cell during infection ( Zeng et al . , 2010; Trinh et al . , 2017 ) .", "The timing of DNA injection from phage is what determines the onset of viral genome replication because both phage DNA injection and DNA replication processes occur on comparable timescales ( Mackay and Bode , 1976 ) .", "In turn , replication dynamics of injected phage genomes determines how phages interact with one another ( Trinh et al . , 2017 ) when multiple phage particles are infecting a single cell .", "It was shown that lysogeny is more likely to occur when infecting phages interact cooperatively during cell infection ( Kourilsky , 1973 ) .", "Synchronized injections result in immediate presence of DNA from multiple phages in a cell that can be integrated into cell’s chromosome without competition for the progeny ( Zeng et al . , 2010; Trinh et al . , 2017 ) .", "Lysogeny results in the absence of selection , which allows various mutants to integrate and persist in the lysogenic state , leading to diversification of phage genes and adaptation to poor or new growth conditions ( Zeng et al . , 2010; Trinh et al . , 2017 ) .", "On the contrary , when DNA injections are desynchronized , few phage particles are delivering their genes faster than the rest of the phage population , resulting in competitive interactions .", "If first phage to deliver its genome is lytic , it will override lysogeny , rapidly replicate itself using the cell’s limited resources and therefore take a larger share of phage progeny .", "As mentioned above , the asynchronous infection delays decrease the chance of lysogeny by also lowering CII viral transcription factor levels in the cell , where CII controls cell’s decision ( Cortes et al . , 2017 ) .", "It was shown that the cutoff time , where delayed phage infections of one cell no longer affect the lytic-lysogenic cell decision varies from few minutes to tens of minutes , depending on the MOI ( higher MOI results in shorter cutoff time ) ( Cortes et al . , 2017 ) , comparable to timescales for overall ejection delays observed in our measurements .", "This suggests that ejection delays resulting from solid-like DNA state in the capsid will incease cell lysis probability .", "The lytic-lysogenic decision is one of the most crucial factors determining virus population dynamics .", "In general , it was observed that limited conditions for phage replication favor lysogeny ( Zeng et al . , 2010; Kourilsky , 1973 ) .", "This is explained by the fact that lysis is metabolically more ‘expensive’ and therefore would not be successful if the cells are not sufficiently healthy .", "Here , we found that ionic composition in the surrounding medium of bacterial cells acts as an on-off switch for DNA structural transition in λ-capsids leading to either synchronized or desynchronized ejection events at the optimum temperature for infection ( 37°C ) .", "It was previously shown that starvation of Mg2+ stimulates lysogenization ( Kourilsky , 1973 ) .", "Our study shows that , at low Mg-concentration , intracapsid DNA stress is high , leading to a DNA transition from solid- to fluid-like at temperatures well below that of infection temperature ( 37°C ) .", "This DNA transition results in synchronized ejections at 37°C , which indeed stimulate lysogenization , as we have discussed immediately above .", "Remarkably , both of these in vitro and in vivo ( Kourilsky , 1973 ) observations are in good agreement , suggesting that DNA injection dynamics from phage into cells may play a significant role in the cell fate decision .", "However , while the probability of lysogeny depends inversely on phage infection delays ( Cortes et al . , 2017 ) , the infection timings alone cannot be used to predict the outcome of either lysogenic or lytic behavior since other factors ( e . g . cell volume [St-Pierre and Endy , 2008] ) have also been shown to play a role .", "Furthermore , we have limited understanding of how many host functions ( e . g . HflKC , cAMP , etc ) feed into the decision ( Casjens and Hendrix , 2015 ) .", "Mapping all host functions into a single axis of rich/poor conditions is not supported by experiments .", "Thus , further interpretation of the effect of desynchronized and synchronized infections on the cell decision process would require a new model for circuit dynamics .", "In conclusion , these in vitro findings suggest a new paradigm affecting virus population dynamics—mechano-signaling mediated by packaged DNA structure in phage λ .", "The environmental factors regulating the mechanical transition between solid- and fluid-like intracapsid DNA states strongly influence the timing of genome ejections .", "In vivo , desynchronized ejections from a phage population lead to competition between phage genomes , affecting the rate of phage gene replication and transcription as well as the lysogeny-lytic cell decision .", "Given similarities between phageλ and Herpesviruses in their mechanism of pressure-driven DNA ejection ( where a temperature-induced solid-to-fluid like DNA transition was observed in both viral systems [Sae-Ueng et al . , 2014; Bauer et al . , 2013] ) , the new insights into the lysogeny-lytic decision process for phage provide intriguing prospects for understanding of latency mechanism in Herpesviruses .", "Indeed , analogies between phage and Herpesviruses in other regulatory factors that affect viral latency have been previously observed ( Pai and Weinberger , 2017; Weller and Sawitzke , 2014 ) ." ], [ "Following previous protocols ( Evilevitch et al . , 2003 ) , phage λ strain λ cI857 with a wild type ( wt- ) genome length of 48 . 5 kbp were produced by thermal induction of lysogenic Escherichia coli ( E . coli ) strain AE1 derived from S2773 strain .", "After cell harvest , phage particles were precipitated in 10% polyethylene-glycol ( PEG ) 8000 and purified by CsCl equilibrium centrifugation .", "Later on phages were dialyzed against TM-MgSO4 buffer ( 10 mM MgSO4 , 50 mM Tris HCl , pH7 . 4 ) or TM-MgCl2 ( 10 mM MgCl2 , 50 mM Tris HCl , pH7 . 4 ) for overnight at 4°C .", "The receptor protein LamB was expressed and purified from pop-154 , an E . coli K12 strain in which the LamB gene was transduced from Shigella sonnei 3070 .", "The detailed preparation method was described earlier ( Ivanovska et al . , 2007 ) .", "Isothermal titration calorimetry ( ITC ) was used to measure the ejection enthalpy of λ genome , ΔHej .", "All ITC measurements in this study were performed using the MicroCal iTC200 system manufactured by Malvern .", "The principles of measuring DNA ejection enthalpy in phages λ were described before ( Jeembaeva et al . , 2010 ) .", "In ITC experiment , 2 . 69 µL λ particles at 1012 - 1013 pfu/mL ( 10 – 100 nM ) were titrated into 200 µL LamB solution in the sample cell ( reference cell was always filled with MilliQ water ) .", "LamB is at a concentration of 0 . 2 - 0 . 5 mg/mL ( 1 . 4 - 3 . 5 µM ) .", "The molar ratio between LamB trimmers and λ particles in the sample cell is always kept above 104:1 ( up to 3 titrations ) to ensure the maximum ejection efficiency of λ with no delay time .", "Both LamB and λ particles were in the same dilution buffer containing TM ( 10mM MgCl2 or MgSO4 , as indicated , 50mM Tris , pH 7 . 4 ) and 1% oPOE .", "The dilution heat of phage particles , LamB particles were measured separately and excluded from the final value of ΔHej ( Jeembaeva et al . , 2010 ) .", "The ejection enthalpy , ΔHej is a good estimate of the internal energy of λ genome , Uencap_DNA .", "In an ITC experiment , ΔHej is measured as an enthalpy change associated with the genome release process , ΔH=ΔU+pΔV , where ΔU is the change in internal energy of the system during DNA ejection ( ΔU=Uempty_cap−Ufilledcap=Uencap_DNA ) , p is the atmospheric pressure acting on the system and ΔV is the change in volume of the solution surrounding the phages during DNA release .", "Since this volume change in solution , ΔV is negligible , ΔH≈ΔU .", "Time-resolved small angle x-ray scattering ( SAXS ) measurements were carried out at the 12-ID B station at the Advanced Photon Source ( APS ) at Argonne National Laboratory .", "A 12KeV x-ray beam was used to illuminate the sample with a short sample-to-detector distance ( 1 m ) and an overall q range from 0 . 013 to 1 . 90 Å−1 .", "To monitor the DNA ejection process , we mixed 60 µL of phage solution with 120 µL of LamB solution in a three-way static mixing tee , the mixture was then sent to a flow-through glass capillary perpendicular to the incident x-ray .", "The mixing ratio between phage and LamB was kept as 1 to 200 .", "To reduce photon damage , the sample solution was oscillating during the SAXS measurement with a flow rate of 10 μL/s .", "The SAXS pattern was recorded every 2 to 10 s with an x-ray exposure time of 1 s .", "After converting the 2D SAXS pattern to 1D Intensity versus q plot , the DNA peak between 0 . 18 to 0 . 32 Å−1 was truncated and analyzed .", "Since LamB and 1% oPOE micelles also contribute to the overall scattering intensity , it is important to properly subtract their intensity before analyzing the DNA peak .", "The SAXS profiles for TM buffer , 1% oPOE in TM buffer , undiluted LamB in 1% oPOE TM buffer were collected at different temperatures .", "The background was constructed using a linear combination of TM buffer , 1% oPOE and LamB .", "After background subtraction , the DNA peak was fitted to a Gaussian plus a linear function .", "Detailes for DNA peak fitting procedure can be found in our previous publications ( Liu et al . , 2014 ) .", "Solution SAXS provides structural information that reveals base pair ( bp ) ordering and average DNA-DNA spacing of the encapsidated genome ( Earnshaw and Harrison , 1977; Liu et al . , 2014 ) .", "However , time-resolved SAXS measurements of DNA structure in phage capsids during the LamB-triggered DNA ejection process can directly reflect the ejection dynamics .", "Figure 3—figure supplement 1A shows integrated scattering intensity , I , versus scattering vector q for wt DNA phage λ particles .", "In the lower q region ( 0 . 007 to 0 . 1 Å−1 ) , the scattering profile originates from the highly symmetrical icosahedral phage capsids .", "At higher q ( between 0 . 2 to 0 . 3 Å−1 ) , the single peak with the small oscillating ripples on its top is due to the diffraction from the encapsidated ordered DNA strands .", "The short-range DNA-DNA interaxial spacings determine the position of the DNA diffraction peak ( Earnshaw and Harrison , 1977 ) .", "The area of DNA peak indicates how well the DNA strands are aligned relative to each other since it is directly proportional to the total number of ordered DNA bp in the capsid ( Earnshaw and Harrison , 1977; Liu et al . , 2014 ) .", "During DNA ejection from phage , DNA diffraction peak area is therefore progressively decreasing due to reduced bp ordering resulting from less coherent diffraction .", "( The position of the peak should then also gradually shift to lower q values due to increasing distance between packaged DNA strands ) .", "To investigate DNA ejection dynamics’ dependence on temperature , we measured DNA diffraction peak area versus time when phage λ is instantly mixed with LamB in a stopped-flow SAXS chamber .", "This reflects the number of remaining DNA-filled phage particles over time .", "As discussed above , DNA ejection dynamics was previously evaluated by measuring total forward scattering intensity over time , I ( 0 ) , using LS ( Figure 2A in the main text ) ( Freeman et al . , 2016 ) since I ( 0 ) depends on density of DNA packaged in the capsid .", "However , the measured decay in I ( 0 ) versus time also depends on diffusive relaxation time of DNA coils that initially remain condensed in solution immediately after ejection ( due to DNA ejection rate being faster than DNA diffusive relaxation in bulk solution ) ( Freeman et al . , 2016; Löf et al . , 2007 ) .", "One of the advantages of following genome ejection dynamics by using SAXS to measure the decrease in DNA diffraction peak area ( instead of measuring change in forward scattering intensity ) , is in the reduced signal contribution from this secondary process of slow diffusion-relaxation dynamics of ejected DNA coils .", "This is due to the fact that DNA diffraction peak area depends mainly on bp ordering with defined DNA-DNA spacing inside the capsid ( bp ordering is mainly lost once DNA is ejected ) , as opposed to forward scattering intensity reflecting condensed DNA density ( rather than ordering ) both inside and outside the capsid .", "The difference in measured DNA ejection dynamics determined by LS ( forward scattering I ( 0 ) ) , SAXS ( forward scattering I ( 0 ) ) and decay in SAXS-measured DNA diffraction area is illustrated in Figure 1 in the main text ( a comparison is also made for derivatives of these three types of data compared with ITC DP curve in Figure 1 inset ) .", "Figure 3—figure supplement 1B shows time-resolved DNA diffraction peak for the DNA ejection process from wt DNA length phageλ in TM-buffer at 25°C ( below the DNA transition temperature of ∼33°C ) .", "When the majority of phage particles have ejected their genomes , the DNA diffraction peak disappears due to lack of ordered DNA .", "As we observed with ITC-measured dynamics above , the decay time for DNA diffraction peak area at 25°C is much longer than the time for DNA translocation from a single phage particle ( ∼10 s ) , suggesting that DNA ejection events are occurring in both a synchronized and desynchronized manner .", "Thus , on the timescale of this measurement , the positon of DNA diffraction peak remains essentially unchanged with time since it mainly shows the decay in number of completely DNA-filled phage capsids .", "In order to estimate the energy of activation for phage populations with synchronized and desynchronized ejections , we analyzed the Arrhenius’ dependence of the rate of ejection events on temperature .", "The natural log of DNA diffraction peak area was plotted versus time in Figure 3—figure supplement 1C for the DNA ejection process at 25 , 30 , and 37°C .", "Interestingly , inset in Figure 3—figure supplement 1C shows that , during the first ∼20 s , the ejection rate has almost no temperature dependence .", "However , at longer times , strong temperature dependence is observed ( Figure 3—figure supplement 1C ) .", "As we found with ITC in Figure 3 in the main text , the first 20 s of the DNA ejection process are dominated by synchronized ejection dynamics from a phage population with intracapsid DNA in a fluid-like state .", "The lack of SAXS-measured ejection rate dependence on temperature during the first 20 s suggests that ejection dynamics initially does not display a measurable activation energy barrier for DNA ejection once the capsid portal is opened through receptor adsorption .", "Such a scenario is plausible , since packaged DNA is in a fluid-like state , where it has low interstrand friction leading to low activation energy required to initiate ejection .", "This also suggests that activation energy for portal plug opening in the capsid is relatively small , being strongly reduced when LamB binds to the phage tail , as we previously observed ( Freeman et al . , 2016; Bauer et al . , 2015 ) .", "However , at time > 20 s , strong temperature dependence is observed in SAXS-measured DNA ejection rate ( Figure 3—figure supplement 1C ) , suggesting measurable energy of activation is required for initiation of DNA ejection from phage .", "In agreement with this observation , we found with ITC ( Figure 3 in the main text ) that , at > 20 s , DNA ejection events are desynchronized below DNA transition temperature ( ∼33°C ) , corresponding to the second exothermic DP peak originating from a phage population with DNA packaged in a solid-like state .", "These observations suggest that measured activation energy is mainly associated with DNA-DNA friction that must be overcome to initiate genome ejection from the capsid ( Gabashvili and Grosberg , 1992; Berndsen et al . , 2014 ) .", "To quantify energies of activation for a phage population with DNA in solid-like and fluid-like states , in Figure 3—figure supplement 1D we plotted natural logarithms of the ejection rate constants ( k ) ( corresponding to the slopes of the linear fits in Figure 3—figure supplement 1C ) as a function of inverse temperature .", "In order to separate the rates for synchronized and desynchronized ejection dynamics , the rate constants in Figure 3—figure supplement 1D are plotted separately for the fast population at time interval < 20 s , and for the slow population at times > 20 s .", "The ejection rates for the slow population ejection dynamics ( > 20 s ) show exponential rate dependence on inverse temperature ( i . e . Arrhenius dependence ) , evidenced by the linearity of the fits in Figure 3—figure supplement 1D .", "Energy of activation derived from the linear fit for the slow population is ∼1 . 2 × 10−19 J/virion corresponding to ∼29 kT at 25°C .", "Thus , activation energy to initiate ejection from phage λ with intracapsid DNA in a solid-like state is relatively high ( ∼20 times higher than molecular thermal energy ) due to a significant DNA-DNA sliding friction during initiation of the ejection process when interstrand separation is small ( Berndsen et al . , 2014; Liu et al . , 2014 ) .", "This in turn leads to stochastic delays in ejection events or even complete arrest of the ejection process at lower temperatures , as has been previously observed in vivo ( Mackay and Bode , 1976 ) ( e . g . no ejection occurs when phage λ is preadsorbed to E . coli cells at 4°C ) .", "Figure 3—figure supplement 1D also shows that , for a fast population with synchronized ejection dynamics and intracapsid DNA in a fluid-like state ( at times < 20 s ) , the ejection rate dependence on inverse of temperature shows essentially no or little temperature dependence .", "It can be noted here that slight temperature dependence of DNA ejection rate below 20 s can be attributed to activation energy associated with portal opening ( Freeman et al . , 2016 ) .", "However , for a slow population with desynchronized ejection dynamics ( where intracapsid DNA is in a solid-like state ) , this is not the dominant component of activation energy ( Bauer and Evilevitch , 2015 ) , since the main contribution comes from DNA-DNA sliding friction , as suggested by this data .", "Some ITC titration curves ( DP versus time ) in our study exhibit bimodal population dynamics with not fully resolved exothermic titration peaks .", "We can mathematically deconvolute the experimental curve into two asymmetric Gaussian functions with an exponential damping function .", "These Gaussians were termed as Exponentially Modified Gaussian distribution ( EMG ) described by a2b⋅exp⁡ ( c22b2+d−tb ) ⋅[erf ( t−dc2−c2b ) +1] ( Grushka , 1972 ) .", "This deconvolution method was previously described as high precision method ( Goodman and Brenna , 1994 ) .", "Equation parameter a denotes the peak area from the deconvoluted Gaussian peak , b denotes the exponential damping term ( exponent relaxation time ) , with b = 0 corresponding to a symmetric Gaussian curve .", "c indicates the width of the Gaussian , which provides the error bars in our figures .", "d is the position of an individual Gaussian peak .", "The estimated population mean of EMG is shifted slightly from the peak position .", "It is the peak position plus the exponential relaxation time μ = d + b .", "The population standard derivation is s2 = c2 + b2 ( Grushka , 1972 ) .", "Skewness of the distribution is defined as γ = 2b3 ( c2+b2 ) 3/2 .", "The median of the distribution equals to μ - 1/3sγ ( according to Pearson’s second skewness law ) .", "ITC measured exothermic titration peaks can be nicely fit with asymmetric Gaussian functions .", "As explained in the manuscript , the first exothermic DP peak results from synchronized DNA ejections for a phage population .", "The shape of this peak will depend on enthalpy change associated with simultaneous DNA translocation from multiple phage particles .", "It has been shown that DNA translocation speed increases with ejection time at the beginning due to a decrease in the DNA-DNA frictional force as DNA packing density decreases in the capsid ( Grayson et al . , 2007 ) .", "However , the translocation speed starts to decrease towards the end of the ejection process due to decrease in the DNA pressure driving its ejection from the capsid .", "This variation in DNA translocation rate is reflected by the rate of internal energy dissipation ( heat flow measured by ITC ) during the ejection process which in turn is fitted by an asymmetric Gaussian function .", "The asymmetric Gaussian shape of the second exothermic DP peak on the titration curves , corresponding to stochastic DNA ejection events results likely from both distribution of delayed particle ejection modes and non-uniform rates of DNA translocation ." ] ]

[ "The cell decision between lytic and lysogenic infection is strongly influenced by dynamics of DNA injection into a cell from a phage population , as phages compete for limited resources and progeny .", "However , what controls the timing of viral DNA ejection events was not understood .", "This in vitro study reveals that DNA ejection dynamics for phages can be synchronized ( occurring within seconds ) or desynchronized ( displaying minutes-long delays in initiation ) based on mobility of encapsidated DNA , which in turn is regulated by environmental factors , such as temperature and extra-cellular ionic conditions .", "This mechano-regulation of ejection dynamics is suggested to influence viral replication where the cell’s decision between lytic and latent infection is associated with synchronized or desynchronized delayed ejection events from phage population adsorbed to a cell .", "Our findings are of significant importance for understanding regulatory mechanisms of latency in phage and Herpesviruses , where encapsidated DNA undergoes a similar mechanical transition ." ]

[ "Viruses are tiny ‘parasites’ that smuggle their genetic material inside a cell and then hijack its resources for their own benefit .", "A viral infection can either be lytic or latent .", "In a lytic cycle , viruses make their host produce many copies of themselves , ultimately killing the cell .", "In contrast , during a latent infection , the viruses go ‘dormant’: for instance , some of them can insert their genetic material into the DNA of their host , which then gets passed on as the cell divides .", "Certain viruses are capable of both lytic and latent infections .", "One example is the lambda phage , which targets Escherichia coli bacteria .", "In the first stage of infection , the genetic material ‘shoots out’ of the virus and gets injected inside the bacterium .", "The dynamics of the ejection process determine the type of infection that will follow .", "If multiple phages release their genomes quickly and within seconds of each other into the same cell , the bacterium tends to incorporate the viral DNA into its own genome , leading to a latent cycle .", "If the infections take place more slowly and not all at the same time , the cell is more likely to go through a lytic phase .", "However , the mechanism behind the different injection behaviors is still unknown; in particular , it is unclear which factors control the specificities of the ejection process in the first place .", "Here , Alex Evilevitch demonstrates that the mechanical state of the phage DNA just before ejection dictates how the genetic material will then be injected in the bacteria .", "The experiments measured the stiffness of the DNA and the amount of heat given off during infection .", "Like fluid toothpaste , if the DNA is more liquid and flexible , it gets ejected quickly and simultaneously from several phages .", "Then , the genetic information of these viruses can be incorporated in the genome of the bacteria .", "On the other hand , if the DNA is more solid , it is likely to ‘stick’ and take time before it can be squeezed out: the injections become unsynchronised , which leads to a lytic phase .", "Evilevitch then shows that the environment can influence the properties of the phages’ genome .", "A little more heat , or certain chemicals , can make the DNA more fluid inside the viruses , and change the way it can be injected inside the bacteria .", "Many viruses that cause diseases in humans – from cold sores to glandular fever – can switch between the lytic and latent cycles .", "For the first time , these results show that the mechanical properties of the DNA inside a virus influence the ‘decision’ between the two types of infection .", "This knowledge could help us prevent infections from becoming lytic and ultimately allow us to control the spread of disease ." ]

[ "Introduction", "Smoothness and the neural code", "Model", "Deep learning networks", "Discussion" ]

[ "neuroscience" ]

[ [ "Neuroimaging and especially functional magnetic resonance imaging ( fMRI ) has come a long way since the first experiments in the early 1990s .", "These impressive findings are curious in light of fMRI’s limitations .", "The blood-oxygen-level-dependent ( BOLD ) response measured by fMRI is a noisy and indirect measure of neural activity ( Logothetis , 2002 , 2008; O'Herron et al . , 2016 ) from which researchers try to infer neural function .", "The BOLD response trails neural activity by 2 s , peaks at 5 to 6 s , and returns to baseline around 10 s , whereas neural activity occurs on the order of milliseconds and can be brief ( Huettel et al . , 2009 ) .", "In terms of spatial resolution , the BOLD response may spill over millimeters away from neural activity due to contributions from venous signals ( Turner , 2002 ) .", "Likewise , differences in BOLD response can arise from incidental differences in the vascular properties of brain regions ( Ances et al . , 2009 ) .", "Such sources of noise can potentially imply differences in neural activity in regions where there should not be .", "The data acquisition process itself places limits on fMRI measurement .", "Motion artefacts ( e . g . , head movements by human subjects ) and non-uniformity in the magnetic field reduce data quality .", "In analysis , three-dimensional images are constructed from slices acquired at slightly different times .", "Once collected , fMRI data are typically smoothed during analyses ( Carp , 2012 ) .", "All these factors place limits on what fMRI can measure .", "Despite these weaknesses , fMRI has proved to be an incredibly useful tool .", "For example , we now know that basic cognitive processes involved in language ( Binder et al . , 1997 ) and working memory ( Pessoa et al . , 2002 ) are distributed throughout the cortex .", "Such findings challenged notions that cognitive faculties are in a one-to-one correspondence with brain regions .", "Advances in data analysis have increased what can be inferred by fMRI ( De Martino et al . , 2008 ) .", "One of these advances is multivariate pattern analysis ( MVPA ) , which decodes a pattern of neural activity in order to assess the information contained within ( Cox and Savoy , 2003 ) .", "Rather than computing univariate statistical contrasts , such as comparing overall BOLD activity for a region when a face or house stimulus is shown , MVPA takes voxel patterns into account .", "Using MVPA , so-called ‘mind reading’ can be carried out — specific brain states can be decoded given fMRI activity ( Norman et al . , 2006 ) , revealing cortical representation and organization in impressive detail .", "For example , using these analysis techniques paired with fMRI we can know whether a participant is being deceitful in a game ( Davatzikos et al . , 2005 ) , and we can determine whether a participant is reading an ambiguous sentence as well as infer the semantic category of a word they are reading ( Mitchell et al . , 2004 ) .", "Representational similarity analysis ( RSA ) , another multivariate technique , is particularly suited to examining representational structure ( Kriegeskorte et al . , 2008; Kriegeskorte , 2009 ) .", "We will focus on RSA later in this contribution , so we will consider this technique in some detail .", "RSA directly compares the similarity ( e . g . , by using correlation ) of brain activity arising from the presentation of different stimuli .", "For example , the neural activity arising from viewing a robin and sparrow may be more similar to each other than between a robin and a penguin .", "These pairwise neural similarities can be compared to those predicted by a particular theoretical model to determine correspondences .", "For example , Mack et al . ( 2013 ) identified brain regions where the neural similarity structure corresponded to that of a cognitive model of human categorization , which was useful in inferring the function of various brain regions .", "The neural similarities themselves can be visualized by applying multidimensional scaling to further understand the properties of the space ( Davis et al . , 2014 ) .", "RSA has been useful in a number of other endeavors , such as understanding the role of various brain areas in reinstating past experiences ( Tompary et al . , 2016; Mack and Preston , 2016 ) .", "Given fMRI’s limitations in measuring neural activity , one might ask how it is possible for methods like RSA to be successful .", "The BOLD response is temporally and spatially imprecise , yet it appears that researchers can infer general properties of neural representations that link sensibly to stimulus and behavior .", "The neural code must have certain properties for this state of affairs to hold .", "What kinds of models or computations are consistent with the success of fMRI ?", "If the brain is a computing device , it would have to be of a particular type for fMRI to be useful given its limitations in measuring neural activity ." ], [ "The BOLD response summates neural activity over space and time , which places hard limits on what fMRI can measure .", "To make an analogy , 3+5 and 6+2 both equal 8 through different routes .", "If different ‘routes’ of neural activity are consequential to the neural code and are summated in the BOLD response , then fMRI will be blind to representational differences .", "To capture representational differences , voxel response must be inhomogeneous both between voxels and within a voxel across time .", "Consider the fMRI analogues shown in Figure 1; paralleling neurons with pixels and voxels with the squares on the superimposed grid .", "The top-left image depicts neural activity that smoothly varies such that the transitions from red to yellow occur in progressive increments .", "Summating within a square , i . e . , a voxel , will not dramatically alter the high-level view of a smooth transition from red to yellow ( bottom-left image ) .", "Voxel response is inhomogeneous , which would allow decoding by fMRI ( cf . Kamitani and Tong , 2005; Alink et al . , 2013 ) .", "Altering the grid ( i . e . , voxel ) size will not have a dramatic impact on the results as long as the square does not become so large as to subsume most of the pixels ( i . e . , neurons ) .", "This result is in line with basic concepts from information theory , such as the Nyquist-Shannon sampling theorem .", "The key is that the red and yellow pixels/neurons are topologically organized: their relationship to each other is for all intents and purposes invariant to the granularity of the squares/voxels ( for more details see: Chaimow et al . , 2011; Freeman et al . , 2011; Swisher et al . , 2010 ) . 10 . 7554/eLife . 21397 . 003Figure 1 . The activity of neurons in the top-left panel gradually changes from left to right , whereas changes are more abrupt in the top-middle and top-right panels . Each square in the grid represents a voxel which summates activity within its frame as shown in the bottom panels .", "For the smoother pattern of neural activity , the summation of each voxel ( bottom left ) captures the changing gradient from left to right depicted in the top-left , whereas for the less smooth representation in the middle panel all voxels sum to the same orange value ( bottom middle ) .", "Thus , differences in activation of yellow vs . red neurons are detectable using fMRI for the smooth case , but not for the less smooth case because voxel response is homogenous .", "Improving spatial resolution ( right panels ) by reducing voxel size overcomes these sampling limits , resulting in voxel inhomogeneity ( bottom-right panel ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 21397 . 003 In contrast , the center-top image in Figure 1 involves dramatic representational changes within voxel .", "Each voxel ( square in the grid ) , in this case , will produce a homogenous orange color when its contents are summated .", "Thus , summating the contents of a voxel in this case obliterates the representational content: red and yellow; returning instead squares/voxels that are all the same uniform color: orange .", "This failure is due to sampling limits that could be addressed by smaller voxels ( see rightmost column ) .", "Unfortunately , arbitrarily small voxels with high sampling rates is not a luxury afforded to fMRI .", "The success of fMRI given its sampling limits is consistent with proposed neural coding schemes , such as population coding ( Averbeck et al . , 2006; Panzeri et al . , 2015; Pouget et al . , 2000 ) in cases where neurons with similar tunings spatially cluster ( e . g . , Maunsell and Van Essen , 1983 ) .", "In population coding , neurons jointly contribute to represent a stimulus in much the same way as pixels were contributing to represent different colors in the leftmost column of Figure 1 .", "When this inhomogeneity breaks down , similarity structures should be difficult to recover using fMRI .", "Indeed , a recent study with macaque monkeys which considered both single-cell and fMRI measures supports this viewpoint — stimulus aspects which were poorly spatially clustered in terms of single cell selectivity were harder to decode from the BOLD response ( Dubois et al . , 2015 ) .", "The same principles extend from the spatial to the temporal domain .", "The BOLD response will be blind to the differences between representations to the extent that the brain relies on the precise timing of neural activity to code information .", "For example , in burstiness coding , neural representations are distinguished from one another not by their average firing rate but by the variance of their activity ( Fano , 1947; Katz , 1996 ) .", "Under this coding scheme , more intense stimulus values are represented by burstier units , not units that fire more overall .", "Neural similarity is not recoverable by fMRI under a burstiness coding scheme .", "Because the BOLD signal roughly summates through time ( Boynton et al . , 1996 ) , firing events will sum together to the same number irrespective of their burstiness .", "Likewise , BOLD activity may be a composite of synchronized activity at multiple frequencies .", "Although gamma-band local field potential is most associated with BOLD response , oscillations at other frequency bands may also contribute to the BOLD response ( Magri et al . , 2012; Scheeringa et al . , 2011 ) .", "If so , fMRI would fail to distinguish between representational states that are differentiated by the balance of contributions across bands , much like the arithmetic example at the start of this subsection in which different addends yield the same sum .", "As before , basic concepts in information theory , such as the Nyquist-Shannon sampling theorem , imply that temporally demanding coding schemes will be invisible to fMRI ( cf . Nevado et al . , 2004 ) .", "The success of fMRI does not imply that the brain does not utilize precise timing information , but it does mean that such temporally demanding coding schemes cannot be the full story given fMRI’s successes in revealing neural representations .", "Instead , the neural code must include in its mixture at least some coding schemes that are consistent with fMRI's successes .", "For example , rate coding ( Adrian and Zotterman , 1926 ) in which the frequency at which neurons fire is a function of the intensity of a stimulus is consistent with the success of fMRI because changes in firing rate for a population of cells should be recoverable by fMRI as more blood flows to more active cells ( O'Herron et al . , 2016 ) .", "These examples make clear that the neural code must be somewhat spatially and temporally smooth with respect to neural activity ( which is several orders of magnitude smaller than voxels ) for fMRI to be successful .", "Whatever is happening in the roughly one million neurons within a voxel ( Huettel et al . , 2009 ) through time is being partially reflected by the BOLD summation , which would not be the case if each neuron was computing something dramatically different for in-depth discussion , see: ( Kriegeskorte et al . , 2010 ) .", "One general conclusion is that important aspects of the neural code are spatially and temporally smooth .", "In a sense , this notion of smoothness is trivial as it merely implies that changes in neural activity must be visible in the BOLD response ( i . e . , across-voxel inhomogeneity ) for fMRI to be successful .", "In this section , we focus on a more subtle sense of smoothness that must also be satisfied , namely functional smoothness .", "Neighboring voxels predominantly contain similar representations ( Norman et al . , 2006 ) , i . e . , they are topologically organized like in Figure 1 .", "However , super-voxel smoothness is neither necessary nor sufficient for fMRI to succeed in recovering similarity structure .", "Instead , a more general notion of functional smoothness must be satisfied in which similar stimuli map to similar internal representations .", "Although super-voxel and functional smoothness are both specified at the super-voxel level , these distinct concepts should not be confused .", "A function f that maps from some input x to output y is functionally smooth if and only if ( 1 ) sim ( x1 , x2 ) ∝sim ( y1 , y2 ) , where f⁢ ( x1 ) =y1 and f⁢ ( x2 ) =y2 .", "For example , x and y could be the beta estimates for voxels in two brain regions and sim could be Pearson correlation .", "To measure functional smoothness , the degree of proportionality between all possible similarity pairs sim ( x1 , x2 ) and sim ( y1 , y2 ) also could be assessed by Pearson correlation ( i . e . , does the similarity between a y1 and y2 pair increase as the similarity increases between the corresponding x1 and x2 pair ) .", "By definition , functional smoothness needs to be preserved in the neural code for fMRI to recover similarity correspondences ( as in RSA ) , whether these correspondences are between stimuli ( e . g . , xs ) and neural activity ( e . g . , ys ) , multiple brain regions ( e . g . , xs and ys ) , or model measure ( e . g . , xs ) and some brain region ( e . g . , ys ) .", "From this definition , it should be clear that functional smoothness is distinct from super-voxel smoothness .", "For example , a brain area that showed smooth activity patterns across voxels for each individual face stimulus but whose activity did not reflect the similarity structure of the stimuli would be super-voxel smooth , but not functionally smooth with respect to the stimulus set .", "Conversely , a later section of this contribution discusses how neural networks with random weights are functionally but not super-voxel smooth .", "To help introduce the concept of functional smoothness , we consider two coding schemes used in engineering applications , factorial and hash coding , which are both inconsistent with the success of fMRI because they do not preserve functional smoothness .", "In the next section , we consider coding schemes , such as deep learning networks , that are functionally smooth to varying extents and are consistent with the success of fMRI ." ], [ "The first in this line of models considered is vector space coding ( i . e . , ℝn ) , in which stimuli are represented as a vector of real-valued features .", "Representing concepts in multidimensional spaces has a long and successful history in psychology ( Shepard , 1987 ) .", "For example , in a large space , lions and tigers should be closer to each other than lions and robins because they are more similar .", "The kinds of operations that are naturally done in vector spaces ( e . g . , additions and multiplications ) are particularly well suited to the BOLD response .", "For example , the haemodynamic response to individual stimuli roughly summates across a range of conditions ( Dale and Buckner , 1997 ) and this linearity seems to extend to representational patterns ( Reddy et al . , 2009 ) .", "In this neural coding scheme , each item ( e . g . , a dog ) is represented as the set of values in its input vector ( i . e . , a set of numbers with range [-1 , 1] ) .", "This means that for a given stimulus , the representation this model produces is identical to the input .", "In this sense , vector space coding is functionally smooth in a trivial sense as the function is identity .", "As shown in Figure 2 , neural similarity gradually falls off with added distortion ( i . e . , noise ) .", "Therefore , this very simple coding scheme creates representational spaces that would be successfully detected by fMRI .", "Building on the basic vector space model , this scheme encodes each input vector by passing it through a monotonic non-linear function , the hyperbolic tangent function ( tanh ) , which is functionally smooth .", "This results in each vector element being transformed , or squashed , to values between [-1 , 1] .", "Such functions are required by artificial neural networks ( and perhaps the brain ) for gain control ( Priebe and Ferster , 2002 ) .", "The practical effect of this model is to push the values in the model’s internal representation toward either -1 or 1 .", "As can be seen in Figure 2 , neural similarity is well-captured by the gain control neural coding model .", "This model performs more sophisticated computations on the input stimuli .", "In line with early connectionism and Rescorla-Wagner modeling of conditioning , this model receives an input vector and performs matrix multiplication on it , i . e . , computes the weighted sums of the inputs to pass on to the output layer ( Knapp and Anderson , 1984; Rescorla and Wagner , 1972 ) .", "These simple one-layer neural networks can be surprisingly powerful and account for a range of complex behavioral findings ( Ramscar et al . , 2013 ) .", "As we will see in later subsections , when a non-linearity is added ( e . g . , tanh ) , one-layer networks can be stacked on one another to build deep networks .", "This neural coding scheme takes an input stimulus ( e . g . , an image of a dog ) and multiplies it by a weight matrix to create an internal representation , as shown in Figure 3 .", "Interestingly , as shown in Figure 3 , the internal representation of this coding scheme is completely nonsensical to the human eye and is not super-voxel smooth , yet it successfully preserves similarity structure ( see Figure 2 ) .", "Matrix multiplication maps similar inputs to similar internal representations .", "In other words , the result is not super-voxel smooth , but it is functionally smooth which we conjecture is critical for fMRI to succeed . 10 . 7554/eLife . 21397 . 006Figure 3 . The effect of matrix multiplication followed by the tanh function on the input stimulus .", "The output of this one-layer network is shown , as well as the outcome of applying a non-linearity to the output of the matrix multiplication .", "In this example , functional smoothness is preserved whereas super-voxel smoothness is not .", "The result of applying this non-linearity can serve as the input to the next layer of a multi-layer network . DOI: http://dx . doi . org/10 . 7554/eLife . 21397 . 006 The preceding coding scheme was a single-layer neural network .", "To create multi-layer networks , that are potentially more powerful than an equivalent single-layer network , a non-linearity ( such as tanh ) must be added to each network layer post-synaptically .", "Here , we consider a single-layer network with the tanh non-linearity included ( see Figure 3 ) .", "As with matrix multiplication previously , this neural coding scheme is successful ( see Figure 2 ) with ‘similar inputs lead[ing] to similar outputs’ ( Rummelhart et al . , 1995 , p . 31 ) .", "The basic network considered in the previous section can be combined with other networks , creating a potentially more powerful multi-layered network .", "These multi-layered models can be used to capture a stream of processing as is thought to occur for visual input to the ventral stream , shown in Figure 2B ( DiCarlo and Cox , 2007; Riesenhuber and Poggio , 1999 , 2000; Quiroga et al . , 2005; Yamins and DiCarlo , 2016 ) .", "In this section , we evaluate whether the similarity preserving properties of single-layer networks extend to deeper , yet still untrained , networks .", "The simulations consider networks with 2 to 8 layers .", "The models operate in a fashion identical to the perceptron neural coding model considered in the previous section .", "The perceptrons are stacked such that the output of a layer serves as the input to the next layer .", "We only perform simulated fMRI on the final layer of each model .", "These simulations consider whether the representations that emerge in multi-layered networks are plausible given the success of fMRI in uncovering similarity spaces see also: ( Cox et al . , 2015; Cowell et al . , 2009; Edelman et al . , 1998; Goldrick , 2008; Laakso and Cottrell , 2000 ) .", "Such representations , as found in deep artificial neural network architectures , are uncovered by adding layers to discover increasingly more abstract commonalities between inputs ( Graves et al . , 2013; Hinton et al . , 2006; Hinton , 2007; Hinton et al . , 2015; LeCun et al . , 2015 ) .", "As shown in Figure 2 , the deeper the network the less clear the similarity structure becomes .", "However , even the deepest network preserves some level of similarity structure .", "In effect , as layers are added , functional smoothness declines such that small perturbations to the initial input result in final-layer representations that tend to lie in arbitrary corners of the representational space , as the output takes on values that are +1 or -1 due to tanh .", "As layers are added , the network becomes potentially more powerful , but less functionally smooth , which makes it less suitable for analysis by fMRI because the similarity space breaks down .", "In other words , two similar stimuli can engender near orthogonal ( i . e . , dissimilar ) representations at the most advanced layers of these networks .", "We measured functional smoothness for a large set of random input vectors using Equation 1 with Pearson correlation serving as both the similarity measure and measure of proportionality .", "Consistent with Figure 2’s results , at layer 1 ( equivalent to the perceptron coding model ) functional smoothness was 0 . 86 , but declined to 0 . 22 by the eighth network layer .", "These values were calculated using all item pairs consisting of a prototype and one of its distortions .", "In the Discussion section , we consider the theoretical significance of these results in tandem with the deep learning network results ( next section ) ." ], [ "The DLN we consider , Inception-v3 GoogLeNet , is a convolutional neural network ( CNN ) , which is a type of DLN especially adept at classification and recognition of visual inputs .", "CNNs excel in computer vision , learning from huge amounts of data .", "For example , human-like accuracy on test sets has been achieved by: LeNet , a pioneering CNN that identifies handwritten digits ( LeCun et al . , 1998 ) ; HMAX , trained to detect objects , e . g . , faces , in cluttered environments ( Serre et al . , 2007 ) ; and AlexNet , which classifies photographs into 1000 categories ( Krizhevsky et al . , 2012 ) .", "The high-level architecture of CNNs consists of many layers ( Szegedy et al . , 2015a ) .", "These are stacked on top of each other , in much the same way as the stacked multilevel perceptrons described previously .", "A key difference is that CNNs have more variety especially in breadth ( number of units ) between layers .", "In many CNNs , some of the network’s layers are convolutional , which contain components that do not receive input from the whole of the previous layer , but a small subset of it ( Szegedy et al . , 2015b ) .", "Many convolutional components are required to process the whole of the previous layer by creating an overlapping tiling of small patches .", "Often convolutional layers are interleaved with max-pooling layers ( Lecun et al . , 1998 ) , which also contain tile-like components that act as local filters over the previous layer .", "This type of processing and architecture is both empirically driven by what works best , as well as inspired by the visual ventral stream , specifically receptive fields ( Fukushima , 1980; Hubel and Wiesel , 1959 , 1968; Serre et al . , 2007 ) .", "Convolutional and max-pooling layers provide a structure that is inherently hierarchical .", "Lower layers perform computations on small localized patches of the input , while deeper layers perform computations on increasingly larger , more global , areas of the stimuli .", "After such localized processing , it is typical to include layers that are fully-connected , i . e . , are more classically connectionist .", "And finally , a layer with the required output structure , e . g . , units that represent classes or a yes/no response as appropriate .", "Inception-v3 GoogLeNet uses a specific arrangement of these aforementioned layers , connected both in series and in parallel ( Szegedy et al . , 2015b , 2015a , 2016 ) .", "In total it has 26 layers and 25 million parameters inclusive of connection weights ( Szegedy et al . , 2015b ) .", "The final layer is a softmax layer that is trained to activate a single unit per class .", "These units correspond to labels that have been applied to sets of photographs by humans , e . g . , ‘space shuttle’ , ‘ice cream’ , ‘sock’ , within the ImageNet database ( Russakovsky et al . , 2015 ) .", "Inception-v3 GoogLeNet has been trained on millions of human-labeled photographs from 1000 of ImageNet’s synsets ( sets of photographs ) .", "The 1000-unit wide output produced by the network when presented with a photograph represents the probabilities of the input belonging to each of those classes .", "For example , if the network is given a photograph of a moped it may also activate the output unit that corresponds to bicycle with activation 0 . 03 .", "This is interpreted as the network expressing the belief that there is a 3% probability that the appropriate label for the input is ‘bicycle’ .", "In addition , this interpretation is useful because it allows for multiple classes to co-exist within a single input .", "For example , a photo with a guillotine and a wig in it will cause it to activate both corresponding output units .", "Thus the network is held to have learned a distribution of appropriate labels that reflect the most salient items in a scene .", "Inception-v3 GoogLeNet , achieves human levels of accuracy on test sets , producing the correct label in its five most probable guesses approximately 95% of the time ( Szegedy et al . , 2015b ) .", "We consider whether functional smoothness declines as inputs are processed by the more advanced layers of Inception-v3 GoogLeNet .", "If so , fMRI should be less successful in brain regions that instantiate computations analogous to the more advanced layers of such networks .", "Unlike the previous simulations , we present novel photographs of natural categories to these networks .", "The key question is whether items from related categories ( e . g . , banjos and guitars ) will be similar at various network layers .", "The 40 photographs ( i . e . , stimuli ) are divided equally amongst 8 subordinate categories: banjos , guitars , mopeds , sportscars , lions , tigers , robins , and partridges , which in turn aggregate into 4 basic-level categories: musical instruments , vehicles , mammals , and birds; which in turn aggregate into 2 superordinates: animate and inanimate .", "We consider how similar the internal network representations are for pairs of stimuli by comparing the resulting network activity , which is analogous to comparing neural activity over voxels in RSA .", "Correlations for all possible pairings of the 40 stimuli were calculated for both a mid and a later network layer ( see Figure 4 ) . 10 . 7554/eLife . 21397 . 007Figure 4 . Similarity structure becomes more difficult to recover in the more advanced layers of the DLN .", "( A ) The similarity structure in a middle layer of a DLN , Inception-v3 GoogLeNet .", "The mammals ( lions and tigers ) and birds ( robins and partridges ) correlate forming a high-level domain , rendering the upper-left quadrant a darker shade of red .", "Whereas the vehicles ( sportscars and mopeds ) and musical instruments ( guitars and banjos ) form two high-level categories .", "( B ) In contrast , at a later layer in this network , the similarity space shows high within-category correlations and weakened correlations between categories .", "While some structure between categories is preserved , mopeds are no more similar to sportscars than they are to robins . DOI: http://dx . doi . org/10 . 7554/eLife . 21397 . 007 The middle layer ( Figure 4A ) reveals cross-category similarity at both the basic and superordinate level .", "For example , lions are more like robins than guitars .", "However , at the later layer ( Figure 4B ) the similarity structure has broken down such that subordinate category similarity dominates ( i . e . , a lion is like another lion , but not so much like a tiger ) .", "Interestingly , the decline in functional smoothness is not a consequence of sparseness at the later layer as the Gini coefficient , a measure of sparseness ( Gini , 1909 ) , is 0 . 947 for the earlier middle layer ( Figure 4A ) and 0 . 579 for the later advanced layer ( Figure 4B ) , indicating that network representations are distributed in general and even more so at the later layer .", "Thus , the decline in functional smoothness at later layers does not appear to be a straightforward consequence of training these networks to classify stimuli , although it would be interesting to compare to unsupervised approaches that can perform at equivalent accuracy levels ( no such network currently exists ) .", "These DLN results are directly analogous to those with random untrained networks ( see Figure 2 ) .", "In those simulations , similar input patterns mapped to orthogonal ( i . e . , dissimilar ) internal representations in later layers .", "Likewise , the trained DLN at later layers can only capture similarity structure within subordinate categories ( e . g . , a tiger is like another tiger ) which the network was trained to classify .", "The effect of training the network was to create equivalence classes based on the training label ( e . g . , tiger ) such that members of that category are mapped to similar network states .", "Violating functional smoothness , all other similarity structure is discarded such that a tiger is no more similar to a lion than to a banjo from the network’s perspective .", "Should brain regions operate in a similar fashion , fMRI would not be successful in recovering similarity structure therein .", "In the Discussion , we consider the implications of these findings on our understanding of the ventral stream and the prospects for fMRI ." ], [ "Neuroscientists would rightly prefer a method that had both excellent spatial and temporal resolution for measuring brain activity .", "However , as we demonstrate in this article , the fact that fMRI has proven useful in examining neural representations , despite limitations in both its temporal and spatial resolution , says something about the nature of the neural code .", "One general conclusion is that the neural code must be smooth , both at voxel ( such that voxel responses are inhomogeneous across time and space ) and functional levels .", "The latter notion of smoothness is often overlooked or confused with super-voxel smoothness , but is necessary for fMRI to recover similarity spaces in the brain .", "Coding schemes , such as factorial and hash coding , are useful in numerous real-world applications and have an inverse function ( i . e . , one can go backwards from the internal representation to recover the unique stimulus input ) .", "However , these schemes are incompatible with the success of fMRI because they are not functionally smooth .", "For example , if the brain solely used such coding schemes , the neural representation of a robin would be no more similar to that of a sparrow than to that of a car .", "The fact that such neural similarities are recoverable by fMRI suggests that the neural code differs from these schemes in many cases .", "In contrast , we found that the types of representations used and generated by artificial neural networks , including deep learning networks , are broadly compatible with the success of fMRI in assessing neural representations .", "These coding schemes are functionally smooth in that similar inputs tend toward similar outputs , which allows item similarity to be reflected in neural similarity ( as measured by fMRI ) .", "However , we found that functional smoothness breaks down as additional network layers are added .", "Specifically , we have shown that multi-layer networks eventually converge to something akin to a hash function , as arbitrary locations in memory correspond to categories of inputs .", "These results take on additional significance given the recent interest in deep artificial neural networks as computational accounts of the ventral stream .", "One emerging view is that the more advanced the layers of these models correspond to more advanced regions along the ventral stream ( Cadieu et al . , 2014-12; Dubois et al . , 2015; Guclu and van Gerven , 2015; Hong et al . , 2016; Khaligh-Razavi and Kriegeskorte , 2014; Yamins et al . , 2014; Yamins and DiCarlo , 2016 ) .", "If this viewpoint is correct , our results indicate that neural representations should progressively become less functionally smooth and more abstract as one moves along the ventral stream ( recall Figure 2 ) .", "Indeed , neural representations appear to become more abstract , encoding whole concepts or categories , as a function of how far along the ventral stream they are located ( Bracci and Op de Beeck , 2016; DiCarlo and Cox , 2007; Riesenhuber and Poggio , 1999 , 2000; Yamins and DiCarlo , 2016 ) .", "For example , early on in visual processing , the brain may extract so-called basic features , such as in broadly-tuned orientation columns ( Hubel and Wiesel , 1959 , 1968 ) .", "In contrast , later on in processing , cells may selectively respond to particular individual stimulus classes i . e . , Jennifer Aniston , grandmother , concept , or gnostic cells ( Gross , 2002; Konorski , 1967; Quiroga et al . , 2005 ) , irrespective of orientation , etc .", "Likewise , we found that Inception-v3 GoogLeNet’s representations became symbol-like at advanced network layers such that items sharing a category label ( e . g . , tigers ) engendered related network states , while items in other categories engendered orthogonal states ( recall Figure 4 ) .", "Our simulations of random networks also found reduced functional smoothness at advanced network layers , suggesting a basic geometric property of multi-layer networks .", "The effect of training seems limited to creating network states in which stimuli that share the same label ( e . g . , multiple viewpoints of Jennifer Aniston ) become similar and items from all other categories ( even if conceptually related ) become orthogonal .", "If so , areas further along the ventral stream should prove less amenable to imaging ( recall Figure 2 ) .", "Indeed , a recent object recognition study found that the ceiling on observable correlation values becomes lower as one moves along the ventral stream ( Bracci and Op de Beeck , 2016 ) .", "Here , we focused on using fMRI to recover non-degenerate similarity spaces ( i . e . , where there are similarities beyond self-similarities ) .", "However , functional smoothness is also important for other analysis approaches .", "For example , MVPA decoders trained to classify items ( e . g . , is a house or a face being shown ? ) based on fMRI activity will only generalize to novel stimuli when functional smoothness holds .", "Likewise , univariate clusters ( e . g . , a house or face area ) will most likely be found and generalize to novel stimuli when functional smoothness holds because functional smoothness implies similar activation profiles for similar stimuli .", "Functional smoothness should be an important factor in determining how well classifiers perform and how statistically robust univariate clusters of voxels are .", "In cognitive science , research is often divided into levels of analysis .", "In Marr’s levels , the top level is the problem description , the middle level captures how the problem is solved , and bottom level concerns how the solution is implemented in the brain ( Marr , 1982 ) .", "Given that the ‘how’ and ‘where’ of cognition appear to be merging , some have questioned the utility of this tripartite division ( Love , 2015 ) .", "Our results suggest another inadequacy of these three levels of description , namely that the implementation level itself should be further subdivided .", "What is measured by fMRI is at a vastly more abstract scale than what can be measured in the brain .", "For example , major efforts , like the European Human Brain Project and the Machine Intelligence from Cortical Networks project ( Underwood , 2016 ) , are chiefly concerned with fine-grained aspects of the brain that are outside the reach of fMRI ( Chi , 2016; Frégnac and Laurent , 2014 ) .", "Likewise , models of spiking neurons e . g . , ( Wong and Wang , 2006 ) are at a level of analysis lower than where fMRI applies .", "Nevertheless , fMRI has proven useful in understanding neural representations that are consequential to behavior .", "Perhaps this success suggests that the appropriate level for relating brain to behavior is close to what fMRI measures .", "This does not mean lower-level efforts do not have utility when the details are of interest .", "However , fMRI’s success might mean that when one is interested in the nature of computations carried out by the brain , the level of analysis where fMRI applies may be preferred .", "To draw an analogy , one could construct a theory of macroeconomics based on quantum physics , but it would be incredibly cumbersome and no more predictive nor explanatory than a theory that contained abstract concepts such as money and supply .", "Reductionism , while seductive , is not always the best path forward ." ] ]

[ "The success of fMRI places constraints on the nature of the neural code .", "The fact that researchers can infer similarities between neural representations , despite fMRI’s limitations , implies that certain neural coding schemes are more likely than others .", "For fMRI to succeed given its low temporal and spatial resolution , the neural code must be smooth at the voxel and functional level such that similar stimuli engender similar internal representations .", "Through proof and simulation , we determine which coding schemes are plausible given both fMRI’s successes and its limitations in measuring neural activity .", "Deep neural network approaches , which have been forwarded as computational accounts of the ventral stream , are consistent with the success of fMRI , though functional smoothness breaks down in the later network layers .", "These results have implications for the nature of the neural code and ventral stream , as well as what can be successfully investigated with fMRI ." ]

[ "We can appreciate that a cat is more similar to a dog than to a truck .", "The combined activity of millions of neurons in the brain somehow captures these everyday similarities , and this activity can be measured using imaging techniques such as functional magnetic resonance imaging ( fMRI ) .", "However , fMRI scanners are not particularly precise – they average together the responses of many thousands of neurons over several seconds , which provides a blurry snapshot of brain activity .", "Nevertheless , the pattern of activity measured when viewing a photograph of a cat is more similar to that seen when viewing a picture of a dog than a picture of a truck .", "This tells us a lot about how the brain codes information , as only certain coding methods would allow fMRI to capture these similarities given the technique’s limitations .", "There are many different models that attempt to describe how the brain codes similarity relations .", "Some models use the principle of neural networks , in which neurons can be considered as arranged into interconnected layers .", "In such models , neurons transmit information from one layer to the next .", "By investigating which models are consistent with fMRI’s ability to capture similarity relations , Guest and Love have found that certain neural network models are plausible accounts of how the brain represents and processes information .", "These models include the deep learning networks that contain many layers of neurons and are popularly used in artificial intelligence .", "Other modeling approaches do not account for the ability of fMRI to capture similarity relations .", "As neural networks become deeper with more layers , they should be less readily understood using fMRI: as the number of layers increases , the representations of objects with similarities ( for example , cats and dogs ) become more unrelated .", "One question that requires further investigation is whether this finding explains why certain parts of the brain are more difficult to image ." ]

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

[ "biochemistry and chemical biology" ]

[ [ "The CRISPR-Cas system is an adaptive immune system present in many archaeal and bacterial species .", "It provides immunity against viruses and other mobile genetic elements mediated through sequence homology-directed detection and destruction of foreign nucleic acid species ( reviewed in Sorek et al . , 2013; Barrangou and Marraffini , 2014; van der Oost et al . , 2014 ) .", "Organisms with an active CRISPR-Cas system encode one or more CRISPR loci in their genomes .", "These comprise a leader sequence followed by an array of short , direct , often palindromic repeats interspersed by short ‘spacer’ sequences , together with a variable number of CRISPR associated ( cas ) genes .", "Spacers are derived from mobile genetic elements and constitute a ‘memory’ of past infections .", "The CRISPR locus is transcribed from the leader , generating pre-CRISPR RNA ( pre-crRNA ) that is subsequently processed into unit length crRNA species and loaded into large ribonucleoprotein complexes .", "These complexes , known as ‘Interference’ , ‘Effector’ or ‘Surveillance’ complexes , utilize crRNA to detect and degrade cognate invading genetic entities , providing immunity against previously encountered viruses and plasmids .", "The process of foreign DNA capture and integration into the CRISPR locus is known as ‘Acquisition’ or ‘Adaptation’ ( reviewed in Fineran and Charpentier , 2012; Heler et al . , 2014 ) .", "This process underpins the whole CRISPR-Cas system , but is the least well understood aspect .", "Fundamentally , acquisition can be separated into two steps: the capture of new DNA sequences from mobile genetic elements , followed by the integration of that DNA into the host genome .", "New spacers are incorporated proximal to the leader sequence and result in the duplication of the first repeat ( Goren et al . , 2012; Yosef et al . , 2012 ) .", "The leader sequence proximal to the repeat is important for integration , but transcription of the locus is not required ( Yosef et al . , 2012 ) .", "New spacers are frequently incorporated in both possible orientations ( Shmakov et al . , 2014 ) .", "The integration process in Escherichia coli results in staggered cleavage of the first CRISPR repeat , where the 3′-end of one strand of the incoming DNA is joined to the end of the CRISPR repeat in a trans-esterification ( TES ) reaction , with another TES reaction occurring on the other strand ( Diez-Villasenor et al . , 2013 ) ( Figure 1 , numbered yellow arrows ) .", "Intermediates in this reaction have been observed in E . coli , and the sequence of the leader-repeat1 junction is important for the activity ( Arslan et al . , 2014 ) .", "The order of the two integration steps shown in Figure 1 is not yet known , although it has been suggested that the reaction on the minus strand ( site", "2 ) occurs first in E . coli ( Nuñez et al . , 2015 ) .", "The sequence at site 2 is flanked by the end of the first repeat and the first spacer , and is therefore inherently less well conserved .", "In E . coli , the last nucleotide of the newly copied repeat is derived from the first nucleotide of the incoming spacer , which imposes further sequence requirements on that system ( Swarts et al . , 2012 ) . 10 . 7554/eLife . 08716 . 003Figure 1 . CRISPR spacer acquisition and Cas1 . ( A ,", "1 ) The 3′-end of an incoming protospacer attacks the chromosomal CRISPR locus at the boundary between the leader sequence and repeat 1 .", "A trans-esterification ( TES ) reaction ( yellow arrow", "1 ) catalyzed by Cas1 joins the protospacer to the 5′ end of repeat 1 .", "For many integrases a ( reverse ) disintegration reaction can be observed in vitro .", "( 2 ) Another TES reaction ( yellow arrow", "2 ) joins the other strand of the protospacer to the 5′ end of repeat 1 on the bottom ( minus ) strand , resulting in the formation of a gapped DNA duplex .", "( 3 ) The gapped duplex is repaired by the host cell DNA replication machinery , resulting in the addition of a new spacer at position 1 and replication of CRISPR repeat 1 .", "( B ) Sequences flanking the two TES reaction sites at repeat 1 in Sulfolobus solfataricus and Escherichia coli are shown .", "The leader is in blue , repeat in black and spacer 1 in teal .", "The number of central nucleotides of the repeat omitted from the sequence is shown in parentheses .", "( C ) Structure of Cas1 from Pyrococcus horikoshii ( PDB 4WJ0 ) with subunits coloured blue and cyan , showing the dimeric ‘butterfly’ conformation with the active site residues highlighted in green . DOI: http://dx . doi . org/10 . 7554/eLife . 08716 . 003 Although shown in Figure 1 as fully double-stranded , the incoming spacer DNA could have a partially single-stranded character .", "Recent work by Sorek and colleagues has shown that the E . coli RecBCD helicase–nuclease complex , which processes DNA double-strand breaks for recombination and degrades foreign DNA , is implicated in the generation of viral DNA fragments captured by Cas1 and incorporated into the CRISPR locus as new spacers ( Levy et al . , 2015 ) .", "This confirms previous observations linking Cas1 with RecBCD ( Babu et al . , 2011 ) and raises some intriguing questions as RecBCD generates ssDNA fragments asymmetrically , generating fragments tens to hundreds of nucleotides long from the 3′ terminated strand and much longer fragments from the 5′ terminated strand ( reviewed in Dillingham and Kowalczykowski , 2008 ) .", "The Cas4 enzyme , which is a 5′ to 3′ ssDNA exonuclease ( Zhang et al . , 2012; Lemak et al . , 2013 ) , may provide ssDNA fragments for Cas1 in systems lacking RecBCD .", "However , it is difficult to see how two integration reactions could occur without two 3′ hydroxyl termini ( Figure", "1 ) and half-site integration is not observed with a ssDNA substrate ( Nuñez et al . , 2015 ) .", "Potentially , the ssDNA fragments generated by these nucleases may re-anneal and experience further processing to generate partially duplex molecules of defined size prior to integration by Cas1 .", "Adaptation requires the products of the cas1 and cas2 genes in vivo and these are the most universally conserved of all the cas genes ( Makarova et al . , 2006 ) .", "Cas1 is a homodimeric enzyme with a two-domain structure and a canonical metal dependent nuclease active site in the large domain formed by a cluster of highly conserved residues ( Wiedenheft et al . , 2009 ) ( Figure 1C ) .", "E . coli Cas1 has nuclease activity in vitro , with activity observed against double- and single-stranded DNA and RNA substrates ( Babu et al . , 2011 ) .", "Some specificity was observed for branched DNA substrates , in particular for DNA constructs resembling replication forks ( Babu et al . , 2011 ) .", "Initial biochemical analyses of a panel of archaeal Cas2 enzymes revealed an endonucleolytic activity against ssRNA substrates that could be abrogated by mutation of conserved residues ( Beloglazova et al . , 2008 ) .", "In contrast , Cas2 from Bacillus halodurans has been shown to be specific for cleavage of dsDNA substrates ( Nam et al . , 2012 ) .", "Recently however , Doudna and colleagues demonstrated that E . coli Cas1 and Cas2 form a stable complex in vitro and that the ‘active site’ of Cas2 was not required for spacer acquisition , suggesting that Cas2 may not have a catalytic role in spacer acquisition in vivo ( Nuñez et al . , 2014 ) .", "It is probable that Cas2 acts as an adaptor protein , either bringing two Cas1 dimers together or mediating interactions with other components necessary for spacer acquisition .", "Recently , Nunez et al . demonstrated that E . coli Cas1 can integrate a protospacer into a supercoiled plasmid in vitro , in a reaction stimulated by Cas2 .", "Integration events were observed at the boundaries of most CRISPR repeats and in other areas of the DNA close to palindromic regions , implicating a role for palindromic DNA structure in the adaptation process ( Nuñez et al . , 2015 ) .", "In order to further mechanistic understanding of the spacer acquisition process , we have undertaken a systematic analysis of the underlying biochemistry .", "We demonstrate that Cas1 catalyses TES of branched DNA substrates efficiently in vitro in a reaction that represents the reverse- or disintegration of an incoming spacer from the CRISPR locus .", "The disintegration reactions catalysed by diverse integrases have proven a powerful model system for the understanding of the mechanism of integration .", "For Cas1 , the reaction is strongly sequence dependent with the specificity matching the predicted integration site 1 for both E . coli and Sulfolobus solfataricus Cas1 , and does not require Cas2 ." ], [ "The Cas1 and Cas2 proteins from S . solfataricus ( CRISPR-Cas subtype IA ) and E . coli ( CRISPR-Cas subtype IE ) were expressed in E . coli with N-terminal polyhistidine tags and purified to homogeneity by metal affinity and gel filtration chromatography .", "Previously , it was demonstrated that E . coli Cas1 ( EcoCas1 ) cleaved branched DNA substrates preferentially ( Babu et al . , 2011 ) .", "We investigated the activity of S . solfataricus Cas1 ( SsoCas1 ) against a DNA substrate with a 5′-flap structure ( Figure 2 ) .", "By labelling the single-stranded 5′-flap of the downstream duplex at the 5′-end with radioactive phosphorus , we observed cleavage of the flap by SsoCas1 , releasing an 18 nt product ( Figure 2B ) .", "A variant of SsoCas1 where the active site metal ligand glutamic acid 142 was changed to an alanine ( E142A ) was inactive , implicating the canonical nuclease active site of Cas1 in the reaction .", "This result appeared consistent with the earlier studies for EcoCas1 ( Babu et al . , 2011 ) and suggested that the ssDNA flap was removed at or close to the branch point .", "The activity was independent of the presence or absence of SsoCas2 , suggesting that Cas2 is not involved in this nuclease activity in vitro . 10 . 7554/eLife . 08716 . 004Figure 2 . Disintegration of a branched DNA substrate by SsoCas1 . Denaturing gel electrophoresis was used to analyse the products generated by SsoCas1 with a branched DNA substrate ( Substrate 1 ) .", "The 5′ flap ( 18 nt ) was released when the phosphodiester backbone was attacked by the 3′-hydroxyl group at the branch point .", "The reaction required active Cas1 and was independent of Cas2 .", "DNA lengths are shown in blue ( nt ) .", "The TES site is indicated with a yellow arrow and the labelled strand with a red star .", "( A ) Shows reactions with the continuous strand ( black ) labelled; ( B ) with the flap ( grey ) strand labelled and ( C ) with the upstream ( green ) strand labelled , each on the 5′ end .", "Lanes: 1 , control with no added protein; 2 , WT Cas1; 3 , Cas2; 4 , Cas1 + Cas2; 5 , Cas1 E142A variant; 6 , E142A Cas1 + Cas2 .", "( D ) The 5′-flap strand was labelled on the 3′ end with a fluorescein moiety , and the flap reduced to 14 nt ( Substrate 1-FAM ) .", "Cas1 catalyses the TES reaction generating a 53 nt labelled strand .", "Lane C: control incubation in absence of Cas1 .", "( E ) TES reactions were carried out with SsoCas1 , or the E142A active site mutant , on a fork substrate containing a nicked SacI restriction site spanning the branch point ( SacI substrate ) .", "A TES product of 53 nucleotides is visible in lane 2 containing Cas1 .", "The right-hand panel shows the effect of adding SacI restriction enzyme after the TES reaction .", "The TES product is no longer visible , but a shorter product of 25 nucleotides is present indicating regeneration of the SacI recognition sequence by the TES reaction . DOI: http://dx . doi . org/10 . 7554/eLife . 08716 . 004 We proceeded to label the other strands of the substrate to follow the reaction products in more detail .", "The continuous ( black ) strand was not cleaved by SsoCas1 ( Figure 2A ) .", "However , when the 23 nt ( green ) strand of the upstream duplex presenting a 3′-hydroxyl end at the junction point was labelled we observed the formation of a new , larger DNA species ( Figure 2C ) .", "This observation was consistent with a joining or TES of the upstream DNA to the downstream DNA strand by Cas1 .", "By switching to a label at the 3′ end of the downstream duplex we confirmed that the reaction catalyzed by Cas1 involved the joining of the upstream and downstream DNA duplexes with concomitant loss of the 5′-flap ( Figure 2D ) .", "The lack of evidence for any shorter DNA species in Figure 2D was consistent with the conclusion that we were observing a TES rather than a nuclease reaction .", "Again , the TES reaction was unaffected by the presence or absence of Cas2 and was dependent on the wild-type active site of Cas1 , as the E142A variant was inactive .", "In order to define the product of the TES reaction in more detail , we modified the sequence of the branch point to introduce an interrupted site for the restriction enzyme SacI across the junction .", "A precise TES reaction at the branch point to generate duplex DNA would result in the ‘repair’ of the restriction site , generating a substrate for SacI .", "SsoCas1 processed this substrate generating the 53 bp TES reaction product .", "On addition of the SacI restriction enzyme , the 53 bp species was converted to a 25 bp product ( Figure 2E ) .", "This confirmed that the Cas1-mediated reaction resulted in the formation of a functional SacI site in vitro , consistent with a precise TES reaction at the branch point .", "It is likely that the TES reaction catalyzed by Cas1 with branched DNA substrates in vitro represents the reverse or disintegration of an integration intermediate , as observed recently for EcoCas1 ( Nuñez et al . , 2015 ) .", "We therefore tested EcoCas1 with the same set of branched substrates , showing that manganese , magnesium and cobalt all supported the same robust disintegration activity in the absence of Cas2 , with no nuclease activity observed ( Figure 3A ) .", "Given that Eco and SsoCas1 are divergent members of the Cas1 family , this suggests that the disintegration activity is likely to be a general property of Cas1 enzymes .", "Experiments where the concentration of Cas1 was titrated against a fixed concentration of substrate ( 50 nM ) , showed that maximal activity plateaued above 250 nM for EcoCas1 and was maximal from 100 to 500 nM for SsoCas1 ( Figure 3B , C ) . 10 . 7554/eLife . 08716 . 005Figure 3 . TES activity of E . coli and S . solfataricus Cas1 . ( A ) E . coli Cas1 also catalyses an efficient metal dependent disintegration reaction .", "TES reactions were carried out under standard conditions , using Substrate 3 and varying the divalent metal ion as indicated .", "EcoCas1 showed robust TES activity in the presence of cobalt , magnesium and manganese .", "Each of the three strands of the substrate was labelled individually as for Figure 2 ( 5′ label indicated by a star ) .", "Lanes were: c , substrate alone; substrate incubated with Cas1 and 5 mM of E , EDTA; Co , cobalt chloride; Ca , calcium chloride; Mg , magnesium chloride; Mn , manganese chloride for 30 min at 37°C .", "( B ) Concentration dependence of Cas1 TES activity .", "Substrate 3 ( 50 nM ) was incubated with the indicated concentration of Sso or EcoCas1 for 30 min under standard assay conditions and the reactants were analysed by denaturing gel electrophoresis and phosphorimaging .", "SsoCas1 showed maximal activity at 250 nM , representing a fivefold molar excess of enzyme over substrate , with a decline in activity above 500 nM enzyme .", "EcoCas1 had maximal activity that plateaued above 250 nM enzyme .", "( C ) Quantification of the data ( raw data provided in Figure 3—source data 1 ) .", "These data are representative of duplicate experiments . DOI: http://dx . doi . org/10 . 7554/eLife . 08716 . 00510 . 7554/eLife . 08716 . 006Figure 3—source data 1 . Concentration Cas1 . DOI: http://dx . doi . org/10 . 7554/eLife . 08716 . 006 To characterise the specificity of the disintegration reaction in more detail , we examined SsoCas1 activity for a variety of substrates differing in the nature of the 5′-flap or duplex arm released by the reaction ( Figure 4 ) .", "SsoCas1 was indifferent to the presence of duplex or single-stranded DNA in the 5′-flap , processing a nicked 3-way junction with a similar efficiency to the 5′-flap substrate .", "The disintegration reaction required the presence of the 3′-hydroxyl moiety at the branch point as a three-way ( or Y ) junction was not a substrate for Cas1 .", "To confirm this observation we studied a 5′-flap substrate with a phosphate moiety at the 3′ end of the upstream strand in place of a hydroxyl group .", "As expected , this substrate did not support disintegration activity for either Sso or EcoCas1 , with no larger TES product detected ( right hand lanes ) .", "Tellingly , neither enzyme cleaved the 5′-flap of this substrate to generate shorter products ( left hand lanes ) , confirming that Cas1 catalyses a concerted TES reaction rather than a sequential ‘cut and join’ activity . 10 . 7554/eLife . 08716 . 007Figure 4 . Importance of flap and 3′ terminus structure . The importance of the released 25 nt 5′-flap structure was investigated by varying the length of duplex DNA in that arm from 0 ( canonical 5′-flap ) to a full 25 bp ( nicked 3-way junction ) ( left hand panel , all based on substrate 19 ) .", "All supported robust disintegration activity by SsoCas1 .", "An intact Y- junction did not support TES activity .", "Lanes: C , substrate alone ( 1 , 5 , 9 , 13 , 17 ) ; E , SsoCas1 E142A variant 30 min incubation ( 2 , 6 , 10 , 14 , 18 ) ; incubation with wild-type SsoCas1 for 10 and 30 min ( other lanes ) .", "The right hand panel shows the effect of replacing the attacking 3′-hydroxyl moiety at the branch point with a phosphate group ( 3′ phos substrate ) no TES or nuclease activity was observed for either Sso or EcoCas1 .", "C , substrate alone; E , SsoCas1 E142A variant . DOI: http://dx . doi . org/10 . 7554/eLife . 08716 . 007 Disintegration reactions are commonly seen for integrases and transposases , and represent a very useful means to study the underlying integration mechanism ( Chow et al . , 1992; Delelis et al . , 2008 ) as the local arrangement of the DNA in the integrase active site is typically the same for the two reactions ( Gerton et al . , 1999 ) .", "One prediction of this hypothesis is that the disintegration reaction could demonstrate some sequence preference if integration , which must happen at a specific , defined site in the CRISPR locus , is partly driven by the sequence specificity of Cas1 .", "We therefore designed a family of related disintegration substrates by systematically varying the nucleotides flanking the TES site and tested how efficiently Cas1 could disintegrate these substrates .", "Having demonstrated conclusively that we could follow the progress of the disintegration reaction by monitoring the liberation of a displaced DNA strand from a 5′-flap substrate , we used this assay for all subsequent investigations .", "We first tested the importance of the nucleotide acting as an acceptor for the attacking 3′-hydroxyl of the incoming DNA strand ( the +1 position ) by varying the nucleotide at this position in the model substrates ( Figure 5 ) .", "In vivo , this acceptor nucleotide should represent the position in the CRISPR locus where new spacers are joined to the end of the repeat .", "The S . solfataricus CRISPR repeat has a guanine at one end and a cytosine at the other , each of which are predicted to act as acceptors for a new phosphodiester bond formed with the incoming spacer ( Figure 1B ) .", "Consistent with this , we observed the most efficient disintegration activity with SsoCas1 when substrates had a guanine at the +1 position ( Figure 5A ) .", "Assays were carried out in triplicate and the reaction products quantified , confirming the qualitative observation of a preference for guanine , followed by cytosine , adenine and thymine , with rates of 0 . 06 , 0 . 013 , 0 . 0058 and 0 . 0009 min−1 , respectively ( Figure 5B ) . 10 . 7554/eLife . 08716 . 008Figure 5 . Sequence specificity of the disintegration reaction at the +1 position . The nucleotide at the acceptor ( +1 ) position was varied systematically to assess the sequence dependence of the disintegration reaction carried out by Cas1 from S . solfataricus ( A , B ) and E . coli ( C , D ) ( Substrates 3 , 6 , 7 , 8 ) .", "In the gels on the left ( A , C ) each substrate was incubated with Cas1 for 1 , 2 , 3 , 5 , 10 , 15 , 20 and 30 min in reaction buffer prior to electrophoresis to separate the cleaved 5′-flap from the intact substrate .", "C–control with no Cas1 added .", "The plots on the right ( B , D ) show quantification of these assays .", "Data points represent the means of triplicate experiments with standard errors shown ( raw data provided in Figure 5—source data 1 and Figure 5—​source data 2 ) .", "The data were fitted to an exponential equation , as described in the ‘Materials and methods’ , and for EcoCas1 a variable floating end point was included to allow fitting as the reaction did not go to completion .", "The effect of Cas2 ( 150 nM ) on EcoCas1 ( 150 nM ) sequence specificity for substrates ( 50 nM ) varying at position +1 ( substrates 3 , 6 , 7 , 8 ) was also tested ( E ) .", "The second panel from the right is a composite image from two phosphorimages of the same time course as indicated by a black line . DOI: http://dx . doi . org/10 . 7554/eLife . 08716 . 00810 . 7554/eLife . 08716 . 009Figure 5—source data 1 . Nucleotide at +1 position . DOI: http://dx . doi . org/10 . 7554/eLife . 08716 . 00910 . 7554/eLife . 08716 . 010Figure 5—source data 2 . Nucleotide at +1 position . DOI: http://dx . doi . org/10 . 7554/eLife . 08716 . 010 For E . coli , the first nucleotide of the repeat is a guanine .", "Although the corresponding position at the other end of the repeat on the minus strand is a cytosine , it has been demonstrated that the new spacer joins at the penultimate residue , which is also a guanine ( Swarts et al . , 2012 ) .", "EcoCas1 displayed a striking preference for a guanine at the +1 position for the disintegration reaction , with all three alternative nucleotides strongly disfavoured at this position ( Figure 5C ) , in good agreement with the prediction based on the repeat sequence .", "For EcoCas1 the reaction did not go to completion and accordingly we fitted the data with a variable end-point as described in the ‘Materials and methods’ ( Figure 5D ) .", "Although the reaction rates could not be determined accurately , guanine at position +1 supported rates at least 10-fold higher than any other nucleotide .", "We also tested the effect of inclusion of Cas2 on the sequence specificity of EcoCas1 , and observed that Cas2 had no effect , with strong preference for a guanine at +1 still observed ( Figure 5E ) .", "We proceeded to investigate the sequence dependence of the nucleotide at the 3′-end of the attacking DNA strand ( the −1 position ) in the disintegration reactions .", "For the first integration site , this should correspond to the last nucleotide of the leader sequence , which is an adenine in both S . solfataricus and E . coli .", "Although both Cas1 enzymes catalyzed the disintegration of substrates with an adenine at this position , clear sequence preference was not apparent ( Figure 6 ) .", "For S . solfataricus , the −1′ position on the minus strand is variable in sequence .", "However , in E . coli , site 2 occurs at the penultimate nucleotide of the repeat and therefore has a cytosine at the −1′ position ( Swarts et al . , 2012 ) .", "In this situation , the incoming 3′ terminal nucleotide of the spacer has to be a cytosine in order to complete the original repeat sequence .", "To mimic the TES substrate at this site more closely , we tested substrates where the first nucleotide of the 5′ flap , equivalent to the incoming nucleotide in the forward reaction , was a cytosine , but a cytosine at position −1 was still not favoured by EcoCas1 ( Figure 6C ) .", "This may suggest that the disintegration reaction is not a good model for integration at site 2 , which is further discussed later . 10 . 7554/eLife . 08716 . 011Figure 6 . Sequence specificity of the disintegration reaction at the −1 position . The nucleotides participating in the disintegration reaction were varied systematically at the −1 position ( substrates 3 , 9 , 10 , 11 ) .", "For SsoCas1 ( A ) there was some preference for adenine at this position , consistent with integration site 1 .", "For EcoCas1 ( B , C ) , a cytosine at position −1 was disfavoured over all other possibilities , even when the residue equivalent to the ‘incoming’ nucleotide was also a cytosine ( substrates 15 , 16 , 17 , 18 ) .", "Each substrate was incubated with Cas1 for 5 , 10 and 30 min in reaction buffer prior to electrophoresis .", "C–control with no Cas1 added . DOI: http://dx . doi . org/10 . 7554/eLife . 08716 . 011 The nucleotide at the −2 position , which is part of the conserved leader sequence for integration site 1 , is also a potential determinant of integration specificity .", "Accordingly , we varied this residue systematically and investigated the efficiency of the disintegration reaction for both Cas1 enzymes ( Figure 7 ) .", "In S . solfataricus , the −2 position in the leader is a cytosine , which supported the strongest disintegration activity ( Figure 7A ) .", "In E . coli , the −2 position in the leader is a guanine .", "A clear preference for guanine over all other nucleotides was observed for EcoCas1 ( Figure 7B ) , confirmed by a more detailed kinetic analysis ( Figure 7C ) which was fitted as for Figure 5D .", "These data are consistent with a role for sequence discrimination at the −2 position by both enzymes , which is relevant for integration site 1 but not site 2 , where this position varies depending on the sequence of the last spacer inserted . 10 . 7554/eLife . 08716 . 012Figure 7 . Sequence specificity of the disintegration reaction at the −2 position . The nucleotides participating in the disintegration reaction were varied systematically at the −2 position , which is a cytosine ( Sso ) or guanine ( Eco ) at integration site 1 , and variable at integration site 2 ( substrates 10 , 12 , 13 , 14 ) .", "( A ) SsoCas1; ( B ) EcoCas1 .", "Each substrate was incubated with Cas1 for 5 , 10 and 30 min in reaction buffer prior to electrophoresis .", "C–control with no Cas1 added .", "( C ) For EcoCas1 , the clear preference for guanine at position −2 was confirmed by more detailed kinetic analysis ( raw data provided in Figure 7—source data 1 ) as described for Figure 5 . DOI: http://dx . doi . org/10 . 7554/eLife . 08716 . 01210 . 7554/eLife . 08716 . 013Figure 7—source data 1 . Nucleotide at −2 position . DOI: http://dx . doi . org/10 . 7554/eLife . 08716 . 013 We next checked for specificity at the 3′ end of the 5′ flap in the disintegration product , which corresponds to the 3′ end of the incoming spacer during integration .", "No sequence preference was detected for SsoCas1 ( data not shown ) , which is consistent with the essentially random nature of the incoming DNA .", "During adaptation in E . coli , the incoming nucleotide at integration site 1 is expected to be random , but at site two is always a cytosine , where it completes the repeat sequence ( Swarts et al . , 2012 ) .", "For EcoCas1 , an adenine or cytosine was strongly favoured over guanine and particularly thymine ( Figure 8 ) , suggesting discrimination by EcoCas1 at this position . 10 . 7554/eLife . 08716 . 014Figure 8 . Sequence specificity of the EcoCas1 disintegration reaction for the incoming nucleotide . The nucleotide corresponding to the incoming 3′ end of the new spacer , which is the nucleotide at the 3′ end of the 5′-flap in the disintegration substrate , was varied systematically to determine its effect on the disintegration reaction catalysed by EcoCas1 ( substrates 2 , 3 , 4 , 5 ) .", "C–control with no Cas1 added .", "Time points were 5 , 10 and 30 min . DOI: http://dx . doi . org/10 . 7554/eLife . 08716 . 014 The substrates examined so far in this study do not correspond to the actual sequences encountered by EcoCas1 during integration .", "Accordingly , we constructed a pair of substrates that correspond to the products of integration when spacer 3 in the E . coli CRISPR array is integrated at site 1 ( top strand ) or site 2 ( bottom strand ) ( Figure 9 ) .", "These were constructed from oligonucleotides as before to generate disintegration substrates with a 5′ flap .", "Disintegration was analysed by denaturing gel electrophoresis , phosphorimaging and quantification of triplicate experiments .", "EcoCas1 disintegrated the site 1 substrate quickly , with the reaction reaching approximately 75% conversion in 3 min , which compares favorably with the best model sequence studied .", "Site 2 was also a good disintegration substrate , though it was converted significantly more slowly than site 1 , perhaps due to the presence of a disfavored cytosine at position −1 . 10 . 7554/eLife . 08716 . 015Figure 9 . Disintegration of authentic E . coli integration intermediates . Disintegration substrates corresponding to the expected site 1 and site 2 integration products arising from the integration of spacer 3 into the CRISPR array were constructed and tested ( spacer 3-1 and spacer 3-2 substrates ) .", "EcoCas1 processed both , with the rate of reaction significantly higher for the substrate corresponding to site 1 ( the top strand ) at the leader-repeat junction .", "Data points represent the means of triplicate experiments with standard errors shown ( raw data provided in Figure 9—source data 1 ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 08716 . 01510 . 7554/eLife . 08716 . 016Figure 9—source data 1 . E .", "coli Site 1 vs Site 2 time course . DOI: http://dx . doi . org/10 . 7554/eLife . 08716 . 016" ], [ "Studies of the CRISPR spacer acquisition process in vivo have yielded many key insights , but they are complicated by the fact that it is very difficult to separate the two distinct steps of spacer capture and spacer integration .", "Consequently we still do not have a clear understanding of the roles of Cas1 , Cas2 and host proteins in the acquisition mechanism .", "In this study we investigated the integration reaction by focusing on the biochemistry of the Cas1 protein from S . solfataricus and E . coli .", "Efficient TES of branched DNA substrates with a 5′-flap or duplex arm is clearly possible for both S . solfataricus and E . coli Cas1 in vitro .", "This is a very precise reaction requiring attack by a 3′-hydroxyl at the branch point , generating a perfect DNA duplex .", "The reaction almost certainly represents the disintegration reaction that is the reverse of the spacer integration step , as observed for many integrases and transposases where it represents a very useful means to study the underlying integration mechanism ( Gerton et al . , 1999 ) .", "Evidence for a disintegration activity was recently described by Doudna and colleagues for EcoCas1 , but the activity observed was relatively weak , most likely because the branched substrate studied had a non-optimal DNA sequence around the branch point ( Nuñez et al . , 2015 ) .", "For the CRISPR adaptation process in vivo , integration occurs at the junction between the first repeat and the leader sequence , which immediately suggests a role for sequence specificity in the reaction .", "It has also been suggested that CRISPR repeat sequences , which are often palindromic , form four-way DNA junctions in supercoiled DNA , acting as a recognition signal for Cas1 , a possibility that is supported by the observation that EcoCas1 can cut four-way DNA junctions in vitro ( Babu et al . , 2011 ) , and the finding that spacer integration in a plasmid lacking a CRISPR locus occurs preferentially next to a palindromic site ( Nuñez et al . , 2015 ) .", "However , a palindrome alone is not sufficient to support spacer insertion in E . coli in vivo ( Arslan et al . , 2014 ) , and this also holds for the type II CRISPR system of Streptococcus thermophilus ( Wei et al . , 2015 ) , suggesting that local sequence helps determine the integration site .", "Furthermore , some CRISPR repeat families , including many in archaea , have little or no palindromic nature and thus cannot form stable hairpin structures ( Kunin et al . , 2007 ) .", "Cas1 therefore might be expected to act as a sequence specific integrase , although local DNA structure could also play a part .", "In support of this hypothesis , both S . solfataricus and E . coli Cas1 catalyse a disintegration reaction with distinct , sequence specific properties .", "In particular , there is a clear preference for a guanine at position +1 , corresponding to the first nucleotide of the repeat , suggesting that this residue is recognised specifically in the active site of Cas1 .", "The specificity is particularly strong for EcoCas1 , consistent with the presence of a guanine in the repeat sequence at the +1 site in both plus and minus strands .", "Preference for a guanine at the +1 nucleotide for EcoCas1 catalysed integration events has also been observed ( Nuñez et al . , 2015 ) .", "For SsoCas1 , a guanine at this position was preferred over a cytosine , which is the nucleotide present at the +1′ position on the minus strand , by a factor of five .", "Although the −1 position might also be expected to play a role in the selection of integration sites , deep sequencing data for integration catalysed by EcoCas1 revealed no sequence preference at this position ( Nuñez et al . , 2015 ) .", "In agreement with this finding , we observed little evidence for sequence discrimination at the −1 position for the disintegration reactions catalysed by either enzyme , with the exception that EcoCas1 disfavours cytosine at this position ( Figure 6B ) .", "A cytosine at position −1 is the expected residue on the minus strand , suggesting that the disintegration reaction may better reflect the reversal of integration at site 1 in the leader-repeat junction .", "Deep sequencing data for integration reactions catalysed by EcoCas1 in vitro did reveal a marked preference for a guanine at position −2 in the integration site ( Nuñez et al . , 2015 ) .", "This corresponds well with the −2 position in the plus strand , which is part of the leader sequence and is a guanine in E . coli , but cannot hold for the minus strand where the −2′ position is inherently variable in nature .", "Disintegration of substrates mimicking the integration intermediates relevant for the integration of spacer 3 into the E . coli CRISPR array reinforce these conclusions , with site 1 on the plus strand processed significantly more quickly than site 2 on the bottom strand ( Figure 9 ) .", "Taken together , both the disintegration specificity and the deep sequencing data for integration support the hypothesis that integration is targeted to the leader-repeat 1 junction on the plus strand at least in part by the inherent sequence specificity of Cas1 , which presumably involves specific recognition of these bases within the active site of the enzyme ( Figure 10 ) . 10 . 7554/eLife . 08716 . 017Figure 10 . Reaction scheme for spacer integration and disintegration by E . coli Cas1 . The Cas 1-2 complex integrates new spacers via two joining reactions ( 1 and 2 ) at either end of the first CRISPR repeat , which differ in their sequence context .", "Disintegration activity by E . coli Cas1 shows clear preference for the sequence at site 1 , with the guanines at position +1 and −2 particularly important .", "At site 2 , the sequence context is not optimal for disintegration in vitro , leading to slower reaction rates .", "In the active site of Cas1 , these nucleobases likely make specific interactions with catalytic residues , and the DNA duplex structure may be distorted . DOI: http://dx . doi . org/10 . 7554/eLife . 08716 . 017 For E . coli integration reactions in vitro , a marked preference for cytosine over thymine at the 3′ end of the protospacer was observed ( Nuñez et al . , 2015 ) .", "Furthermore , protospacers with a cytosine at the 3′ end were preferentially incorporated into the minus strand at the junction between repeat 1 and spacer 1 .", "These data are consistent with the requirement for protospacers to supply the final cytosine of the repeat on the minus strand during integration ( Swarts et al . , 2012 ) .", "The deep sequencing data also revealed a marked preference for adenine over thymine at the 3′ end of the protospacer ( Nuñez et al . , 2015 ) .", "For disintegration by EcoCas1 , we observed a clear preference for adenine or cytosine at the equivalent position , whilst thymine did not support the disintegration reaction ( Figure 8 ) .", "Thus EcoCas1 appears to recognise the nucleotide at the 3′ end of the incoming DNA , although no such discrimination was observed for SsoCas1 .", "A dinucleotide sequence ‘AA’ motif over-represented at the 3′ end of protospacers incorporated in E . coli strain BL21 has been described previously ( Yosef et al . , 2013 ) .", "Although the CRISPR spacer integration system has been compared to the integration and transposition reactions carried out by mobile genetic elements , there is one key difference in the two processes—the length of DNA integrated .", "The persistence length of DNA , the distance over which it behaves as a fairly rigid rod , is estimated as 35–50 nm ( 100–150 bp ) under conditions found in cells ( Hagerman , 1988; Brinkers et al . , 2009 ) .", "This means that the two ends of a viral genome of several kilobases can be looped around and brought close together relatively easily , but a new spacer of 30–40 base pairs of dsDNA cannot be manipulated in the same way .", "Considering the scheme in Figure 1 , the molecular origami required to achieve the second TES reaction looks challenging .", "Several related enzymes , including Mu transposase ( Savilahti et al . , 1995 ) , V ( D ) J recombinase ( Ramsden et al . , 1996 ) and HIV integrase ( Gerton et al . , 1999 ) are known to disrupt base pairing of DNA substrates and make sequence-specific contacts during the integration reaction .", "It is likely that Cas1 also manipulates the local DNA duplex structure , which may help in positioning the DNA strands correctly for the TES reaction .", "The observation that the incoming DNA ( the 5′-flap in the disintegration reaction ) can be single stranded , partially or fully duplex in nature suggests that there is some flexibility in the recognition of the incoming spacer .", "This is also consistent with the recent observation of a link between RecBCD , which generates ssDNA products , and Cas1 in E . coli ( Levy et al . , 2015 ) .", "There is no formal requirement that the protospacer should be fully duplex in nature , although current understanding of the integration reaction requires that new spacers have two intact 3′-ends for the two integration reactions so must be at least partially duplex in nature .", "Many integrases process the ends of the integrated DNA using a nuclease activity , which occurs at the same active site as the integrase activity ( Gerton et al . , 1999 ) .", "There is no reason to expect that Cas1 will differ in that regard , and indeed the reaction products of the RecBCD nuclease are on average much longer than the DNA molecules integrated by Cas1 , suggesting the requirement for further processing .", "In conclusion , we have shown that Cas1 from both S . solfataricus and E . coli have robust TES activities in vitro which reflect the reversal , or disintegration , of the integration reaction .", "Disintegration is strongly sequence specific , and the specificity fits with the expected sequence for the plus strand at the leader-repeat junction ( Figure 9 ) .", "This site is the logical place for the initiation of integration , as it has a unique , defined sequence , in contrast to the repetitive and more variable sequence found at the second integration site on the minus strand .", "Doudna and colleagues recently proposed a model based on an initial attack at site 2 on the minus strand ( Nuñez et al . , 2015 ) .", "However , this preference was only significant for spacers with a cytosine at the 3′ terminus , and does not explain the marked preference observed by the authors for a guanine at position +2 , which is a feature of the positive strand .", "Future studies of both the forward and reverse integration reactions catalyzed by Cas1 will help to address these issues and delineate the mechanism of spacer integration in the CRISPR system ." ], [ "The sso1450 ( cas1 ) gene and sso1450a ( cas2 ) genes were amplified from S . solfataricus P2 genomic DNA by PCR using primer pairs ( 5′-ATATAACCATGGCAAGCGTGAGGACTT; 5′-TATTGGATCCTCACATCACCAACTTGAAACCC ) and ( 5′-GCGCCATGGTTACACTAACCATTCCTCTAATC; 5′-GGCCGGATCCTTGAAATTATTGGTAGTATATGAC ) , respectively .", "The amplified genes were cloned into the pEHisTEV vector ( Liu and Naismith , 2009 ) downstream of a cleavable His6-tag using NcoI and BamHI restriction sites .", "Site-directed mutagenesis was carried out on the vector containing sso1450 to mutate active site residue glutamic acid 142 to an alanine using the oligonucleotide sequence 5′-GTTGGATAAGGATGCACCGGCTGCTGCTAG .", "Standard site-directed mutagenesis protocols ( QuikChange , Stratagene , La Jolla , CA , United States ) were followed .", "Mutations were confirmed by sequencing ( GATC Biotech , Constance , Germany ) .", "The constructs were expressed in C43 ( DE3 ) E . coli cells grown in LB ( Luria-Bertani ) medium supplemented with 35 µg/ml kanamycin to an OD600 of 0 . 6–0 . 8 at 37°C .", "Expression was then induced by the addition of 0 . 4 mM isopropyl-β-D-thiogalactopyranoside ( IPTG ) and overnight incubation with shaking at 25°C .", "Cells were harvested ( 8000×g , 20 min ) and resuspended in lysis buffer ( 4 . 5 mM NaH2PO4 , 15 mM Na2HPO4 [pH 7 . 5] , 500 mM NaCl , 30 mM imidazole , 1% Triton-X and protease inhibitors [Roche Applied Science , Basel , Switzerland] ) .", "Cells were lysed by sonication , the lysate cleared by ultracentrifugation ( 90 , 000×g , 35 min ) and the supernatant filtered though a 0 . 22 µm syringe filter and loaded on to a HP HisTrap 5 ml column ( GE Healthcare , Little Chalfont , United Kingdom ) equilibrated in buffer A ( 4 . 5 mM NaH2PO4 , 15 mM Na2HPO4 [pH 7 . 5] , 500 mM NaCl , 30 mM imidazole ) .", "The His-tagged protein of interest was eluted with a linear gradient from 30 to 500 mM imidazole .", "Fractions containing Cas1 ( or Cas2 ) were concentrated and buffer exchanged into buffer A , using centrifugal concentrators ( 30 kDa cutoff , Vivaspin , Sartorius Stedim Biotech GmbH , Goettingen , Germany ) .", "His-tags were cleaved by the addition of TEV protease ( 1:10 ratio of TEV:protein ) and overnight incubation at room temperature .", "The cleaved protein was passed through a HisTrap column in buffer A , and the cleaved protein collected in the flow through .", "The final purification step was gel filtration on a 26/60 Superdex 200 prep grade column ( GE Healthcare ) in buffer C ( 20 mM Tris-HCL ( pH 7 . 5 ) , 500 mM NaCl , 0 . 5 mM DTT , 1 mM EDTA , 10% glycerol ) .", "Purified and concentrated protein samples were flash frozen and stored at −80°C .", "Genes encoding E . coli K-12 MG1655 Cas1 ( ygbT ) and Cas2 ( ygbF ) were amplified by PCR from genomic DNA using the PCR primer pairs ( 5′-GGAATTCCATATGACCTGGCTTCCCCTTAATC; 5′-GGAATTCTCAGCTACTCCGATGGCCTGC ) and ( 5′-GGAATTCCATATGAGTATGTTGGTCGTGGTCAC; 5′-GGAATTCTCAAACAGGTAAAAAAGACAC ) , respectively .", "Each PCR product was subcloned into protein expression plasmid pET14b using restriction enzyme NdeI and EcoRI .", "EcoCas1 and Cas2 proteins were over-produced individually in strain BL21 AI ( Life Technologies , Carlsbad , CA , United States ) , each with N-terminal ( His ) 6 tags .", "Cells were grown at 37°C to OD600 0 . 5–0 . 6 in LB broth containing ampicillin ( 50 µg/ml ) and induced using IPTG ( 1 mM ) and arabinose ( 0 . 2% wt/vol ) , with growth continued for 3 hr after induction .", "Cas1 or Cas2 expressing cells were harvested for re-suspension in buffer H ( 20 mM Tris . HCl pH7 . 5 , 500 mM NaCl , 5 mM imidazole , 10% glycerol ) and flash frozen in liquid nitrogen for storage at −80°C until required .", "The first purification step was identical for both Cas1 and Cas2: sonicated biomass was clarified by centrifugation ( 90 , 000×g , 25 min ) and soluble extract was passed into a 5 ml Hi-Trap Nickel chelating column ( GE Healthcare ) equilibrated with buffer H . Cas1 or Cas2 eluted at 70–100 mM imidazole in a linear imidazole gradient .", "Sodium chloride was reduced to 50 mM by dialysis against buffer S ( 20 mM Tris . HCl pH7 . 5 , 50 mM NaCl , 1 mM DTT , 10% glycerol ) .", "Cas1 was loaded onto a 5 ml Hi-Trap heparin column and eluted in a gradient of NaCl at 200–300 mM in buffer S . Pooled Cas1 fractions were loaded directly onto a S300 size exclusion column equilibrated in buffer S with 250 mM NaCl .", "Cas1 fractions were pooled for storage at −80°C in buffer S containing 250 mM NaCl and 40% glycerol .", "Desalted Cas2 eluted from Ni-NTA was dialyzed into buffer S containing 1 . 5 M NaCl and applied to a 5 ml Hi-Trap butyl-Sepharose column ( GE Healthcare ) , eluting in the flow through .", "Cas2 fractions were pooled and loaded directly onto a S300 size exclusion column equilibrated in buffer S with 250 mM NaCl .", "Following isocratic elution , Cas2 fractions were pooled and stored as for Cas1 .", "Substrates were purchased from Integrated DNA Technologies ( Coralville , IA , United States ) either unlabeled or with a 3′-fluorescein label ( Table 1 ) .", "Oligonucleotides were 5′-32P-radiolabelled and gel purified as described previously ( Hutton et al . , 2010 ) .", "Labelled oligonucleotides were annealed with complementary strands by heating with an excess of unlabelled strands at 95°C for 5 min and then slow cooling to room temperature overnight in a heating block .", "The assembled substrates ( Table", "2 ) were purified by native polyacrylamide ( 12% ) gel electrophoresis with 1× Tris-borate-EDTA ( TBE ) buffer , followed by band excision , gel extraction and ethanol precipitation before being resuspended in nuclease free water , as described previously ( Hutton et al . , 2010 ) .", "The final substrate concentration was measured using the extinction coefficient and absorbance at 260 nm in a UV-Vis spectrophotometer ( Varian Cary , Agilent , Santa Clara , CA , United States ) and DNA diluted to ∼50 nM or ∼200 nM final concentration for use in assays . 10 . 7554/eLife . 08716 . 018Table 1 . Sequence of oligonucleotides used for substrate constructionDOI: http://dx . doi . org/10 . 7554/eLife . 08716 . 018StrandSequence 5′→3′Length1aTAGTAAGAGATTAATAAACCCTCAGATAATCTCTTATAGAATTGAAAGTTCGG531bTTTTTTTTTTTTTTTTTTATTATCTGAGGGTTTATTAATCTCTTACTA481cCCGAACTTTCAATTCTATAAGAG232aTAGTAAGAGATTAATAAACCCTCAGATAACCTCTTATAGAATTGAAAGTTCGG532bTTTTTTTTTTTTTTTTTTGTTATCTGAGGGTTTATTAATCTCTTACTA483bTTTTTTTTTTTTTTTTTAGTTATCTGAGGGTTTATTAATCTCTTACTA484bTTTTTTTTTTTTTTTTTCGTTATCTGAGGGTTTATTAATCTCTTACTA485bTTTTTTTTTTTTTTTTTGGTTATCTGAGGGTTTATTAATCTCTTACTA486aTAGTAAGAGATTAATAAACCCTCAGATAAGCTCTTATAGAATTGAAAGTTCGG536bTTTTTTTTTTTTTTTTTACTTATCTGAGGGTTTATTAATCTCTTACTA487aTAGTAAGAGATTAATAAACCCTCAGATAAACTCTTATAGAATTGAAAGTTCGG537bTTTTTTTTTTTTTTTTTATTTATCTGAGGGTTTATTAATCTCTTACTA488bTTTTTTTTTTTTTTTTTAATTATCTGAGGGTTTATTAATCTCTTACTA489aTAGTAAGAGATTAATAAACCCTCAGATAACATCTTATAGAATTGAAAGTTCGG539cCCGAACTTTCAATTCTATAAGAT2310aTAGTAAGAGATTAATAAACCCTCAGATAACTTCTTATAGAATTGAAAGTTCGG5310cCCGAACTTTCAATTCTATAAGAA2311aTAGTAAGAGATTAATAAACCCTCAGATAACGTCTTATAGAATTGAAAGTTCGG5311cCCGAACTTTCAATTCTATAAGAC2312aTAGTAAGAGATTAATAAACCCTCAGATAACTCCTTATAGAATTGAAAGTTCGG5312cCCGAACTTTCAATTCTATAAGGA2313aTAGTAAGAGATTAATAAACCCTCAGATAACTGCTTATAGAATTGAAAGTTCGG5313cCCGAACTTTCAATTCTATAAGCA2314aTAGTAAGAGATTAATAAACCCTCAGATAACTACTTATAGAATTGAAAGTTCGG5314cCCGAACTTTCAATTCTATAAGTA23SacI-aTAGTAAGAGATTAATAAACCCTCAGATGAGCTCTTATAGAATTGAAAGTTCGG53SacI-bTTTTTTTTTTTTTTCTCATCTGAGGGTTTATTAATCTCTTACTA441b-3′-FAMTTTTTTTTTTTTTTATTATCTGAGGGTTTATTAATCTCTTACTA-FAM4819aCCTCGAGGGATCCGTCCTAGCAAGCCGCTGCTACCGGAAGCTTCTGGACC5019bGCTCGAGTCTAGACTGCAGTTGAGAGCTTGCTAGGACGGATCCCTCGAGG5019cGGTCCAGAAGCTTCCGGTAGCAGCG2520d-10AGTCTAGACTCGAGC1520d-5ACTGCAGTCTAGACTCGAGC2020dTCTCAACTGCAGTCTAGACTCGAGC2525c-dGGTCCAGAAGCTTCCGGTAGCAGCGTCTCAACTGCAGTCTAGACTCGAGC501c-3′PCCGAACTTTCAATTCTATAAGAG-phos25Sp3-1aCTGGCGCGGGGAACTCTCTAAAAGTATACATTTGTTCTT39Sp3-1bTGTAATTGATAATGTTGAGAGTTCCCCGCGCCAG34Sp3-1cAAGAACAAATGTATACTTTTAGA23Sp3-2aCCAGCGGGGATAAACCGTTTGGATCGGGTCTGGAATTTC39Sp3-2bTGTTCCGACAGGGAGCCCGGTTTATCCCCGCTGG34Sp3-2cGAAATTCCAGACCCGATCCAAAC2310 . 7554/eLife . 08716 . 019Table 2 . DNA constructs used in this studyDOI: http://dx . doi . org/10 . 7554/eLife . 08716 . 019SubstrateOligonucleotide componentsJunction sequence−2−11ICSubstrate 11a , 1b , 1cAGATSubstrate 1-FAM1a , 1b-3′-FAM , 1cAGATSacI substrateSacI-a , SacI-b , 1cAGCTSubstrate 22a , 2b , 1cAGGTSubstrate 32a , 3b , 1cAGGA3′-phos substrate2a , 3b , 1c-3′PAGGASubstrate 42a , 4b , 1cAGGCSubstrate 52a , 5b , 1cAGGGSubstrate 66a , 6b , 1cAGCASubstrate 77a , 7b , 1cAGTASubstrate 81a , 8b , 1cAGAASubstrate 99a , 3b , 9cATGASubstrate 1010a , 3b , 10cAAGASubstrate 1111a , 3b , 11cACGASubstrate 1212a , 3b , 12cGAGASubstrate 1313a , 3b , 13cCAGASubstrate 1414a , 3b , 14cTAGASubstrate 152a , 4b , 1cAGGCSubstrate 1611a , 4b , 11cACGCSubstrate 1710a , 4b , 10cAAGCSubstrate 189a , 4b , 9cATGCSubstrate 1919a , 19b , 19cCGGAGap1019a , 19b , 19c , 20d-10CGGAGap519a , 19b , 19c , 20d-5CGGANick19a , 19b , 19c , 20dCGGAY-junction19a , 19b , 20c-dCGGASpacer 3-1 substrateSp3-1a , Sp3-1b , Sp3-1cGAGASpacer 3-2 substrateSp3-2a , Sp3-2b , Sp3-2cACGCThe sequence of the central portion of the junction ( positions −2 , −1 , 1 and the incoming nucleotide ( IC ) ) for each substrate is shown .", "The oligonucleotides used to assemble the complete substrate are indicated .", "Reactions were typically carried out under single turnover conditions .", "Titration of SsoCas1 ( Figure 3B , C ) showed evidence for inhibition at enzyme:substrate ratios above 10:1 .", "For standard assays , 2 μM Cas1 protein was mixed with 200 nM substrate in cleavage buffer ( 20 mM Tris [pH 7 . 5] , 10 mM NaCl , 1 mM DTT and 5 mM MnCl2 ) and incubated at 55°C ( for SsoCas1 ) or 37°C ( for EcoCas1 ) .", "For reactions with Cas1 and Cas2 , the proteins were mixed in equimolar concentration and incubated together at either 37 or 55°C for 30 min before being added to the reaction .", "At indicated times , reactions were quenched by the addition of EDTA to 20 mM final concentration and 1 μl 20 mg/ml Proteinase K ( Promega , Madison , WI , United States ) and incubation at 37°C for 30 min .", "The DNA was then separated from the reaction by phenol chloroform extraction .", "60 μl neutral phenol:chloroform:isoamyl alcohol ( Sigma–Aldrich , St . Louis , MO , United States ) was added and the reaction vortexed for 30 s .", "The sample was then centrifuged ( 15 , 000×g , 1 min ) and the upper aqueous phase , containing the DNA , collected .", "Formamide loading dye ( 100% formamide with 0 . 25% bromophenol blue , 0 . 25% xylene cyanol ) was added ( 5 µl ) and the sample heated at 95°C for 2 min before being chilled on ice .", "Reaction products were resolved on a pre-run 20% denaturing ( 7 M urea ) polyacrylamide gel containing 1× TBE in 1× TBE buffer .", "Gels were run at 80 W , 45°C for 90 min before overnight exposure to a phosphorimaging plate and imaging with a FLA-5000 Imaging System ( Fujifilm Life Science , Düsseldorf , Germany ) .", "Assays with the SacI junction substrate were carried out under standard conditions with SsoCas1 for 30 min .", "FastDigest SacI enzyme ( 1 μl ) and 1 μl FastDigest Buffer ( Thermo Scientific , Waltham , MA , United States ) were then added and the reaction incubated at 37°C for 30 min .", "Product extraction , separation and visualization was then carried out as described above .", "For the time course assays , the concentration of DNA substrates was 50 nM and the concentration of Cas1 protein 50 nM for SsoCas1 or 500 nM for EcoCas1 .", "Reactions were performed as described above with the omission of the Proteinase K step .", "Following phosphorimaging , substrates and products were quantified using Image Gauge software ( Fujifilm ) and the reaction course was plotted using Kaleidagraph ( Synergy Software , Reading , PA , United States ) .", "Experiments were carried out in triplicate and the mean and standard error calculated for each point .", "For SsoCas1 , the data were fitted using a single exponential ( Niewoehner et al . , 2014 ) .", "For EcoCas1 the reactions did not go to completion and were therefore fitted with a floating end-point , as described previously ( Niewoehner et al . , 2014 ) ." ] ]

[ "The adaptive prokaryotic immune system CRISPR-Cas provides RNA-mediated protection from invading genetic elements .", "The fundamental basis of the system is the ability to capture small pieces of foreign DNA for incorporation into the genome at the CRISPR locus , a process known as Adaptation , which is dependent on the Cas1 and Cas2 proteins .", "We demonstrate that Cas1 catalyses an efficient trans-esterification reaction on branched DNA substrates , which represents the reverse- or disintegration reaction .", "Cas1 from both Escherichia coli and Sulfolobus solfataricus display sequence specific activity , with a clear preference for the nucleotides flanking the integration site at the leader-repeat 1 boundary of the CRISPR locus .", "Cas2 is not required for this activity and does not influence the specificity .", "This suggests that the inherent sequence specificity of Cas1 is a major determinant of the adaptation process ." ]

[ "In most animals , the adaptive immune system creates specialized cells that adapt to efficiently fight off any viruses or other pathogens that have invaded .", "Bacteria ( and another group of single-celled organisms called archaea ) also have an adaptive immune system , known as CRISPR-Cas , that combats viral invaders .", "This system is based on sections of the microbes' DNA called CRISPRs , which contain repetitive DNA sequences that are separated by short segments of ‘spacer’ DNA .", "When a virus invades the cell , some viral DNA is incorporated into the CRISPR as a spacer .", "This process is known as adaptation .", "CRISPR-associated proteins ( or ‘Cas’ proteins ) then use this spacer to recognize and mount an attack on any matching invader DNA that is later encountered .", "Exactly how a spacer is inserted into the correct position in the CRISPR array during adaptation remains poorly understood .", "However , it is known that two CRISPR proteins called Cas1 and Cas2 play essential roles in this process .", "Rollie et al . took Cas1 proteins from a bacterial cell ( Escherichia coli ) and an archaeal species ( Sulfolobus solfataricus ) and added them to branched DNA structures in the laboratory .", "These experiments revealed that Cas1 from both organisms can break the DNA down into smaller pieces .", "Cas2 , on the other hand , is not required for this process .", "This ‘disintegration’ reaction is the reverse process of the ‘integration’ step of adaptation where the CRISPR proteins insert the invader DNA into the CRISPR array .", "Rollie et al . also found that the disintegration reaction performed by Cas1 takes place on specific DNA sequences , which are also the sites where Cas1 inserts the spacer DNA during adaptation .", "Therefore , by examining the disintegration reaction , many of the details of the integration step can be deduced .", "Overall , Rollie et al . show that selection by Cas1 plays an important role in restricting the adaptation process to particular DNA sites .", "The next step will be to use the disintegration reaction to examine the DNA binding and manipulation steps performed by Cas1 as part of its role in the adaptation of the CRISPR system ." ]

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

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

[ [ "SARS-CoV-2 is the virus causing the global pandemic ‘coronavirus diseases 2019’ ( COVID-19 ) .", "This is a zoonotic beta-coronavirus from bats that causes severe acute respiratory syndrome ( Lu et al . , 2020 ) .", "Its effects on human physiology extends well beyond the lung as it unleashes a cytokine storm to alter immune response ( Chen et al . , 2020 ) and cause disseminated intravascular coagulation ( Lillicrap , 2020 ) .", "It also exhibits multiorgan tropism that impacts kidney , liver , heart and brain function ( Puelles et al . , 2020 ) .", "Among its structural elements , the SARS-CoV-2 Spike protein is critical for viral attachment , fusion and entry into host ( Hoffmann et al . , 2020a; Li et al . , 2003; Lan et al . , 2020 ) .", "The RBD region of Spike enables binding to its primary human cellular receptor , angiotensin-converting enzyme-2 ( ACE2 ) , which is ubiquitously expressed on epithelial , endothelial and blood cells ( Li et al . , 2003; Hamming et al . , 2004 ) .", "A role for heparan sulfates in viral entry has also been proposed ( Clausen et al . , 2020 ) .", "Once bound , viral fusion and entry depends on two proteolysis sites , the furin/RRAR-S site located between the Spike S1 and S2 subunits , and a second S2’/SKR-S site ( Belouzard et al . , 2009 ) .", "A variety of enzymes including TMPRSS2 ( transmembrane protease , serine 2 ) and cathepsin-L may aid viral entry ( Hoffmann et al . , 2020b; Matsuyama et al . , 2005; Shang et al . , 2020 ) .", "While inhibitors of viral binding , RNA transcription and protease activity are being tested to reduce viral load , this manuscript suggests an orthogonal approach based on glycan engineering .", "Both the viral Spike-protein and ACE2 receptor are extensively glycosylated , with a majority of the 22 N-glycosylation sites of Spike and 7 N-glycosylation sites of ACE2 bearing carbohydrates ( Walls et al . , 2020; Wrapp et al . , 2020; Shajahan et al . , 2020a; Figure 1 ) .", "The exact distribution of the oligomannose , hybrid , complex , sialylated , and fucosylated structures in these macromolecules is likely dictated both by the protein structure and host expression system ( Shajahan et al . , 2020b; Watanabe et al . , 2020 ) .", "O-linked glycans are also reported on both proteins ( Shajahan et al . , 2020a; Shajahan et al . , 2020b ) .", "A variety of functions have been proposed for these glycans including immunological shielding ( Watanabe et al . , 2020 ) , direct regulation of Spike-ACE2 binding ( Bernardi et al . , 2020 ) , and control of Spike up/down conformation ( Casalino et al . , 2020 ) .", "Experimental data supporting these concepts is currently lacking and thus our understanding of the impact of glycosylation on SARS-CoV-2 function is incomplete .", "We address the above gaps in knowledge in the current manuscript .", "Our results suggest that both the O- and N-linked glycans of the SARS-CoV-2 Spike protein , including sialylated glycan epitopes , have a relatively minor role in regulating direct Spike-ACE2 binding interactions .", "However , these glycans control the rates of viral entry into HEK293T cells expressing human ACE2 .", "Here , blocking N-glycosylation on SARS-CoV-2 pseudovirus at the high-mannose stage using CRISPR-Cas9 and also a small molecule inhibitor ( kifunensine ) resulted in extensive cleavage/shedding of the viral Spike protein at the time of production due to enhanced proteolysis at the S1-S2 interface .", "Our data also suggest that glycans may contribute to additional aspects of Spike proteolysis during viral entry .", "Due to these effects , viral entry into human ACE2 expressing cells was reduced by >95% when virus was produced in the CRISPR-Cas9 MGAT-1 knockout cells lacking complex N-glycans , and by 85–90% upon production in the presence of kifunensine .", "These observations support the need to screen chemical inhibitors of glycosylation for the treatment of COVID-19 ." ], [ "In order to study the impact of N- and O-linked glycosylation on SARS-CoV-2 function , we engineered cells over-expressing full-length Spike-protein and ACE2 ( Figure 2A ) .", "We also histidine-tag purified soluble , dimeric Fc-his fusion proteins for the Spike S1 region , RBD and extracellular portion of ACE2 .", "The molecules were extensively glycosylated with their apparent molecular mass being greater than their theoretical mass based on peptide alone .", "Thus , RBD-Fc was ~60 kDa instead of a theoretical mass of 51 kDa , S1-Fc was ~150 kDa rather than 101 kDa , and ACE2-Fc was ~140 kDa instead of 110 kDa ( Figure 2B ) .", "RBD-Fc and S1-Fc readily bound to HEK293T cells upon human ACE2 over-expression , confirming that they are functionally active ( Figure 2C ) .", "ACE2-Fc also specifically bound Spike-protein expressed on 293Ts .", "Baseline RBD-Fc and S1-Fc binding to Spike expressing 293 T cells ( i . e . 293 T/S ) was even lower than that of wild-type 293T ( red line , Figure 2C ) .", "This suggests expression of low amounts of Spike binding proteins on WT-293Ts .", "These basal Spike receptors may engage cell-surface Spike on 293 T/S cell ( via cis-interactions ) causing RBD-Fc and S1-Fc binding to be lower in 293 T/S cells compared to WT-293Ts .", "Sialidase treatment of Spike-protein expressed on 293Ts did not affect ACE2-Fc binding ( Figure 2D ) .", "Sialidase treatment of ACE2 expressed on 293Ts , however , increased RBD-Fc and S1-Fc binding by 26% and 56% respectively .", "Control lectin binding studies confirmed the high activity of the sialidase enzyme used in this study ( Figure 2—figure supplement 1A ) .", "The relatively small effect of the sialidase in these studies was not due to the absence of sialic acid on either S1-Fc or ACE2-Fc .", "In this regard , independent glycoprotemics mass spectrometry ( MS ) data showed that ~80–100% of the N-glycans expressed at specific sites of ACE2-Fc and ~40–100% of the S1-Fc glycopeptides expressed complex-type glycans ( K . C . , et al . , manuscript in preparation ) .", "Up to ~60% of the antennae on these complex structures on ACE2 and ~20% of the structures on S1-Fc were terminated by sialic acid .", "To complement the above binding studies , we measured SARS-CoV-2 pseudovirus entry into stable 293T/ACE2 cells .", "This assay provides an aggregate measure of both molecular binding and viral fusion/entry in the context of the physiological Spike trimeric configuration ( Tai et al . , 2020 ) .", "Here , the control VSVG ( Vesicular stomatitis virus G-protein ) pseudotyped virus displayed broad tropism both for wild-type HEK293T and stable 293T/ACE2 ( Figure 2E , Figure 2—figure supplement 1B ) .", "Wild-type SARS-CoV-2 Spike-protein virus ( ‘Spike-WT’ ) only entered 293T/ACE2 cells confirming the strict dependence on cell-surface ACE2 receptor .", "A ‘Spike-mutant’ pseudovirus , where the ‘RRAR’ furin-site was swapped with an ‘SRAS’ sequence , also only entered 293T/ACE2 ( 22 ) .", "This mutation results in reduced proteolysis at the S1-S2 interface , as discussed later , likely due to the presence of a single arginine site at the S1-S2 interface rather than the polybasic ( ‘RRAR’ ) sequence .", "To study the role of sialic acid on viral entry , we titered the Spike-WT and Spike-mutant virus using p24 ELISA ( Figure 2—figure supplement 2 ) .", "The viruses were then treated with a pan-sialidase from Arthrobacter ureafaciens , and the same amount of Spike-WT and Spike-mutant particles were applied to stable 293T/ACE2 cells ( Figure 2F , Figure 2—figure supplement 1C ) .", "Here , Spike-mutant pseudotyped virus was ~5–10 times more effective at stimulating DsRed-reporter expression compared to Spike-WT .", "Thus , the efficiency of cleavage at the S1-S2 interface can fine-tune SARS-CoV-2 infectivity ( Hoffmann et al . , 2020b; Shang et al . , 2020; Walls et al . , 2020 ) .", "Sialidase treatment of virus did not impact viral entry , consistent with the notion that Spike sialic acid does not regulate molecular recognition .", "Additionally , sialidase treatment of 293T/ACE2 did not alter either VSVG or Spike-WT pseudovirus entry ( Figure 2G , Figure 2—figure supplement 1D ) .", "A partial increase in Spike-mutant viral entry was measured upon sialidase treatment ( p=0 . 08 ) , suggesting a minor inhibitory role for ACE2 sialic acid when viral infectivity is high .", "Glycoproteomics analysis of purified pseudovirus produced in HEK293T cells showed that ~45–100% of the viral N-glycans were complex-type , with up to ~55% of their antennae being terminated by sialic acid ( K . C . , et al . manuscript in preparation ) .", "Thus , the pseudovirus used in this study was processed by glycoenzymes that are typically found in the cellular medial and trans-Golgi compartments .", "Together , the binding and pseudovirus data suggest that ACE2 sialic acids may modestly shield virus/Spike binding in some biological contexts and perhaps some cell systems .", "To determine if other aspects of N- and O-linked glycosylation may impact SARS-CoV-2 function , we applied CRISPR-Cas9 technology to develop isogenic HEK293T clones that lack either the core-1 O-glycan forming galactosyltransferase C1GalT1 or the N-glycan branching β1 , 2GlcNAc-transferase MGAT1 ( Figure 3A; Stolfa et al . , 2016; Chugh et al . , 2018 ) .", "Here , knocking out C1GalT1 blocks O-glycan biosynthesis at the GalNAcα-Ser/Thr ( +/- sialic acid ) stage as it is not elaborated to form the core-1 structure ( Galβ1 , 3GalNAcα ) .", "These cells are termed ‘[O]-293T’ .", "Knocking out MGAT1 blocks N-glycan biosynthesis at the Man-5 stage , and the resulting clone is called ‘[N]-293T’ .", "Sanger sequencing confirmed that all three C1GalT1 alleles of [O]-293T contained indels ( Figure 3B ) .", "Frameshift mutation was also noted at the single MGAT1 allele in [N]-293T .", "As anticipated , VVA-lectin ( Vicia villosa agglutinin ) binding to GalNAcα was dramatically augmented in the [O]-293Ts ( Figure 3C ) .", "Complex glycans were absent in [N]-293Ts as they did not engage PHA-L ( lectin from Phaseolus vulgaris ) .", "Additional data with a panel of fluorescent lectins further confirm specific blockade of N-glycan complex structures and lactosamine chains in [N]-293T , and inhibition of O-glycan extension in the [O]-293 T cells ( Figure 3—figure supplement 1 ) .", "Together , the findings confirm the absence of O- and N-glycan elaboration in the [O]-293T/C1GalT1-KO and [N]-293T/MGAT1-KO cells , respectively .", "Wild-type 293Ts and the glycoenzyme knockouts were transiently transfected to express Spike and ACE2 for binding studies .", "Both full-length proteins expressed well and at equal levels in the different cell systems ( Figure 3—figure supplement 2A ) .", "In order to quantify the effect of glycosylation on ACE2-Spike binding , ACE2-Fc was applied to Spike bearing cells , either 293T or the O/N-glycan knockouts ( Figure 3D ) .", "Similarly , either S1-Fc ( Figure 3E ) or RBD-Fc ( Figure 3F ) were applied to ACE2 bearing 293T or the N-/O-glycan knockouts .", "These Fc-proteins were applied at sub-saturation concentrations in the cytometry binding studies in order to quantitatively evaluate differences in receptor-ligand-binding .", "Here , we observed a 40% decrease in ACE2-Fc binding to cell-surface Spike expressed on [N]-293Ts , and a slight 20% increase when Spike was expressed on the [O]-293Ts ( Figure 3D ) .", "Glycan engineering of ACE2 in the [O]- and [N]-293Ts did not impact either S1-Fc ( Figure 3E ) or RBD-Fc binding ( Figure 3F ) .", "Here , RBD-Fc bound ~10 times more avidly to ACE2 compared to S1-Fc .", "Together , the data suggest a modest role for Spike-protein glycosylation on direct Spike-ACE2 binding .", "Our observation that the binding of RBD for ACE2 was substantially higher than S1-ACE2 interactions is consistent with a previous report ( Shang et al . , 2020 ) .", "This highlights the need to carefully consider RBD presentation/conformation in the context of the full protein when quantifying molecular affinity to ACE2 .", "We evaluated the impact of ACE2 and Spike glycosylation on viral entry .", "To determine the impact of ACE2 glycosylation , we transiently expressed this protein on wild-type 293Ts , [O]-293Ts and [N]-293Ts .", "The entry of the pseudovirus ( VSVG , Spike-WT and Spike-mutant ) to these three cell types was then evaluated ( Figure 3G , Figure 3—figure supplement 2B ) .", "Here , the pattern of viral entry was similar in all cell systems .", "Lower entry was measured for Spike-WT and Spike-mutant pseudovirus infection of [O]-293T/ACE2 cells , but this was also noted for the control VSVG-virus .", "Thus , this reduced infectivity in the O-glycan knockout is not exclusively due to ACE2 glycans .", "We conclude that ACE2 glycans may not regulate viral entry .", "In contrast to ACE2 , glycan engineering of viral Spike protein dramatically affected viral entry ( Figure 4 ) .", "To study this , VSVG , Spike-WT and Spike-mutant pseudotyped virus were generated in wild-type 293T , [O]-293T and [N]-293T ( Figure 4A ) .", "All nine viruses were used to infect stable 293T/ACE2 cells .", "When produced in [O]-293T , Spike-WT and Spike-mutant pseudovirus resulted in a 70–85% loss in DsRed fluorescence ( Figure 4B ) , and a partial reduction ( 35–70% ) in the fraction of DsRed positive cells ( Figure 4C ) .", "In stark contrast , knocking out N-glycans in both pseudoviruses abrogated viral infection into 293T/ACE2 cells by >95% ( Figure 4B and C ) .", "Dose studies confirmed loss of viral function upon inhibiting N-glycan elaboration over a range of viral titers ( Figure 4D ) .", "This was observed for the Spike-mutant and also the Spike-WT pseudovirus ( data not shown ) .", "Western blot with anti-S2 pAb ( polyclonal antibody ) suggest that both the Spike-WT and Spike-mutant virus expressed comparable levels of Spike protein , regardless of the glycosylation mutation ( Figure 4E ) .", "The molecular mass of Spike expressed in [N]-293T was lower than that of other virus consistent with the extensive N-glycosylation of this protein .", "Additionally , the data suggest more extensive cleavage of Spike-WT compared to Spike-mutant , consistent with the presence of the proteolysis-sensitive polybasic furin-site in Spike-WT .", "The Spike pseudovirus produced in [N]-293Ts also appeared to have lower intact Spike protein .", "To examine this more closely as the anti-S2 pAb may have preference for binding the S2-domain over full Spike , we measured the FLAG-epitope at the C-terminus of Spike-mutant using an anti-FLAG mAb ( monoclonal antibody ) .", "This may provide more uniform recognition of both the full Spike and the S2-subunit ( Figure 4F ) .", "This blot suggests that N-glycan loss may trigger precipitous ( 95% , based on densitometry ) loss of intact Spike protein due to enhanced cleavage at the S1-S2 interface .", "Consistent with the viral entry assay , we also noted greater proteolysis of virus produced in [O]-293Ts ( 52% ) compared to that produced in wild-type 293Ts ( 32% ) .", "Together , the data suggest that both Spike N-glycans , and possibly also O-glycans , may play a role in regulating SARS-CoV-2 Spike-protein stability .", "Remarkably , the same observations as seen in the viral N-glycan knockouts could be reproduced upon producing viral particles in cells cultured with the potent mannosidase-I alkaloid inhibitor kifunensine ( KI ~25–125 nM , Elbein et al . , 1990; Figure 5A ) .", "This water-soluble inhibitor reduced the formation of complex N-glycans on cultured cells within 24–48 hr with no change in cell viability ( Figure 5—figure supplement 1A ) .", "Virus produced in culture with 15 µM kifunensine displayed ~85–90% reduction in infection , as measured using DsRed-reporter in microscopy ( Figure 5B , Figure 5—figure supplement 1B ) and cytometry assays ( Figure 5C and D ) .", "The reduction in Spike molecular mass upon addition of this small molecule was less apparent compared to that in [N]-293Ts ( Figure 5E ) , since blockade of Mannosidase-I preferentially gives rise to Man7-9 glycans while the MGAT1/[N]-293Ts knockouts produce Man-5 .", "Here also , the Spike-protein proteolysis was more extensive upon kifunensine addition though it was not complete , as observed in the anti-FLAG blot .", "In order to determine if the enhanced S1-S2 site proteolysis of Spike protein upon N-glycan inhibition is the exclusive reason for reduced viral entry , we produced virus with a Spike variant ( ‘Spike-delta’ ) that contained the C-terminal FLAG-epitope but that lacked the furin-site ( Figure 5F ) .", "The substitution of the ‘RRAR’ sequence with a single Alanine ( ‘A’ ) resulted in viral Spike that was not extensively cleaved both in the absence and presence of kifunensine .", "Robust viral entry into 293T/ACE2 cells was observed using this furin resistant virus , and this too could be partially blocked by kifunensine .", "Thus , in addition to proteolysis at the S1-S2 site , N-glycans may have additional roles during viral entry ." ], [ "This manuscript undertook a series of studies in order to comprehensively describe the role of O- and N-linked glycans on SARS-CoV-2 Spike binding to human ACE2 ( Figure 6A ) , and related viral entry ( Figure 6B ) .", "It demonstrates only a minor role for carbohydrates in regulating Spike-ACE2 direct binding .", "In this regard , we observed some enhancement in Spike binding and viral entry upon treating 293T/ACE2 with a pan-sialidase .", "Based on the published crystal structure and molecular modeling , the critical glycans regulating this process are likely located at Asn-90 or Asn-322 , proximal to the Spike binding interface ( Lan et al . , 2020; Figure 1 ) .", "The effect was nevertheless small compared to that reported for other sialic acid dependent viruses like influenza ( Stencel-Baerenwald et al . , 2014 ) , reovirus , parovirus ( Löfling et al . , 2013 ) and coronavirus like the Middle-East Respiratory Syndrome virus/MERS ( Li et al . , 2017 ) .", "The studies performed with CRISPR-Cas9 cell lines containing disrupted O- and N-linked glycans also support the concept that glycosylation is not a critical regulator of ACE2-Spike binding .", "Here , blocking O- and N-glycan biosynthesis on ACE2 did not impact either viral entry or Spike-Fc binding .", "Thus , the functional change observed upon enzymatically removing sialic acid on ACE2 is likely a steric effect , as it can be offset by other modifications .", "Blocking elaboration of O- and N-glycans on Spike , also , only had a partial effect on ACE2-Fc binding .", "This observation is consistent with previous cryo-EM ( Walls et al . , 2020; Wrapp et al . , 2020 ) and molecular dynamics simulation results ( Grant et al . , 2020 ) , which show that the active/‘up’ form of Spike RBD is largely exposed with few glycans at the binding interface .", "It is now well appreciated that this lack of glycan shielding , makes the exposed RBD a prime target for vaccine development .", "In contrast to the modest effect of glycosylation on receptor-ligand-binding , we observed a much more profound role for glycans in regulating viral entry in part due to their impact on Spike proteolysis at the S1-S2 interface .", "In this regard , we observed that blocking N-glycan elaboration using both genetic approaches ( i . e . MGAT1-KO/[N]-293T ) and the natural product mannosidase-I inhibitor , kifunensine , dramatically reduced viral entry into 293T/ACE2 cells .", "This was observed for two virus types: Spike-WT and Spike-mut .", "In the case of Spike-mut , enzymatic and small molecule inhibition resulted in enhanced proteolysis of Spike-protein during pseudovirus production .", "These data suggest an inverse relation between furin cleavage and viral entry into 293T/ACE2 cells .", "Consistent with this , Spike-mut displayed reduced S1-S2 proteolysis compared to Spike-WT , and higher viral infectivity .", "Others have also noted a reduction in viral entry , upon increasing furin cleavage potential by addition of more basic residues ( ‘RRRKR’ mutation ) at the S1-S2 interface ( Hoffmann et al . , 2020b ) .", "Finally , a recent study observed that the introduction of the D614G mutation in Spike reduced furin cleavage , and this resulted in enhanced viral infectivity ( Korber et al . , 2020 ) .", "SARS-CoV-2 containing the D614G mutation is currently the dominant strain worldwide ( >80% prevalence , Zhang et al . , 2020 ) , and the possibility that this is due to altered furin cleavage potential is hotly debated .", "Overall , our studies with MGAT1-KO , C1GalT1-KO and kifunensine suggest that both N- and O-glycans may modulate the rates of proteolysis at the S1-S2 interface , thus affecting viral entry ( Figure 6C ) .", "While some S1 may be associated with Spike even after partial cleavage at the S1-S2 interface ( Belouzard et al . , 2009 ) , this may potentiate a quantitative reduction in RBD presentation on viral surface , resulting in reduced ACE2 binding and lower viral entry .", "Indeed several N-glycans are located proximal to the furin-site: N603 and N657 , and also N61 ( Watanabe et al . , 2020 ) .", "O-glycans have also been hypothesized to exist in this region at S673 , T678 and S686 ( Andersen et al . , 2020 ) , and some of these have been experimentally verified ( Sanda et al . , 2020 ) .", "How this site-specific glycosylation affects SARS-CoV-2 entry , remains to be determined .", "In addition to its role in regulating RBD presentation , our data suggest that N-glycans may also have other roles in regulating viral entry .", "In this regard , we noted that even the robust entry of Spike-delta pseudovirus into 293T/ACE2 cells could be partially inhibited by kifunensine ( Figure 6C ) , and this is independent of S1-S2 proteolysis .", "Others have noted that the effect of ‘Spike-delta’ mutation may be cell type specific in that this mutation results in lower infectivity in some cell types like Calu-3 which rely on the enzyme TMPRSS2 for viral fusion , while this mutation does not affect entry into other cell types like VeroE6 which lacks TMPRSS2 ( Hoffmann et al . , 2020b ) .", "Based on these observations , we speculate that additional glycosylation sites proximal to S2’ may also modulate viral entry , perhaps because some proteases like TMPRSS2 have both ligand-binding and protease activity .", "The N-glycans proximal to S2’ include N801 , and to a lesser extent N616 and N657 .", "Additional studies with more cell types and mutant virus are necessary in order to fully elucidate the molecular mechanism by which O- and N-glycans regulate host-cell specific viral entry response .", "In addition to basic science understanding , our findings have translational potential as it suggests that both O- and N-glycan truncation may be used to reduce viral entry .", "In this regard , it would be attractive to test additional N-glycosylation inhibitors , like Swainsonine , which has a demonstrated safety profile in humans ( Goss et al . , 1997 ) .", "Additional potential inhibitors that may modulate viral entry include α-mannosidase inhibitors ( deoxymannojirimycin , mannostatin A ) , α-glucosidase inhibitors ( N-butyl deoxynojirimycin , N-nonyl-deoxynojirimycin , castanospermine , celgosivir ) ( Clarke et al . , 2020 ) , and finally compounds like SNAP ( Del Solar et al . , 2020; Wang et al . , 2018 ) and 4F-GalNAc ( Marathe et al . , 2010 ) which block various aspects of N- and O-glycan biosynthesis .", "These potentially represent off-the-shelf drugs and compounds that may be repurposed to reduce viral load and ameliorate SARS-CoV-2 related respiratory symptoms .", "Studies are currently underway in our laboratory to test these concepts , and extend the assay to authentic SARS-CoV-2 strains .", "Overall , while SARS-CoV-2 has developed a natural ability to enhance infectivity , we propose that glycoengineering may provide a strategy to take advantage of these evolutionary features to regulate viral entry and reduce disease transmission ." ], [ "Human embryonic kidney 293 T cells ( ‘293T’ ) and Lenti-X 293 T cells were purchased from Clontech/Takara Bio ( Mountain View , CA ) .", "Stable HEK293T/ACE2 cells were kindly provided by Michael Farzan ( Scripps Research , Jupiter , FL ) .", "All cells were mycoplasma negative .", "These cell types were maintained in culture using Dulbecco’s Modified Eagle Medium ( DMEM ) containing 10% fetal bovine serum , 1% Pen-Strep and 1% GlutaMAX supplement .", "All lectins were from Vector labs .", "These include VVA ( Vicia Villosa lectin , binds GalNAcα on O-glycans ) , SNA ( Sambucus nigra lectin , binds primarily α2 , 6 sialic acid ) , ECL ( Erythrina cristagalli lectin , binds desialylated lactosamine Galβ1 , 4GlcNAc chains ) and PHA-L ( Phaseolus vulgaris Leucoagglutinin , binds Galβ1 , 4GlcNAcβ1 , 6 ( GlcNAcβ1 , 2Manα1 , 3 ) Manα1 , 3 on complex N-glycans ) .", "Goat anti-human ACE2 polyclonal antibody ( AF933 ) and mouse anti-human ACE2 mAb MAB9332 were available from R and D Systems ( Minneapolis , MA ) .", "Rabbit anti-SARS-CoV-2 RBD ( 40592-T62 ) and anti-S2 ( 40590-T62 ) pAbs were from Sino Biologicals ( Beijing , China ) .", "Anti-FLAG clone L5 was from Biolegend ( San Diego , CA ) .", "All secondary antibodies ( Abs ) were from Jackson ImmunoResearch ( West Grove , PA ) .", "When necessary , lectins and Abs were labelled by addition of 25-fold molar excess succinimidyl ester coupled Alexa-405 , Alexa-488 , Alexa-555 or Alexa-647 dye ( Fluoroprobes , Scottsdale , AZ ) to protein suspended in phosphate buffered saline ( PBS , pH 7 . 4 ) for 1 hr at room temperature ( RT ) .", "Following this , the reaction was quenched with 1/10th volume 1 M Tris , and unreacted Alexa-dye was removed using 7 kDa molecular mass cutoff Zeba desalting spin columns ( Thermo ) .", "Unless mentioned otherwise , all other reagents were from either Thermo-Fisher or Sigma Chemicals .", "The ACE2-RBD complex structure ( 6LZG , Wang et al . , 2020 ) was superimposed to the trimeric spike-protein state ( 6VYB Walls et al . , 2020 ) , in which one of the RBD domains is ‘open’ so that ACE2 is associated with the RBD domain in the open conformation .", "Missing residues in the structure were modeled using PyRosetta ( Chaudhury et al . , 2010 ) .", "N-glycans were then added to the structure using Glycan Reader within CHARMM-GUI ( Jo et al . , 2008; Jo et al . , 2011 ) .", "The identity of the glycans was assigned based on a published work ( Watanabe et al . , 2020 ) .", "The final structure contained glycan modifications at 54 spike Asn and 6 ACE2 Asn .", "All simulations were performed using NAMD2 ( Phillips et al . , 2005 ) using the CHARMM36 parameters ( Vanommeslaeghe et al . , 2010 ) .", "The complex was first placed in a water box of sufficient size to ensure a minimum 12 Å separation from the wall .", "Net charge in the molecule was neutralized by adding 841 K+ and 743 Cl- ions , resulting in 867 , 166 atoms total .", "Protein and glycan atoms were first fixed while water ( TIP3 ) molecules were energy minimized for 10 , 000 steps .", "Next , the entire system was relaxed for another 10 , 000 steps , and the system temperature was gradually increased to 310 K in increments of 1 K with 120 fs of equilibration at each temperature .", "A time step of 2 fs was used .", "Constant temperature and pressure were maintained using the Langevin framework ( Martyna et al . , 1994 ) .", "Long range electrostatic interactions were treated using the particle mesh Ewald method ( Darden et al . , 1993 ) .", "The simulation was continued for 26 . 5 ns .", "Intermediate structures were saved every 10 ps .", "A plasmid containing the Spike protein ( 2019-nCov_pcDNA3 . 1 ( + ) -P2A-eGFP [v1] ) was kindly provided by Dr . Haisheng Yu ( Institute of Laboratory Animal Science , Peking Union Medical College ) .", "To create plasmids [v3] and [v4] , the full S1 domain coding region ( M1 to R682 ) or RBD coding region ( R319 to F541 ) was PCR amplified and cloned into the NheI/XbaI or AgeI/XbaI sites , respectively of pCSCG-19FcHisP2AdTom ( Lo et al . , 2013 ) .", "Human ACE2 plasmid was obtained from Addgene ( #1786 , Li et al . , 2003 ) .", "The entire ACE2 coding region was amplified in two segments to silence an EcoRI cutting site in the coding region and joined by overlap extension PCR .", "The resulting PCR fragment was cloned into the EcoRI/NheI site of pKLV2-CMVpuroBFP vector to obtain [v2] .", "Additionally , [v5] was created by PCR amplifying the region encoding ACE2 extracellular domain ( Q18 to S740 ) and cloning into the AgeI/XbaI sites of pCSCG-19FcHisP2AdTom plasmid .", "Plasmids [v2]-[v5] will be distributed via Addgene .", "HEK293T CRISPR-Cas9 cells were generated by transfecting wild-type 293 T cells with pX330-U6-Chimeric_BB-CBh-hSpCas9 vector ( Addgene , Plasmid# 42230 ) carrying single-guide RNA ( sgRNA ) targeting either the Core-1 β3Gal-T ( C1GalT1 , target site: GCAGATTCTAGCCAACATAA ) or β1 , 2 GlcNAc-transferase ( MGAT1 , GTGGGGCGCTATCCTCTTTGTGG ) .", "While knocking out the former gene results in cells expressing O-glycans truncated at the Tn-antigen stage ( GalNAcα±sialic acid ) , the latter prevents the N-glycan processing beyond the oligomannose/Man5 stage ( Stolfa et al . , 2016 ) .", "Isogenic clones of both cell lines were obtained by single-cell flow cytometry sorting using fluorescently conjugated VVA and PHA-L for selection .", "Knockouts were confirmed by Sanger sequencing of genomic DNA .", "For convenience , cells with disrupted O- and N-glycoprotein processing are termed ‘[O]-293T’ and ‘[N]-293T’ , respectively .", "All Fc-his tag proteins ( vectors [v3] , [v4] and [v5] ) were expressed by transient transfection of HEK 293 T cells using the calcium phosphate method ( Buffone et al . , 2013 ) , in 3–4 150 mm cell culture Petri dishes per protein .", "Protein secreted into serum-free media were passed through a 1 mL HisTrap FF column ( Cytiva , Marlborough , MA ) at 1 mL/min .", "Following extensive washing with 20 mM sodium phosphate buffer containing 0 . 5 M NaCl and 10 mM imidazole ( pH7 . 2 ) , the bound protein was eluted by collecting 0 . 5 mL fractions upon step increasing imidazole concentration in the above buffer to 100 mM ( 8 mL ) followed by 500 mM ( 8 mL ) .", "Protein concentration in each fraction was determined using a flow cytometry bead assay .", "To this end , briefly , 5 µm Polybead carboxylate microspheres ( Polysciences , Warrington , PA ) were covalently coupled with goat anti-human pAb using 1-Ethyl-3- ( 3-dimethylaminopropyl ) carbodiimide/EDC chemistry ( Marathe et al . , 2008 ) .", "1 μL of each fraction collected during HisTRAP elution was incubated with these beads for 10 min at RT , before washing and addition of 1:200 diluted FITC conjugated secondary Ab for an additional 10 min .", "Bead-bound Fc-protein was then quantified using flow cytometry .", "All fractions containing the Fc-fusion proteins were then pooled and spin concentrated to ~150 μL volume .", "Buffer was exchanged to HEPES buffer ( 30 mM HEPES , 110 mM NaCl , 10 mM KCl , 10 mM glucose , 2 mM MgCl2 ) using 7 kDa Zeba desalting columns .", "Final protein concentration was determined in ELISA format by using a calibrant Fc-protein of known concentration that was verified to be >95% pure based on silver stain analysis .", "Here , a common anti-human HRP conjugated Ab was used to quantify both the Fc- proteins described in this manuscript and the calibrant Fc in the same assay .", "Identity of expressed protein and also viral Spike was determined using western blotting with anti-Fc ( Jackson ) , anti-RBD ( Sino Biologicals ) , anti-S2 ( Sino Biologicals ) and anti-ACE2 ( R and D Systems ) pAbs .", "Anti-FLAG mAb L5 was also used for western blotting of Spike-mutant virus as it carried a C-terminal FLAG tag .", "Standard flow cytometry methods were applied in some instances to measure cell-surface protein expression and lectin binding , using directly conjugated fluorescent reagents ( Stolfa et al . , 2016 ) .", "Here the lectins/Abs were added to cells for 15 min , prior to performing a quick wash followed by cytometry analysis .", "These data are presented as Geometric Mean Fluorescence Intensity ( ‘GMFI’ ) .", "Full-length Spike-protein and human ACE2 were expressed in 293 T cells ( wild-type , [O]-293T and [N]-293T ) by transient transfection using the calcium phosphate method ( Buffone et al . , 2013 ) .", "These cells are called ‘293 T/S’ or ‘293T/ACE2’ when expressed on wild-type HEK293T cells , and ‘[O]-293 T/S’ , ‘[O]-293T/ACE2’ , ‘[N]-293 T/S’ or ‘[N]-293T/ACE2’ when expressed in the glycosylation-KO mutants .", "Here , just prior to functional studies , cells were released from the culture substrate 48–72 hr after transfection using PBS containing 5 mM EDTA , washed and re-suspended in HEPES buffer containing 1 . 5 mM CaCl2 .", "During the binding studies , 0–4 μg/mL S1-Fc , RBD-Fc or ACE2-Fc were added to 20 × 106 cells/mL at 4°C for 15 min .", "Following this , 1:200 diluted Alexa-647 ( Ax647 ) conjugated goat anti-human-Fc ( Fab' ) 2 Ab was added for an additional 10 min .", "The cells were then washed , resuspended and read immediately using a 4-laser BD Fortessa flow cytometer .", "Transfected cells were gated based on EGFP expression for Fc-protein binding studies performed with 293T/S-protein cells , and BFP expression for studies using 293T/ACE2 cells .", "cell-surface expression of ACE2 and Spike on transiently transfected cells was also monitored on experimental day using flow cytometry .", "These studies used Alexa-647 conjugated anti-ACE2 ( MAB9332 , R and D systems ) and anti-RBD ( Sino Biologicals ) antibodies .", "In some cases , binding studies were also performed with stable 293T/ACE2 cells , in which case fluorescence gating was not necessary .", "In some assays , cells were treated with sialidase ( 200 U/mL Arthrobacter ureafaciens α2–3 , 6 , 8 , 9-Neuraminidase , New England BioLabs ) for 1 hr at 37°C , and washed using HEPES buffer prior to the binding assay .", "Sialidase activity was verified based on SNA and ECL staining .", "In this case , control cells were treated identically , except that sialidase was withheld .", "SNA was directly conjugated with Alexa-555 and ECL was conjugated with Alexa-647 in order to simplify the implementation of dual color cytometry measurements .", "SARS-CoV-2 Spike-protein virus was generated by replacing the classical Vesicular stomatitis virus ( VSV-G ) envelope protein of 3rd generation lentivirus with either wild-type Spike protein ( ‘Spike-WT’ , [v1] ) or a mutant Spike plasmid ( ‘Spike-mutant’ ) containing an ‘SRAS’ sequence in place of furin sensitive ‘RRAR’ ( gift from Michael Farzan , Quinlan et al . , 2020 ) .", "In an additional variant called ‘Spike-delta’ , the ‘SRAS’ site in Spike-mutant was replaced by a single ‘A’ .", "To produce these viruses , Lenti-X 293 T cells ( Takara Bio , MountainView , CA ) were transiently transfected with 17 . 8 µg of pLKO . 1", "TRC-DsRed , 22 µg of psPAX2 and 5 . 8 µg of VSVG ( or 9 . 9 µg of v1 or 8 . 6 µg of Spike-mutant/Spike-delta ) plasmid in a 15 cm dish using the calcium phosphate method ( Buffone et al . , 2013 ) .", "Six hours post-transfection , the medium was changed to virus collection medium ( OPTIMEM + 10% FBS ) .", "The first batch of virus was collected 18–20 hr thereafter .", "Virus collection medium was then supplemented with 10 mM sodium butyrate .", "The second virus batch was collected 18–20 hr later .", "Both virus batches were pooled , filtered through 0 . 45 μm filter and ultra-centrifuged at 50 , 000 × g to concentrate the particles .", "The viral pellet was resuspended in OPTIMEM + 10% FBS , aliquoted and stored at −80°C until use .", "Following the above protocol , 14 additional virus types were generated in this manuscript .", "This includes virus with envelope composed of: i .", "VSVG , ii .", "Spike-WT or iii .", "Spike-mutant that were produced in either:", "a . wild-type HEK293T ,", "b . [O]-293 T", "c . [N]-293T or", "d . wild-type HEK293T being cultured in the presence of 15 µM kifunensine ( Toronto Research Chemicals , Canada ) .", "In addition , Spike-delta was produced in the presence and absence of 15 µM kifunensine .", "For many of the production lots , viral titer was determined using the lentiviral p24 ELISA kit from Takara Bio ( MountainView , CA ) following manufacturer’s instructions .", "Virus concentration was also verified by western blotting with anti-S2 pAb or anti-FLAG to detect equivalent amounts of Spike protein in each of the preparations .", "In order to transduce cells , virus was added at various titers indicated in the main manuscript to either wild-type 293Ts , stable 293T/ACE2 cell lines or various glycoengineered variants that transiently overexpressed ACE2 ( at 48 hr post-transfection ) .", "Such infection was performed in the presence of 8 µg/ml polybrene as described previously ( Buffone et al . , 2013 ) .", "In some cases , either the virus or cells were treated with sialidase for 1 hr at 37°C under conditions described above , prior to transduction .", "Virus was removed and fresh medium was added 8 hr post-transduction .", "DsRed fluorescence was monitored on a daily basis .", "All flow cytometry and microscopy data presented in this manuscript correspond to the 72 hr time point .", "Here , DsRed positive cells and the arithmetic mean fluorescence intensity ( ‘MFI’ ) of all cells in the PE-channel were also quantified using flow cytometry ( BD Fortessa X-20 ) .", "Microscopy images were acquired using a Zeiss AxioObserver instrument ( 10X/0 . 25 NA or 20X/0 . 4 NA objective ) .", "All data are presented as mean ± standard deviation .", "Dual comparisons were performed using the two-tailed Student’s t-test .", "ANOVA followed by the Tukey post-test was used for multiple comparisons .", "p<0 . 05 was considered to be statistically significant .", "Number of repeats are specified in individual panels ." ] ]

[ "The Spike protein of SARS-CoV-2 , its receptor-binding domain ( RBD ) , and its primary receptor ACE2 are extensively glycosylated .", "The impact of this post-translational modification on viral entry is yet unestablished .", "We expressed different glycoforms of the Spike-protein and ACE2 in CRISPR-Cas9 glycoengineered cells , and developed corresponding SARS-CoV-2 pseudovirus .", "We observed that N- and O-glycans had only minor contribution to Spike-ACE2 binding .", "However , these carbohydrates played a major role in regulating viral entry .", "Blocking N-glycan biosynthesis at the oligomannose stage using both genetic approaches and the small molecule kifunensine dramatically reduced viral entry into ACE2 expressing HEK293T cells .", "Blocking O-glycan elaboration also partially blocked viral entry .", "Mechanistic studies suggest multiple roles for glycans during viral entry .", "Among them , inhibition of N-glycan biosynthesis enhanced Spike-protein proteolysis .", "This could reduce RBD presentation on virus , lowering binding to host ACE2 and decreasing viral entry .", "Overall , chemical inhibitors of glycosylation may be evaluated for COVID-19 ." ]

[ "COVID-19 is an infectious disease caused by the virus SARS-CoV-2 .", "To access the internal machinery necessary for its replication , the virus needs to latch onto and then enter host cells .", "Such processes rely on specific ‘glycoproteins’ that carry complex sugar molecules ( or glycans ) , and can be found at the surface of both viruses and host cells .", "In particular , the viral ‘Spike’ glycoprotein can attach to human proteins called ACE2 , which coat the cells that line the inside of the lungs , heart , kidney and brain .", "Yet the roles played by glycans in these processes remains unclear .", "To investigate the role of Spike and ACE-2 glycans , Yang et al . designed a form of SARS-CoV-2 that could be handled safely in the laboratory .", "How these viruses infect human kidney cells that carry ACE2 was then examined , upon modifying the structures of the sugars on the viral Spike protein as well as the host ACE2 receptor .", "In particular , the sugar structures displayed by the virus were modified either genetically or chemically , using a small molecule that disrupts the formation of the glycans .", "Similar methods were also applied to modify the glycans of ACE2 .", "Together , these experiments showed that the sugars present on the Spike protein play a minor role in helping the virus stick to human cells . However , they were critical for the virus to fuse and enter the host cells .", "These findings highlight the important role of Spike protein sugars in SARS-CoV-2 infection , potentially offering new paths to treat COVID-19 and other coronavirus-related illnesses .", "In particular , molecules designed to interfere with Spike-proteins and the viral entrance into cells could be less specific to SARS-CoV-2 compared to vaccines , allowing treatments to be efficient even if the virus changes ." ]

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

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

[ [ "Assembly and maturation of HIV-1 and other retroviruses is driven by the viral structural polyprotein , Gag , which consists of a series of independently folding domains , called MA ( matrix ) , CA ( capsid ) , and NC ( nucleocapsid ) ( reviewed in Bush and Vogt , 2014; Ganser-Pornillos et al . , 2012; Lingappa et al . , 2014 ) .", "During assembly , Gag makes a spherical , immature capsid shell that packages the viral genome , directs interactions with the plasma membrane to promote envelopment , and recruits cellular machinery to release the new virus particle from the host cell .", "The Gag shell has hexagonal paracrystalline lattice symmetry , wherein the central CA region makes two layers of interactions through its two domains ( NTD and CTD , for N-terminal and C-terminal domain ) .", "During maturation , Gag is cleaved by the viral protease at specific sites .", "This results in disassembly of the Gag shell and release of new structural proteins , which then rearrange into the mature , infectious virion .", "In the mature virion , the genome is repackaged inside a cone-shaped capsid made up of the CA protein , with the NTD forming hexameric or pentameric rings that are connected by the CTD .", "As with other animal viruses , the large-scale capsid rearrangements that occur during retroviral maturation are associated with the operation of structural switches that undergo dramatic conformational changes .", "In HIV-1 , two such putative switches flank the CA region of Gag .", "One switch spans the MA-CA junction , which is in an extended configuration in the immature virion ( Kelly et al . , 2006; Tang et al . , 2002 ) .", "Upon proteolysis , the new CA N-terminus folds into a β-hairpin stabilized by a buried salt bridge between the new N-terminal proline and a conserved aspartate residue and promotes assembly of the mature capsid ( Gitti et al . , 1996; von Schwedler et al . , 1998 ) .", "A second putative switch spans the junction between CA and the first spacer peptide ( SP1 ) ( Gross et al . , 2000 ) .", "The CA-SP1 junction is predicted to fold into an α-helix ( Accola et al . , 1998; Liang et al . , 2002 ) , and forms the binding site of so-called 'maturation inhibitors , ' as exemplified by 3-O- ( 3’ , 3’-dimethylsuccinyl ) betulinic acid ( bevirimat ) ( reviewed in Adamson et al . , 2009 ) .", "These inhibitors delay proteolysis of the CA-SP1 junction and induce aberrant maturation .", "The importance of the CA-SP1 junction as a regulator of proteolysis during maturation is well established ( Kräusslich et al . , 1995; Pettit et al . , 1994; Wiegers et al . , 1998 ) , but the molecular basis of this function has been unknown .", "Mutagenesis studies also indicate that the CA-SP1 junction is important for assembly of the immature HIV-1 Gag shell ( Accola et al . , 1998; Al-Mawsawi et al . , 2014; Datta et al . , 2011; Guo et al . , 2005; Kräusslich et al . , 1995; Liang et al . , 2002; Liang et al . , 2003; Melamed et al . , 2004; Pettit et al . , 1994; Rihn et al . , 2013; von Schwedler et al . , 2003 ) .", "Low resolution electron microscopy studies indicate that the junction forms what appears to be a pillar of density or helical bundle , and so is likely to stabilize the Gag lattice ( Schur et al . , 2015a; Schur et al . , 2015b; Wright et al . , 2007 ) .", "However , neither the SP1 spacer nor the putative helical bundle is a universal feature of retroviral Gag shells ( Bharat et al . , 2012 ) .", "This suggests that the SP1 spacer is not generally important for Gag assembly , or that perhaps the CA-SP1 junction has another , as yet structurally undefined assembly determinant .", "Structural studies of the HIV-1 CA-SP1 junction in context of longer Gag fragments ( CTD-SP1 and CTD-SP1-NC ) have shown that the junction is primarily disordered ( Newman et al . , 2004; Worthylake et al . , 1999 ) .", "Nevertheless , the backbone NMR chemical shifts of junction residues deviate from expected random coil values , indicating a small propensity towards an α-helical conformation ( Newman et al . , 2004 ) .", "Indeed , the isolated SP1 peptide displays concentration dependent secondary structure in aqueous solution , and folds into an α-helix at the millimolar protein concentrations found in virions ( Datta et al . , 2011 ) .", "Nucleic acids ( both RNA and DNA ) can promote Gag assembly in vitro , presumably because of proximity-induced interactions between multiple copies of Gag that bind to the same nucleic acid molecule ( Campbell and Rein , 1999; Campbell and Vogt , 1995; Gross et al . , 2000 ) .", "It therefore seems that nucleic acid-induced Gag clustering might also promote folding of the SP1 helix .", "To learn how the CA-SP1 junction functions as a molecular switch during HIV-1 assembly and maturation , we determined the structure of its immature , assembled form by X-ray crystallography at a resolution of 3 . 27 Å .", "Our analysis elucidates how local conformational changes in the CA-SP1 switch promote Gag assembly , drive large-scale capsid rearrangements , and regulate proteolysis during maturation .", "We also gained new insights on the mechanism of action of maturation inhibitors ." ], [ "In the immature HIV-1 Gag shell , the NTD , CTD , and SP1 regions form three layers of lattice-stabilizing interactions ( Schur et al . , 2015b; Wright et al . , 2007 ) .", "The CTD-SP1 fragment of HIV-1 Gag contains the minimal information required to assemble the immature lattice , whereas the NTD is dispensable ( Accola et al . , 2000 ) .", "Accordingly , we found that purified CTD-SP1 protein assembled in vitro into flat sheets with the subunits organized into a hexagonal lattice with the expected unit cell spacing of the CTD layer of the immature Gag lattice ( ~74 Å ) ( Wright et al . , 2007 ) ( Figure 1—figure supplement 1 ) .", "We then performed crystallization screens using a large number of commercial and in-house precipitants .", "We obtained many crystal hits , but invariably these were of the mature-like dimer form of the CTD with disordered SP1 tails , as observed previously ( Worthylake et al . , 1999 ) .", "Using electron diffraction , we identified small and scarce plate crystals that gave a hexagonal diffraction pattern and unit cell spacing close to 70 Å ( not shown ) .", "Upon optimization , these crystals diffracted X-rays to about 3 . 27 Å resolution ( mean I/σI ≥ 1 ) , and we determined the crystal structure to R/Rfree values of 0 . 246/0 . 278 ( Table 1 and Figure 1—figure supplement 2 ) .", "Our post hoc analysis is that these crystals were rare because the CA-SP1 junction residues had to fold and become ordered during crystallization .", "We speculate that this rate limiting step of crystallization reflects the behavior of the junction during assembly of HIV-1 Gag . 10 . 7554/eLife . 17063 . 003Table 1 . Structure statistics for HIV-1 Gag CTD-SP1 . DOI: http://dx . doi . org/10 . 7554/eLife . 17063 . 003DiffractionBeamlineAPS 22IDWavelength ( Å ) 1 . 0Processing programHKL2000Space groupC2Cell dimensionsa = 70 . 96 Åb = 122 . 73 Åc = 85 . 41 Åα = γ = 90° , β = 94 . 3°Resolution range , Å50-3 . 27 ( 3 . 42-3 . 27 ) Rmerge / Rpim0 . 22 ( 0 . 74 ) / 0 . 11 ( 0 . 47 ) Mean I/σ<I>5 . 99 ( 1 . 28 ) Completeness , %87 . 0 ( 66 . 4 ) Average redundancy3 . 7 ( 2 . 5 ) Wilson B-factor , Å285 . 21RefinementRefinement programPHENIXResolution range42 . 59-3 . 27 ( 3 . 45-3 . 27 ) No .", "of unique reflections9 , 710 ( 908 ) Reflections in free set1 , 009 ( 88 ) Rwork0 . 246 ( 0 . 369 ) Rfree0 . 278 ( 0 . 408 ) No .", "of nonhydrogen atoms protein3 , 865 solvent0Average B-factor , Å2 protein84 . 09 solventn/aCoordinate deviations bond lengths , Å0 . 003 bond angles , °0 . 513Validation and DepositionRamachandran plot favored , %98 . 9 outliers , %0MolProbity clash score0 . 13PDB ID5I4TValues in parenthesis are for the highest resolution shell .", "The CTD-SP1 plate crystals were made up of stacked sheets of flat hexagonal lattices , with each sheet consisting of CTD-SP1 hexamers connected by CTD dimer linkages ( Figure 1A ) .", "Our structure therefore represents a flattened version of the immature HIV-1 Gag lattice .", "The entire CTD-SP1 hexamer – including both the main CTD fold and ordered SP1 residues – gave an excellent fit to an 8 . 8 Å resolution cryoEM map of the immature HIV-1 Gag lattice ( Schur et al . , 2015b ) ( Figure 1—figure supplement 3A ) .", "The dimer also gave a good fit ( Figure 1—figure supplement 3B ) , but in this case the SP1 helix was more laterally displaced from its corresponding density in the cryoEM map ( asterisk in Figure 1—figure supplement 3B ) .", "This is likely because the cryoEM structure is of a curved Gag lattice , whereas our crystal structure is of a flat lattice .", "To gain insight on how loss of curvature in our crystal lattice is accommodated by the subunits , we superimposed the crystal structure with the model reported by Schur et al . , which was created by flexible fitting of the main CTD fold ( but not the CA-SP1 junction ) into the cryoEM map ( PDB 4USN ) ( Schur et al . , 2015b ) .", "We found that superposition of the hexamer units resulted in an average root mean square displacement of 1 . 92 Å over equivalent Cα atoms , whereas superposition of the dimers resulted in a comparable displacement of 1 . 93 Å .", "We therefore surmise that any changes in tertiary or quaternary structure that were caused by flattening of the lattice in our crystals have been dispersed across the subunits .", "Unlike the mature capsid , which contains sharp pentameric declinations and a variably curving lattice ( Ganser et al . , 1999; Pornillos et al . , 2011; Zhao et al . , 2013 ) , the immature HIV-1 Gag shell is a more smoothly curving sphere that is interrupted by large discontinuities ( Briggs et al . , 2009; Keller et al . , 2011; Wright et al . , 2007 ) .", "It therefore seems that generation of Gag curvature does not necessarily require the kinds of conformational variations observed in the mature capsid subunits . 10 . 7554/eLife . 17063 . 004Figure 1 . Crystal structure of the immature HIV-1 Gag CTD-SP1 lattice .", "( A ) Top view of the lattice , with symmetry equivalent subunits in the same color .", "A hexameric unit is outlined in red , and a dimeric unit is outlined in blue .", "( B , C )", "Side views of the hexamer .", "The structural elements that make intermolecular contacts are colored and labeled: MHR ( red ) , helix 9/10 loop ( orange ) , GVGGP β-turn motif ( yellow ) , and junction helix ( blue/magenta ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 17063 . 00410 . 7554/eLife . 17063 . 005Figure 1—figure supplement 1 . Initial characterization of CTD-SP1 . ( A ) Schematic diagram of the HIV-1 Gag protein , with the different domains indicated .", "The polyhistidine-tagged CTD-SP1 construct used in these studies is indicated beneath .", "( B ) Merged projection map of two-dimensional CTD-SP1 crystals at about 9 Å resolution .", "Density features consistent with a 6-helix bundle are encircled in red . DOI: http://dx . doi . org/10 . 7554/eLife . 17063 . 00510 . 7554/eLife . 17063 . 006Figure 1—figure supplement 2 . Maps and model building .", "( A ) Initial maps ( mesh ) calculated after molecular replacement with the CTD and rigid body refinement in PHENIX .", "The maps are of high quality , as evidenced by the appearance of unbiased densities for the 6-helix bundle ( indicated in left ) and sidechains ( encircled in right ) .", "( B ) Initial unbiased 6-fold NCS map ( blue mesh ) of the junction helix region , which was used to build a polyalanine model ( sticks ) .", "( C ) After several rounds of refinement and rebuilding , a feature-enhanced map ( FEM , magenta mesh ) was calculated with model phases ( Afonine et al . , 2015 ) .", "Sidechains densities are evident in the FEM map ( encircled ) , which provided a unique solution to the helix registry .", "( D ) The model ( sticks ) was rebuilt into the FEM map , with the helical registry assigned .", "( E ) Validation of the final model ( yellow ) with a composite simulated annealing ( SA ) omit map ( blue mesh , 1σ ) shown in stereoview .", "Close-up views of the SA omit map are also shown in Figure 3D and Figure 4A . DOI: http://dx . doi . org/10 . 7554/eLife . 17063 . 00610 . 7554/eLife . 17063 . 007Figure 1—figure supplement 3 . Comparison with the 8 . 8 Å cryoEM map of the immature HIV-1 Gag hexamer .", "( A ) Fitting of the CTD-SP1 hexamer as a single unit into the 8 . 8 Å resolution cryoEM map of the immature HIV-1 lattice ( Schur et al . , 2015b ) .", "( B ) Fitting of the CTD-SP1 dimer .", "Asterisk indicates the CA-SP1 junction helix that is laterally displaced from its corresponding density in the cryoEM map . DOI: http://dx . doi . org/10 . 7554/eLife . 17063 . 007 In our crystal structure , each subunit contains the main CTD fold ( amino acids 281–351 in the HIV-1 Gag numbering scheme ) and the CA-SP1 switch region ( residues 352–370 ) .", "The CTD adopts the canonical fold: a short 310 helix , followed by a strand/turn element called the major homology region ( MHR ) , and then four α-helices ( helices 8–11 ) ( Gamble et al . , 1997; Worthylake et al . , 1999 ) ( Figure 2A ) .", "The switch region immediately follows the main CTD fold , and consists of two parts: a type II β-turn ( residues 352–355 ) and an α-helix that spans the CA-SP1 junction ( residues 356–370 ) .", "This bipartite character was not anticipated but could explain the apparent structural variability in the C-terminal switch regions of immature retroviral capsids , which were discerned from subnanometer resolution cryoEM maps ( Bharat et al . , 2012; Schur et al . , 2015a; 2015b ) . 10 . 7554/eLife . 17063 . 008Figure 2 . Comparison of immature and mature forms of the CTD-SP1 switch .", "( A ) The immature CTD-SP1 subunit .", "Helices are shown as colored ribbons .", "Absolutely conserved MHR residues that mediate a salt bridge/hydrogen bond network are shown as yellow sticks , as are contributing residues from the helix 9/10 loop .", "( B ) Equivalent view of the mature CTD ( PDB 1A43 ) ( Worthylake et al . , 1999 ) .", "Disordered residues at the C-terminus are in dashes .", "A pronounced kink in the dimerization helix ( H9 , pink ) is absent in the immature form ( black arrows ) .", "( C ) Sequence conservation in the CTD-SP1 junction , derived from the curated Los Alamos HIV sequence database ( Kuiken et al . , 2003 ) ( 6824 sequences ) .", "Secondary structure of the immature and mature forms are shown above , with dashes indicating disordered regions .", "Proteolytic processing sites are marked by red arrowheads . DOI: http://dx . doi . org/10 . 7554/eLife . 17063 . 00810 . 7554/eLife . 17063 . 009Figure 2—figure supplement 1 . Quasi-equivalent conformations of the immature and mature dimers .", "( A , B )", "Top views of the CTD-SP1 dimer in context of: ( A ) the planar immature lattice from this study and ( B ) the CTD dimer in context of the planar mature lattice ( PDB 4XFX ) ( Gres et al . , 2015 ) .", "The interfacial Trp316 sidechains ( Trp184 in mature ) are encircled for reference .", "( C ) Superposition of the two structures as dimeric units emphasizes the different configurations of the subunits across the same interface . DOI: http://dx . doi . org/10 . 7554/eLife . 17063 . 009 Comparison of the immature CTD-SP1 subunit with the mature CTD subunit reveals a local conformational change ( presence or absence of a kink ) in the dimerization helix , as noted previously ( Bartonova et al . , 2008 ) ( arrows in Figure 2A , B ) .", "This correlates with the different orientations of the subunits across the immature and mature dimer interfaces ( Figure 2—figure supplement 1 ) ( Bartonova et al . , 2008; Gres et al . , 2015 ) .", "Another difference is that , in the mature CTD , residues downstream of helix 11 are disordered ( Bartonova et al . , 2008; Gamble et al . , 1997; Worthylake et al . , 1999 ) ( Figure 2B , C ) .", "These observations provide further support for the presumption that the C-terminal tail of CA becomes unfolded upon maturation and has no apparent role in assembly of the mature capsid .", "The CTD-SP1 hexamer looks like a goblet , with the main CTD fold forming the cup and the CA-SP1 junction forming the stem ( Figure 1B , C ) .", "Lateral packing between the subunits is quite loose along the body of the cup , and close contacts involving the main CTD fold occur only where the cup meets the stem .", "In this region , the MHR loops line the bottom inner surface of the cup ( Figure 1B , C , colored in red ) , whereas the loops connecting helices 9 and 10 ( 9/10 loop , orange ) line the outer surface of the cup and contact the tops of the junction helices in the stem ( blue/magenta ) .", "Underneath the cup , the junction helices form a 6-helix bundle as predicted ( Accola et al . , 1998; Wright et al . , 2007 ) .", "All of the aliphatic sidechains in the junction helix make 'knobs-in-holes' interactions similar to classical coiled-coils ( Figure 3A–C ) . 10 . 7554/eLife . 17063 . 010Figure 3 . The junction 6-helix bundle .", "( A ) Top view of the 6-helix bundle .", "The helical backbone is in ribbons , and sidechains that mediate 'knobs-in-holes' type packing are shown as sticks .", "Yellow spheres indicate the scissile peptide bond between Leu363 and Ala364 ( CA residue Leu231 and SP1 residue Ala1 ) .", "( B ) Same top view , with the subunits rendered in surface representation .", "( C ) Side view , with alternating subunits rendered as ribbons or surfaces .", "'Knobs-in-holes' sidechains are shown as orange sticks and labeled .", "( D ) Close-up of the scissile bond , which is sequestered at the bottom of a pocket occupied by the Met367 sidechain .", "Mesh shows a composite simulated annealing omit map ( 1σ ) .", "( E ) Comparison of the CA-SP1 junction in context of the 6-helix bundle ( left ) and bound to the viral protease active site ( right ) ( PDB 1 KJH ) ( Prabu-Jeyabalan et al . , 2000 ) .", "Sidechains that mediate both types of interactions are encircled , with equivalent residues in the same color . DOI: http://dx . doi . org/10 . 7554/eLife . 17063 . 010 During HIV-1 maturation , the CA-SP1 junction is one of ten cleavage sites in the precursor Gag and Gag-Pol polyproteins that are cleaved by the viral protease ( PR ) .", "Proteolytic processing occurs at different rates , with the CTD-SP1 junction processed with the slowest rate , and the downstream SP1-NC junction the fastest ( reviewed in Lee et al . , 2012 ) .", "The C-terminal boundary of the junction helix tracks precisely with sequence conservation; downstream residues that are disordered in our crystal structure are not conserved ( Figure 2C ) .", "This is consistent with the notion that the downstream SP1-NC junction has the fastest rate of processing because it is disordered in the immature virion .", "In our structure , the scissile bond ( between Leu363 and Ala364 ) is located inside the helical barrel ( yellow spheres in Figure 3A ) , where it is sequestered within a pocket covered by a methionine sidechain ( Met367 ) ( Figure 3D ) .", "When the CA-SP1 junction binds to PR , it adopts a fully extended , β-strand configuration wherein sidechains on either side of the scissile bond occupy sub-pockets within the enzyme active site ( Prabu-Jeyabalan et al . , 2000 ) ( Figure 3E , right ) .", "In the CTD-SP1 hexamer , these same sidechains mediate the 'knobs-in-holes' interactions of the 6-helix bundle ( Figure 3E , left ) .", "Therefore , for PR to gain access to its substrate , the 6-helix bundle must unfold .", "Since proteolysis rates for the different cleavage sites are comparable when measured with peptide substrates ( Lee et al . , 2012 ) , the rate limiting step for the CA-SP1 site is unfolding of the 6-helix bundle .", "This explains why the CA-SP1 junction displays the slowest rate of proteolysis and is the final trigger that elicits HIV-1 maturation .", "Our structure revealed a second structural component of the CA-SP1 switch , which consists of five amino acid residues that connect the main fold of the CTD and the junction helix ( 352GVGGP356 ) ( Figure 4A ) .", "This motif makes a right-handed type II β-turn that buries the Val353 sidechain within a shallow sub-pocket at the bottom of the CTD , which is made of the MHR and 9/10 loops .", "In context of the hexamer , the entire β-turn is almost completely buried within a larger pocket made up of two MHR loops , two 9/10 loops , and the junction helices from three adjacent subunits ( Figure 4B , C ) .", "Thus , the β-turn brings together multiple structural elements to generate the Gag hexamer .", "This key structural role indicates that the β-turn is a critical Gag assembly determinant . 10 . 7554/eLife . 17063 . 011Figure 4 . The β-turn 'clasp' motif .", "( A ) Close-up view of the GVGGP motif in context of a single subunit .", "An i , i+3 hydrogen bond characteristic of a right-handed β-turn is indicated , as is the sub-pocket occupied by the Val353 sidechain .", "Mesh shows a composite simulated annealing omit map ( 1σ ) .", "( B ) Stereo view of the β-turn and its surrounding pocket , which is made up of two MHR loops ( red ) , two helix 9/10 loops ( orange ) , and the N-terminal ends of three junction helices ( blue ) .", "The hexamer is rendered as a translucent surface .", "( C ) The six β-turns ( yellow sticks ) in context of the hexamer .", "The surrounding MHR , 9/10 loops , and junction helices are colored as in ( B ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 17063 . 01110 . 7554/eLife . 17063 . 012Figure 4—figure supplement 1 . Comparison of the HIV and MPMV switch regions . The sequence alignment spans the CTD-SP1 fragment of HIV-1 Gag and CTD of MPMV Gag .", "Unlike HIV-1 , MPMV does not have a spacer peptide in between its CA and NC domains .", "Despite the absence of a junction helix , the MHR , helix 9/10 loop , and putative 'clasp' motif of MPMV are highly similar in sequence to HIV-1 . DOI: http://dx . doi . org/10 . 7554/eLife . 17063 . 012 Numerous mutagenesis studies have established the importance of the CA-SP1 switch in assembly of the HIV-1 Gag protein ( Accola et al . , 1998; Datta et al . , 2011; Guo et al . , 2005; Kräusslich et al . , 1995; Liang et al . , 2002; Liang et al . , 2003; Melamed et al . , 2004; Pettit et al . , 1994 ) .", "Assembly defects in previous studies were assessed in different ways , however , so we performed our own alanine scan to obtain a uniform dataset .", "Mutations were made in context of the previously described △MA-CA-SP1-NC HIV-1 Gag construct , which displays pH-dependent assembly behavior upon dialysis with a single-stranded DNA template ( Gross et al . , 2000 ) .", "At high pH , △MA-CA-SP1-NC assembles into thick-walled immature virus-like particles , whereas at low pH , it assembles mature-like tubes , cones , and spheres .", "The immature △MA-CA-SP1-NC VLPs are nearly identical to the immature Gag shell of authentic virions ( Briggs et al . , 2009; Gross et al . , 2000 ) .", "When we dialyzed 2 mg/mL of wildtype ( WT ) △MA-CA-SP1-NC with a 26-mer DNA oligonucleotide at pH 8 , we obtained immature VLPs as expected , with little evidence of off-pathway assemblies or aggregation ( Figure 5B and Figure 5—figure supplement 1 ) .", "Under the same conditions , we obtained three mutant phenotypes ( which are color coded in Figure 5A and summarized in Table 2; a representative image of each mutant is shown in Figure 5—figure supplement 1 ) : WT-like mutants assembled immature VLPs ( green , + ) ( Figure 5B ) , 'non-assembling' mutants primarily produced aberrant particles or aggregates ( magenta , – ) ( Figure 5C ) , and intermediate mutants formed both immature VLPs and aberrant particles/aggregates ( yellow , +/– ) ( Figure 5D ) .", "We also observed mature-like tubes for some of the mutants , but these were relatively rare ( indicated by asterisks in Table 2; representative images in Figure 5—figure supplement 1 ) . 10 . 7554/eLife . 17063 . 013Figure 5 . Summary of structure-based alanine scanning mutagenesis .", "( A–D )", "Phenotypes of HIV-1 Gag △MA-CA-SP1-NC assembled with DNA .", "The ribbon diagram is of two opposing subunits in the hexamer with Cα atoms shown as spheres and color-coded according to assembly phenotypes as shown in panels B , C , and D . ( E–H ) Phenotypes of △MA-CA-SP1-NC assembled with tartrate .", "Scale bars = 150 nm . DOI: http://dx . doi . org/10 . 7554/eLife . 17063 . 01310 . 7554/eLife . 17063 . 014Figure 5—figure supplement 1 . Alanine scanning mutagenesis of CA-SP1 . Shown are representative images of △MA-CA-SP1-NC proteins assembled in vitro through dialysis with a single-stranded DNA template .", "Boxes are colored according to assembly phenotypes as indicated in Figure 5A–D and Table 2 .", "Insets show a representative immature particle for each particular mutant , if observed .", "Scale bars = 150 nm . DOI: http://dx . doi . org/10 . 7554/eLife . 17063 . 01410 . 7554/eLife . 17063 . 015Figure 5—figure supplement 2 . Alanine scanning mutagenesis of CA-SP1 . Shown are representative images of △MA-CA-SP1-NC proteins assembled in vitro by incubating with tartrate .", "Boxes are colored according to assembly phenotypes as indicated in Figure 5E–H and Table 2 .", "Scale bars = 150 nm . DOI: http://dx . doi . org/10 . 7554/eLife . 17063 . 01510 . 7554/eLife . 17063 . 016Table 2 . In vitro assembly phenotypes of HIV-1 △MA-CA-SP1-NC Gag . DOI: http://dx . doi . org/10 . 7554/eLife . 17063 . 016MutationLocationDNA template1Tartrate2Wildtype++R286AMHR strand++/mP289AMHR loop–mK290AMHR loop–mR294AMHR helix++P328A9/10 loop–mD329A9/10 loop–mM347AHelix 11+/–+Q351AHelix 11–mG352Aβ-turn–mV353Aβ-turn–mG354Aβ-turn–mG355Aβ-turn–mP356Aβ-turn /Junction helix–mG357AJunction helix+/–+/mH358AJunction helix–mK359AJunction helix++/mA360VJunction helix–mR361AJunction helix–+/mV362AJunction helix–mL363AJunction helix–mA364VJunction helix–mE365AJunction helix–*mA366VJunction helix–mM367AJunction helix–mS368AJunction helix+/–mQ369AJunction helix–mV370AJunction helix–*mT371ADisordered+/–*+/mN372ADisordered+/–+/mP373ADisordered+/–*mA374VDisordered+/–+/mT375ADisordered+/–+/mI376ADisordered+/–+/mM377ADisordered+/–+/m1 + , as in Figure 5B; – , as in Figure 5C; +/– , as in Figure 5D2 + , as in Figure 5F; m , as in Figure 5G; +/m , as in Figure 5H* rare mature tubes As expected from the structure , mutations in the main fold of the CTD that are located within the loosely packed cup region of the hexamer ( R286A and R294A ) were WT-like , whereas CTD mutations closer to the tightly packed junction region ( M347A and Q351A ) were more disruptive of assembly ( Table 2 and Figure 5—figure supplement 1 ) .", "In contrast , virtually all of the 19 single-point mutations in the structured region of the CA-SP1 switch ( residues 352–370 ) resulted in aberrantly assembled particles and/or protein aggregates ( Figure 5A , Table 2 , and Figure 5—figure supplement 1 ) .", "Two exceptions were G357A and K359A , both of which are located near the N-terminal end of the junction helix .", "The G357A phenotype was not unexpected because this is a polymorphic position ( Figure 2C ) .", "K359A , on the other hand , replaced a lysine residue lining the inside of the helical barrel with a hydrophobic residue , and the substitution may have eliminated clashes between like charges inside the 6-helix bundle .", "Thick-walled immature VLPs were also obtained with the S368A mutation near the C-terminal end of the junction helix .", "Apart from residues close to the C-terminal end of the junction helix , many of the switch mutants appeared to behave in an 'all-or-none' manner ( i . e . , they either assembled immature VLPs or aberrantly ) , indicating that both the β-turn and junction helix must be functional for assembly to occur in vitro .", "Such a behavior was unexpected because in high-order assembly systems , local defects in protein-protein interactions are typically buffered by cooperativity .", "Therefore , the 'all-or-none' behavior must be due to a defect in nucleation .", "To test this idea more rigorously , we used tartrate salts to induce assembly of the △MA-CA-SP1-NC VLPs ( Figure 5E ) .", "The absence of nucleic acids ensured that nucleation was triggered only by protein-protein interactions .", "In this format , most of the CA-SP1 switch mutants again failed to form immature VLPs , but now instead assembled efficiently into thin-walled mature-like particles ( Figure 5E–H , Table 2 , and Figure 5—figure supplement 2 ) .", "The same phenotype is observed when the entire SP1 spacer is deleted ( Gross et al . , 2000 ) .", "Since junction residues downstream of the main CTD fold are not required for assembly of the mature lattice and are almost certainly unfolded in the thin-walled particles , these results further indicate that nucleation of the immature particles requires folding of both the β-turn and junction helix .", "Some of the mutants formed both immature VLPs and mature particles in the presence of tartrate , in similar proportions ( R286A , G357A , K359A , R361A ) ( Figure 5H and Figure 5—figure supplement 2 ) .", "Examination of well-separated particles indicated that each particle was either immature-like or mature-like , i . e . , we did not observe ones that were partly thin-walled and partly thick-walled .", "We believe that this is an important observation , because it indicates that the identity of the particle is established at nucleation ( Gross et al . , 2000 ) , and is more supportive of a disassembly/reassembly pathway of HIV-1 capsid maturation than a displacive or condensation mechanism ( Keller et al . , 2013 ) .", "Another notable mutant was K359A , which assembled into immature VLPs in the presence of DNA ( Figure 5—figure supplement 1 ) , but assembled into predominantly mature-like particles by the tartrate method ( Figure 5—figure supplement 2 ) .", "These results indicate that , although the HIV-1 Gag protein does not require nucleic acid to assemble into immature particles in vitro , folding of the CA-SP1 switch can be promoted by nucleic acid , likely due to clustering .", "We then extended our alanine scan to include SP1 residues downstream of the junction helix ( residues 371–377 ) .", "As expected , all of these mutants were competent to form immature VLPs in the presence of DNA , but surprisingly , were still significantly impaired in assembly efficiency ( Table 2 and Figure 5—figure supplement 1 ) .", "By the tartrate method , the mutants formed both mature and immature VLPs , with the exception of P373A , which was predominantly mature ( Table 2 and Figure 5— figure supplement 2 ) .", "Our interpretation of these results is that , even though the C-terminal end of SP1 is not highly conserved in sequence ( Figure 2C ) , the mutants were still defective in folding of the CA-SP1 junction , and again , nucleic acid ( DNA ) can partially compensate for the folding defect .", "Alternatively , it is possible that the helical character of SP1 extends downstream all the way to the NC domain , as previously proposed ( Morellet et al . , 2005 ) .", "However , our crystal structure , the cryoEM reconstruction ( Figure 1 —figure supplement 3 ) ( Schur et al . , 2015a ) , and sequence conservation ( Figure 2C ) appear to be in good agreement as to the boundaries of the junction helix .", "We therefore conclude that our structure defines the core helical bundle , although an extended helix may occur in a fraction of Gag molecules , for example the ones that nucleate assembly .", "Finally , we tested mutations at the MHR loop ( P289A , K290A ) and the 9/10 loop ( P328A , D329A ) that surround the Val353 pocket at the bottom of the CTD .", "These mutants behaved in a similar way as the β-turn and junction helix mutations ( Figure 5—figure supplement 1 , Figure 5—figure supplement 2 , and Table 2 ) .", "These results confirm that binding of Val353 to the bottom of the CTD – and by inference , proper folding of the β-turn – is a critical step in nucleation of the immature VLPs .", "We speculate that the valine pocket at the bottom of the CTD functions as a folding chamber or 'chaperone' for the β-turn ." ], [ "In a concurrent publication , Schur et al . report a cryoEM structure of the immature HIV-1 Gag lattice at near-atomic resolution ( Schur et al . , 2016 ) .", "They also conclude that the CA-SP1 boundary folds into a 6-helix bundle that is important for assembly of Gag and its proteolytic maturation ." ], [ "CTD-SP1 ( residues 281–377 of HIV-1 NL4-3 Gag ) was cloned with a non-cleavable His-tag ( His6-Gly2 ) into pET30a ( Novagen/EMD Millipore , Germany ) .", "The construct also contained the P373T substitution , which is a natural sequence variant ( Kuiken et al . , 2003 ) .", "Protein was expressed in E . coli BL21 ( DE3 ) cells by induction with 1 mM IPTG for 4 hr at 25°C in shake cultures .", "Bacteria were harvested by centrifugation and resuspended in 50 mM Tris , pH 8 . 3 , 1 M LiCl , 10 mM β-mercaptoethanol ( βME ) supplemented with 0 . 3% ( w/v ) deoxycholate and protease inhibitor tablets ( Roche ) .", "Cells were lysed by incubation with lysozyme and sonication .", "Lysates were clarified by centrifugation and then incubated with Ni-agarose resin ( Qiagen , Germany ) for 30 min at 4°C .", "Bound fractions were washed and eluted with a step gradient of 15–300 mM imidazole .", "The protein was purified to homogeneity using anion exchange and size exclusion chromatography in 20 mM Tris , pH 8 . 0 , 0 . 5 M NaCl , 20 mM βME .", "Pure proteins were concentrated to 15–20 mg/mL .", "Screening for 2D crystals was performed as described ( Yeager et al . , 2013 ) .", "CTD-SP1 ( 1 mM ) was mixed with an equal volume of 0 . 4 M sodium-potassium tartrate and incubated overnight at room temperature .", "Samples were placed on a carbon-coated grid , washed with 0 . 1 M KCl , and preserved with 2% glucose in 0 . 1 M KCl .", "Low-dose images of vitrified samples were recorded with a Titan Krios transmission electron microscope ( Philips/FEI , Hillsboro , OR ) operating at 120 kV .", "A merged projection map ( Figure 1—figure supplement 1 ) was calculated from 7 images , using the program 2dx ( Gipson et al . , 2007 ) .", "A B-factor of -500 Å2 was imposed to sharpen the map .", "Screening for three-dimensional crystals was performed using a large number of commercial and in-house precipitants .", "Plate crystals that formed in 0 . 1 M Bis-Tris propane , pH 7–8 , 0 . 8–1 . 0 M LiSO4 were initially identified by electron diffraction as being composed of stacked hexagonal sheets .", "Crystals for X-ray diffraction experiments were optimized in sitting drops , which were set up at a 1:2 protein:precipitant ratio .", "We found that the best diffracting crystals formed when drops were made with freshly purified protein .", "Ethylene glycol ( 25% ) in mother liquor was used as cryoprotectant .", "Diffraction data were collected from a single crystal at beamline 22-ID at the Advanced Photon Source , and processed with HKL2000 ( Otwinowski and Minor , 1997 ) .", "The phase problem was solved by molecular replacement with an immature CTD hexamer model ( PDB 4USN ) ( Schur et al . , 2015b ) .", "Upon rigid body refinement , unbiased densities for the 6-helix bundle were readily observed in model-phased maps ( Figure 1—figure supplement 2A ) .", "Multiple rounds of iterative model building and refinement were performed with the programs PHENIX ( version 1 . 9–1692 ) ( Adams et al . , 2010 ) and Coot ( Emsley et al . , 2010 ) .", "Due to the small size of the crystal ( ~20 microns in the longest dimension ) , the diffraction data were weak ( mean I/σ<I> = 6 and completeness = 87%; Table 1 ) .", "Nevertheless , we obtained very high quality maps for model building due to the fortuitous existence of 6-fold non-crystallographic symmetry ( NCS ) , and through the use of modern density modification techniques implemented in PHENIX .", "To obtain the best unbiased map for building the CTD-SP1 junction , we first extensively refined the main CTD fold using reference model restraints ( to PDB 3DS2 ) ( Bartonova et al . , 2008 ) .", "A 6-fold NCS averaged map was then calculated , which clearly revealed helical densities ( unbiased ) for the junction ( Figure 1 — figure supplement 2B ) .", "The junction helix was built into these densities as a polyalanine model using the 'Place Helix Here' command in Coot .", "After additional rounds of building and refinement , a feature-enhanced map was calculated with PHENIX ( Afonine et al . , 2015 ) , which gave a unique solution to the helical registry ( Figure 1 —figure supplement 2C , D ) .", "At low contour levels ( ~0 . 5 σ ) , residual densities that appeared to correspond to N-terminal His-tag residues were also observed , but these were left unmodeled .", "Secondary structure hydrogen bonding restraints , riding hydrogens , and local ( torsion angle ) 6-fold NCS restraints were used throughout the refinement process , as were structure validation tools implemented in both PHENIX and Coot .", "The current model was also validated with a composite simulated annealing omit map , shown in Figure 3D , Figure 4A , and Figure 1—figure supplement 2E .", "Structure statistics are summarized in Table 1 .", "For in vitro assembly assays , we used the △MA-CA-SP1-NC construct , which is a well-validated model system for the immature HIV-1 Gag shell ( Briggs et al . , 2009; Gross et al . , 2000 ) .", "WT and mutant proteins were expressed and purified as described ( Gross et al . , 2000 ) .", "Assembly reactions were set up as follows .", "DNA templated assembly: Purified protein ( 148 μL at 2 mg/mL ) was mixed with 26-mer single-stranded DNA oligonucleotide ( 5’-GGGAGTGGGGGGACTGAAGCAATGAG-3’ ) ( 2 μL at 1 mM ) and dialyzed for 2 hr at 4°C into 1 L of 50 mM Tris , pH 8 . 0 , 0 . 1 M NaCl , 1 mM EDTA , 2 mM βME .", "Particles were concentrated by centrifugation in a microcentrifuge at maximum speed for 10 min at 4°C , and resuspended in 15 μL of dialysis buffer .", "Tartrate-induced assembly: Protein ( 50 μL at 10–15 mg/mL ) was mixed with 19 . 25 μL of 1 . 5 M tartrate and 7 . 7 μL of 1 M Tris , pH 7 . 5 and incubated for 2 hr at 37°C .", "This second approach was more efficient at promoting assembly , and so the centrifugation step was omitted .", "For electron microscopy , each sample ( 3 μL ) was applied to a glow-discharged , continuous carbon-coated grid for 2 min .", "Excess liquid was blotted off by touching the edge of the grid with filter paper .", "The grid was washed with distilled water , blotted , stained with 2% ( w/v ) uranyl acetate for 2 min , and blotted again .", "Images of the negatively stained samples were recorded using a Tecnai F20 transmission electron microscope ( Philips/FEI ) operating at 120 kV .", "The assembly experiments were performed with two independent protein preparations for each mutant .", "In the second set of experiments , the grids were randomized so that the individual who acquired the images was unaware of the identity of the samples .", "Coordinates and structure factors are deposited in the Protein Data Bank under accession number 5I4T ." ] ]

[ "Virus assembly and maturation proceed through the programmed operation of molecular switches , which trigger both local and global structural rearrangements to produce infectious particles .", "HIV-1 contains an assembly and maturation switch that spans the C-terminal domain ( CTD ) of the capsid ( CA ) region and the first spacer peptide ( SP1 ) of the precursor structural protein , Gag .", "The crystal structure of the CTD-SP1 Gag fragment is a goblet-shaped hexamer in which the cup comprises the CTD and an ensuing type II β-turn , and the stem comprises a 6-helix bundle .", "The β-turn is critical for immature virus assembly and the 6-helix bundle regulates proteolysis during maturation .", "This bipartite character explains why the SP1 spacer is a critical element of HIV-1 Gag but is not a universal property of retroviruses .", "Our results also indicate that HIV-1 maturation inhibitors suppress unfolding of the CA-SP1 junction and thereby delay access of the viral protease to its substrate ." ]

[ "Viruses like HIV must undergo a process called maturation in order to successfully infect cells .", "Maturation involves a dramatic rearrangement in the architecture of the virus .", "That is to say , the virus’s internal protein coat – called the capsid – must change from an immature sphere into a mature cone-shaped coat .", "Notably , this maturation process is what is disrupted by the protease inhibitors that are a major component of anti-HIV drug cocktails .", "Structural changes in small portions of the capsid protein , termed molecular switches , commonly trigger the viral capsids to reorganize .", "The HIV capsid has two of these switches , and Wagner , Zadrozny et al . set out to understand how one of them – called the CA-SP1 switch – works .", "Solving the three-dimensional structure of the immature form of the CA-SP1 switch revealed that it forms a well-structured bundle of six helices .", "This helical bundle captures another section of the capsid protein that would otherwise be cut by a viral protease .", "The CA-SP1 switch therefore controls how quickly the protein is cut and the start of the maturation process .", "Wagner , Zadrozny et al . then discovered that other small molecule inhibitors of HIV , called maturation inhibitors , work by binding to and disrupting the transformation of the CA-SP1 switch .", "Finally , further experiments showed that the formation of the CA-SP1 helical bundle controls when the immature capsid shell forms and coordinates the process with the capsid gaining the genetic material of the virus .", "The new structure means that researchers now know what the HIV capsid looks like at the start and end of maturation .", "The next challenge will be to figure out exactly how the capsid changes from one form to the next as HIV matures ." ]

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

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

[ [ "The responses of many sensory neurons reflect multiple stages of computational transformation , arising from the functional and anatomical organization of underlying neural circuitry .", "Although much of our knowledge of these circuits comes from direct measurements of responses during sensory stimulation , experimental and analytical techniques for inferring the properties of intermediate stages remain limited .", "In particular , in many neural circuits , the critical computations performed by interneurons are difficult to measure directly .", "In the retina , cones , bipolar cells , and retinal ganglion cells ( RGCs ) form a two-tier cascade of linear and nonlinear processing that is thought to underlie many important visual functions ( see Schwartz and Rieke , 2011; Gollisch , 2012 ) .", "A notable example is the nonlinear subunit computation ( Hochstein and Shapley , 1976 ) observed in the responses of several types of RGCs , the output neurons of the retina .", "In these cell types , the circuitry composing the RGC receptive field does not linearly integrate cone photoreceptor inputs over space , as assumed by simplified descriptive models ( Chichilnisky , 2001; Keat et al . , 2001; Pillow et al . , 2008; Field et al . , 2010 ) .", "Instead , cone signals are first combined by bipolar interneurons , whose outputs are rectified at the synapse onto RGCs ( Demb et al . , 1999 , 2001; Borghuis et al . , 2013 ) .", "This nonlinear architecture endows the RGC receptive field with localized subunits that permit the representation of finer spatial detail than would be expected given the overall receptive field size ( Hochstein and Shapley , 1976; Lee et al . , 1995; Demb et al . , 2001; Baccus et al . , 2008; Crook et al . , 2008; Schwartz et al . , 2012 ) .", "Subunits have also been implicated in the processing of features like object motion and looming ( Olveczky et al . , 2007; Münch et al . , 2009 ) , and some of their functional characteristics have been inferred for various cell types and species ( Enroth-Cugell and Robson , 1966; Hochstein and Shapley , 1976; Victor and Shapley , 1979a , 1979b; Enroth-Cugell and Freeman , 1987; Bölinger and Gollisch , 2012; Schwartz et al . , 2012 ) .", "However , the spatial organization of convergent cone input onto nonlinear subunits has never been visualized , nor mapped across a population of RGCs .", "We combined large-scale parallel recordings of the responses of RGC populations to high resolution visual stimulation in order to characterize RGC subunits at the resolution of photoreceptors .", "Independent stimulation of individual cones and pairs of cones revealed significant nonlinear interactions within the receptive fields of OFF midget RGCs .", "A cascade model and an associated fitting method were developed to capture these nonlinear response properties .", "The model revealed the spatial structure of nonlinear interactions in the individual cone inputs to each OFF midget RGC , and enabled adaptive generation of validation stimuli targeted to specific cones during experiments .", "When fitted to entire populations of OFF midget RGCs , the model revealed a spatial organization of nonlinear subunits consistent with the known anatomical convergence of cones to midget bipolar cells ." ], [ "Targeted stimulation of cone pairs within the receptive field of RGCs revealed nonlinear interactions .", "Specifically , increments and decrements of light were presented in regions that illuminated individual cones or pairs of cones ( Figure 1 , see ‘Materials and methods’ ) .", "If a RGC linearly combines cone inputs , its response should be eliminated , or strongly reduced , when inputs of opposite polarity are presented to two separate cones that are similarly weighted within the receptive field .", "To test this prediction , we selectively stimulated pairs of cones that were close in space , but sufficiently distinct to ensure independent stimulation ( see ‘Materials and methods’ ) . 10 . 7554/eLife . 05241 . 003Figure 1 . Failure of linear integration in OFF midget retinal ganglion cells ( RGCs ) .", "( A ) Above , spatial spike-triggered average derived from white-noise analysis with high-resolution pixel stimulation .", "Black lines indicate regions of pixels independently stimulating individual cones ( identified online ) .", "White line , scale bar ( 8 . 4 microns ) .", "Below , rasters of responses to repeated brief presentations of uniform luminance within each cone region .", "Each row is a trial , each point is a spike .", "Black line , average firing rate across trials .", "Gray line , stimulus presentation ( 250 ms ) .", "Stimulation in cone regions was either paired increments and decrements of light , or decrements alone , as shown in insets .", "( B ) Another RGC showing failure of cancellation .", "( C ) A RGC exhibiting cancellation . DOI: http://dx . doi . org/10 . 7554/eLife . 05241 . 003 Many cells exhibited non-linear responses to opposite polarity stimulation of cone pairs ( Figure 1A , B ) .", "Specifically , many OFF midget cells responded robustly to decrements of light presented to either of a pair of cones individually , but also responded robustly when a decrement of light was presented to one cone and an increment of the same contrast was presented simultaneously to the other cone ( for 73/78 cone pairs tested from 13 RGCs , response to paired stimulations was at least 50% of the response to single cone decrements alone ) .", "Only a small number ( 5/78 ) of cone pairs yielded response cancellation ( see example in Figure 1C ) .", "These failures of cancellation indicate significant spatial nonlinearities within OFF midget cell receptive fields .", "These examples are consistent with the following interpretation suggested by preceding work in other species and cell types: OFF midget bipolar cells ( which carry cone signals to OFF midget RGCs ) ( Kolb and Marshak , 2003 ) linearly combine convergent cone input , but introduce a rectifying nonlinearity at the synapse with RGCs ( Demb et al . , 2001; Bölinger and Gollisch , 2012; Schwartz et al . , 2012 ) .", "In this interpretation , the cancellation of opposing signals from a pair of cones indicates that the cones converge on a single midget bipolar cell , while failure of cancellation indicates that the cones provide input to distinct midget bipolar cells .", "The analyses and experiments described below use a model-based approach that provides further evidence for this interpretation .", "The paired stimulation approach described above is not suitable for characterizing large populations of cones and RGCs , because it relies on testing a small number of cone pairs , in a small number of RGCs per retina .", "Testing all cone pairs in all RGCs is infeasible .", "It is possible , however , to identify the two key properties of interest—the presence of an intermediate nonlinearity , and the specific pairs ( or groups ) of cone signals that combine linearly in a common subunit—by fitting a single hierarchical model to the responses of RGCs under random stimulation with spatiotemporal noise .", "Across many random stimulus presentations , different spatial patterns will engage different degrees of linear and nonlinear spatial integration within the receptive field .", "Thus , finding the model that best predicts the RGC response can , in principle , reveal the structure of the nonlinearities .", "We developed a hierarchical model with two stages of linear integration and an intervening static nonlinearity ( Figure 2 ) mirroring the known elements of the underlying circuitry ( Figure 2 ) .", "First , subsets of one or more cones are assigned to ‘subunits’ , which are assumed to be non-overlapping to ensure unique solutions during fitting , that is , a cone cannot provide input to multiple subunits ( see ‘Materials and methods’ ) ; this assumption is biologically sensible given evidence for minimal overlap in bipolar dendritic trees ( Wässle et al . , 1994 ) and little divergence from cones to OFF midget bipolar cells ( Kolb and Marshak , 2003 ) .", "Signals from cones within a subunit are weighted and linearly combined .", "Second , a common nonlinear transformation is applied to the outputs of these subunits ( Ahrens et al . , 2008 ) .", "Finally , the model computes a weighted sum of subunit responses , meant to reflect the convergence of bipolar cells onto the ganglion cell , and this summed response is subjected to an output nonlinearity that produces a non-negative spike rate .", "This model builds on previous functional models of nonlinear spatial integration in RGCs ( Victor and Shapley , 1979a , 1979b; Enroth-Cugell and Freeman , 1987 ) by describing the subunit computation directly with components that resemble elements of the circuit , and by making predictions about their organization at the resolution of individual cones .", "It also bears resemblance to two-stage subunit models of V1 complex cells ( Adelson and Bergen , 1985; Rust et al . , 2005; Chen et al . , 2007 ) , and recently developed methods of fitting such models to data ( Ahrens et al . , 2008; Vintch et al . , 2012; Lochmann et al . , 2013; McFarland et al . , 2013 ) . 10 . 7554/eLife . 05241 . 004Figure 2 . OFF midget RGC anatomy and modeling framework .", "( A ) Bipolar cells receive convergent input from cones , and ganglion cells receive convergent input from bipolar cells .", "( B ) A linear–nonlinear ( LN ) model describes the RGC response with one stage of linear integration of cone inputs , followed by a nonlinearity .", "White disks indicate cones , black line indicates integration weights .", "( C ) The subunit model describes the RGC response with two stages of linear integration and nonlinearity; subunits perform initial integration following by a nonlinearity ( common to all subunits ) .", "Red , subunits receiving input from two cones; black , subunits receiving input from one cone .", "Cones are indexed by c , subunits by s .", "xc is the input to each cone .", "asc is the weight from cone c to subunit s .", "bs is the weight on each subunit .", "f and g are the subunit and spiking nonlinearities , respectively .", "See ‘Materials and methods’ for details on parameterization and fitting . DOI: http://dx . doi . org/10 . 7554/eLife . 05241 . 004 The model was fitted to hundreds of simultaneously recorded OFF midget RGCs by measuring responses to high-resolution spatio-temporal noise ( Field et al . , 2010 ) ( 15 retinas total , see ‘Materials and methods’ ) .", "First , cone locations were identified using a previously described fitting procedure ( Field et al . , 2010 ) ( see ‘Materials and methods’ ) , and the noise stimulus was then spatiotemporally filtered for each cell to describe the stimulus in terms of cone inputs rather than pixel intensities .", "Second , model parameters ( the two nonlinearities , and the summation weights over the cones and over the subunits ) were adjusted to maximize the likelihood of the spiking responses of each cell , assuming spikes arose from an inhomogeneous Poisson process ( Simoncelli et al . , 2004; Pillow et al . , 2008 ) .", "Specifically , coordinate ascent was used to infer the weights and nonlinearities at both stages of the model , and the assignment of cones to subunits was determined by an iterative greedy merging method , initializing to a single-cone subunit model ( one cone per subunit ) , and then iteratively merging the pair of subunits that yielded the largest improvement in response likelihood ( see ‘Materials and methods’ ) .", "The fitted functional model for each cell is depicted graphically ( Figure 3A ) .", "For most cells , the estimated subunit nonlinearity approximated half-wave rectification , consistent with the failures of cancellation observed in paired stimulation .", "Subunits were generally small .", "Many OFF midget RGCs had only single-cone subunits ( 48 ± 10% , percentage ±s . e . m . across retinas ) .", "The remaining cells generally had subunits with two ( 27 ± 6% OFF midgets ) or three ( 13 ± 4% OFF midgets ) cones .", "The performance of the model was assessed by computing the percent of firing rate variance explained ( R2 ) on a portion of the data not used in the fitting process ( see ‘Materials and methods’ ) .", "Although the model was fitted by maximizing likelihood under a Poisson spiking model , R2 provides a more easily interpretable measure of performance .", "Model improvement was assessed relative to a standard linear–nonlinear ( LN ) cascade model ( Chichilnisky , 2001 ) , containing only one stage of linear integration followed by a nonlinearity , which was fitted and evaluated using identical methods .", "Results from each retina were summarized with the average nonlinearities ( see Figure 3B for example subunit nonlinearities ) , as well as the improvement in predictive accuracy of the subunit model over the LN model ( percent increase in cross-validated R2 , quantified as the slope of the best-fitting regression line relating the performance of the two models , Figure 3C ) .", "Note that the subunit model can mimic an LN model when all subunits contain single cones and the subunit nonlinearity is linear , and in this case , it cannot underperform the LN model unless the data are insufficient to constrain it ( i . e . , ‘overfitting’ ) .", "We found an improvement of 18 ± 3% ( mean ± s . e . m . ) across 15 retinas ( Figure 3D ) . 10 . 7554/eLife . 05241 . 005Figure 3 . Model fits .", "( A ) Recovered model fit for an example OFF midget RGC .", "Top left , spatial spike-triggered average .", "Identified cone locations indicated by black circles .", "White line , scale bar ( 8 . 4 microns ) .", "Top right , diagram of model fit .", "Light gray regions indicate subunits , including those containing single cones .", "Within a subunit , color saturation indicates the weight on each cone .", "Red indicates that the subunit has two cones , orange , three .", "Line intensities indicate weights used in summing over subunits .", "Bottom left , recovered subunit and output nonlinearity .", "Bottom right , explanatory power ( R2 , see ‘Materials and methods’ ) of LN and subunit ( ‘Sub’ ) models .", "R2 was computed either for all presented stimuli , or for maximally differentiated stimuli , defined as those stimuli for which the predictions of the two models differed the most ( see ‘Materials and methods’ ) ; in both cases models were evaluated on data not used for model fitting .", "( B ) Summary of subunit and output nonlinearities for a population of 104 OFF midgets from one retina .", "Each line corresponds to a single RGC .", "Nonlinearities only shown for RGCs with R2 exceeding 0 . 2 .", "( C ) Summary of model performance for 76 OFF midget RGCs from an example retina .", "Above , R2 for subunit and LN models on all stimuli; below , R2 for subunit and LN models on maximally differentiating stimuli ( see panel A , and ‘Materials and methods’ ) .", "Each corresponds to a single RGC .", "( D ) Histogram of model improvement ( subunit/LN ) across multiple retinas , quantified for each retina as the slope of the best-fitting regression line to the points shown in panel C ( forced through the origin ) .", "The data point for OFF midgets from one outlying retina ( improvement = 5 ) is not shown . DOI: http://dx . doi . org/10 . 7554/eLife . 05241 . 00510 . 7554/eLife . 05241 . 006Figure 3—figure supplement 1 . In three separate simulations , spiking responses were simulated using a known set of parameters ( derived from the model fit to a real OFF midget RGC ) , and simulation duration was either 4 , 8 , or 16 min ( typical experiments were 30 min ) .", "The model was then fit to these simulated responses .", "The process was repeated five times for each simulation and duration , yielding five sets of recovered parameters: subunit assignments , weights ( on cones and subunits ) , and nonlinearities .", "In cases where the fitted subunit assignments did not match the true assignment , the true assignment was used to infer weights and nonlinearities beacuse otherwise comparing to the true values is not well defined .", "Fitted subunits depicted as in Figures 3 , 6 , 7 .", "The recovered parameters in all cases were found to be similar to the parameters used in simulating the data .", "In particular , with 8 or 16 min of data , accurate subunit assignments are nearly always recovered . DOI: http://dx . doi . org/10 . 7554/eLife . 05241 . 006 The improvement in predictive power of the subunit model over the LN model was modest , in part because the predictions were performed over a large ensemble of random noise stimuli that only infrequently and weakly activate cones and subunits in a manner that differentiates the model predictions .", "To obtain a more incisive comparison , for each fitted cell , the model ( fitted to training data ) was used to identify stimuli ( from data not used in fitting ) for which the predictions of the two models differed the most .", "Because independent data were used for training and testing , and because the selection of ‘potent’ stimuli was based only on the magnitude of the difference in predicted response , the improvement is not statistically biased in favor of one model or the other .", "Model accuracies on these ‘maximally differentiating’ test stimuli revealed a larger improvement of 92 ± 26% ( Figure 3C ) .", "Variation in improvement across retinas ( Figure 3D ) may have arisen from differences in physiological state across tissue preparations ( see ‘Discussion’ ) .", "In order to assess model accuracy , responses were averaged over repeated presentations of a short spatio-temporal noise sequence ( Figure 4A ) .", "These were used to assess the accuracy of predictions of both the subunit and LN models , expressed as a fraction of the stimulus-induced variation of the response over time ( R2 adjusted , see ‘Materials and methods’ ) .", "Accuracy for the subunit model was 51 ± 1% R2 adj . ( mean ± s . e . m . across 68 OFF midget RGCs from one retina ) , and improvement over the LN model was 40% ( Figure 4B ) . 10 . 7554/eLife . 05241 . 007Figure 4 . Subunit model predicted responses to repeated white noise .", "( A ) Average firing rate of a single OFF midget RGC to an 8 s white noise stimulus repeated 100 times .", "Purple , prediction of subunit model; green , prediction of LN model .", "Both models were fit to independent data using non-repeated white noise stimuli .", "Bin width , 83 ms . ( B ) Adjusted R2 ( see ‘Materials and methods’ ) for the two models; each point shows performance for one OFF midget RGC . DOI: http://dx . doi . org/10 . 7554/eLife . 05241 . 007 A traditional test of nonlinear integration involves measuring responses to contrast reversing sinusoidal gratings ( Enroth-Cugell and Robson , 1966; Victor and Shapley , 1979a , 1979b; Enroth-Cugell and Freeman , 1987 ) .", "Responses to gratings were measured in 32 OFF midget RGCs from one retina .", "RGCs exhibited clear signs of nonlinear integration ( see Figure 5A for an example cell ) .", "At low spatial frequencies ( period matched approximately to the size of the receptive field ) , responses were modulated at the temporal frequency of the stimulus .", "But at higher spatial frequencies , their responses were modulated at twice the temporal frequency of the stimulus , for at least some spatial phases .", "The rectifying nonlinearities within the fitted subunit model were sufficient to predict this frequency doubling , whereas the LN model exhibited dominant response modulation at the frequency of the stimulus ( Figure 5A ) .", "Across the population , accuracy of the subunit model was 83 ± 1% R2 adj . ( mean ± s . e . m . across 32 RGCs ) , and improvement in predictive performance of the subunit model over the LN model was 28 ± 2% ( mean ± bootstrapped confidence interval ) ( Figure 5B ) .", "Improvement was more pronounced at higher spatial frequencies ( Figure 5C ) , ranging from 10 ± 1% ( at a frequency where one cycle spanned the entire RF ) to 50 ± 6% ( at a frequency where each cycle spanned 1–2 cones ) , and was significant at all frequencies ( paired t-test , p < 0 . 0001 ) .", "A qualitatively similar pattern of results was observed in a second retina ( data not shown ) .", "Especially for the higher spatial frequencies , the difference between models for gratings was qualitatively more pronounced than for noise stimuli ( Figure 3C ) , suggesting that stimuli with extended spatial structure help highlight differences between model predictions . 10 . 7554/eLife . 05241 . 008Figure 5 . Responses to gratings .", "( A ) Sinusoidal gratings temporally modulated in contrast were presented to OFF midget RGCs .", "Top: grating stimuli superimposed on the cone locations of a single RGC , for a set of example combinations of spatial frequency and phase ( with spatial frequency increasing from left to right ) .", "Bottom: cycle-averaged firing rate of the RGC ( black ) ; predictions of the LN model ( green ) ; predictions of the subunit model ( purple ) .", "Both models were fit to independent data from white-noise stimulation .", "( B ) Performance ( adjusted R2 , see ‘Materials and methods’ ) for subunit and LN models , across 32 OFF midget RGCs from a single retina .", "Data point in red corresponds to example shown in panel A . ( C ) Adjusted R2 , computed separately for the two models for different spatial frequencies , averaged across RGCs .", "Error bars indicate s . e . m . across RGCs . DOI: http://dx . doi . org/10 . 7554/eLife . 05241 . 008 Although the subunit model provided large improvements over an LN model , the improvement of the full fitted subunit model over a simpler model with all subunits as single cones was small: roughly 0–10% across all RGCs ( see ‘Materials and methods’ ) .", "The small difference was likely due to the small size and number of multi-cone subunits per RGC—typically no more than a few subunits with two or three cones—and the simplistic spatial structure of the stimuli used .", "This raises the question of whether the multi-cone subunits inferred from model fits truly reflect the function and anatomy of the underlying neural circuitry .", "Anatomical measurements of synapses between cones and OFF midget bipolar cells ( Wässle et al . , 1994 ) provide direct estimates for the expected number of cones that synapse on a bipolar cell as a function of retinal eccentricity .", "The functional connectivity of the fitted model provides , for each recorded retina , a prediction of the number of cones per bipolar cell ( Figure 6 ) .", "This allowed a comparison between the anatomical connectivity and the functional connectivity predicted by the full subunit model .", "Note that comparing the number of subunits rather than cone convergence onto subunits is approximately equivalent , because midget bipolar cells tile the retina with a coverage factor of ∼1 and without overlap ( Wässle et al . , 1994 ) .", "For RGCs at small eccentricities , the fraction of subunits recovered by the model including 1–2 cones was consistent with the fraction expected based on anatomical observations made separately ( Figure 6D ) .", "For larger eccentricities , the model recovered larger subunits , also generally in agreement with anatomical observations .", "Two inconsistencies include some unexpectedly large subunits at high eccentricities ( Figure 6C , [Wässle et al . , 1994] ) and one surprising distribution of subunit sizes at high eccentricity ( Figure 6D ) .", "Despite this , the data generally support the interpretation that the fitted model revealed the structure of interneuron circuitry responsible for RGC responses . 10 . 7554/eLife . 05241 . 009Figure 6 . Recovered subunit structure varies with eccentricity .", "( A–C )", "Model fits recovered for local populations of OFF midget RGCs from retinas at three different retinal eccentricities .", "Model fits for each RGC depicted as in Figure 3 .", "Colors indicate the number of cones in each subunit; white = 1 , red = 2 , orange = 3 , lime = 4 , turquoise = 6 .", "( D ) Probability of occurrence of subunits with different numbers of cone contacts as a function of eccentricity , for morphological and functional data .", "Horizontal bars show estimates derived from morphological data from Wässle et al . , 1994 .", "Circles show estimates from the functional measurements and model fitting for individual retinas . DOI: http://dx . doi . org/10 . 7554/eLife . 05241 . 009 To validate the model fits functionally , we developed a closed-loop experiment to test the specific subunit structure of individual cells recovered by the model .", "The subunit model was fitted to many OFF midget RGCs simultaneously based on responses to noise stimuli , and these fits were then used to guide the selection of RGCs and cone pairs to be tested for linear cancellation .", "This is identical to the experiment presented earlier ( Figure 1 ) , with the important difference that cone pairs were chosen based on predictions from the fitted model , during the experiment , instead of at random .", "Responses to combined increment and decrement stimulation were qualitatively consistent with the predictions of the model: For cones within subunits , RGC responses to inputs of opposite polarity were at least partially cancelled , whereas responses to cones in separate subunits exhibited little cancellation ( Figure 7 ) .", "Quantitatively , across 252 cone pairs tested ( from 21 RGCs ) , the responses were significantly more accurately predicted by the full subunit model ( r2 = 0 . 24 ) than by either a model with single-cone subunits ( r2 = 0 . 11 ) or an LN model ( r2 = 0 . 10 ) ( both p < 0 . 005 , bootstrap test , see ‘Materials and methods’ ) . 10 . 7554/eLife . 05241 . 010Figure 7 . Closed-loop validation of subunit organization .", "( A ) Upper left , spatial spike-triggered average and cone stimulation regions , as in Figure 1 .", "Lower left , diagram of fitted subunit model , as in Figure 3A .", "The three cones ( A , B , C ) were stimulated with increments ( + ) or decrements ( − ) of light .", "Panels on the right show the responses ( as in Figure 1 ) of a single OFF midget RGC to different combinations of light increments and decrements .", "Top row , and left column , show responses to stimulation of single cones .", "Remaining panels show responses to paired stimulation .", "Red indicates the pair of cones found by the model to belong to a single subunit , and thus selected for paired stimulation .", "Gray line , stimulus presentation ( 250 ms ) .", "( B ) Another example , plotted as in panel A . DOI: http://dx . doi . org/10 . 7554/eLife . 05241 . 010" ], [ "Previous work has aimed to identify whether different RGC classes exhibit linear or nonlinear spatial summation .", "In the cat retina , ‘X’ cells were defined as those that sum visual input linearly within the spatial receptive field , whereas ‘Y’ cells were defined as those that exhibited nonlinear behaviors ( notably , frequency-doubling ) ( Enroth-Cugell and Robson , 1966; Hochstein and Shapley , 1976 ) .", "These functionally defined classes have been associated with the morphologically distinct beta and alpha RGCs ( Boycott and Wässle , 1974; Peichl and Wässle , 1981; Wässle et al . , 1981a , 1981b ) .", "Subsequent work aimed to establish a similar dichotomy in the primate retina ( Field and Chichilnisky , 2007 ) .", "The magnocellular-projecting parasol RGCs have been shown to exhibit nonlinearities similar to Y cells .", "In contrast , the parvocellular-projecting midget RGCs have usually been likened to linear X cells ( Shapley et al . , 1981; Kaplan and Shapley , 1982; Derrington and Lennie , 1984; Levitt et al . , 2001 ) .", "The nonlinearities revealed here ( Figures 1 , 4 , 6 ) are inconsistent with this interpretation , as are previous in vivo studies of parvocellular-projecting cells ( Derrington and Lennie , 1984 ) and ex vivo studies of midget RGCs ( Petrusca et al . , 2007; Cafaro and Rieke , 2013 ) , which demonstrated modest but clear frequency-doubling in response to contrast reversing visual stimuli .", "Two factors may explain these discrepancies .", "First , many previous in vivo studies probed midget cell responses in the central retina , where the receptive field center consists of a single cone , and thus , a single subunit .", "In such cases , the entire receptive field is formed by the cone collecting aperture , and would be expected to exhibit the linear summation of incident light intensity performed by the cone .", "Second , in vivo studies were typically performed using anesthetics that could raise the basal release of neurotransmitter from bipolar cells , and low basal release rates are thought to underlie at least part of the rectifying subunit nonlinearity ( Robbins and Ikeda , 1989; Demb et al . , 2001 ) .", "Indeed , variability in basal release rates could account for some of the variability across preparations observed here ( Figures 3D , 6 ) .", "Although OFF midget RGCs exhibit nonlinear spatial summation not found in X cells , they are also different from Y cells .", "The most commonly stated defining characteristic for Y cells is a frequency doubled response to a contrast reversing grating , with a magnitude that is independent of the spatial phase of the stimulus ( Enroth-Cugell and Robson , 1966; Hochstein and Shapley , 1976; Crook et al . , 2008 ) .", "Although midget cells exhibited frequency doubling in response to contrast reversing gratings ( Figure 5 ) , the magnitude was phase dependent ( not shown ) .", "Phase dependence may arise both from the low occurrence of multi-cone subunits , and the fact that midget cells receive input from only one bipolar cell type , which forms a single mosaic with minimal dendritic overlap ( Wässle et al . , 1994 ) .", "In contrast , the ON and OFF parasol RGC populations , which exhibit properties consistent with the Y cell definition ( Petrusca et al . , 2007; Crook et al . , 2008 ) , receive input primarily from two bipolar cell types ( Bordt et al . , 2006; Calkins and Sterling , 2007 ) , each of which receive convergent input from many cones , and overlap each other in space , and thus would be expected to exhibit greater phase invariance .", "Lack of phase independence in midget cells may also arise from differences between feed-forward and crossover inhibition that shapes midget vs parasol RGC responses respectively ( Cafaro and Rieke , 2013 ) .", "A final potential caveat in comparing our findings to previous work is the possibility that the ex vivo preparation used here affected the properties of midget cells .", "The firing rates obtained in the present work are somewhat lower than those found in anesthetized in vivo recordings ( Troy and Lee , 1994 ) .", "Nonlinear spatial summation ( as in Figure 1A , B ) was observed in midget cells exhibiting a range of spontaneous firing rates from 0 to 10 spikes/s ( not shown ) .", "It remains uncertain whether and to what degree these ex vivo recordings , or in vivo recordings under anesthesia , reflect a more natural state with respect to the signals studied .", "The confirmation of the biological relevance of nonlinear signaling by OFF midget cells awaits a decisive test in awake behaving animals .", "The model developed here was inspired by previous subunit models ( Victor and Shapley , 1979b ) , general successes in maximum likelihood fitting of neural models to spiking responses ( Paninski , 2004; Truccolo et al . , 2005; Ahrens et al . , 2008; Pillow et al . , 2008 ) , and recent efforts to fit subunit-like models to V1 complex cells ( Rust et al . , 2005; Vintch et al . , 2012; Lochmann et al . , 2013; McFarland et al . , 2013 ) .", "But it differs in several essential aspects .", "First , the model expresses subunit computations directly in terms of elementary circuit components: photoreceptors , inferred bipolar cells , and ganglion cells .", "Combined with precise experimental control , this approach bridges the gap between abstract functional models and detailed circuit computations , and may thus complement the growing number of tools available for manipulating neurons and circuits .", "Second , unlike some previous efforts to characterize subunits ( Victor and Shapley , 1979b ) , all components of the model used here were fitted based on a single ensemble of stimuli and spiking responses , rather than inferred separately based on responses to specialized stimuli .", "Simultaneous fitting is a generally preferable strategy for optimization , because it allows the parameters to work together to explain observed responses .", "Finally , unlike some approaches ( e . g . , spike-triggered covariance [Strong et al . , 1998; Brenner et al . , 2000; Rust et al . , 2005; Touryan et al . , 2005; Pillow and Simoncelli , 2006; Schwartz et al . , 2006; Chen et al . , 2007] or maximally-informative dimensions [Sharpee et al . , 2004] ) , which have recovered stimulus features that are unique only up to an arbitrary linear transformation , this method recovers a unique solution for the spatial structure of subunits .", "Two recent efforts characterized RGC nonlinearities using novel experimental and anatomical methods that complement the approaches described here .", "One study ( Bölinger and Gollisch , 2012 ) used a closed-loop experiment to find combinations of stimuli eliciting constant RGC responses , and used the shapes of the resulting iso-response contours in stimulus space to dissociate linear and nonlinear integration .", "While elegant , iso-response contour mapping has only proven practically useful with stimuli defined in two or three dimensions ( Bölinger and Gollisch , 2012; Horwitz and Hass , 2012 ) , whereas the current model operated on the full set of cone inputs to each RGC ( typically 5–15 dimensions ) .", "Another study ( Schwartz et al . , 2012 ) developed a model for responses of ON alpha-like mouse RGCs by combining detailed morphological measurements with direct physiological measurements from bipolars and RGCs .", "The functional approach taken here , while lacking the firm anatomical foundation , can extend more readily to describing computations in complete neuronal populations ( Figure 6 ) and in multiple cell types .", "The model developed here does not account for electrical coupling between photoreceptors ( Tsukamoto et al . , 1992; Hornstein et al . , 2004; O'Brien et al . , 2012 ) which could produce cancellation of increment and decrement stimuli delivered to neighboring cones .", "A study of cone electrical coupling in primate retina suggested that signal mixing is modest ( Hornstein et al . , 2004 ) , thus it is probably unable to account for the cancellation observed .", "However , that study was performed in dark-adapted retina , whereas the results reported here were obtained in light-adapted retina; coupling may depend on light adaptation and the circadian cycle ( Bloomfield and Völgyi , 2009 ) ( but see Hornstein et al . , 2004 ) .", "Thus , it is difficult to entirely rule out a contribution of cone coupling .", "However , since many cone pairs tested exhibited little or no cancellation ( e . g . , Figure 1 ) , any effects of adjacent cone coupling are likely to be weak or sporadic .", "In contrast to this prediction , connexin-36 immunolabeling revealed ubiquitous expression at junctions between nearly all neighboring cones , in both central and peripheral primate retina ( O'Brien et al . , 2012 ) .", "Also , the observed eccentricity dependence of the spatial extent of cancellation ( Figure 6 ) implies that for the present results to be explained by cone coupling , the coupling would have to increase substantially with eccentricity .", "The model described here could potentially capture nonlinear computations in other RGC types , such as ON midget or parasol RGCs .", "But in some cases the model would require modification .", "For example , unlike midget RGCs , parasol RGCs receive input from more than one type of bipolar cell , and the receptive fields of these bipolar cells likely exhibit substantial spatial overlap , whereas the current model assumes nonoverlapping subunits ( Figure 2 ) .", "Indeed , preliminary model fits of parasol responses produce subunits smaller than what is expected from anatomical data , as would be expected in the case of two overlapping bipolar populations ( Boycott and Wässle , 1991 ) ( not shown ) .", "Subunit overlap may underlie the reported Y-cell invariance to spatial phase ( see above ) , and is thus an important goal for future modeling .", "Extensions to the model would also be required to capture RGC responses under more natural stimulus conditions .", "For example , the current model does not include receptive field surrounds .", "Insofar as surrounds reflect the contributions of horizontal and amacrine cells ( Mangel , 1991; Cook and McReynolds , 1998; McMahon et al . , 2004; Ichinose and Lukasiewicz , 2005; Davenport et al . , 2008 ) , additions to the model could capture the contribution of these interneuron types and elucidate their computational role .", "Incorporating inhibitory interneurons could also improve the accuracy of subunit estimation , if other components in the current model are indirectly absorbing their effects .", "In some stimulus regimes , it may also be important to incorporate nonlinearities in cone signals .", "Specifically , the logic of the linearity test ( Figure 1 ) , and the structure of the model ( Figure 2 ) , assume linearity of the cone signal , which is a reasonable approximation for weak stimuli ( Schnapf et al . , 1990 ) , but fails for stronger stimuli ( J Angueyra and F Rieke , personal communication ) .", "This may account for the moderately lower predictive accuracy of the model when fit to noise and tested on stronger increment-decrement stimuli ( Figure 7 ) .", "The model would also be enhanced by allowing for temporal nonlinearities ( rather than static ones ) ( Borghuis et al . , 2013 ) , post-spike temporal filtering ( Pillow et al . , 2008 ) , more complex spike generation , and perhaps nonlinearities that vary across subunits .", "More generally , the model should be extended to include mechanisms for luminance and contrast gain control ( Mante et al . , 2008 ) , both of which are essential for natural vision .", "All of these extensions would require new targeted stimulus ensembles and model fitting procedures , and together provide an exciting direction for future research .", "The approach developed here also offers potential opportunities for uncovering the structure and function of other neural circuits .", "Subunit models have been used to explain pooling behaviors in primary and secondary visual cortex ( Hubel and Wiesel , 1959; Rust et al . , 2005; Vintch et al . , 2012; Freeman et al . , 2013; Lochmann et al . , 2013 ) , and may represent a ‘canonical’ computation of sensory processing ( Fukushima , 1980; Douglas et al . , 1989; Heeger et al . , 1996; Riesenhuber and Poggio , 1999 ) .", "New stimulation and measurement techniques that facilitate experimental manipulation of these circuits are proliferating .", "For example , optogenetics enables simultaneous stimulation of many neurons of a given class , while measuring electrical responses of other neurons ( Papagiakoumou , 2013 ) .", "But in most scenarios , the circuits of interest will contain neurons that are neither directly stimulated nor measured , analogous to the bipolar interneurons in the present work .", "Thus , further development of the computational approaches described here may prove an essential tool for inferring the function and connectivity of interneurons in neural circuits ." ], [ "Preparation and recording methods have been described previously ( Chichilnisky and Kalmar , 2002 ) .", "Eyes were enucleated from 15 terminally anesthetized macaque monkeys ( M . mulatta and M . fascicularis ) used by other experimenters in accordance with institutional guidelines for the care and use of animals .", "Five had been used for behavioral and neurophysiological experiments , eight had been experimentally exposed to ethanol ( Wright et al . , 2013 ) , and two had been terminally anesthetized for several days .", "No systematic differences in retinal physiology were observed between categories of animals .", "Immediately after enucleation , the anterior portion of the eye and the vitreous were removed in ambient indoor illumination .", "Segments of peripheral retina ( eccentricity 5 . 5–13 . 8 mm temporal equivalent [Chichilnisky and Kalmar , 2002] ) that were well attached to the pigment epithelium were dissected and placed flat , RGC side down , on a planar array of extracellular microelectrodes .", "The array consisted of 512 electrodes in an isosceles triangular lattice with 30 μm spacing , covering a hexagonal region 450 μm on a side ( Field et al . , 2010 ) .", "Attachment to the pigment epithelium was preserved during dissection .", "In some preparations the choroid was largely removed , up to Bruch's membrane , to ensure even retinal thickness and maximize oxygenation .", "While recording , the retina was perfused with Ames' solution ( 31–36°C ) bubbled with 95% O2 and 5% CO2 , pH 7 . 4 .", "Recordings were analyzed online to isolate the spikes of different cells , as described previously ( Dabrowski et al . , 2004; Field et al . , 2007 ) .", "Briefly , candidate spike events were detected using a threshold on each electrode , and voltage waveforms on the electrode and nearby electrodes around the time of the spike were extracted .", "Clusters of similar spike waveforms were identified as candidate neurons if they exhibited a 1 ms refractory period and accounted for more than 100 spikes in 30 min of recording .", "Duplicate spike trains were identified by temporal cross-correlation and removed .", "Visual stimulation was performed with the optically reduced image from a computer display .", "In 14 experiments a gamma-corrected OLED microdisplay ( eMagin , Bellvue , WA ) refreshing at 60 . 35 Hz was used .", "In one experiment a gamma correct CRT display ( Sony Trinitron ) refreshing at 120 Hz was used .", "The image was focused through the retina onto the photoreceptor outer segments .", "The emission spectrum of each display primary was measured with a spectroradiometer ( PR-701 , PhotoResearch , Chatsworth , CA ) after passing through the optical elements between the display and the retina .", "The mean photoisomerization rates for the L , M and S cones were estimated by computing the inner product of the power scaled emission spectra per unit area with the spectral sensitivity of each opsin , and multiplying by the effective collecting area of primate cones ( 0 . 37 μm2 ) ( Baylor et al . , 1987 ) .", "The power of each display primary was measured at the preparation with a calibrated photodiode ( UDT Instruments , San Diego , CA ) .", "At the mean background illumination level of the OLED display , the photoisomerization rates for the rods , L , M , and S cones were approximately 29 , 120 , 9440 , 9270 , and 2320 photoisomerizations per receptor per second ( 4670 , 2200 , 2200 , 900 for the CRT display ) .", "These estimates were not corrected for the angle of illumination and pigment self screening in the cone outer segments , because the precise angle of illumination and the amount of bleached pigment were unknown .", "The spatial , temporal and chromatic response properties of recorded RGCs were characterized using a movie stimulus composed of an array of square pixels whose values were updated randomly and independently on each frame ( see Chichilnisky , 2001 ) .", "For initial characterization with large pixels , the intensity of each display primary at each pixel location was independently chosen from a binary distribution , yielding a stimulus with chromatic variation .", "For high resolution characterization of individual cones , the intensities of the display primaries were modulated in unison in most experiments ( 13 of 15 ) .", "This yielded a black-white binary intensity modulation with higher variance and thus greater modulation of RGC responses .", "In the other two experiments , the display primaries were modulated independently of one another .", "In both conditions , the contrast of each primary ( difference between the maximum and minimum intensities divided by the sum ) was 96% .", "For low spatial resolution receptive field maps used for classification ( not shown ) , the pixels were 25 . 5 or 34 μm on a side , the stimulus refresh rate was 20 , 30 , or 60 Hz , and recording duration was 30 min .", "For high resolution maps ( Figures 1 , 3 , 4 ) , the pixels were 3 . 4 μm on a side , the stimulus refresh rate was 10 , 12 , or 15 Hz , and recording duration was 30–60 min ( depending on the experiment ) .", "High resolution spatial receptive fields shown in Figures 1 , 3 , 4 were calculated by collapsing the spike-triggered average over time .", "For this purpose , the time course was calculated from the average of a subset of stimulus pixels whose absolute peak intensity exceeded four robust standard deviations of all pixel intensities .", "RGC classification was performed as described previously ( Field et al . , 2007; Petrusca et al . , 2007; Field et al . , 2010 ) .", "OFF midget RGCs were readily identified as those cells with the smallest receptive fields of the appropriate sign , and relatively sustained temporal response properties .", "The identification of distinct cell types was confirmed by the mosaic organization of receptive fields within each type , and the identities of the different types were consistent with anatomical density measurements ( Chichilnisky and Kalmar , 2002 ) .", "Cone finding was performed using a Bayesian approach described previously ( Field et al . , 2010 ) .", "Briefly , the linear spatial receptive field of each cone was modeled as a gaussian , and the linear spatial receptive field of each RGC was modeled as a sum of cones .", "For each retina , a mosaic of cones was greedily chosen to maximize the likelihood of spiking responses across all reliably identified cells ( including multiple types ) .", "After identifying cone locations , the high-resolution noise stimulus was integrated against the Gaussian profile of each cone , to obtain a set of cone input signals for each RGC .", "These signals were then convolved in time with the temporal component of the spike-triggered average , effectively aligning the stimulus with the response in time .", "All modeling subsequently focused only on the spatial component of the response .", "Spikes were binned by the timing of stimulus frames , which across experiments was typically 12 Hz ( 83 ms bins ) .", "This spatial and temporal preprocessing yielded , for each RGC , a C × T stimulus matrix X , where C is the number of cones providing input to that RGC , and T is the number of stimulus frames in the experiment , and a T × 1 vector y of spike counts .", "These preprocessing steps assume a linear model of the ganglion cell response , but linear analysis is adequate for the purpose of cone localization , because it only requires that each cone provides non-zero input to at least one ganglion cell , which will be true for most monotonic non-linearities; the same procedure was also anatomically validated in previous work ( Field et al . , 2010 ) .", "The subunit model describes the transformation of cone signals to the spiking response of a RGC using two stages of computation .", "Formally , the complete model is a linear-nonlinear-linear-nonlinear-poisson cascade , generalizing and extending both LNP models ( Simoncelli et al . , 2004; Pillow et al . , 2008 ) , models with ‘input nonlinearities’ ( Ahrens et al . , 2008; McFarland et al . , 2013 ) , and previous subunit models ( Rust et al . , 2005; Vintch et al . , 2012; Lochmann et al . , 2013 ) .", "The first stage linearly combines cone inputs into ‘subunits’ followed by a instantaneous subunit nonlinearity .", "Let U be the S × C weight matrix connecting C cones to S subunits .", "U is partitioned as I ⊙ A , where I is a binary indicator matrix , A is a continuous-valued weight matrix , and ⊙ is the pointwise product .", "I specifies which cones provide inputs to which subunits , and A captures the weight on each cone within a subunit .", "The response of subunit s at time t isyst=f ( ∑c=1Cuscxct ) , where f ( • ) is the subunit nonlinearity ( see below for parameterization ) , and xct and usc are elements of the matrices X and U . The second stage of the model linearly combines subunit responses followed by an instantaneous output nonlinearity .", "For any given RGC , let w be the 1 × S vector encoding the weights from subunits to that RGC .", "The response ( mean firing rate ) of the RGC at time t iszt=g ( ∑s=1Swsyst ) , where g ( • ) is the output nonlinearity and ws is an element of the vector w .", "The nonlinearities f and g were parameterized as piece-wise polynomials ( specifically , cubic splines ) with eight node points .", "Our optimization procedure sought parameters I , A , w , f , g to maximize the log likelihood of the measured spike counts rt , assuming that the number of spikes in at each time t was given by a Poisson distribution with rate parameter zt:logp ( rt|zt ( xct;I , A , w , f , g ) ) =∑trtlogzt ( xct;I , A , w , f , g ) −∑tzt ( xct;I , A , w , f , g ) The dimensionality of the parameter space is substantial , and the objective function is non-convex and likely to have many local minima .", "To encourage reliable estimation , while remaining sufficiently flexible to capture a variety of nonlinear RGC properties , we introduced several constraints: ( 1 ) Nonlinearity smoothness was enforced through a set of linear constraints on the spline parameters that guarantees first and second-order derivatives are defined at the nodes ( implying that the output of a subunit is smoothly related to its input with no discontinuities ) ; ( 2 ) The binary assignment matrix I was assumed to be orthogonal ( i . e . , the subunits were non-overlapping ) : each cone provided input to exactly one subunit , and the number of subunits was thus constrained to lie between one ( equivalent to an LN model ) and the number of cones ( which we refer to as the single-cone subunit model ) .", "( 3 ) The weights from cones to subunits were assumed to be positive , and the weights within each subunit were assumed to sum to 1 .", "No constraints were imposed on the sign or magnitude of weights in the second stage .", "Even with constraints , the two-stage structure of the model , and the combination of binary assignments and continuous-valued weights ( and nonlinearities ) makes optimization difficult .", "We decomposed the problem into a greedy search over the assignments , and for each assignment performed an optimization over the continuous-valued parameters .", "The optimization over the continuous-valued parameters—the cone weights ( A ) , the subunit weights ( w ) , and the nonlinearities ( f , g ) —was performed using an alternating sequence of simpler gradient descent problems ( known as ‘coordinate decent’ ) .", "First , g was initialized as log ( 1 + exp ( • ) ) , f was initialized as negative half-wave rectification , w and A were initialized to contain values of 1 .", "These parameters were then repeatedly optimized , sequentially , in the following order: w , A ,", "f . The nonlinearity g was only refit once at the end .", "In practice , we found that repeating the full optimization after estimating g did not substantially change the recovered parameters .", "Most of these subproblems correspond closely to estimation problems that have been characterized in previous literature on fitting neural encoding models ( Chichilnisky , 2001; Paninski , 2004; Ahrens et al . , 2008 ) .", "For example , if the weights ( A ) and nonlinearities ( f , g ) are held fixed , estimating the subunit weights ( w ) is identical to fitting a single-stage LNP model , which can be done with gradient descent as long as f is convex and log-concave ( Paninski , 2004 ) .", "In practice , each of the subproblems on their own , as well as the combined procedure , converged to unique solutions from random initial starting points .", "The best-fitting assignment matrix I was found through a greedy procedure .", "I was initialized with the identity matrix ( the ‘single cone’ subunit model ) , all remaining model parameters were estimated ( as described above ) and the maximized likelihood recorded .", "For every possible merger of two subunits into a larger subunit , all the remaining model parameters were re-estimated ( as described above ) , and the maximized likelihood recorded .", "If any of the potential merged pairs yielded an improvement in likelihood over that of the unmerged model , those two subunits were merged .", "This process was repeated , at each step considering a merger of all possible pairs of subunits , until none of the pairings offered an improvement in likelihood .", "All code used for model fitting is available at https://github . com/freeman-lab/subunits and an interactive web visualization is available at https://github . com/freeman-lab/subunits-web .", "Simulations were performed to confirm that the estimation procedure reliably recovered model parameters .", "Spiking responses were generated using a known set of parameters , with values chosen as typical for those recovered from RGCs , and simulation duration was matched approximately to that of typical experiments ( as well as smaller and larger values ) .", "The model was fit to these simulated responses .", "The recovered parameters were found to be nearly identical to the parameters used for simulation ( Figure 3—figure supplement 1 ) .", "The accuracy of the subunit model was compared to a simpler LN model , containing only weights on each cone and an output nonlinearity .", "Formally , the subunit model reduces to the LN model if I and A are identity matrices and f is linear , leaving the parameters w and", "g . ( An alternative parameterization of LN behavior would be to make I a vector of ones , i . e . put all cones in a single subunit ) .", "In some cases the subunit model was also compared to a subunit model that included a subunit nonlinearity ( f ) , but with an identity matrix for I and A; that is , the single cone subunit model .", "In all cases , parameters were fit using similar likelihood maximization methods to those used to fit the full subunit model .", "For every RGC , the model was fit to 80% of the data , and the accuracy of the model was evaluated on the remaining 20% .", "To account for possible nonstationarities in responses , the subsets of data used for fitting were drawn randomly from periods of time distributed throughout the duration of the experiment .", "On the evaluation data , model performance was evaluated asR2=1−∑t ( zt−rt ) 2∑t ( rt−rt¯ ) 2 Although the model was fit by maximizing log likelihood , we used the more intuitive metric of R2 to report performance .", "Comparisons between models were similar when using log likelihood .", "For repeated white noise and grating data ( Figures 5 , 6 ) , R2 was adjusted to account for the reliability of the responses ( see below ) .", "For each retina , the improvement of one model over another ( e . g . , subunit over LN ) was estimated as the slope of the best-fitting regression line ( through the origin ) relating the R2 of the two models across RGCs ( i . e . , the best-fitting line to the scatter plots in Figure 3C , D ) .", "In a small number of cases , when analyzing the maximum differentiated stimuli ( see below ) , R2 for the LN model was negative , due to particularly poor predictions on these stimuli .", "In all such cases , the performance of the subunit model was positive .", "To avoid the influence of these outliers on average performance improvement , they were excluded from the calculation .", "To better differentiate the models , stimuli were selected on the basis of the model predictions .", "Specifically , the models were fit to training data , and then used to generate predicted responses for stimuli from the remaining data .", "From amongst these held out stimuli , we selected the 20% individual stimulus frames for which the squared difference in the prediction of the two models was largest .", "Selecting stimuli by individual frames was appropriate because stimuli were preprocessed to have a one-to-one correspondence with the response at each frame .", "Performance of both models was then compared to actual neural response on these stimuli .", "This selection does not favor either model a priori , because it is not based on their performance in explaining the data , but only on how much their predictions differ .", "For the results of Figure 4 , the model was fit to a single run of independent white noise , and then tested on a 10 s sequence of white noise that was repeated 100 times .", "The repeated presentations allowed prediction accuracy to be computed relative to the inherent reliability of the responses .", "We compared model predictions of the spike count on individual trials of each 10 s stimulus sequence to the actual responses averaged over all remaining runs of the sequence .", "In each case , the performance was quantified as the square of the correlation coefficient .", "This is similar to R2 as defined above , but allows for an arbitrary offset and gain , which was important for some RGCs due to changes in overall firing rate between the two experimental preparations .", "These two R2 values were averaged across all trials , and their ratio yielded an adjusted R2 , representing the fraction of explainable variance accounted for by the model .", "Contrast reversing gratings modulating at 2 Hz were presented at eight spatial phases for each of 10 spatial periods .", "Each presentation of a grating lasted 8 s followed by 2 s of a uniform screen that had an intensity equal to the mean grating intensity over time .", "Each grating of a particular spatial period and phase was presented three times .", "For a RGC , the mean spike rate over one contrast reversal was estimated by averaging responses over the 16 contrast reversals ( 8 s × 2 Hz ) in each of these three grating presentations ( Figure 6A ) .", "The grating spatial periods ranged from 10 to 1305 microns on the retina .", "Data reported here ( Figure 5 ) only for the five spatial periods matched to ( or smaller than ) a typical OFF midget RGC receptive field .", "As for white noise , model accuracy was evaluated as adjusted R2 .", "For each spatial period and phase , the response to each of the 36 repeated presentations of the grating ( 16 contrast reversals × 3 repeats ) was predicted by ( 1 ) the model and ( 2 ) the average across other presentations , and their ratio ( averaged across trials ) yielded an adjusted R2 .", "Cone locations and subunit model fits were obtained online during experiments , computed in parallel across roughly 10 multi-core Linux workstations .", "With parallelization , hundreds of RGCs from an entire retina could be fit in under 10 min .", "Once model fits were obtained for every RGC , several RGCs ( 5–10 ) with high SNR and a variety of subunit configurations were chosen for targeted single cone and paired cone stimulation .", "Chosen RGCs were at least two cell spacings away from each other , to minimize the impact of stimulating cones lying in the receptive field surround of any of the chosen RGCs .", "Typically four cones were chosen per RGC .", "For simplicity , Figure 7 only reports data for the three cones per RGC showing the strongest response to single-cone stimulation , but the quantitative analysis ( see below ) included all cones tested .", "Cone location analyses generated stimulus screen coordinates for the center of every cone found in the mosaic .", "Coordinates of cones within the receptive field of the chosen RGCs were then used to specify regions of the screen selected to stimulate single cones , as follows .", "Depending on cone spacing in each preparation , spots of radius 7 . 65–9 . 35 μm were generated around each cone center location .", "The full native screen resolution was used ( pixels 1 . 7 μm on a side ) .", "For cases in which cone spacing was close enough that the resulting regions would overlap , pixels were assigned to whichever competing cone had the nearest center coordinates .", "Single cones were then stimulated with uniform contrast steps over the defined regions .", "Recording trials were 750 ms long with the stimulus presentation occurring in the first 250 ms . Stimulation region ( i . e . , choice of cone ) and contrast were randomized across trials .", "Control experiments in which light stimuli were presented within the spaces in between cones demonstrated the specificity of this stimulation ( Li et al . , 2014 ) .", "For each RGC , each selected cone was presented with an increment or decrement of light alone , and then separately paired with an increment of light to three other cones .", "For each such selected cone , the firing rate response to single or paired stimulations were averaged across repeated trials ( typically 20–40 ) and within a 300 ms time window following stimulus presentation .", "These four firing rates were then divided by their maximum .", "This normalization served to emphasize the relative effect of pairing increments with a decrement of light , as well as correct for differences in effective cone strength between measurement and model due to , for example , small movement of the retina in between stimulus presentations .", "For each RGC , model predictions were generated by providing negative or positive inputs to the same combination of cones tested in the experiment .", "The predictions were then similarly divided by the maximum predicted firing rate for each cone .", "Finally , the predictive accuracy was computed as the square of the correlation coefficient between predicted and measured response across all cone pairs .", "Given the non-normal estimator of interest ( a difference of correlation coefficients ) , a bootstrap test was used to compare predictions of the different models—full subunit , single-cone subunit , and LN .", "On each iteration of a bootstrap , cone pairs were randomly sampled with replacement across RGCs , accuracies for all models were reestimated ( as above ) , and the differences in accuracy between models were computed .", "The accuracies were considered significantly different if the fifth percentile of the resulting distribution of differences exceeded 0 ." ] ]

[ "The function of a neural circuit is shaped by the computations performed by its interneurons , which in many cases are not easily accessible to experimental investigation .", "Here , we elucidate the transformation of visual signals flowing from the input to the output of the primate retina , using a combination of large-scale multi-electrode recordings from an identified ganglion cell type , visual stimulation targeted at individual cone photoreceptors , and a hierarchical computational model .", "The results reveal nonlinear subunits in the circuity of OFF midget ganglion cells , which subserve high-resolution vision .", "The model explains light responses to a variety of stimuli more accurately than a linear model , including stimuli targeted to cones within and across subunits .", "The recovered model components are consistent with known anatomical organization of midget bipolar interneurons .", "These results reveal the spatial structure of linear and nonlinear encoding , at the resolution of single cells and at the scale of complete circuits ." ]

[ "Light that enters the eye begins the process of vision by activating two types of photoreceptors: rods , which support vision under low light levels , and cones , which are responsible for fine detail and color vision .", "Activation of either type of photoreceptor triggers responses in bipolar cells , which activate the ganglion cells that transmit visual signals to the brain .", "Bipolar cells therefore belong to a class of cells called interneurons , which relay information from certain cell types to others .", "Interneurons play an important role in information processing throughout the brain , but directly accessing them or characterizing their role in neural computation is often difficult .", "To address this problem , Freeman , Field et al . have developed a combined computational and experimental approach to describe the flow of sensory signals through the circuits within the retina of primates .", "Large arrays of electrodes were used to record the responses of many retinal ganglion cells in response to the activation or de-activation of pairs of cones .", "These experiments revealed that the responses of ganglion cells are not simply the sum of the inputs that they receive from cones; specifically , the activation of one cone is not cancelled by the deactivation of another .", "Instead , the data suggest that bipolar cells add cone inputs together and then pass on the total activation ( but not deactivation ) to ganglion cells .", "By analyzing the responses of ganglion cells to numerous random patterns of cone activation , Freeman , Field et al . were able to estimate the locations and arrangements of bipolar cells that connect to them .", "These predicted patterns of connectivity agreed with observations from anatomical studies .", "This work provides detailed insights into how the primate retina works .", "It also suggests that similar approaches may be used to characterize how signals flow across other brain networks in which large-scale recordings are now possible ." ]

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

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

[ [ "Cardiovascular disease including myocardial infarction ( MI ) remains a leading cause of morbidity and mortality in the Western and developing worlds .", "After acute MI , millions of cardiomyocytes ( CM ) are lost by necrosis and apoptosis , and an initially adaptive collagen-rich scar is laid down to preserve chamber geometry and prevent rupture .", "The mammalian heart is regarded as being poorly regenerative as the long-term sequelae in virtually all etiologies of heart disease involve increased wall stiffness , reduced heart function and progression to heart failure .", "However , some inbred strains of mice show surprising cardiac reparative abilities ( Patterson et al . , 2017 ) , and CM renewal and heart regeneration can be stimulated experimentally ( D'Uva et al . , 2015; Mohamed et al . , 2018; Srivastava and DeWitt , 2016; Wang et al . , 2018 ) , garnering optimism that heart regeneration can be achieved in humans .", "Cardiac chamber walls are composed of a complex , interdependent community of interstitial cells , including vascular , fibroblast , immune and neuronal cells , although how they interact in cardiac homeostasis , injury and repair , is relatively unexplored .", "In regenerative systems , connective tissues play key roles in defining positional information , and organizing tissue architecture and niche environments ( Nacu et al . , 2013; Chan et al . , 2013; Greicius et al . , 2018 ) .", "Cardiac fibroblasts represent ~10% of all cardiac cells ( Pinto et al . , 2016 ) and are distributed throughout the cardiac interstitial , perivascular and sub-epicardial spaces , where they are proposed to have sentinel , paracrine , mechanical , extracellular matrix ( ECM ) and electrical functions ( Shinde and Frangogiannis , 2014; Tallquist and Molkentin , 2017 ) .", "After injury , inflammation is principally executed by poly-functional monocytes ( Mo ) and macrophages ( MΦ ) , and is necessary to protect against pathogens and autoimmunity , and to coordinate healing .", "Fibroblasts also participate in inflammation and phagocytosis and are the principal drivers of fibrotic repair ( Shinde and Frangogiannis , 2014; Gourdie et al . , 2016 ) .", "In heart repair , timely resolution of inflammation is necessary for limiting fibrosis and enabling tissue replacement , while uncontrolled inflammation leads to increased fibrosis and chamber wall stiffening , poor electro-mechanical coupling , continued loss of CMs and worsening outcomes ( Mescher , 2017; Lai et al . , 2017; Williams et al . , 2018 ) .", "The general principles of inflammation and fibrosis have been mapped in different organs , and the implementation of specific lineage-tracing tools has provided significant new insights into cardiac leukocyte and fibroblast origins and fate ( Tallquist and Molkentin , 2017; Williams et al . , 2018; Fu et al . , 2018; Ivey et al . , 2018; Kanisicak et al . , 2016; Moore-Morris et al . , 2014; Ensan et al . , 2016; Heidt et al . , 2014; Molawi et al . , 2014; Epelman et al . , 2014; Plein et al . , 2018 ) .", "However , controversies persist around nomenclature , defining markers , origins , heterogeneity and plasticity ( Tallquist and Molkentin , 2017; Epelman et al . , 2015; Swirski and Nahrendorf , 2018 ) .", "Even the question of whether the transitions from quiescent to activated fibroblast , then to myofibroblast , should be seen as differentiation in the classical sense , degrees of a scalable and reversible continuum governed by the injury environment , or a branched dynamic network , is unresolved ( Tallquist and Molkentin , 2017; Ivey and Tallquist , 2016; Travers et al . , 2016 ) .", "One approach to a deeper understanding of cardiac population biology is through single-cell genomics , including single-cell RNA sequencing ( scRNA-seq ) .", "Single-cell methods have the power to overcome the limitations of bulk cell analyses , where insights into complex cell system dynamics are lost ( Tanay and Regev , 2017 ) .", "The rich data generated by single-cell methods allow new computational frameworks for inferring cell dynamics and causality , unencumbered by strict a priori notions of cell identity , hierarchy , trajectory and markers .", "Here , we present the first comprehensive analysis of cellular lineage heterogeneity , dynamics and intercellular communication among immune and stromal ( non-CM ) cells in healthy and injured adult mouse hearts using scRNA-seq .", "Clustering analysis of >30 , 000 cells identified over 30 cell populations across the total non-CM fraction and enriched ( Pdgfra-GFP+ ) fibroblast lineage cells .", "These populations comprised most of the known cell types and their dynamics after injury , as well as novel cell types and their intermediates .", "We describe a novel population of activated fibroblasts present in both sham and injured hearts expressing a strong anti-Wingless-related integration site ( WNT ) transcriptome signature , a putative pre-proliferative state , and three novel myofibroblast subtypes expressing pro-fibrotic or anti-fibrotic ( including anti-WNT ) signatures .", "We were also able to distinguish the major tissue-resident and infiltrating Mo/MΦ , and numerous minor populations .", "Overall , our data reveal dynamic , multi-dimensional lineage trajectories in the injured heart .", "This deep resource will provide novel insights into the inflammatory and fibrotic cascades in the injured mouse heart that may suggest novel molecular or cellular targets for enhancing heart repair and regeneration in man ." ], [ "We performed single-cell expression profiling on the total cardiac interstitial cell population ( TIP ) using the 10x Genomics Chromium platform , from hearts of 8 weeks old male PdgfraGFP/+ mice at days 3 and 7 post-sham or MI surgery .", "To enrich for cells relevant to cardiac ischemic injury and repair , we isolated TIP cells from dissected ventricles and interventricular septum , excluding cells of the atria , annulus fibrosus and atrioventricular valves ( Figure 1—figure supplement 1A ) .", "Transcriptional profiles of 13 , 331 cells were captured after quality control filtering ( sham: 5 , 723; MI-day 3: 3 , 875; MI-day 7: 3 , 733 ) .", "To identify cells with distinct lineage identities and transcriptional states , we performed unbiased clustering on an aggregate of cells using the Seurat R package ( Butler et al . , 2018 ) , with cell populations visualized in t-SNE dimensionality reduction plots ( Materials and methods ) .", "For initial analyses , populations expressing markers of multiple lineages ( hybrids ) were removed; however , select examples are discussed in more detail below .", "TIP cells were represented by a total of 24 populations and nine distinct cell lineages ( Figure 1A–D; Figure 1—figure supplement 1B–F ) .", "Major cell types comprised fibroblasts/myofibroblasts ( Col1a1+Pdgfra+GFP+ ) , endothelial cells ( ECs; Kdr+Pecam1+ ) , mural cells ( Cspg4+Pdgfrb+ ) , Mo and MΦ ( Cd68+Itgam+ ) , dendritic-like ( DC ) cells ( Cd209a+Itgam+ ) , glial cells ( Plp1+Kcna1+ ) , B-cells ( Cd79a+Ms4a1+ ) , T-cells ( Cd3e+Cd3d+Lef1+ ) and natural killer cells ( NKCs; Klrk1+Ccl5+ ) ( Figure 1A–E; Figure 1—figure supplement 1D; Supplementary file 1 ) .", "New lineage markers were identified; for example , Vtn , encoding Vitronectin , was specifically expressed in mural cells , whereas Kcna1 , encoding the potassium voltage-gated channel subfamily A member 1 , was highly specific to glial cells ( Figure 1D and Figure 1—figure supplement 1D ) .", "Within the fibroblast lineage , we identified several sub-populations , each marked by expression of Pdgfra , Pdgfra-GFP , Col1a1 and other canonical fiboblast markers ( Tallquist and Molkentin , 2017; Ivey and Tallquist , 2016 ) ( Figure 1A–D; Figure 1—figure supplement 2A ) .", "We describe these in more detail after enrichment below .", "There were three major sub-populations of ECs ( EC1 , EC2 , EC3 ) , comprising vascular and lymphatic lineages ( Pecam1+Kdr+Ly6a+ ) ( Figure 1D; Figure 1—figure supplement 2B ) .", "The majority EC1 population expressed Ly6a ( encoding SCA1 ) as well as the vascular transcription factor ( TF ) Sox17 , and likely represents microvascular ECs .", "EC2 expressed canonical arterial endothelial markers such as Bmx , Sema3g and Efnb2 , as well as TF genes Sox17 and Hey1 ( Figure 1—figure supplement 2B ) , the latter acting downstream of NOTCH which is required for arterial EC fate .", "EC3 almost uniquely expressed venous EC marker Nr2f2 ( encoding COUPTFII ) and Von Willebrand factor ( Vwf ) , and a minority ( ~3% ) expressed Prox1 and Lyve1 ( Figure 1—figure supplement 2B ) , consistent with a lymphatic identity .", "A small number of ECs were cycling ( Cyc; Figure 1A–C; Figure 1—figure supplement 2C ) .", "Our EC data are broadly consistent with recently published single-cell data ( Zhao et al . , 2018 ) .", "Among lymphocytes , a single B-cell population ( BC ) expressed Cd79a , Ms4a1 and Ly6d ( Figure 1D; Figure 1—figure supplement 2D ) .", "T-cell sub-populations TC1-Cd8 ( Cd8a+ ) and TC2-Cd4 ( Cd4+Lef1+ ) likely represent cytotoxic and helper T-cells , respectively .", "NKCs exclusively expressed Klrk1 and upregulated Lck , Ccl5 and Ctsw ( Figure 1—figure supplement 2D ) .", "We observed major injury-induced cellular responses and flux after MI , including expansion of Mo/MΦ populations at MI-day 3 , as well as myofibroblasts and an additional MΦ population at MI-day 7 ( Figure 1A; Figure 1—figure supplement 1B , C ) .", "To analyze whether changes in the proportion of populations were greater than expected by chance , we developed a novel permutation-based statistical test ( differential proportion analysis; DPA ) that considered sources of variation which could arise from experimental procedures ( such as differing cell numbers and cell-type capture bias ) or in silico analysis ( cluster assignment accuracy ) ( Materials and methods; Figure 1—figure supplement 3A–H ) .", "DPA identified 12 populations showing significant ( p<0 . 01 ) flux between conditions ( Figure 1E; Supplementary file 2 ) ; for example , the fibroblast sub-populations F-SL and F-SH ( see below ) decreased sharply in proportion at MI-day 3 , while M1 and M2 MΦ populations expanded at days 3 and 7 after MI , respectively .", "Cardiac tissue-resident MΦ originate from CX3CR1+ progenitors in the yolk sac and Mo from fetal liver and post-natal bone marrow ( Ensan et al . , 2016 ) , and have roles in immunity , coronary artery and pacemaker development , and heart regeneration ( Lavine et al . , 2014; Hulsmans et al . , 2017 ) .", "Resident MΦ are long lived and self-renewing ( Epelman et al . , 2014; Bajpai et al . , 2018 ) , although some are supplanted by blood-derived Mo with age or injury ( Heidt et al . , 2014; Molawi et al . , 2014; Dick et al . , 2019 ) .", "MI triggers a biphasic cascade of inflammation and remodeling , with the acute phase involving early influx of neutrophils and CCR2+LY6C2high pro-inflammatory M1 Mo/MΦ , which phagocytose debris and secrete pro-inflammatory factors IL-1β , IL-6 and TNFα to amplify the inflammatory response ( Swirski and Nahrendorf , 2018 ) .", "The repair phase begins around MI-day 3 when non-classical LY6C2-F4/80high M2 MΦ accumulate and secrete anti-inflammatory cytokines such as Il-10 and TGF-β , and stimulate angiogenesis ( Epelman et al . , 2015; Swirski and Nahrendorf , 2018 ) .", "In sham hearts , we identified cardiac tissue-resident MΦ with the signature Cx3cr1highAdgre1 ( F4/80 ) highH2-Aa ( MHC-II ) +Itgam ( CD11b ) lowLy6c2lowCcr2- ( MAC-TR; Figure 1A , B; Figure 2A–D ) ( Ensan et al . , 2016; Epelman et al . , 2014; Swirski and Nahrendorf , 2018; Lavine et al . , 2014; Lavine et al . , 2018 ) .", "Recent work using flow cytometry and scRNA-seq has delineated several subsets of cardiac tissue-resident MΦ , including a pro-regenerative population with the signature TIMD4+LYVE1+MHC-IIlowCCR2- , that self-renew and are not replaced by blood monocytes even after injury ( Dick et al . , 2019 ) .", "We could discern this same population at the scRNA-seq level as a subset within MAC-TR , which persisted after injury ( Figure 2—figure supplement 1A; Figure 2B ) .", "The additional major subset of CCR2- tissue-resident MΦ ( Dick et al . , 2019 ) could also be recognised at the scRNA-seq level as the Timd4-Lyve1-H2-Aa ( MHC-II ) highCcr2- subset of MAC-TR – this population has been shown to have a low monocyte dependence during homeostasis but is almost fully replaced by monocytes after MI ( Dick et al . , 2019 ) .", "Among other minor resident Mo/MΦ populations detected , the most abundant ( pale green cells in Figure 1A , B ) clustered with the M2 MΦ present at MI-day 7 .", "In fact , all minor Mo/MΦ populations in sham hearts aligned with adult monocyte-derived Mo/MΦ populations which influx after MI ( Figure 1A , B ) , consistent with recent findings ( Dick et al . , 2019 ) .", "A prominent B-cell , and minor DC , T- and NK cell populations were also present in sham hearts .", "These populations may represent a mixture of resident cells and those involved in homeostatic immunosurveillance ( Lavine et al . , 2018 ) , although we cannot exclude a response to sham operation .", "At MI-day 3 , a major influx population was identified as classical blood-derived M1 Mo , based on the signature Adgre1 ( F4/80 ) +Itgam ( CD11b ) +Fcgr1 ( CD64 ) +Ly6c2highCcr2highH2-Aa ( MHC-II ) low ( M1Mo; Figure 2A–C ) ( Epelman et al . , 2015; Swirski and Nahrendorf , 2018 ) .", "Differentially expressed genes showed Gene Ontology ( GO ) term over-representation for cell migration , inflammation and T cell activation ( Figure 2D ) .", "In FACS , Mo are distinguished from MΦ by having lower size and granularity , and lower levels of MΦ markers including Adgre ( F4/80 ) , Itgam ( CD11b ) and H2-Aa ( MHC-II ) ( Bajpai et al . , 2018; Hilgendorf et al . , 2014 ) .", "M1Mo identified at MI-day 3 were also low or negative for other MΦ markers Siglec1 , Mrc1 , Maf , Trem2 and Mertk , the latter involved in phagocytosis ( Figure 2—figure supplement 1A ) ( Bajpai et al . , 2018 ) , and C1 complement genes C1qa , b and c ( Figure 2C ) , which are involved ( in addition to complement fixation ) in recruitment of new inflammatory cells and protection against autoimmunity ( Emmens et al . , 2017; Thielens et al . , 2017 ) .", "The more abundant population at MI-day 3 was identified as classical Mo-derived M1 MΦ based on the signature Ccr2highAdgre1 ( F4/80 ) +Ly6c2+H2-Aa ( MHC-II ) + ( M1MΦ; Figure 1A , B; Figure 2A–D ) .", "This assignment was supported by expression of the additional MΦ markers cited above , including Mertk and C1q , and hierarchical clustering , which showed M1MΦ to be most closely related to M1Mo ( Figure 1C ) , as for human cognates ( Bajpai et al . , 2018 ) .", "Differentially expressed genes showed GO term over-representation for leukocyte migration and responses to interleukin-1 ( Figure 2D ) .", "The most prominent population at MI-day 7 was identified as non-classical M2 MΦ involved in inflammation resolution and repair , with the signature Ccr2highAdgre1 ( F4/80 ) +H2-Aa ( MHC-II ) highLy6c2- ( M2MΦ; Figure 1A , B; Figure 2A–D ) ( Epelman et al . , 2015; Swirski and Nahrendorf , 2018 ) .", "Differentially expressed genes showed GO term over-representation for antigen presentation via MHC class II ( Figure 2D ) .", "As expected , the non-classical M2 MΦ population increased late during injury repair from <2% of TIP in sham and MI-day 3 hearts , to 16% at MI-day 7 .", "Interestingly , the population dendrogram showed that M2MΦ were most closely related to MAC-TR ( Figure 1C ) , and similarities between resident and subsets of infiltrating Mo/MΦ have been discerned recently using single-cell methods ( Dick et al . , 2019 ) .", "Both MAC-TR and M2MΦ expressed Cx3cr1 , often used to define tissue-resident MΦ ( Figure 2B ) , and both upregulated pro-regenerative genes Igf1 ( Figure 2B ) and Pdgfb/c ( Figure 2—figure supplement 1A ) .", "The majority of M2MΦ were Ccr2high ( important for migration ) ; however , a minor sub-populaion was Ccr2low ( arrows , Figure 2B ) and these expressed the highest levels of Igf1 and lower levels of MHC-II ( Figure 2—figure supplement 1B ) .", "In this sense they are similar to the CCR2-MHC-IIlow subset of tissue-resident MΦ which appear to be yolk sac-derived ( Dick et al . , 2019; Leid et al . , 2016 ) , and which play a role through expression of IGF1 and IGF2 in remodeling of the fetal coronary vascular plexus ( Leid et al . , 2016 ) .", "However , whether in the adult post-MI heart they represent persisting resident cells or an infiltrating population that has matured into a MAC-TR-like MΦ state will require lineage mapping approaches .", "Il10 , associated with the anti-inflammatory functions of M2MΦ , was expressed in only few cells in our dataset and may be at the limit of detection ( Figure 2—figure supplement 1A ) .", "Expression of mouse genes encoding cognates of human CD14 and CD16/FCGR3 , previously used to define classical , non-classical and intermediate Mo in human blood ( Villani et al . , 2017 ) , did not help to discrimate the above Mo/MΦ populations , nor did new markers recently highlighted from CyTOF analysis ( Williams et al . , 2018 ) .", "Moreover , the M2MΦ marker Arg1 ( encoding Arginase 1 ) was more lowly expressed in M2MΦ described here than in M1MΦ , consistent with findings that ARG1 does not always mark M2 cells ( Jablonski et al . , 2015 ) .", "Neither the M2 MΦ , nor any other myeloid population , expressed Col1a genes , likely precluding the presence of myeloid-derived fibroblasts ( Duerrschmid et al . , 2015 ) .", "Diffusion Map ( Angerer et al . , 2016 ) analysis applied to model possible temporal ( pseudotime ) changes in major Mo/MΦ populations ( Figure 2—figure supplement 1C ) revealed a continuum of states resolved into a trajectory from early infiltrating M1Mo ( left ) and inflammatory M1MΦ ( centre ) , to the late peaking M2MΦ ( right ) , similar to a recent scRNA-seq study ( Dick et al . , 2019 ) and consistent with the current model in which M1 Mo differentiate into M2 cells in situ ( Lavine et al . , 2018; Hilgendorf et al . , 2014 ) .", "The Diffusion Map also demonstrated the convergence of M2MΦ present at MI-day 7 with tissue-resident MΦ ( MAC-TR ) in sham hearts , a relationship reflected in the population dendrogram ( Figure 1C; see Discussion ) .", "The minor myeloid populations also showed different expression profiles and dynamics ( Figure 1A–C , E; Figure 2A–D ) .", "For example , MAC6 showed upregulation of granulocyte markers including S100a9 and Csf3r ( Supplementary file 3 ) , with sub-populations expressing markers of neutrophils ( Ly6g ) and eosinophils ( Siglecf ) ( Figure 2—figure supplement 1A ) .", "MAC-IFNIC cells showed strong upregulation of interferon ( IFN ) -induced genes including Ifit3 , Ifit1 and Cxcl10 ( Figure 2C ) , consistent with GO term analysis implicating responses to IFN α , β , and γ ( Figure 2D ) .", "These cells appear to arise from Ccr2+ MΦ as opposed to monocytes ( Dick et al . , 2019 ) , and likely correspond to the recently described inflammatory MΦ subtype that has negative effects on heart repair after MI through promotion of inflammatory cell types , and cytokine and chemokine expression ( King et al . , 2017 ) .", "We constructed cell-cell communication networks with weighted edges reflecting expression fold-changes of ligands and receptors in source and target populations , respectively ( Materials and methods ) .", "Ligand-receptor interactions were derived from a curated map of human ligand-receptor pairs ( Ramilowski et al . , 2015 ) with mouse-specific weights added after reference to the STRING database ( Szklarczyk et al . , 2017 ) .", "Based on permutation testing of randomized network connections , 91 cell-cell relationships with weighted paths higher than expected by chance ( Padj <0 . 01 ) were identified ( Figure 3A–B ) .", "Myofibroblasts ( MYO ) and MΦ populations M1MΦ and MAC-8 exhibited the largest number of outbound connections , with MYO having the highest weight .", "ECs by far showed the largest number and weighting of significant inbound connections .", "Strikingly , fibroblast populations ( F-SH , F-SL , F-Act and F-WntX ) appeared to communicate exclusively with vascular ( ECs and mural ) and glial cells .", "In line with this result , immunofluorescence analysis of sham and MI-day 3 hearts showed that Pdgfra-GFP+ fibroblasts were observed in close spatial relationships or direct contact with CD31+ endothelial cells ( Figure 3—figure supplement 1A–B ) .", "Top-weighted interactions involving MYO were driven mostly by ECM-associated ligands including COL1a1 , COL1a2 , Fibronectin 1 , Pleiotrophin , COL3a1 , COL5a2 , Biglycan , Metalloprotease inhibitor one and COL5a1 , that can engage with receptors expressed in numerous populations ( Figure 3C ) .", "The minority glial cell population expressed canonical neuronal glia markers , including Plp1 , Prnp and Gfra3 ( Figure 1—figure supplement 1D ) , and likely support cardiac sympathetic nerves essential for cardiac regeneration in neonates ( Mahmoud et al . , 2015 ) .", "Glial cells also appeared to communicate with the three EC populations and mural cells ( Figure 3A ) , consistent with the phenomenon of neurovascular congruence in the cardiac sympathetic plexus ( Stubbs et al . , 2009 ) .", "In support of this , we detected eight ligands highly specific to glial cells ( expressed in <5% of other TIP cells ) including Dhh ( Desert Hedgehog ) and Semaphorin genes Sema3b and Sema4f ( Figure 3D ) , involved in both neural and angiogenic development ( Gamboa et al . , 2017 ) .", "Thus , these maps suggest the extent , directionality and complexity of interactions between cardiac cell types in homeostasis and injury .", "We detected five minor populations expressing markers of two lineages ( Figure 1—figure supplement 4A–D ) .", "Such ‘hybrid’ cells may betray trans-differentiation events or doublets in proximity that are resistant to the conditions of dissociation .", "Microdroplet microfluidics platforms are also known to generate a significant number of doublets ( Zheng et al . , 2017a ) ; thus , the provenance of hybrid cells requires independent validation .", "ECs are highly plastic and endothelial-to-mesenchyme transition ( EndMT ) has been reported to generate fibroblasts after cardiac injury ( Kovacic et al . , 2019 ) .", "Conversely , cardiac fibroblasts have been observed to transdifferentiate to ECs ( Ubil et al . , 2014 ) , albeit that this has been disputed ( He et al . , 2017 ) .", "The F-EC hybrid population co-expressed markers of fibroblasts and ECs , and segregated with other fibroblast populations ( Figure 1—figure supplement 4A–C ) .", "To explore this population , we isolated interstitial cells from dissected ventricles of PdgfraGFP/+ mice after sham or MI surgery , and asked whether we could detect GFP+CD31+ cells using flow cytometry and a stringent gating strategy that excluded cell aggregates ( Materials and methods; Figure 1—figure supplement 5A–D ) .", "We detected 2 . 4 ± 0 . 28% of GFP+CD31+ cells in sham hearts , 1 . 51 ± 0 . 26% in MI-day 7 hearts , and none in controls ( Figure 1—figure supplement 6A–E ) - thus , while double positive cells were found , they did not appear responsive to injury .", "An ability of Mo/MΦ to transdifferentiate into endothelial-like cells in different settings has been documented in vitro and in vivo , and has therapeutic implications ( Das et al . , 2015 ) , although a natural plasticity in Mo toward an endothelial cell fate in vivo does not have strong support ( Basile and Yoder , 2014 ) .", "The M2MΦ-EC hybrid population co-expressed markers of ECs ( Kdr+Pecam1+Sox17+ Efnb+Mcam+ ) and M2MΦ ( Ccr2highAdgre1[F4/80]+H2-Aa[MHC-II]highCx3cr1+Mrc1[CD206]+Ly6 c2- ) , and segregated with M2MΦ ( Figure 1—figure supplement 4A , B ) .", "Flow revealed 0 . 56 ± 0 . 02% single live CD31+CD45+ cells in sham-day 7 hearts , increasing to 4 . 04 ± 1 . 03% in MI-day 7 hearts , demonstrating an increase in injury ( Figure 1—figure supplement 7A–C ) .", "Among these , 35 . 67 ± 3 . 01% were F4/80+CD206+ ( a signature of M1 and M2 MΦ ) in sham hearts , increasing to 60 . 03 ± 4 . 60% in MI-day 7 hearts .", "It is well known that the expression of EC markers on the surface of bone-marrow-derived cells is insufficient to define them as ECs , although they can be angiogenesis promoting cells ( Basile and Yoder , 2014 ) .", "While these data do not exclude the possibility of doublet formation in our scRNA-seq experiments , they support the existence of distinct F-EC and M2MΦ-EC populations with hybrid qualities and different responses to injury .", "These warrant further investigation .", "A major subset of fibroblasts in the uninjured adult murine heart express the cell surface stem/progenitor cell markers SCA1 and/or PDGFRα ( Kanisicak et al . , 2016; Asli et al . , 2017; Chong et al . , 2011; Noseda et al . , 2015 ) .", "However , when fibroblasts differentiate into MYO , they reduce these markers and express fibrogenic ( e . g . Periostin; POSTN ) and/or contractile ( e . g . αSmooth Muscle Actin; αSMA ) proteins ( Fu et al . , 2018 ) .", "To circumvent the dominance of immune cells in TIP following MI , which dilute out other cell populations , and to focus on fibroblast sub-populations ( Figure 1A ) , we performed single-cell expression profiling on PDGFRα+CD31- cardiac interstitial cells at days 3 and 7 post-sham or MI .", "GFP fluorescence from PdgfraGFP/+ mice was used as a surrogate lineage tracing tool and enabled us to capture both GFPhigh fibroblasts as well as their derivatives in MI mice , including MYO ( Asli et al . , 2017 ) .", "We sorted for GFP+CD31- cells ( Figure 4—figure supplement 1A ) , although did not use SCA1 as an index marker so as to capture the substantial Pdgfra-GFP+ fibroblast population that is negative or low for SCA1 expression ( Figure 4—figure supplement 1B ) .", "We performed unbiased clustering on an aggregate of the 16 , 787 cells ( Materials and methods ) , identifying 11 sub-populations ( Figure 4A–D; Figure 4—figure supplement 1C–F ) .", "The two sham conditions showed high concordance ( Figure 4—figure supplement 1E , F ) and are displayed merged ( Figure 4A , B ) unless indicated .", "All populations showed expression of canonical fibroblast markers Pdgfra , GFP , Ddr2 and Col1a1 , albeit at varying proportions and levels ( Figure 4E ) , and major changes in cell proportions were seen between conditions ( Figure 4D ) .", "Here , we refer to ‘activated fibroblasts’ and myofibroblasts ( MYO ) as distinct cell entities , without prejudice about their stability , origin , fate or contractile nature .", "In sham conditions , two major fibroblast populations could be distinguished on the basis of scRNA-seq .", "We termed these Fibroblast-Sca1-high ( F-SH ) and Fibroblast-Sca1-low ( F-SL ) , as the highest upregulated gene in F-SH was Ly6a ( Sca1 ) ( Figure 4E; Supplementary file 4 ) .", "F-SH contained the highest frequency of Pdgfra and Ly6a ( Sca1 ) -expressing cells and likely corresponds to the PDGFRα+SCA1+ ( S+P+ ) population previously defined by FACS ( Pinto et al . , 2016; Chong et al . , 2011 ) ( see also Figure 4—figure supplement 1A ) and enriched in cardiac colony-forming mesenchymal stromal cell ( MSC ) -like cells ( cCFU-F ) , which show multi-lineage differentiation and self-renewal in vitro ( Chong et al . , 2011; Noseda et al . , 2015 ) .", "In order to confirm the relationship between F-SH and S+P+ , we performed deeper scRNA-seq on 103 FACS-purified S+P+ cells from uninjured wild-type mice using the Fluidigm platform and predicted cell identity using an iterative Random Forest ( iRF ) classifier ( Basu et al . , 2018 ) trained on populations defined in our GFP+ experiments in sham conditions using the Chromium platform ( Materials and methods; Figure 4—figure supplement 2A ) .", "Approximately 60% of single S+P+ cells analyzed by Fluidigm were predicted to correspond to the F-SH population ( Figure 4—figure supplement 2B ) , compared to <30% among total sham GFP+ cells ( Figure 4D ) , indicating that S+P+ cells are significantly over-represented in F-SH cells ( Fisher’s exact test , p=8 . 13e-11 ) .", "We previously showed that cCFU-F are enriched in the S+P+ population ( Chong et al . , 2011; Noseda et al . , 2015 ) .", "THY1/CD90 is a recognised MSC marker , and Thy1 was upregulated in F-SH with high significance ( p=4 . 48e-176; Figure 4—figure supplement 2C ) .", "Furthermore , FACS-isolated S+Pdgfra-GFP+CD90 . 2high cells isolated from healthy hearts showed a ~ 6 fold enrichment in cCFU-F compared to S+Pdgfra-GFP+CD90 . 2low cells ( Figure 4—figure supplement 2D , E ) .", "Together , these results show that the F-SH population contains a subset of cells expressing Pdgfra , Ly6a ( Sca1 ) and Thy1 ( Cd90 ) that is enriched in cCFU-F , highlighting the distinct expression signatures and functional properties of F-SH and F-SL .", "We calculated differentially expressed ( DE ) genes between F-SL and F-SH in sham conditions ( Supplementary file 5 ) .", "F-SH was characterized by over-representation of genes involved in the biological process ( BP ) cell adhesion , which included cell surface receptor genes Ackr3 ( Cxcr-7 ) , Thy1 ( Cd90 ) , Axl and Cd34 .", "In contrast , F-SL was characterized by GO BP terms signaling and signal transduction ( Supplementary file 6 ) .", "Within the signal transduction category , protein localization prediction with LocTree3 ( Goldberg et al . , 2014 ) indicated an over-represented majority ( 19/28 ) of secreted proteins ( Fisher’s exact test , p=0 . 03 ) , with 10/19 identified as ligands , including APOE , BMP4 and ADM . Thus , F-SL , a major sub-division of fibroblasts , has a unique secretory phenotype distinct from that in F-SH , which is enriched in MSC-like colony forming cells .", "We identified two previously unstudied GFP+ fibroblast populations termed Fibroblast - Wnt expressing ( F-WntX ) and Fibroblast - transitory ( F-Trans ) .", "These were present in both sham and MI hearts , although had diminished significantly by MI-day 7 ( DPA; p<0 . 01; Figure 4D ) .", "In F-WntX , differential gene expression analysis showed that the top upregulated gene was Wif1 , encoding a secreted canonical WNT pathway inhibitor essential for cardiac repair after MI ( Meyer et al . , 2017 ) .", "WIF1 can also antagonize Connective Tissue Growth Factor ( CTGF ) signaling ( Surmann-Schmitt et al . , 2012 ) , which plays a supportive role in cardiac fibrosis ( Travers et al . , 2016 ) .", "Wif1 was almost uniquely expressed in F-WntX in all conditions ( Figure 4E ) .", "A single cell in the Fluidigm data corresponded to the F-Wntx population ( Figure 4—figure supplement 2B ) .", "Multiple other WNT pathway-related genes were upregulated in F-WntX encoding WNT ligands ( WNT5a , WNT16 ) , soluble WNT antagonists ( DKK3 , SFRP2 ) , membrane-bound WNT receptor ( FRZB ) and AXIN2 , a component of the β-catenin destruction complex ( see schematic in Figure 5A; Figure 4—figure supplement 3 ) .", "F-WntX also showed upregulated Fmod , which inhibits fibrillogenesis and sequesters pro-fibrotic factor TGF-β within ECM ( Zheng et al . , 2014; Zheng et al . , 2017b ) ( Supplementary file 4 ) .", "Overall , this signature suggests an anti-WNT , anti-CTGF and anti-TGF-β extracellular and intracellular signaling milieu for F-WntX cells .", "F-WntX cells expressed Postn ( Periostin ) , Acta2 ( αSMA ) , Tagln ( Transgelin ) and Scx ( Scleraxis ) , in both sham and MI conditions ( Figure 5B; Figure 5—figure supplement 1A ) , suggesting an activated state even in the absence of injury ( Tallquist and Molkentin , 2017 ) .", "The adjacent cluster , F-Trans , did not express activation markers , nor the WNT signature identified in F-WntX , or other uniquely identifying markers; however , cell trajectory analysis , described below , allowed us to assign F-Trans as a transitionary population between F-WntX and F-SL fibroblasts .", "We examined DE and GO BP terms in F-WntX compared with F-SL and F-SH combined .", "Notably , negative regulation of Wnt signaling pathway was over-represented in DE genes for F-WntX ( Figure 5C ) , driven by WNT pathway antagonist genes Wif1 , Dkk3 , Frzb and Sfrp2 , discussed above , as well as Apoe , Nkd1 and Wwtr1 , also known to interact with the WNT pathway .", "BP terms related to negative regulation of development/differentiation , extracellular matrix ( ECM ) organization and signaling , were also significant .", "ECM organization terms were over-represented in the adjacent F-Trans ( Supplementary file 6 ) , involving genes also upregulated in F-WntX ( e . g . Eln[Elastin] , Vit[Vitrin] and Mfap4 ) , and others upregulated in F-Trans but not F-WntX , including collagen genes ( Col1a1 , Col3a1 and Col14a1 ) and Fbln1 ( Fibrilin-1 ) .", "The top GO BP term in F-WntX was regulation of cell proliferation ( Figure 5C ) ; however , F-WntX did not express cell cycle markers under any condition .", "Analysis of Molecular Function ( MF ) terms revealed over-representation of signaling receptor binding and growth factor binding ( Figure 5—figure supplement 1B ) , overlapping significantly with regulation of cell proliferation ( Figure 5—figure supplement 1C , D; Fisher’s exact test , p<1e-03 ) .", "In this latter term , there were several cytokine and chemokine genes , including Pdgfa , Tgfb3 , Ptn , Ccl19 and Cxcl12 , some of which bind receptors that were down-regulated in F-WntX , strongly suggesting paracrine functions related to their expression in F-WntX ( Figure 5—figure supplement 1E ) .", "A paracrine function for F-WntX was supported by our ligand-receptor analysis , which indicated that F-WntX cells communicate most significantly with ECs ( Figure 3A ) .", "Analysis of top upregulated ligands in F-WntX connecting to receptors in ECs ( Figure 5D ) identified several factors such as Ptn ( pleiotrophin ) , Myoc ( myocilin ) and Timp3 ( TIMP metallopeptidase inhibitor 3 ) .", "Here again , the corresponding receptor was expressed in ECs but downregulated in F-WntX ( Figure 5E ) .", "To explore the location of F-WntX cells and their behaviour after injury , we examined the expression of WIF1 protein in Pdgfra-GFP sham and MI hearts by immunofluorescence ( IF ) , after first confirming that our chosen antibody detected known sites of Wif1 expression in E14 . 5 embryos ( Figure 6—figure supplement 1 ) .", "Interestingly , we detected WIF1 protein only in the infarct border zone at MI-day 3 , but not in sham hearts or at MI-days 1 or 7 ( Figure 6A–C , Figure 6—figure supplement 2 ) .", "In cardiac cells ( and some embryonic cells ) WIF1 staining was perinuclear , and we demonstrated co-expression of WIF1 and the golgi marker GM130 ( Figure 6D ) , consistent with WIF1 being a secreted protein .", "We found WIF1 in ~4% of total nuclei of the infarct border zone at MI-day 3 , with a fraction of these ( ~5% ) being GFP+ by IF , indicating a fibroblast identity ( Figure 6C , I ) , and overall ~17% were positive for Ki67 ( Figure 6E , I ) .", "WIF1+ cells were negative for CD31 , and negative or very low for αSMA , with rare exceptions ( Figure 6F , F’ , H ) .", "However , WIF1 was also expressed in ~4% of total CD45+ cells in the infarct border zone ( ~15% of WIF1+ cells were CD45+ ) ( Figure 6G , I ) .", "We observed frequent close proximity or contact between WIF1+ cells and CD31+ ECs in tissue sections ( Figure 6H ) , in line with the predicted cell-cell ligand-receptor connection between F-WntX cells and ECs ( Figure 3A ) .", "Such proximity was less obvious for α-SMA+ or CD45+ cells ( Figure 6F , G ) .", "Overall , our data suggest that WIF1 expression is post-transcriptionally regulated and injury-dependent , appearing in the infarct border-zone at MI-day 3 in a subset of fibroblasts and immune cells .", "The temporal window of WIF1 expression overlaps with fibroblast activation and expansion , and the beginning of EC renewal and MYO differentiation .", "MI is associated with appearance of activated fibroblasts and myofibroblasts .", "MI-day 7 was characterized by the appearance of a large population of myofibroblasts ( MYO ) , representing 52% of GFP+ cells at MI-day 7 in our data ( Figure 4A–D ) .", "MYO showed strong upregulation of numerous collagen genes ( e . g . Col1a1 , Col3a1 , Col5a2 ) , as well as Postn ( 99 . 5% ) and Acta2 ( 61% ) at high levels , indicative of an activated state and suggestive of a contractile phenotype for a subset of cells ( Tallquist and Molkentin , 2017; Travers et al . , 2016 ) ( Figure 4E; Figure 4—figure supplement 3 ) .", "Upregulated genes involved in wound healing and cell migration included Fn ( Fibronectin ) and Cthrc1 ( Collagen Triple Helix Repeat Containing I ) , the latter representing a highly specific marker for MYO ( Figure 4F ) .", "MYO showed decreased expression of Pdgfra , Pdgfra-GFP , Ly6a ( Sca1 ) , Thy1 ( Cd90 ) and Cd34 ( Figure 4E; Figure 4—figure supplement 2C; Supplementary file 4 ) , indicating loss of stem/progenitor cell markers .", "Earlier in the injury process , there was a distinct increase in a population with a signature consistent with activated fibroblasts ( Fibroblast: activated; F-Act ) .", "These represented 48% of GFP+ cells at MI-day 3 , before diminishing to 12% at MI-day 7 ( Figure 4A–D ) .", "F-Act expressed Postn at high levels in ~80% of cells ( Figure 4E ) consistent with an activated state ( Tallquist and Molkentin , 2017 ) .", "They expressed Acta2 in 28% and 35% of cells at MI-day 3 and MI-day 7 , respectively , although at much lower levels compared to MYO , suggesting an emerging contractile phenotype in some cells .", "On the population dendrogram , F-Act was most closely related to fibroblast populations ( F-SH , F-SL , F-Trans ) and was more distant from MYO ( Figure 4C ) .", "Whereas F-Act expressed few genes that could be considered highly specific , the top upregulated gene was Cilp ( Figure 4F ) , encoding a matricellular protein and inhibitor of TGF-β1 signaling , consistent with F-Act being a pre-MYO population in which fibrosis is constrained .", "The expansion of F-Act at MI-day 3 correlated with a decrease in the proportion of F-SH and F-SL , whereas the diminishment of the F-Act population at MI-day 7 coincided with the appearance of MYO and an apparent partial restoration of F-SH and F-SL cells ( Figure 1A , E; Figure 4A , D; see Discussion ) .", "A distinct GFP+ population contained fibroblasts undergoing proliferation ( Fibroblast - cycling; F-Cyc ) , comprising 15% of GFP+ cells at MI-day 3 and 3% at MI-day 7 ( Figure 4D ) , consistent with studies showing peak fibroblast proliferation at MI-days 2–4 ( Fu et al . , 2018; Ivey et al . , 2018 ) .", "F-Cyc uniquely expressed a strong cell cycle gene signature , including Ccnb2 ( CyclinB ) , Cdk1 ( Cyclin dependent kinase 1 ) and Mki67 ( Ki67 ) ( Figure 4E; Figure 4—figure supplement 3 ) , and expressed both Postn ( 88% ) and Acta2 ( 76% ) at high levels ( Figure 4E; see below ) .", "To look at potential relationships between the major GFP+ populations , we analyzed cell trajectories using Diffusion Maps ( Materials and methods ) .", "MYO , F-WntX and F-Cyc were represented as three different trajectories along diffusion components 1 , 2 and 3 , respectively ( Figure 7A ) , with the root containing the two large unactivated fibroblast populations F-SH and F-SL , which were most prominent in sham hearts .", "F-Trans was an intermediary population along the trajectory to F-WntX , and F-Act was an intermediary population for both F-Cyc and MYO branches .", "F-Cyc , characterized by expression of a strong cell cycle gene signature , was represented most strongly at MI-day 3 , whereas MYO was exclusively associated with MI-day 7 ( Figure 7B , C ) .", "These data suggest that F-Act expands by proliferation up to MI-day 3 ( F-Cyc trajectory ) and differentiates to MYO during the transition from MI-day 3 to MI-day 7 .", "The presence of some F-Cyc-like cells between the F-Cyc and MYO trajectories at MI-day 7 raises the possibility that a small fraction of F-Act cells differentiate rapidly into MYO after or during division ( see below ) .", "We examined DE and GO BP terms in F-Act , F-Cyc and MYO compared with F-SL and F-SH combined , across all conditions ( Figure 7D; Supplementary file 5; Supplementary file 6 ) .", "DE genes for F-Act were over-represented in terms for collagen fibril organization ( including several collagen genes ) and regulation of wound healing .", "Many of these genes were also expressed in F-Cyc and MYO; however , there was an additional large gene signature strongly upregulated in MYO compared to F-Act ( Figure 7D ) .", "DE genes for MYO demonstrated GO BP terms for collagen fibril organization and cell adhesion , containing collagen genes Col3a1 , Col5a1 , Col11a1 and Col14a1 , and others involved in cell:cell and cell:matrix adhesion including Thbs1 ( encoding Thrombospondin 1 ) and Fbn1 .", "Other terms included angiogenesis and heart development as well as negative regulation of canonical Wnt signaling pathway , containing many genes previously identified in F-WntX .", "Minor GFP+ populations included epicardial cells ( EPI ) , observed only at MI-day 7 .", "These expressed Wt1 ( Figure 4—figure supplement 3 ) and overall were related transcriptionally to dissected adult mouse epicardium ( Bochmann et al . , 2010 ) ( Spearman’s correlation test , p=0 . 014 , r = 0 . 26 ) .", "These are likely to be epicardial-derived fibroblasts that arise after MI as the epicardium reactivates its developmental program ( including Pdgfra expression ) ( Zhou et al . , 2011 ) .", "Consistent with a previous cardiac scRNA-seq analysis on uninjured hearts ( Skelly et al . , 2018 ) , we did not detect epicardial cells in TIP data , suggesting that these cells are under-sampled in our experiments - this is likely technical as epicardial cells have been detected readily in single-nucleus RNA-seq ( Hu et al . , 2018 ) .", "The minor population F-IFNS ( Fibroblast: Interferon stimulated ) , found in all conditions , was negative for Cd45 and expressed high levels of Col1a1 and other fibroblast markers ( Figure 4E; Figure 4—figure supplement 3 ) , demonstrating a fibroblast identity , and interferon-responsive genes ( Figure 4F; Supplementary file 4 ) ( Zhou et al . , 2013 ) .", "Other minor GFP+ populations were EC and MAC ( Figure 4A–C ) , which had EC and MΦ identities , respectively ( Materials and methods ) .", "Several TF genes expressed in GFP+ cells may drive differentiation or responses to environmental stimuli ( Figure 4—figure supplement 4 ) .", "Scleraxis , already mentioned , was expressed in F-WntX cells and MYO .", "The basic helix-loop-helix factor gene , Tcf21 , an accepted marker of cardiac fibroblasts ( Tallquist and Molkentin , 2017 ) , was expressed in most GFP+ populations with the exception of F-WntX cells .", "T-box factor gene Tbx20 , another fibroblast marker , was expressed across all GFP+ cells and upregulated in F-WntX .", "The homeodomain TF gene , Meox1 , which is part of the cardiac fetal gene expression signature reactivated in injured hearts ( Lu et al . , 2018 ) , showed upregulation in subsets of GFP+ cells , most prominently in activated populations ( F-Act , F-Wntx , F-Cyc and MYO ) , and may drive the activated state .", "Csrp2a , Zfp385a and Hmgb2 expression was also restricted among populations , with Csrp2a and Zfp385a showing strikingly complementary patterns .", "Interestingly , the homeodomain TF gene , Prrx1 , which in BM is expressed in a subset of mesenchymal cells with CFU-F and multi-lineage differentiation potential ( Kfoury and Scadden , 2015 ) , was expressed across all GFP+ populations in all conditions .", "We re-clustered the sham and MI datasets at days 3 and 7 individually ( Figure 8A , B ) , and repeated Diffusion Map analysis of GFP+ fibroblast lineages ( Figure 8C , D ) ( Materials and methods ) .", "Whereas most populations identified at day 3 directly corresponded to those identified in the aggregate analysis , we found that F-Cyc could now be sub-divided into two populations , with only one exhibiting a clear cell cycle signature ( Figure 8E , Supplementary file 7; Supplementary file 8 ) .", "An intermediary population ( Fibroblast - Cycling Intermediate; F-CI ) showed upregulation of fibroblast activation markers including Postn , Cthrc1 and Acta2 ( Figure 8—figure supplement 1A ) , but did not express markers of cell cycle , suggesting that it represents an additional population of activated fibroblasts , potentially competent for cell cycle entry .", "F-CI also upregulated genes involved in protein translation , a signature absent in F-Act ( Figure 8E ) .", "The translation signature was maintained , albeit in attenuated form , in F-Cyc .", "Based on an iRF classifier trained to predict MI-day 3 populations ( Figure 8—figure supplement 1B ) , we found no corresponding F-CI cluster at MI-day 7 , indicating its transient nature ( Figure 8—figure supplement 1C , D ) .", "Diffusion Map analysis also lent weight to the hypothesis that F-CI is a transitory population between F-Act and F-Cyc at MI-Day 3 ( Figure 8C ) .", "We next removed genes annotated with the GO term ‘Cell Cycle’ and re-clustered populations .", "At MI-day 3 , 88% of F-Cyc cells merged with F-CI , strongly supporting the hypothesis that F-CI is a pre-proliferative precursor of F-Cyc ( Figure 8—figure supplement 1E ) .", "After removal of cell cycle genes at MI-day 7 , 65% of F-Cyc cells remained as a distinct ( proliferative ) population , although 26% merged with F-Act and 6% merged with the MYO populations ( Figure 8—figure supplement 1F ) .", "Taken together with Diffusion Map analysis , these data indicate that the closest population to the majority F-CI and cycling cells is F-Act rather than MYO , even though both express Acta2 and other MYO markers at high levels ( Figure 4E ) .", "However , a minority of F-Cyc cells at MI-day 7 may be dividing MYO cells or in transition to MYO ( see Discussion ) .", "Our data support the idea that F-CI cells are an activated form of fibroblast closely related to F-Cyc and derivative of F-Act .", "We hypothesize that they are primed for cell cycle entry and differentiation , but this requires further investigation .", "In the MI-day 7 re-analysis , we found that MYO could be sub-divided into three clusters - MYO-1 , MYO-2 and MYO-3 ( Figure 8B , F; Supplementary file 9; Supplementary file 10 ) , and comparing these clusters we noted genes corresponding to the contrasting functions of fibrosis inhibition and promotion .", "MYO-2 upregulated Tgfb1 ( encoding TGF-β1 ) , which is one of the strongest and most studied drivers of MYO formation ( Figure 8G; Supplementary file 9 ) , Scx ( Scleraxis ) , which regulates ECM production and the myofibroblast phenotype downstream of TGF-β ( Bagchi et al . , 2016 ) and Thbs4 ( Thrombospondin 4 ) , a regulator of cardiac fibrosis ( Frolova et al . , 2012 ) .", "We sourced RNA-seq data from cultured mouse cardiac fibroblasts untreated or treated with TGF-β ( Schafer et al . , 2017 ) and extracted DE genes .", "As expected , the highest positive correlations with TGF-β treatment ( log2 fold-changes ) were in MYO and other activated fibroblast populations ( Supplementary file 11 ) .", "For inter-MYO comparisons , we found a significant positive correlation with TGF-β treatment in DE genes comparing MYO-2 v MYO-1 ( Spearman’s correlation test , r = 0 . 26 , Padj = 1 . 93e-12 ) and MYO-3 v MYO-1 ( Spearman’s correlation test , r = 0 . 25 , Padj = 1 . 75e-10 ) , supporting the conclusion that MYO-2 and MYO-3 are pro-fibrotic .", "In contrast , MYO-1 showed strong upregulation of anti-fibrosis genes included Wisp2 , encoding matricellular protein CCN5 which can reverse established fibrosis ( Jeong et al . , 2016 ) , and Sfrp2 encoding a soluble WNT receptor and antagonist of canonical WNT signalling , which is pro-fibrotic ( Mirotsou et al . , 2007 ) ( Figure 8G ) .", "MYO-1 upregulated genes showed significant GO terms negative regulation of growth factor stimulus and negative regulation of cell proliferation ( Figure 8F; Supplementary file 10 ) , which included Sfrp1 , implicated in inhibition of fibroblast proliferation and fibrosis ( Sklepkiewicz et al . , 2015 ) , and Htra1 and Htra3 , implicated in TGF-β signaling inhibition ( Tocharus et al . , 2004 ) .", "Diffusion Map analysis of MYO sub-populations showed that MYO-1 and MYO-2 were distinct clusters with some overlap , suggesting a continuum of states , whereas MYO-3 did not appear to be a distinct population ( Figure 8—figure supplement 1G ) ." ], [ "The mammalian heart is composed of a complex interdependent community of cells , although their interactions and flux are poorly characterized .", "Here , we present scRNA-seq data on >30 , 000 individual cardiac interstitial cells from sham , and MI days 3 and 7 hearts .", "Our interrogation of both the total interstitial population ( TIP ) and flow-sorted Pdgra-GFP+CD31- fibroblast lineage cells has given us a high-resolution map of cell lineage , state and flux in the healthy and injured heart , considerably extending preliminary studies ( Kanisicak et al . , 2016; Skelly et al . , 2018; Gladka et al . , 2018 ) .", "On the basis of these data , resident fibroblasts could be segregated into two major sub-populations denoted Sca1high ( F-SH ) and Sca1low ( F-SL ) , both expressing canonical fibroblast markers such as Pdgfra , Pdgfra-GFP , Ddr2 and Col1a1 .", "F-SH cells were enriched in S+P+ ( SCA1+PDGFRα+ ) fibroblasts and clonal colony-forming units ( Pinto et al . , 2016; Chong et al . , 2011; Noseda et al . , 2015 ) , and F-SH and F-SL showed distinct adhesive and secretory phenotypes , highlighting the likely functional differences between them .", "We describe a novel activated fibroblast population , F-WntX , in sham hearts , which persists after MI .", "The related F-Trans is an intermediary population between F-SL and F-WntX ( Figure 9 ) .", "Aside from a low proportion of activated fibroblasts ( F-Act ) present in sham hearts , no other sham population ( >75% of GFP+ cells ) showed an activated phenotype .", "When we re-analyzed the scRNA-seq data of Skelly et al . on interstitial cells from uninjured hearts ( Skelly et al . , 2018 ) , we identified all of the main fibroblast populations that we describe here in sham hearts ( Figure 4—figure supplement 5A–C ) .", "F-Act and F-WntX expressed activation marker Postn in both studies; however , in contrast to our study , all populations identified by Skelly et al . , including endothelial and immune cells , expressed the contractile marker Acta2 .", "The reason for this is unclear , but is likely technical .", "Gladka et al . profiled cardiac populations after ischemia-reperfusion injury using scRNA-seq and highlighted Ckap4 as a novel marker of activated fibroblasts ( Gladka et al . , 2018 ) ; however , our data shows Ckap4 expression across virtually all cardiac stromal populations ( Figure 4—figure supplement 5D ) , a discrepancy that may relate to the relatively low number of cells profiled in the Gladka et al . study .", "Among fibroblasts , F-WntX uniquely expressed Wif1 , encoding a canonical and non-canonical WNT signaling antagonist ( Meyer et al . , 2017; Bányai et al . , 2012 ) .", "WNT pathways play complex roles in cardiac biology and disease , impacting immune , vascular and pro-fibrotic pathways , and many drugs inhibiting WNT signaling are under investigation for their impacts on heart repair ( Foulquier et al . , 2018; Palevski et al . , 2017 ) .", "WIF1 additionally inhibits signaling through CTGF , a polyfunctional matricellular protein and positive driver of fibrosis ( Travers et al . , 2016; Surmann-Schmitt et al . , 2012 ) .", "WIF1 is a tumor suppressor inhibiting tumour angiogenesis through both WNT and VEGF pathways ( Ko et al . , 2014; Hu et al . , 2009 ) .", "Wif1 knockout mice show inhibition of Mo differentiation and abnormal chamber remodeling after MI ( Meyer et al . , 2017 ) , while un-regulated transgenic WIF1 expression causes dilated cardiomyopathy ( Lu et al . , 2013 ) , collectively indicating that correctly regulated WIF1 positively contributes to cardiac repair .", "One source of WNT proteins is inflammatory macrophages , and myeloid-specific deletion of the essential WNT transporter Wntless leads to improved cardiac functional recovery after MI involving an increase in reparative M2-like macrophages and angiogenesis ( Palevski et al . , 2017 ) .", "In addition to WIF1 , F-WntX cells showed upregulation of other WNT and TGFβ pathway antagonists ( Figure 5A ) , overall flagging F-WntX cells as paracrine mediators of an anti-WNT/CTGF/TGFβ signaling milieu essential for cardiac repair .", "WIF1 protein expression occurred in the border zone at MI-day 3 , but not in sham or MI-days 1 and 7 hearts , consistent with previous findings ( Meyer et al . , 2017 ) .", "We also detected WIF1 in ~4% of CD45+ immune cells infiltrating the border zone .", "We acknowledge that our IF studies may have underestimated the number of WIF1+ cells; for example , if we were unable to detect cells actively secreting WIF1 but lacking protein accumulation in the golgi .", "Contrary to our results , Meyer et al . showed WIF1 levels peaking at MI-day 1 and diminished by MI-day 7 using western blotting ( Meyer et al . , 2017 ) .", "Whereas these differences need resolving , en face our data suggest that WIF1 expression is post-transcriptionally regulated with a peak around MI-day 3 , consistent with our proposed function for WIF1 and the F-WntX population generally in inhibiting fibrosis and angiogenesis , and promoting differentiation of Mo , during the transition between the inflammatory and fibrotic phases of heart repair .", "At MI-day 3 , both F-SH and F-SL were significantly diminished , and we hypothesize that this occurs as they convert to an activated state ( F-Act; Postn+Acta2negative-low ) .", "The scale of conversion suggests that fibroblasts remote from the infarct also become activated .", "Whereas F-Act upregulated genes that were associated with collagen fibrils and wound healing in common with MYO , F-Act cells are by far more closely related to resident fibroblasts than to MYO .", "A proportion of F-Act cells were actively proliferating at MI-day 3 and to a lesser extent at MI-day 7 , consistent with the known peak of fibroblast proliferataion ( Fu et al . , 2018; Ivey et al . , 2018 ) .", "At MI-day 3 , we also identified an activated , non-proliferating fibroblast population ( F-CI ) situated between F-Act and F-Cyc in trajectory plots , showing strong upregulation of genes supporting protein translation , and we hypothesize that this secondary activated state occurs in readiness for cell division and differentiation .", "MYO was evident only at MI-day 7 , where they represented >50% of total GFP+ cells .", "This may be an underestimate , as the Chromium microfluidic device biases against larger cells , and some MYO cells may be GFP- .", "Highlighting the limitations of using the contractile marker αSMA to define MYO ( Tallquist and Molkentin , 2017 ) , a proportion of F-WntX , F-Act , F-Cyc and MYO cells expressed its gene , Acta2 .", "MYO cells massively upregulated a distinct network of genes related to cell adhesion , collagen fibril organization and angiogenesis , consistent with their known roles ( Shannon et al . , 2003; Tallquist and Molkentin , 2017 ) .", "Top ECM genes in these categories such as Postn , Fn1 and Col8a1 were expressed in virtually all MYO cells .", "However , our stage specific analysis of GFP+ cells at MI-day 7 allowed us to discern three distinct MYO sub-populations expressing pro-fibrotic ( MYO-2 and MYO-3 ) or anti-fibrotic ( MYO-1 ) states .", "MYO-1 expressed anti-TGF-β , anti-WNT and anti-proliferative signatures .", "Consistent with the view that MYO differentiation involves multiple cellular states , Fu et al . ( 2018 ) recently described a population of fibrobast-derived 'matrifibrocytes' , quiescent cells which persist in the mature scar after MI .", "The three major trajectories predicted by our Diffusion Map pseudotime analysis offer new insights into cardiac homeostasis and repair .", "The directionality of trajectories is suggested by the fact that all appear rooted in the resident F-SH and F-SL fibroblasts .", "One major trajectory arises specifically from F-SL and transits through F-Trans to F-WntX as the terminal state ( Figure 9 ) .", "Neither F-Trans nor F-WntX proliferate and may be primed for involvement in an injury response without the need for expansion .", "Up to MI-day 3 , F-Act cells proliferate , but do not differentiate to MYO , showing that these events can be uncoupled .", "Differentiation to MYO occurs after MI-day 3 , and our trajectory data suggest that this is largely from non-proliferating F-Act cells .", "We found no evidence for significant proliferation of MYO , although earlier time points need to be analyzed .", "Resident fibroblasts F-SH and F-SL were depleted at MI-day 3 , likely as a result of their activation and proliferation , and showed an apparent restoration by MI-day 7 in both TIP and GFP+ cells ( falling just short of our stringent p-value of 0 . 01 for DPA ) ( Figure 1A , E; Figure 4A , D; Figure 8A , B ) .", "A caveat of pseudotime trajectories is that they may be bi-directional .", "If confirmed , the restoration of F-SH and F-SL between MI-days 3–7 ( after the main fibroblast proliferative period [Fu et al . , 2018; Ivey et al . , 2018] ) points to renewal involving phenotypic regression .", "Regression of myofibroblasts to a less activated state was proposed recently ( Kanisicak et al . , 2016 ) , although our data suggest that the Postn-Cre lineage tracing mice used in that study to mark myofibroblasts labels most if not all F-Act cells ( Figure 4E ) .", "Certainly , the mechanism of activation , proliferation , self-renewal , differentiation and de-differentiation of cardiac fibroblasts warrants deeper investigation .", "The Mo/MΦ lineages of the heart have diverse functions , including in immunity , removal of debris and protection against autoimmunity during the early phases of MI , while promoting repair in latter phases , remodeling of the fetal coronary tree , neonatal heart regeneration and atrioventricular conduction ( Lavine et al . , 2014; Hulsmans et al . , 2017; Leid et al . , 2016; Aurora et al . , 2014; Nahrendorf et al . , 2007 ) .", "The adult heart , like other organs , contains resident MΦ , some of which have their origins in erythromyeloid progenitors in the embryonic yolk sac and fetal liver ( Leid et al . , 2016 ) and which self-renew during homeostasis and injury ( Heidt et al . , 2014; Epelman et al . , 2014; Dick et al . , 2019 ) .", "Other resident populations have differing degrees of monocyte dependence and some may eventually be supplanted during injury and aging by blood-born monocytes ( Molawi et al . , 2014; Dick et al . , 2019 ) .", "These resident MΦ likely have roles in defense against infection , antigen presentation and stimulation of T-cell responses , as well as efferocytosis ( Epelman et al . , 2014 ) .", "In the neonatal heart , they are though to be essential for regeneration through stimulation of CM proliferation and angiogenesis ( Lavine et al . , 2014 ) , and in the adult may be important for limiting fibrosis ( Lavine et al . , 2014; Dick et al . , 2019 ) , functions similar to pro-reparative M2 MΦ .", "Our scRNA-seq data identified most of the known Mo/MΦ populations highlighted by targeted scRNA-seq of cardiac Mo/MΦ cells ( Dick et al . , 2019 ) , including Ccr2+ and Ccr2- tissue resident MΦ , pro-inflammatory M1 Mo and MΦ at MI-day 3 , and non-classical M2 MΦ at MI-day 7 , as well as several minor populations and inflammatory fibroblasts .", "These data , combined with communication maps , provide a preliminary framework for further analysis of their relationships and flux in homeostasis , different disease models and after therapeutic interventions .", "Our Diffusion Maps lineage trajectory shows a continuum of states from M1Mo through M1MΦ to M2MΦ across the injury response , consistent with the recognised plasticity of blood born Mo ( Lavine et al . , 2018; Nahrendorf and Swirski , 2016 ) .", "However , whether these states are determined exclusively within the injury environment remains to be determined .", "Our trajectory also shows convergence of M2MΦ with tissue-resident MΦ ( Figure 2—figure supplement 1C ) .", "An emerging theme in the field is the similarity between yolk sac-derived tissue-resident MΦ and subsets of blood-born MΦ that appear during the reparative phase of MI ( Dick et al . , 2019 ) .", "However , the latter may not fully adopt the gene expression signature of resident cells , nor fully compensate for their proposed functions , as suggested in the context of genetic ablation of Cc3cr1+ tissue-resident MΦ , although this may relate to the timing of their deployment ( Dick et al . , 2019 ) .", "Irrespective of whether such cells are identical , it is noteworthy that pro-repairative macrophages appear to have multiple developmental origins , as found for virtually all other major adult heart lineages including CMs , ECs , SMCs , fibroblasts and adipocytes .", "Our scRNA-seq data can be mined for expression of genes implicated in other forms of heart disease .", "For example , of 167 genes proximal to single nucleotide polymorphisms implicated in GWAS studies in atrial fibrillation ( AF ) ( Roselli et al . , 2018 ) , 119 ( 71% ) showed expression in our TIP single-cell data ( for examples see Figure 1—figure supplement 8 ) , suggesting possible involvement of stromal cells in AF risk .", "We have identified substantial non-linear dynamics in the interactive cell communities of the heart ( Figure 9 ) , which , in this new light , can be further analyzed with lineage and functional tools .", "There remains a compelling clinical and economic rationale for finding new therapies for controlling the inflammation and fibrosis that accompanies virtually all forms of adult heart disease ( Gourdie et al . , 2016 ) .", "In the long term , scRNA-seq analysis of cardiac homeostasis and disease will provide new entry points for discovering novel drugs and interventions supporting heart repair and regeneration ." ], [ "8-12 weeks old male mice were used in all experiments unless otherwise stated .", "For the single-cell RNA sequencing experiment , mice had a H2B-eGFP fusion gene knock-in at the endogenous Pdgfra locus ( Pdgfratm11 ( EGFP ) Sor; PdfgraGFP/+ ) .", "To induce acute MI , mice were anaesthetized by intraperitoneal injection of a combination of ketamine ( 100 mg/kg ) and xylazine ( 20 mg/kg ) , and intubated .", "Hearts were exposed via a left intercostal incision followed by ligation of the left anterior descending coronary artery .", "Sham operated mice underwent surgical incision without ligation .", "Hearts were harvested for paraffin-embedding , or FACS analysis at 3 or 7 days post-surgery , as indicated in Results .", "TIP were isolated as previously described ( Chong et al . , 2011 ) .", "Briefly , hearts were minced and incubated in collagenase type II ( Worthington , USA ) at 37°C before filtering through 40 μm strainers .", "Cells were resuspended in red cell lysis buffer , followed by dead cell removal , immunostaining for 15 min on ice with fluorophore-conjugated antibodies and two times wash with FACS buffer ( 1x PBS containing 2% fetal bovin serum ) before acquisition .", "We employed very stringent gating strategies to exclude doublets in the FACS analysis: FSC-H vs FSC-A , FSC-H vs FSC-W and SSC-H vs SSC-W cytograms were used to discriminate and gate out doublets/cell aggregates during sorting or from the analysis ( Figure 1—figure supplement 5 ) .", "To account for the autofluorescence generated by MI , we used a wild-type MI mouse as control to set up the gating strategy ( Figure 1—figure supplement 6 ) .", "For the TIP fraction , total DAPI-negative live single cells were sorted in FACS buffer .", "For the GFP+ fraction , GFP+CD31-cells were sorted from the DAPI-negative live single cells .", "For Fluidigm experiments , SCA1+PDGFRα+CD31- ( S+P+ ) cells were isolated and sorted as described above .", "For each sample , at least 10 , 000 final gate events were collected and stored for the later analyses .", "TIP cells were isolated as described and stained with indicated antibodies .", "FACS sorted primary cells were seeded at a clonogenic density of 50 cells/cm2 ( 500 cells per well of 6-well plate ) and were cultured in α-Minimal Essential Medium ( α-MEM ) containing 20% FBS+1% Pen/Strep at 37°C in a humidified 2% O2 and 5% CO2 incubator , with medium changes every 2–3 days .", "After 8-day culture , colonies were rinsed with phosphate-buffered saline ( PBS ) , fixed with 2% paraformaldehyde ( PFA ) and stained with 0 . 05% ( v/v ) crystal violet dye in water .", "Differences in colony number and size were evaluated by a two-tailed one-sample t-test to test for variability between individual samples .", "The single-cell library preparation relied on a commercially available droplet method , the 10x Genomics Chromium System ( 10x Genomics Inc San Francisco , CA ) .", "The number of cells loaded on the system was calculated based on the desired number of captured cells following manufacturer’s instructions .", "scRNA-seq libraries were generated following capture .", "Chromium GFP+ day 3 samples were sequenced on the Illumina HiSeq 2500 and the remainder on the Illumina NextSeq 500 high output .", "This experiment was performed using the Fluidigm C1 platform ( Fluidigm , San Francisco , CA ) .", "Immediately after cell sorting and counting , cells were loaded on the integrated fluidics circuits ( IFC ) C1 chip .", "Each capture site was carefully examined under a Leica fluorescence microscope in bright field , Red and Green fluorescence channels for cell doublets and viability , and to ensure the capture rate was satisfactory before cell lysis and cDNA preparation .", "The reverse transcription was performed on the chip using Clontech SMARTer Ultra Low Input RNA Kit V4 ( Takara-Clontech , USA ) .", "After running the C1 Fluidigm system , single cell RNA libraries were generated from 100 to 300 pg ( picogram ) of cDNA , using the low throughput Nextera XT DNA library prep kit ( Illumina , USA ) .", "Individual barcoded libraries were sequenced by Illumina HiSeq 2500 ( 125bp ) .", "Raw scRNA-seq data was processed using the 10x Genomics CellRanger software ( version 1 . 3 . 0 ) .", "The BCL files obtained from the Illumina NextSeq platform were processed to Fastq files using the CellRanger mkfastq program .", "The Fastq files were mapped to the mm10 version 1 . 2 . 0 reference , downloaded from the 10x Genomics website , with the sequence for H2B-eGFP appended to the reference .", "The CellRanger count program was run on individual Fastq data-sets from the different conditions .", "The aggr program was run to generate aggregate unique molecular identifier ( UMI ) count matrices for the following experimental data-sets analyzed in this study: ( 1 ) TIP: sham-day 3 , MI-day 3 and MI-day 7; ( 2 ) GFP+: sham-day 3 , sham-day 7 , MI-day 3 and MI-day 7 , ( 3 ) GFP+ day 3: sham-day 3 and MI-day 3; ( 4 ) GFP+ day 7: sham-day 7 and MI-day 7 .", "Bioinformatics processing of the scRNA-seq data was performed in R ( R Development Core Team , 2018 ) using the Seurat package ( Butler et al . , 2018 ) with figures primarily generated using ggplot2 ( Wickham , 2009 ) .", "R scripts containing the steps used for processing and clustering the data for each individual data-set ( TIP aggregate , GFP+ aggregate , GFP+ day 3 and GFP+ day 7 ) are available in Source code", "1 . For all data-sets , initial quality control filtering metrics were applied as follows .", "Cells with fewer than 200 detected expressed genes were filtered out .", "Genes that were expressed in less that 10 cells were filtered out .", "In order to control for dead or damaged cells , cells with over 5% of raw UMIs mapping to mitochondrial genes were filtered out .", "To further control for potential doublets in our data-sets , we visualized the distribution of expressed genes and UMI numbers and filtered out cells which were clear outliers .", "UMIs were normalized to counts-per-ten-thousand , log-transformed , and a set of highly variable genes was identified by gating for mean expression level and dispersion level in a per-data-set manner .", "The log-normalized data was scaled , with variation due to total number of UMIs regressed out using a linear model .", "Principal component analysis was run on the scaled data for the set of previously defined highly variable genes .", "In order to identify the number of principal components ( PCs ) to use for clustering , we ran the JackStraw procedure implemented in Seurat that identifies statistically significant PCs .", "Based on running the JackStraw procedure with 1000 permutations , we defined significant PCs as those up to p<0 . 001 , which were used as input to the Seurat graph-based clustering program , FindClusters .", "We experimented with modifying the number of PCs used for clustering but found that varying the number of PCs used caused only minor impact on the clustering solutions .", "The resolution parameter for FindClusters , which determined the number of returned clusters , was decided on a per-data-set basis after considering clustering output from a range of resolutions .", "The cells and clusters were visualized on a t-SNE dimensionality reduction plot generated on the same set of PCs used for clustering .", "We inspected the clusters for hybrid gene expression signatures that could indicate captured cell doublets , or a signature of stress/apoptosis that could indicate cells damaged during the process of cell sorting and capture in the microfluidics device .", "Within TIP , our initial clustering analysis returned 29 clusters; within these we identified five minor clusters exhibiting hybrid gene expression signatures: F-EC , M2MΦ-EC , EC-L1 , EC-L2 and BC-TC ( Figure 1—figure supplement 4A–D ) .", "The small size of these populations meant that we could not exclude the possibility of doublets; the five hybrid clusters were therefore removed and all subsequent analysis ( e . g . differential expression analysis ) was performed on the remaining 24 clusters .", "Within the GFP+ data-set , we identified one cluster where GO analysis suggested cells in a stressed state; these cells were removed prior to down-stream analyses of the GFP+ data-sets .", "We found when clustering the GFP+ day 7 data-set that the clustering solutions , depending on the resolution parameter , tended to either under-cluster ( too few clusters ) or over-cluster ( too many clusters ) the data when compared to clustering solutions for the full GFP+ aggregate or GFP+ day 3 data-set .", "In order to achieve a clustering solution that was directly comparable to the GFP+ aggregate and GFP+ day 3 data , we first over-clustered the data then ran an iterative procedure to ‘collapse’ transcriptionally similar clusters .", "We first generated a dendrogram of cluster similarity using the Seurat BuildClusterTree program , which builds a phylogenetic tree by first calculating average RNA expression across clusters , then performed hierarchical clustering on a distance matrix calculated from the averaged RNA profiles .", "We then ran AssessNodes to identify the clusters that were the most transcriptionally similar according to the Random Forest out-of-bag error calculations .", "The most similar clusters were merged and the process was repeated .", "We found four iterations of the above procedure produced clustering results directly comparable to the GFP+ aggregate and GFP+ day 3 clustering solutions .", "Fastq files were mapped to the Gencode mouse mm10 reference using STAR aligner ( Dobin et al . , 2013 ) ( version 2 . 5 . 2a ) .", "Reads marked unaligned by STAR were mapped using Bowtie 2 ( Langmead and Salzberg , 2012 ) ( version 2 . 3 . 1 ) with parameters –local –very-sensitive-local .", "BAM files generated by STAR and Bowtie2 were merged and read counts generated on the merged BAM using the Subread featureCounts ( Liao et al . , 2014 ) program with parameters -p -T 12 t exon -g gene_name .", "Count data was normalized to counts per-million ( CPM ) and transformed to Log2 ( CPM + 1 ) .", "Percent of RNA mapped to mitochondrial genes per cell was calculated .", "We filtered out cells that had below 1 million reads or greater than 20% of reads mapped to mitochondrial genes .", "This yielded 52 cells for experiment 1 and 52 cells for experiment", "2 . In order to circumvent the difficulties associated with clustering small numbers of cells , we instead used classification to map cell population identities from our analysis of the Chromium GFP+ experiment to Fluidigm .", "Due to significant differences in sequencing depth between the experiments , we built an iterative Random Forest ( iRF ) classifier trained on relative gene rankings ( i . e . ranking genes in each cell from highest to lowest expressed ) , hypothesising that using ranks instead of expression should alleviate some of the difficulties in comparing data-sets of different sequencing depths .", "The classifier was built as follows .", "As the Fluidigm S+P+ experiment was performed on healthy hearts , we first removed the injury conditions from the GFP+ data-set and retained the populations most representative of the sham conditions: F-SL , F-Act , F-SH , F-Trans and F-WntX .", "We re-calculated a set of 706 highly variable genes for GFP+ by gating for genes with higher dispersion and mean expression and took the overlap with expressed genes ( CPM > 0 in at least 1 cell ) in Fluidigm .", "The expression matrix of highly variable genes was converted to a rank matrix , which was used as training data for an iRF classifier with 1000 trees and 3 iterations .", "We evaluated the accuracy of the classifier with 10-fold cross-validation and receiver operating characteristic ( ROC ) analysis .", "We found high accuracy across populations with area under the ROC curve ( AUC ) over 0 . 95 for all populations ( Figure 4—figure supplement 2A ) .", "The classifier was applied to the Fluidigm data by first converting the expression matrix ( highly variable genes ) to ranks and returning the most probable cluster assignment .", "This was done for the two individual Fluidigm experiments ( Figure 4—figure supplement 2B ) .", "We compared the clusters identified in the GFP+ day 3 to GFP+ day 7 data-sets using two approaches .", "First was to build a multi-class iRF classifier for the GFP+ day 3 populations as above but including both sham and MI conditions .", "The classifier was trained on a set of 914 overlapping highly variable genes between the data-sets .", "We found the iRF classifier maintained reliable prediction accuracy even when including the injury conditions as determined by cross-validation and evaluation with multiple metrics including AUC , sensitivity , specificity and precision ( Figure 8—figure supplement 1B ) .", "The GFP+ day 7 expression matrix was then converted to ranks and the iRF was applied to obtain population probabilities for each cell ( Figure 8—figure supplement 1C ) .", "We additionally counted the number of cells with iRF score >0 . 5 ( Figure 8—figure supplement 1D ) .", "For calculating DE , we first identified genes expressed in at least 25% of cells for at least one of the populations being compared and with an absolute log2 fold-change difference of 0 . 5 ( including a pseudo-count of 1 ) .", "We then assigned p-values using the ‘bimodal’ test for DE ( McDavid et al . , 2013 ) implemented in the Seurat FindMarkers program .", "A Bonferroni-adjusted p-value of 1e-05 was used to determine significantly DE genes .", "Trajectory analysis was performed using Diffusion Maps implemented in the Destiny R package ( Angerer et al . , 2016 ) .", "For the set of populations being tested , we took the top upregulated genes for each population and the corresponding log-normalized expression matrix was input to the DiffusionMap program with default parameters .", "A fold-change ( log2 ) cutoff of 1 was used to select upregulated genes with the exception of the GFP+ day 7 inter-MYO analysis , where a cutoff of 0 . 5 was used .", "We tested the stability of the DiffusionMap output by altering the numbers of input genes ( i . e . modulating fold-change cutoff and using the full set of highly variable genes ) but found the resulting trajectories to be consistent .", "Over-representation of GO terms in gene lists was calculated using the PANTHER web-service ( Mi et al . , 2017 ) .", "The set of expressed genes in the relevant experiment was used as background .", "A false-discovery rate cut-off of 0 . 05 was used to determine statistical significance .", "The EC ( Endothelial Cell ) population , identified by Pecam1 ( CD31 ) expression , were observed in all conditions ( Figure 4—figure supplement 3 ) and their transcriptome showed a strong positive correlation with previously isolated adult cardiac CD31+ ECs ( Pearson’s correlation test , p-value=3 . 6e-15 , p=0 . 48 ) ( Quaife-Ryan et al . , 2017 ) .", "Detection of ECs was surprising , given the FACS gates to exclude CD31+ cells .", "Thus , the EC cluster may have low cell-surface CD31 .", "The MAC population , unique to MI-day 3 , were Cd45+Cd68+ MΦ ( Figure 4—figure supplement 3 ) and likely correspond to the minority population of Pdgfra-GFP+CD45+ cells identified at day 5 post-MI ( Asli et al . , 2017 ) .", "We were also able to identify Pdgfra-GFP+CD45+ cells in MI-day 3 samples , as shown in Figure 6G .", "Very few cells in EC and MAC clusters showed GFP expression ( Figure 4E ) and were likely detected because of GFP perdurance .", "It is unclear at present whether they derive from intra-cardiac or extra-cardiac GFP+ cells .", "We developed an approach for detecting changes in population proportions across conditions ( Source code 1 ) .", "For some number of conditions to be compared , clustering is first performed on an aggregate of all cells across conditions .", "Cells are assigned two labels: a group ( G ) label representing experimental group/condition and a cluster label ( L ) .", "A count table is generated for each cluster per-condition , which can be converted to a proportion table .", "We define a statistic for the differential proportion test , Δpj , as the difference in cluster proportions between two conditions; that is Δpj=p1j-p2j for some cluster j and corresponding proportions in experiments 1 and", "2 . This workflow is illustrated in Figure 1—figure supplement 3A .", "We next construct a null distribution for Δpj by randomly permuting cluster labels L for some w proportion of n total cells .", "Specifically , w*n cells are randomly selected , and their cell-type labels are replaced by a random sub-sample of labels drawn from the labels from all the cells ( sampling without replacement ) .", "A new count and proportion table is then generated from this randomized data ( Figure 1—figure supplement 3B ) .", "This process is repeated t times , and the resulting Δpj across the randomized data forms the null distribution .", "We then calculate empirical p-values representing either an increase or decrease in Δpj such thatPincrease= 1t∑i=1tIΔpi≥ΔpjPdecrease= 1t∑i=1tIΔpi≤Δpj Where I ( • ) is the indicator function .", "A final p-value , Pj is determined as the minimum of Pincrease and Pdecrease .", "The most important parameter that needs to be set for DPA is w , where lower values will trend towards a stricter test ( fewer significant hits ) and higher values trend towards higher numbers of significant hits .", "For the following tests , and throughout this paper , we use w = 0 . 1 .", "As a negative control experiment , we evaluated DPA on our two GFP+ sham experiments ( Figure 1—figure supplement 3C ) , which would be expected to demonstrate no major differences in population composition .", "We compared the results of DPA ( w = 0 . 1 , t = 100 , 000 ) to performing Fisher’s exact tests .", "We found Fisher’s exact test returned 7/11 of the populations as having significantly different proportions between the two sham experiments with p<0 . 05 ( Figure 1—figure supplement 3D ) .", "In contrast , DPA identified only one population with significant proportion change between conditions with p=0 . 03 ( Figure 1—figure supplement 3D ) .", "As p=0 . 03 represented the most significant change between sham conditions , we used a conservative p-value cutoff of 0 . 01 for comparing injury time-points as presented in Results .", "We further evaluated DPA using simulation testing .", "We first designed a simulation experiment involving two replicate scRNA-seq experiments performed on one biological system with 10 cells types; that is where the underlying cell proportions are the same in both experiments .", "We simulated varying degrees of noise ( e . g . experimental error or biological variability ) by introducing an error rate e .", "Error was introduced when creating a simulated profile of each cell-type per-experiment by randomly adjusting each proportion , p , by ±e*p with the proportions finally readjusted to sum to", "1 . We randomly drew 5000 and 3000 cells from experiment 1 and experiment 2 , respectively and performed DPA and Fisher’s exact test for each population .", "The procedure was repeated 100 times with error rates of 0 . 01 , 0 . 05 , 0 . 1 , 0 . 15 and 0 . 2 .", "We compared specificity measurements between Fisher’s exact test and DPA and found that DPA consistently made fewer false-positive calls than Fisher’s exact test , with difference in specificity between the two methods increasing with higher error rates ( Figure 1—figure supplement 3E ) .", "We next simulated a control vs condition experiment with 10 cell populations , where six populations change proportionally in the condition and four remain the same .", "We performed simulated experiments including error as above , drawing 4000 and 6000 cells for the control and condition experiments , respectively .", "For this experiment we have both true changes ( positives ) and non-changes ( negatives ) and could therefore evaluate both true-positive and false-positive detection rates .", "We found that both Fisher’s exact test and DPA correctly identified all true population changes with a sensitivity of 1 across all error rates ( Figure 1—figure supplement 3F ) .", "When considering false-positives , DPA again outperformed Fisher’s exact test in specificity and precision measurements ( Figure 1—figure supplement 3G , H ) .", "These results demonstrate that while DPA can identify true proportion changes with comparable sensitivity to Fisher’s exact test , it better controls for the detection of false-positive changes .", "In order to represent cell-cell communication networks via ligand-receptor interactions , we implemented a directed , weighted network with four layers of nodes as follows .", "The top layer of nodes refers to a set of source cell populations , defined as the cell populations expressing ligands .", "The second layer of nodes represents the set of ligands expressed by the source populations .", "A weighted edge connects Source:Ligand where the weight is the Log2 fold-change of the ligand in the source compared to the remaining populations .", "The third layer of nodes is the receptor targets of the ligands .", "These are determined from a map of ligand-receptor pairs , collated using both known ligand-receptor interactions and interactions predicted though protein localization and protein-protein interaction ( PPI ) information ( Ramilowski et al . , 2015 ) .", "As this map contains human ligand-receptor interactions , we add a weight using mouse-specific protein-protein associations from the STRING database ( Szklarczyk et al . , 2017 ) ; these are represented as values between 0 and", "1 . The fourth layer of nodes represents the target cell populations .", "Receptors are connected to target populations where they are expressed with weight again determined by Log2 fold-change .", "We did not normalize the three edge weights so as to ensure that gene expression provides the greatest weighting .", "A path weight connecting a source to target via a ligand:receptor pair is calculated as the sum of weights along that path .", "For our analysis , we initially considered all ligand:receptor pairs expressed in at least 10% of cells in a population .", "We then built a network using all 24 sub-populations identified in the TIP data-set .", "In order to filter out downregulated ligand-receptor connections , we set a minimum path weight of 1 . 5 .", "An overall weight describing the strength of connection between a source and target population , ws:t , could then be calculated by summing all path weights between the source and target .", "In order to identify Source:Target connections that have significantly higher summed path weights than would be expected by chance we generated random networks as follows .", "Given a Source:Target connection we identified the number of total unfiltered paths ( T ) , the set of paths with weight greater than 1 . 5 ( Gs:t ) and the subsequent summed path weights ( ws:t ) for Gs:t .", "We then generated random networks by retaining the Ligand:Receptor edges ( i . e . PPI connections ) but randomly selecting T number of Source:Ligand edges and Receptor:Target edges and re-calculating Gs:t and ws:t ( i . e . sub-sampling the fold-changes ) .", "This process was repeated m = 100 , 000 times and empirical p-values were calculated asPw= 1m∑i=1mIwi≥ws:t As considering all possible combinations of Source:Target paths yields a large number of tests , we adjusted p-values with the Benjamini-Hochberg correction method and considered all edges with adjusted Pw <0 . 01 to be significant .", "Significant Source:Target edges were visualized as a hierarchical graph using Cytoscape ( Shannon et al . , 2003 ) with edge thickness determined by ws:t .", "The code for performing the above analysis on the TIP scRNA-seq is available in Source code 1 ." ] ]

[ "Besides cardiomyocytes ( CM ) , the heart contains numerous interstitial cell types which play key roles in heart repair , regeneration and disease , including fibroblast , vascular and immune cells .", "However , a comprehensive understanding of this interactive cell community is lacking .", "We performed single-cell RNA-sequencing of the total non-CM fraction and enriched ( Pdgfra-GFP+ ) fibroblast lineage cells from murine hearts at days 3 and 7 post-sham or myocardial infarction ( MI ) surgery .", "Clustering of >30 , 000 single cells identified >30 populations representing nine cell lineages , including a previously undescribed fibroblast lineage trajectory present in both sham and MI hearts leading to a uniquely activated cell state defined in part by a strong anti-WNT transcriptome signature .", "We also uncovered novel myofibroblast subtypes expressing either pro-fibrotic or anti-fibrotic signatures .", "Our data highlight non-linear dynamics in myeloid and fibroblast lineages after cardiac injury , and provide an entry point for deeper analysis of cardiac homeostasis , inflammation , fibrosis , repair and regeneration ." ]

[ "In our bodies , heart attacks lead to cell death and inflammation .", "This is then followed by a healing phase where the organ repairs itself .", "There are many types of heart cells , from muscle and pacemaker cells that help to create the beating motion , to so-called fibroblasts that act as a supporting network .", "Yet , it is still unclear how individual cells participate in the heart's response to injury .", "All cells possess the same genetic information , but they turn on or off different genes depending on the specific tasks that they need to perform .", "Spotting which genes are activated in individual cells can therefore provide clues about their exact roles in the body .", "Until recently , technological limitations meant that this information was difficult to access , because it was only possible to capture the global response of a group of cells in a sample .", "A new method called single-cell RNA sequencing is now allowing researchers to study the activities of many genes in thousands of individual cells at the same time .", "Here , Farbehi , Patrick et al . performed single-cell RNA sequencing on over 30 , 000 individual cells from healthy and injured mouse hearts .", "Computational approaches were then used to cluster cells into groups according to the activities of their genes .", "The experiments identified over 30 distinct sub-types of cell , including several that were previously unknown .", "For example , a group of fibroblasts that express a gene called Wif1 was discovered .", "Previous genetic studies have shown that Wif1 is essential for the heart's response to injury .", "Further experiments by Farbehi , Patrick et al . indicated that this new sub-type of cells may control the timing of the different aspects of heart repair after damage .", "Tens of millions of people around the world suffer from heart attacks and other heart diseases .", "Knowing how different types of heart cells participate in repair mechanisms may help to find new targets for drugs and other treatments ." ]

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

[ "microbiology and infectious disease" ]

[ [ "Current treatment for HIV infection requires an individual to undergo lifelong daily cART administration , as interruptions in dosing usually result in the rapid recrudescence of virus .", "This viral recrudescence is thought to result from periodic activation of viral production from cells , including latently infected cells ( Pinkevych et al . , 2015; Fennessey et al . , 2017 ) , allowing the virus to spread to uninfected target cells no longer protected from infection by cART .", "A variety of approaches are currently being studied to reduce the RCR or the frequency of activation leading to virus production , in order to produce anti-retroviral free remission in treated patients ( Deeks et al . , 2016; Davenport et al . , 2019 ) .", "A variety of assays have been developed to measure HIV DNA ( total or integrated virus , intactness of viral genomes , or replication competence in outgrowth assays ) , levels of HIV-RNA production ( spliced or unspliced ) , or the ability to drive viral production through in vitro or in vivo stimulation of cells ( Eriksson et al . , 2013; Procopio et al . , 2015; Finzi et al . , 1999; Lorenzi et al . , 2016; Metcalf Pate et al . , 2015; Barton and Palmer , 2016; Bruner et al . , 2015 ) .", "These measures usually demonstrate a very wide range of values between individuals , varying between 100-fold and 1 , 000-fold for different individuals , and different measures are often poorly correlated ( Eriksson et al . , 2013 ) .", "Moreover , although these are often described as measures of the ‘viral reservoir’ , their contribution to the actual post-ART viral recrudescence is unclear .", "While complete eradication of the HIV RCR would prevent viral recrudescence , greatly decreasing the frequency of cells producing infectious virus may provide prolonged remission and the possibility of functional ‘cure’ ( Davenport et al . , 2019; Cromer et al . , 2017; Hill et al . , 2016 ) .", "In human studies , time-to-detectable viremia can be used as a rough estimate of the size of the RCR in a cohort of individuals ( Pinkevych et al . , 2015 ) and has been used to compare the size of the RCR between groups ( Bar et al . , 2016; Colby et al . , 2018; Li et al . , 2015 ) .", "However , this approach has very limited statistical power , due in part to variability of the exact time first detectable viremia occurs and wide confidence intervals within individual groups .", "It has been estimated that groups of >100 individuals may be required to convincingly demonstrate even quite substantial changes in the reservoir size with treatment ( Pinkevych et al . , 2015; Moore et al . , 2019 ) .", "Therefore , the efficacy of these interventions is often quantified by surrogate measures such as the levels of various forms of HIV DNA , cell-associated HIV RNA , or the ability of latent cells to be reactivated ex-vivo ( Rasmussen et al . , 2014; Søgaard et al . , 2015; Elliott et al . , 2014 ) .", "Understanding how these surrogate measures relate to the frequency of HIV reactivation and the resulting duration of viral rebound free remission after treatment interruption would greatly facilitate the accurate evaluation of novel therapies to reduce the RCR ( Eriksson et al . , 2013; Sharaf and Li , 2017 ) .", "The frequency of individual infected cells able to activate and produce sufficient viral progeny to allow for a spreading infection is postulated to be determined by a number of factors , including;", "( i ) the number of infected cells harbouring HIV DNA ,", "( ii ) the fraction of that DNA representing replication-competent proviruses ,", "( iii ) the viral integration site and level of cellular activation and epigenetic and transcriptional states of individual proviruses ( which together may help determine the per cell frequency of reactivation of virus; Hill , 2017; Yukl et al . , 2018 ) , and", "( iv ) the level of immune control of viral reactivation .", "A number of studies have reported that levels of integrated HIV DNA are a poor predictor of time-to-recrudescence of virus .", "Williams et al . ( 2014 ) investigated a cohort of 154 individuals who initiated treatment in the chronic phase of infection and underwent treatment interruption , and found that integrated HIV DNA was not a significant predictor of time-to-recrudescence ( defined as time to reach 50 copies/ml of virus in plasma; Williams et al . , 2014 ) .", "In a study by Calin et al . ( 2016 ) , patients selected for having very low HIV DNA levels failed to show a delayed time-to-rebound viremia .", "A recent study by Colby et al . ( 2018 ) showing time-to-recrudescence of virus in patients with very low HIV DNA who were treated within weeks of infection ( in Fiebig stage 1 ) suggests relatively little change in the frequency of HIV reactivation from latency compared to cohorts treated in chronic infection .", "However , other studies have suggested that early ART treatment in combination with lower levels of HIV DNA at interruption may be associated with delayed time-to-HIV-relapse ( Li et al . , 2015; Williams et al . , 2014; Assoumou et al . , 2015; Wen et al . , 2018; Steingrover et al . , 2008 ) .", "Therefore , the relationship between surrogate measures of HIV ‘reservoir’ ( in peripheral blood ) and the later time-to-viral-rebound remains unclear .", "Moreover , in studies of the efficacy of candidate latency reducing strategies , it is unclear if changes measured in peripheral blood will be associated with meaningful changes in post-treatment time to viral rebound ( Petravic et al . , 2017 ) .", "In this study , we used a macaque model of AIDS virus infection , employing the barcoded SIVmac239M virus to investigate the dynamics of SIV rebound after treatment interruption , and assess what host factors might correlate with or predict cellular activation and subsequent viral rebound .", "The use of the barcoded virus allows estimation of the frequency of SIV reactivation from latency in individual animals ( Fennessey et al . , 2017 ) .", "We studied viral reactivation rates in macaques in which cART was initiated at different times after infection ( between day 4 and day 27 ) , allowing for differing extents of viral dissemination reflected by different levels of viral DNA ( vDNA ) ( spanning two logs ) in PBMC .", "SIV DNA levels decayed under treatment and when cART was stopped after 43–68 weeks of treatment , we were able to estimate the frequency of SIV reactivation following treatment interruption .", "While SIV DNA level in PBMC just before treatment interruption was a predictor of SIV reactivation , this relationship appeared extremely ‘flat’ .", "That is , an ≈100 fold increase in SIV DNA levels was associated with only a two-fold difference in reactivation rates during a analytic treatment interruption ( ATI ) .", "We also investigated a variety of other factors that could plausibly affect the relationship between vDNA levels and the frequency of reactivation from latency .", "This work suggests that the rebound competent reservoir may be saturable and can be seeded early in the course of infection Subsequent increases in viral DNA due to a delay in therapy only marginally alters the dynamics of post-ATI rebound ." ], [ "We previously developed a barcoded SIVmac239 virus , designated SIVmac239M , containing ≈10 , 000 clonotypes , present at approximately equal proportions ( Fennessey et al . , 2017 ) .", "Infection of macaques with a suitable inoculum of this virus creates an essentially isogenic and phenotypically equivalent population of viruses containing diverse barcodes in both the circulating plasma virus and the SIV DNA of infected cells .", "cART leads to prolonged suppression of virus in this model , and treatment interruption is followed by rapid viral rebound .", "We previously reported studies of a cohort of rhesus macaques , infected intravenously with 2 . 2 × 105 IU of SIVmac239M , and treated with cART initiated on day four post-infection for 300–478 days followed by treatment interruption ( Fennessey et al . , 2017 ) ( Figure 1a , n = 6 ) .", "In the present study , we extended this work to investigate macaques in which cART was initiated on day 10 ( n = 4 ) or day 27 ( n = 5 ) post-infection ( hereafter referred to as ‘day 10 treated’ and ‘day 27 treated’ respectively ) .", "Animals were treated for between 310 and 476 days , followed by treatment interruption .", "The plasma viral loads for the nine animals treated beginning on days 10 and 27 are shown ( Figure 1c , e ) .", "Following treatment interruption , we measured the growth rate of total plasma viral RNA and calculated the proportional contribution of individual SIVmac239M barcode clonotypes ( obtained using high throughput sequencing of rebound virus ) to the total rebound viremia ( Figure 1b , d , f ) .", "We then used a mathematical modelling approach to estimate the average frequency of reactivation from latency in individual animals ( Pinkevych et al . , 2015; Fennessey et al . , 2017 ) ( Figure 1g–i ) .", "For the four macaques treated starting on day 10 post-infection , the estimated frequency of reactivation from latency varied between 1 . 63 and 2 . 94 reactivations per day ( mean = 2 . 29 , SD = 0 . 47 , n = 4 ) .", "For animals treated beginning on day 27 , the frequency varied between 0 . 75 and 3 . 10 reactivations per day ( mean 1 . 33 , SD = 0 . 90 , n = 5 ) and was not significantly different from day 10 animals p=0 . 19 ( Mann Whitney ) .", "The frequency of reactivation of animals treated on or after peak infection ( pooled day 10 and day 27 groups ) was significantly higher ( Mann Whitney’s p=0 . 0028 ) than the frequency of reactivation previously measured for animals treated at day four post-infection , which were between 0 . 40 and 0 . 87 per day ( mean = 0 . 61 , SD = 0 . 17 , n = 6 ) ( Figure 1h ) .", "Longer duration of treatment also appeared to be associated with declining frequency of reactivation ( half-life 216 days ) , although this was not significant ( p=0 . 064 , linear mixed effects ( LME ) model ) ( Figure 1i ) .", "In order to understand the association between peripheral blood virologic measurements and the estimated frequency of SIV reactivation after ATI , we first measured cell-associated ( CA ) viral gag DNA levels during cART treatment in macaques starting at 4 , 10 , or 27 dpi ( Figure 2a ) .", "These data showed an early phase of rapid decline , followed by a slower decay with prolonged treatment .", "We then focused on the SIV gag DNA level in PBMC at the time of treatment interruption as a predictor of the frequency of reactivation .", "The levels of SIV gag DNA at interruption varied over a wide range between cohorts , ranging from <3 . 2 copies/106 PBMC among macaques treated beginning on day four post-infection , to as many as 1000 DNA copies/106 PBMC among animals treated beginning on day 10 ( Figure 2b ) .", "When we correlated the level of log SIV DNA at treatment interruption with the log frequency of reactivation , we found a significant linear correlation ( linear regression slope = 0 . 20 , R2 = 0 . 56 , p=0 . 0019 ) ( Figure 2b ) .", "Although individual SIV genomes may vary greatly in their probability of reactivation ( for example due to replication competence , integration site , or cell phenotype , activation state , and epigenetic or transcriptional blockades ) , if SIV DNA measured in the animals treated on different days had on average a similar probability of contributing to SIV reactivation from latency , then a doubling of DNA would equate to a doubling of reactivation ( and we should expect a 1:1 correlation in this log:log plot ) .", "Instead , the slope was only 0 . 20 .", "Although the day 4 and day 27 treated animals were treated for a similar length of time ( Figure 1b ) , the day 27 treated animals had at least 116 fold higher SIV DNA level at interruption , but only an approximately 2-fold difference in the frequency of reactivation with a mean of 0 . 61 ( SD 0 . 17 , n = 6 ) versus 1 . 33 ( SD 0 . 90 , n = 5 ) reactivations per day for day 4 and day 27 treated animals respectively .", "To compare the ‘per cell’ frequency of reactivation between the groups , we estimated reactivations per DNA copy ( Figure 2c , d ) .", "This analysis showed that treatment later in infection was associated with a declining frequency of reactivation per DNA copy measured at treatment interruption ( Figure 2c ) .", "In addition , within the d4 treated and d27 treated animals , increased duration of treatment also showed a trend towards reduced per cell frequency of reactivation ( Figure 2d ) .", "Thus , although SIV CA-DNA quantity in PBMC increases greatly from day 4 to day 27 post-infection , the frequency of reactivation per DNA copy declines in this time , apparently dependent on other factors .", "Differences in the per-cell probability of activating and producing progeny virus may explain the unexpected relationship between post-ATI reactivation frequency and SIV DNA copies in PBMC .", "Unspliced viral RNA transcription on cART has previously been used as a potential measure of the level of active transcription of infected cells ( Elliott et al . , 2014; Petravic et al . , 2017 ) , and some previous studies in HIV have suggested that levels of CA-RNA is a better predictor of time-to-recrudescence than CA-DNA ( Li et al . , 2015 ) .", "Therefore , we also explored the relationship between the levels of unspliced SIV gag RNA throughout the course of treatment , including immediately prior to ATI , and the subsequent frequency of reactivation .", "A steep decline in CA-SIV-RNA is observed soon after treatment , which then slows at around day 100 .", "This likely reflects the early , rapid loss of highly productive cells ( with high RNA per DNA ) due to viral cytopathic effects or immune clearance and subsequent survival of cells with a more restricted viral expression phenotype ( low RNA per DNA ) ( Figure 3a ) .", "Comparing reactivation rates with CA-RNA levels immediately prior to ATI , we find that CA-RNA is not a significant predictor of post-ATI reactivation frequency ( Pearson r of log-log transformed data is 0 . 37 , p=0 . 18 , n = 15 ) ( Figure 3b ) .", "Moreover , comparing between groups we again found that this was not a 1:1 relationship where a doubling of RNA would predict a doubling of reactivation .", "That is , the day 4-treated and d27-treated animals differed by 84 fold in their levels of SIV-CA RNA , despite only a two-fold difference in reactivation rate ( Figure 3b ) .", "We also investigated the frequency of reactivation per RNA copy , and again found major differences between the groups ( Figure 3c ) .", "As with the viral CA-DNA , CA-RNA is not proportionate to the frequency of post-ATI reactivation in vivo .", "One possibility to explain these data is that SIV CA-RNA is a poor measure of cellular activation leading to virus production and that other measures of immune activation may better predict the per cell SIV reactivation frequency .", "We therefore investigated other indicators of immune activation , studying CD4+ and CD8+ T cell expression of the activation markers CD38 , HLA-DR , and Ki67 ( these are generally correlated , so we limit our discussion to Ki67 expression in CD4+ T cells ) .", "Comparing animals treated beginning at day 4 , 10 and day 27 , we found that the animals treated beginning at day 10 had higher immune activation just prior to treatment interruption than those treated on day 4 and 27 ( day 4 - mean ( + /- SD ) Ki67 = 1 . 12% , ( + /- 0 . 38% ) , n = 6; day 10 – mean = 4 . 54% , ( + /- 1 . 87% ) , n = 4; day 27 – mean = 1 . 72% , ( + /- 0 . 39% ) , n = 5; however , only day 4 and day 10 groups were significantly different , Dunn's multiple comparisons test’s p=0 . 005 ) ( Figure 3d ) .", "Overall , cellular activation in PBMC did not seem a major driver of reactivation frequency , since reactivation per SIV DNA copy was negatively correlated with the level of Ki67 expression ( Spearman r −0 . 56 , p=0 . 034 ) .", "Our approach to estimating the average frequency of SIV reactivation from latency requires measuring the proportional contribution of different SIVmac239M clonotypes to the pool of total rebound plasma viremia after cART discontinuation .", "Thus , it estimates the frequency of successful reactivation events , and if some reactivating cells were targeted by immune responses , this would lead to an underestimation of the frequency of reactivation .", "Thus , it is possible that the observed differences in per cell reactivation frequency may be explained by increased immune control of a proportion of attempted reactivation events .", "To explore this hypothesis , we measured T cell responses ( Figure 4a ) using intracellular cytokine staining .", "We analysed IFN-γ , TNF-α , IL-2 , CD107a and MIP1β responses to pooled peptides from SIV Env , Gag , Pol and accessory ( ACC ) proteins ( see Materials and methods ) .", "The proportion of CD8+ T cells expressing CD107a in response to pooled SIV peptides ( measured at the time of treatment interruption ) was negatively correlated with the peak viral load observed during treatment interruption ( Spearman r = −0 . 62 , p=0 . 015 , n = 15 ) .", "This suggests that CD8+ T cells are able to mediate some level of immune control of SIV viral growth during rebound ( Figure 4c ) .", "We discuss here only the CD107a responses , while other responses are summarised in Figure 4—figure supplement 1 , 2 and 3 .", "Across the pooled animals from the day 4 , day 10 and day 27-treatment initiation groups , there was a negative correlation between peripheral immune response and reactivation per DNA copy ( Spearman r = −0 . 57 , p=0 . 03 , n = 15 ) ( Figure 4d ) .", "However , this largely appeared driven by the differences between the groups; day four treated animals had lower immune responses , and they had higher reactivation per DNA than the day 27 treated .", "However , if immune control directly reduced the frequency of reactivation , then we might also expect to see a similar trend within the groups , so that the animals with the highest immune response in each group would also have the lowest reactivation rate .", "Instead , we see that the animal in the day four group with the highest immune response also had the highest frequency of reactivation per DNA copy .", "Similarly , the trend within the day 27 treated animals is towards higher immune responses being associated with higher per-latent-cell reactivation .", "There is also one animal in each of the day 10 and d27 groups with immune responses as low as the d4 treated animals , but 60 and 100-fold lower reactivation per DNA copy .", "Although the small number of animals limits these comparisons , overall this suggests that mechanism of CD8+ T cell immunity blocking or controlling a proportion of reactivation events is unlikely to explain the observed differences in per cell reactivation frequency .", "One potential explanation for our observed relationship between SIV DNA and reactivation frequency is that the proportion of SIV that is replication competent changes rapidly during early infection as a consequence of accumulation of deletions and or hypermutations .", "For example , if SIV DNA were 100% replication competent at day 4 , and 1% replication competent at day 27 , this could explain a 100-fold change in reactivation rates .", "To investigate this , we sequenced near full length SIV DNA from PBMCs taken just prior to ATI from animals treated on day 10 ( n = 4 ) and day 27 ( n = 5 ) ( we note that this data has also been included as part of another study; Long et al . , 2019 ) ( Figure 5 ) .", "We found that 50/60 sequences from the day 10 treated and 86/101 sequences from day 27 treated animals were intact and presumptively replication competent ( mean of 84% intact , range of 75–100% in individual animals ) .", "Since the majority of sequences were intact , this suggests that differences in reactivation frequency cannot be explained by differences in replication competence .", "Thus far , we have investigated a variety of individual factors that might explain post-ATI reactivation frequency .", "In order to determine whether a combination of these factors could better predict reactivation frequency , we performed a multiple regression analysis that included all potential variables ( listed in Table 1 ) .", "We found that none of the peripheral immune or cytokine variables contributed significantly to the regression model , indicating that these variables cannot accurately predict reactivation frequency .", "The best model to predict reactivation frequency included both treatment duration and DNA levels at interruption ( adjusted R2 = 0 . 73 ) .", "Previous studies have demonstrated that latency is established very early after SIV infection ( Whitney et al . , 2014; Whitney et al . , 2018; Okoye et al . , 2018 ) , consistent with our observations that seeding of the rebound competent viral reservoir is already established in macaques infected with a high-dose of virus and starting cART by day four post inoculation .", "However , our study suggests that the frequency of reactivation from latency does not increase much beyond early infection , even though large increases in SIV CA-DNA and CA-RNA are observed accumulating in PBMC .", "This raises the question of how the level of reactivation can saturate so early , while delayed treatment clearly allows much higher levels of infection and persistent SIV DNA ?", "It seems likely that the majority of SIV reactivation events observed derive from a relatively small portion of the total proviral population , which can be established extremely early in infection .", "This ‘reactivation initiating’ population increases only slightly with ongoing viral replication , while infected cell frequency and CA-DNA levels accumulate , but does not proportionally contribute to post-ATI reactivation frequency .", "One additional possible explanation is that there may be limited and saturable cellular or anatomic compartments that are preferred for harbouring reactivatable viruses and include cells infected early after infection which persist during treatment .", "To investigate this , we adapted a standard model of HIV infection to investigate the effects of a ‘susceptible subset’ of cells on latency and reactivation .", "The model included a subclass of cells that was both more susceptible to infection ( and thus infected earlier after inoculation ) , and more prone to later reactivation from latency ( see Materials and methods ) .", "A subset of 0 . 4% of cells , that is 100-fold more susceptible to infection , and 500-fold more prone to reactivation ( or a variety of combinations of these factors ) could recapitulate the observed dynamics of DNA accumulation and post-ATI reactivation from the data ( Figure 6 ) ." ], [ "A major question in HIV ‘cure’ research is whether biomarkers in peripheral blood correlate with and can be used to predict the duration of HIV remission after treatment interruption ( Margolis and Deeks , 2019 ) .", "We used variable timing of the initiation of cART , allowing different extents of viral dissemination before starting suppressive treatment , to produce SIV reservoirs of different sizes .", "The levels of SIV DNA at the time of ATI varied by >100 fold between different animals , from <3 . 2 copies/million PBMC in animals treated beginning on day 4 , to >1000 copies in some animals starting treatment on day 10 .", "The frequency of successful SIV reactivation after ATI also varied by ≈8-fold ( from 0 . 4 to 3 . 10 reactivations per day ) .", "This is remarkable when one considers the different levels of exposure to infection .", "The peak viral load varied ≈350 fold between animals starting treatment on day 4 ( geometric mean = 1 . 5×105 copies ml−1 , CI of geo .", "mean ( 4 . 9 × 104 , 4 . 4 × 105 ) , n = 6 ) and animals treated on day 27 ( geometric mean = 5 . 2×107 copies ml−1 , CI of geo .", "mean ( 1 . 8 × 107 , 1 . 5 × 108 ) , n = 5 ) .", "Similarly , the ‘area under the curve’ of viral exposure varied by >4 , 000 fold ( geometric means 5 . 2 × 104 , CI of geo .", "mean ( 1 . 8 × 104 , 1 . 5 × 105 ) n = 6 , vs 2 . 2 × 108 , CI of geo .", "mean ( 1 . 3 × 108 , 3 . 8 × 108 ) , n = 5 ) .", "However , although the prolonged viral replication led to an increase in SIV CA-DNA ( Archin et al . , 2012 ) , the additional exposure to virus between day 4 and 27 was associated with only approximately double the frequency of reactivation .", "This suggests that most of the additional infected cells , reflected in increased SIV CA-DNA generated between day 4 and day 27 did not significantly contribute to rebound viremia .", "The results suggest that key anatomic and/or cellular compartments that represent the major contributors to the rebound viremia may be limited and saturable as early as d4 after a high dose intravenous inoculation .", "The fact that very large changes in SIV CA-DNA are associated with only small changes in the frequency of reactivation perhaps goes some way to explaining why HIV DNA is a relatively poor predictor of time-to-reactivation after treatment interruption in many studies ( Li et al . , 2015; Williams et al . , 2014; Calin et al . , 2016; Assoumou et al . , 2015 ) .", "One potential explanation for our observations that this SIV DNA that accumulates later in infection is not as replication competent as DNA integrated earlier in infection .", "Sequencing studies suggest that only 2 . 4% of HIV proviral DNA is intact in patients starting cART in chronic infection ( Hiener et al . , 2017; Bruner et al . , 2016; Bruner et al . , 2019 ) .", "Thus , for example , if SIV proviral DNA integrated at day 4 was 100% replication competent , but DNA integrated after this were 99% defective , this might explain the observed relationship between reactivation frequency and SIV DNA copies .", "However , near full length viral sequencing from animals treated on day 10 and day 27 revealed that the vast majority ( >80% ) of sequences were intact .", "Studies of SIVmac251 DNA in animals treated in chronic infection ( 95 weeks post-infection ) for 36 weeks revealed that 28% remained intact ( Bender et al . , 2019 ) .", "Thus , it appears that at least over the first year or two of infection , SIV DNA remains intact at much higher rates than HIV .", "Taken together , it is clear that the observed differences in reactivation frequency between animals treated on different days cannot be explained by different proportions of replication competent virus .", "Previous work has suggested that extremely early treatment can either delay or prevent post-ATI SIV viral recrudescence ( Whitney et al . , 2014; Whitney et al . , 2018; Okoye et al . , 2018 ) .", "In a study by Whitney et al , intrarectal inoculation of 500 TCID50 of SIVmac251 and 6 months of cART begun on day three post-infection ( when animals did not have detectable plasma virus ) resulted in delayed viral rebound .", "However , animals treated for 6 months beginning on days 7 , 10 and 14 post infection showed similar rebound times ( Whitney et al . , 2014 ) .", "Okoye et al . ( 2018 ) infected macaques with 500 IU of SIVmac239 , and found that animals treated for nearly 2 years beginning on day 4–5 post infection ( with viral loads of 60–1100 copies/ml at treatment initiation ) failed to rebound after treatment interruption ( Okoye et al . , 2018 ) .", "By contrast , our study involved intravenous inoculation of 2 . 2 × 105 IU of SIVmac239M viral stock , and animals treated on day four had maximal viral loads of between 3 . 8 × 104 and 9 . 1 × 105 ( geometric mean of 1 . 5 × 105 ) copies/ml of plasma virus .", "Taken together , these data suggests that extremely early treatment ( with viral loads at treatment <1000–10 , 000 copies/ml ) may severely restrict the establishment of the RCR .", "However , once plasma viremia passes around 10 , 000 copies/ml the subsequent post-ATI frequency of reactivation is established .", "Extending the duration of infection prior to starting cART ( from day 4 to day 27 ) may greatly increase the level of proviral DNA ( Archin et al . , 2012 ) and cell-associated RNA in PBMC , but does comparatively little to increase the frequency of reactivation .", "Although animal models of infection provide the ability to control factors such as time and duration of treatment , it is not clear whether these same lessons are applicable to HIV infection .", "The RCR of SIV may differ from that in HIV , either because of the cells targeted for infection , or because of differences in infected cell behaviour .", "For example , cellular entry using alternative receptors such as CXCR6 and GPR15 , or differences in host restriction factors may lead to differences in infected cell phenotypes in SIV ( Riddick et al . , 2016; Reynolds et al . , 2011 ) .", "Similarly , although coinfection of cells with multiple copies of HIV is thought to be low ( Josefsson et al . , 2011 ) , rates of SIV coinfection of cells were not determined in our study and could play a role in determining the frequency of reactivation per DNA copy observed .", "However , recent studies have suggested that integrations sites and the propensity for clonal expansion of latent virus are similar between SIV and HIV ( Ferris et al . , 2019 ) .", "The initial inoculum of virus in our studies is several orders of magnitude higher than that estimated in HIV ( Keele et al . , 2008 ) , and thus this may disproportionately contribute to the early establishment and potential saturation of the SIV RCR .", "However , since viral loads increase around 10-fold per day in early infection ( Figure 1a , c , e ) , it is unlikely that this initial inoculum virus equates to >0 . 1% of virus present even at day four post-infection ( the timing of earliest treatment ) .", "Thus , it seems unlikely that the higher inoculum in SIV infection could be a major contributor to the SIV reservoir measured later during treatment .", "Our finding of early ‘filling’ of the RCR appear consistent with the more limited observations of reactivation in early HIV infection .", "In particular , although some studies have shown delayed post-ATI rebound in HIV patients treated early in infection and with small reservoirs ( Li et al . , 2015; Steingrover et al . , 2008 ) , the size of the reservoir appears only weakly associated with the time to recrudescence of HIV after treatment interruption ( Li et al . , 2015; Williams et al . , 2014 ) .", "A number of recent studies have shown that even in individuals treated extremely early and/or with a very small reservoir , the time to viral recrudescence can be very similar to that observed in individuals treated beginning in chronic infection and with a presumably larger reservoir ( Calin et al . , 2016; Assoumou et al . , 2015 ) .", "So while the case study of the Mississippi baby suggests that extremely early treatment can significantly delay viral rebound ( Luzuriaga et al . , 2015 ) , Colby et al have recently shown that individuals treated very early during Fiebig stage one have only slightly longer average time to detection of plasma viral rebound than individuals starting treatment in chronic infection ( median 26 vs . 14 days ) , despite considerably lower HIV DNA levels ( Colby et al . , 2018 ) .", "This strongly supports our observations with SIV , in that the frequency of post-ATI viral reactivation is not only established extremely early in infection , but once established does not increase much from early primary infection to chronic infection .", "A significant limitation of our study is that we were not able to perform quantitative viral outgrowth assays ( QVOA ) ( Finzi et al . , 1999 ) on our macaque samples , due to limitations in available cell numbers in rhesus macaques .", "Comparison of HIV sequences between circulating provirus and ex vivo-inducible virus show that there is a significant sequence overlap between these two compartments ( Lu et al . , 2019 ) .", "However , a recent study showed that despite the overlap between provirus and QVOA , there was no overlap of either compartment with post-ATI rebound virus ( Lu et al . , 2019 ) .", "This was taken to suggest that rebound virus originates from a different pool of cells to that reflected in circulating provirus .", "Combined with our SIV results , it is clear that most current measures of the HIV reservoir are in fact measuring total CA-HIV or even presumptively intact proviruses from PBMCs ( Bullen et al . , 2014 ) but not the major contributor to post-ATI rebound viremia .", "Further work is clearly required to focus attention on what must be a small proportion of the total latently infected cells ( compared to total provirus in PBMC ) , that appear to be the major drivers of rebound .", "The observation that DNA levels do not predict reactivation frequency between individuals does not answer the question of whether it might be a useful predictor of the impact of anti-latency therapies within an individual .", "Thus , for example , if the RCR declines ( or is reduced by treatment ) in proportion to the total HIV DNA , then monitoring total DNA might still be useful to assess the impact of anti-latency treatments on reactivation frequency within an individual patient over time .", "The stability of the HIV reservoir has been measured in humans by measuring the decay of both HIV DNA and ex vivo inducible HIV , which were both found to decay with a half-life of >4 years ( Besson et al . , 2014; Siliciano et al . , 2003 ) .", "Our study suggests that , after 6 months of therapy , SIV DNA decays with a half-life of around 345 days and SIV CA-RNA decays faster ( half-life = 86 days , p<0 . 016 , LME model ) .", "The frequency of reactivation declines slightly faster than SIV-DNA with a half-life of ≈216 days ( although not significantly different , p=0 . 53 , LME model ) .", "Thus , reactivation frequency appears to decay at the same or faster rate than DNA levels in PBMC .", "It is important to understand the mechanisms of latency formation , maintenance and reactivation when designing interventions aimed to induce long-term remission .", "Our work suggests that the RCR can be established very early following infection and provides some explanation as to why treating early may only have a small effect on subsequent time-to-recrudescence of virus ( even if total HIV DNA levels continue to increase ) .", "A major impediment in HIV remission studies is finding a proxy measure that can predict the time-to-recrudescence of virus , without patients undergoing the risks of treatment interruption ( Julg et al . , 2019 ) .", "This may be challenging , as our studies suggest that total PBMC SIV CA-DNA is not the main determinant of post-treatment rebound .", "Our work also suggests that efforts to develop optimal interventions to prevent HIV reactivation following treatment interruption will require a more thorough understanding of viral latency ." ], [ "Nine purpose-bred Indian-origin male rhesus macaques ( Macaca mulatta ) were housed at the National Institutes of Health ( NIH ) and cared for in accordance with the Association for the Assessment and Accreditation of Laboratory Animal Care ( AAALAC ) standards in an AAALAC-accredited facility and all procedures were performed according to protocols approved by the Institutional Animal Care and Use Committee of the National Cancer Institute ( Assurance #A4149-01 ) .", "Animal care was provided in accordance with the procedures outlined in U . S . NIH Guide for Care and Use of Laboratory Animals .", "Reference numbers associated with the ethical approval are AVP047 and AVP058 .", "At the start of the study , all animals were free of cercopithecine herpesvirus 1 , simian immunodeficiency virus ( SIV ) , simian type-D retrovirus , and simian T-lymphotropic virus type 1 .", "In total , 15 animals were intravenously infected with of transfection produced SIVmac239M as previously described ( Fennessey et al . , 2017 ) .", "9 of 15 animals were infected with 2 . 2 × 105 IU ( 1 mL ) and treated with cART at 4 and 27dpi , while the remaining four animals were infected with 1 × 104 IU ( 1 mL ) and treated with cART at 10dpi .", "cART regimen for d27 and d4 animals consisted of a co-formulated preparation containing the reverse transcriptase inhibitors tenofovir ( TFV: ( R ) −9- ( 2-phosphonylmethoxypropyl ) adenine ( PMPA ) , 20 mg/kg ) and emtricitabine ( FTC; 50 mg/kg ) administered by once-daily subcutaneous injection , plus raltegravir ( RAL; 150–200 mg ) given orally twice daily .", "Day four animal were additionally treated with the protease inhibitor indinavir ( IDV; 120 mg BID ) and ritonavir ( RTV; 100 mg BID ) for the first 9 months ( Fennessey et al . , 2017 ) .", "Day 10 animals received a co-formulated preparation of emtricitabine ( FTC , 40 mg/kg ) , tenofovir disoproxil fumarate ( TDF , 5 . 1 mg/kg ) , and dolutegravir ( DTG , 2 . 5 mg/kg ) administered by once-daily subcutaneous injection .", "Sequence analysis was used to enumerate the number of detectable barcodes measured during primary infection prior to cART and during rebound after cART interruption .", "Viral nucleic acid was sequenced using next generation sequencing as previously described ( Fennessey et al . , 2017 ) .", "Plasma viral load determinations for SIV RNA were performed over the duration of the study using quantitative real-time PCR as described previously ( Li et al . , 2016 ) .", "The limit of detection of this assay is 15 vRNA copies/mL .", "Quantitative assessment of cell-associated viral DNA and RNA in PBMC pellets was determined by the hybrid real-time/digital RT-PCR and PCR assays essentially as described previously ( Hansen et al . , 2011 ) but specifically modified to accommodate cell pellets .", "100 μL of TriReagent ( Molecular Research Center , Inc ) was added to cell pellets in standard 1 . 7 mL microcentrifuge tubes and the tubes sonicated in a Branson cup horn sonicator ( Emerson Electric , St . Louis ) for 15 s at 60% amplitude to disrupt the pellet .", "Additional TriReagent was added to a final volume of 1 mL and the remainder of the protocol was carried out as described previously ( Hansen et al . , 2011 ) .", "Limit of detection is evaluated on a sample by sample basis , dependent on the number of diploid genome equivalents of extracted DNA assayed .", "Antibodies and reagents were obtained from BD Biosciences , unless indicated otherwise , and data analysis was performed using FCS Express ( De Novo Software ) .", "Antibody panel validation and population gating were performed using fluorescence-minus-one and corresponding biological controls .", "Compensation was performed using goat-anti-mouse Ig ( Spherotech ) , anti-Rat Ig and amine-reactive ( Invitrogen ) compensation bead , single-color controls , under identical sample treatment conditions .", "For activation immunophenotyping , 100 μl EDTA-anti-coagulated whole blood or 1 × 106 quick-thawed cryopreserved PBMC were incubated with the following antibody panel: CD4 Pacific Blue ( OKT4 , BioLegend ) , CD8 BV510 ( SK1 ) , CD14 BV605 ( M5E2 , BioLegend ) , CD69 BV650 ( FN50 ) , CD163 BV711 ( GHI/61 ) , CCR5 PerCP-Cy5 . 5 ( 3A9 ) , CD38 PE ( OKT10; NIH Nonhuman Primate Reagent Resource ) , CD28 ECD ( CD28 . 2 , Beckman Coulter ) , CD95 PE-Cy5 ( DX2 ) , HLA-DR Alexa Fluor 700 ( L243 , BioLegend ) and CD3 APC-Cy7 ( SP34-2 ) .", "Samples were lysed with 1X BD FACS Lyse buffer , washed and then fixed and permeabilized with BD Cytofix/Cytoperm reagents , according to manufacturer instructions .", "Samples were incubated with an intracellular staining panel containing Ki67 BV786 ( B56 ) , MNDA FITC ( 3C1 , Beckman Coulter ) , CD68 PE-Cy7 ( Y1/82A , BioLegend ) and CD66 APC ( TET2 , Miltenyi Biotech ) , washed and approximately 200 , 000 CD3+ T-cells were acquired for each sample using a BD LSR-II flow cytometer .", "For intracellular cytokine staining , 1 × 106 quick-thawed , DNase I-treated ( 30 U/ml , Roche ) cryopreserved PBMC were stimulated for 8 hr in 96-well polypropylene round-bottom plates ( Costar ) with pools of 2 μg/ml overlapping 15mer SIVmac239 accessory ( combination of nef , rev , tat , vif , vpr and vpx ) , gag , pol and env peptides ( NIH AIDS Reagent Program ) , in the presence of CD107a BV785 antibody ( H4A3 , BioLegend ) .", "Phorbol 12-myristate 13-acetate with Ionomycin ( Sigma ) and DMSO in media were used as positive and negative controls , respectively , and 5 μg/ml brefeldin A ( Sigma ) mixed with 0 . 14 μl/well BD GolgiStop was added after 1 hr of stimulation to block protein transport .", "Samples were cultured in a DigiTherm Unibator ( Tritech Research ) at 37°C , 5% CO2 and immediately after the 8 hr incubation , rapidly cooled and maintained at 4°C until the following morning .", "Samples were surface stained with an antibody panel containing Yellow Fluorescent Reactive Dye ( Invitrogen ) , CD4 Pacific Blue , CD28 ECD , CD95 PE-Cy5 and CD8α PE-Cy7 , fixed and permeabilized with BD Cytofix/Cytoperm reagents and then incubated with an intracellular staining panel containing CD3 APC-Cy7 , IFNγ FITC ( B27 ) , MIP-1β PE ( D21-1351 ) , TNF-α BV711 ( MAb11 , BioLegend ) and IL-2 APC ( MQ1-17H12 ) .", "Cells were washed and approximately 200 , 000 live CD3+ T-cells were acquired for each sample using an HTS-equipped BD LSR-II cytometer .", "Genomic DNA was purified from PBMC from SIV-infected rhesus macaques using QIAamp DNA Mini Kit ( Qiagen ) .", "SGA sequencing was performed by diluting template DNA such that the majority of wells contain no template and the wells with template most likely contain only a single copy ( Keele et al . , 2008 ) .", "Nested PCR was performed using the following primers: SIVnFL . F1 5’-GAT TGG CGC CYG AAC AGG GAC TTG-3’; SIVnFL . R1 5’-CCC AAA GCA GAA AGG GTC CTA ACG-3’ for the first round PCR and SIVnFL . F2 5’-GTG AAG GCA GTA AGG GCG GCA GG-3’; SIVnFL . R2 5’-CCA GGC GGC GRC TAG GAG AGA TGG-3’ for the second round .", "The PCR reaction consisted of 1x SuperFi buffer ( Invitrogen ) , 0 . 2 mM each of dNTPs , 1x SuperFi GC enhancer ( Invitrogen ) , 0 . 02 U/uL Platinum SuperFi DNA Polymerase ( Invitrogen ) , 0 . 5 uM forward primer , 0 . 5 uM reverse primer , and template DNA .", "Thermal cycling conditions were as follows: 95°C for 2 min; 35x ( 95°C for 10 s , 68°C ( round", "1 ) or 72°C ( round", "2 ) for 10 s , 68°C ( round", "1 ) or 72°C ( round", "2 ) for 5 min; and a final extension of 5 min followed by 4°C hold .", "The higher annealing and extension temperatures ( 72°C ) in round two were used to avoid mispriming .", "SGA PCR products were directly sequenced using BigDye Terminator Sanger Sequencing ( Life Technologies ) with the primers previously described ( Lopker et al . , 2016 ) .", "In order to estimate the reactivation rate , we used a method that incorporates the ratio of the number of copies of different barcoded clonotypes , and the growth rate of virus ( Fennessey et al . , 2017 ) .", "We assumed that all barcodes have approximately the same growth rate and , thus , the reactivation rate ( RR ) can be estimated using ( 1 ) RR=g ( n−1 ) ∑i=1n−1 ( ln Si+1−lnSi ) , where g is the estimate of the growth rate of the single barcode , Sii = 1 , .", ". , n is the number of sequences for each barcode .", "The schematic explanation of this method is presented in the Figure 1d .", "The ratio of number of copies of each clonotype was estimated from Illumina sequencing of plasma virus , and growth rate was estimated as the maximal growth rate between any two viral load measurements in each animal .", "We assumed the exponential growth of virus between two neighbouring measurement as shown below ( 2 ) V ( ti+1 ) =V ( ti ) egi ( ti+1−ti ) , \\ i=1 , . . . , m−1 , where m is the number of measurements of viral load , V ( ti ) is the viral load at time ti .", "Thus the estimate of the growth rate on the interval can be found using formula ( Deeks et al . , 2016 ) ( 3 ) gi= ln V ( ti+1 ) −lnV ( ti ) ti+1−ti , i=1 , .", ". , m−1 , and the maximal viral load in subject is: ( 4 ) g=maxi=1 , .", ". , m−1gi Note that the maximal two-point growth gives slightly higher growth rates than fitting of multiple timepoints , but allowed for consistency across all groups , to avoid issues related to different sampling times .", "In order to estimate the area under the curve we approximated the trajectory of viral load by a piecewise function ( Fennessey et al . , 2017 ) where growth rates on the interval can be found using formula ( 3 ) .", "Having this simple approximation , we can find the integral of this function between any timepoints .", "This was implemented in Wolfram Mathematica 11 . 2 , Wolfram Research Inc , Champaign , IL , USA .", "All statistical tests were performed in GraphPad Prism 7 . 04 , GraphPad Software , La Jolla , CA , USA .", "In order to estimate the decay rate of CA RNA and SIV DNA in chronic infection , we estimated decay rates after approximately 6 months of treatment .", "We also measured the frequency of reactivation ( using the Materials and method described above ) in monkeys treated for different times .", "We fitted the following linear mixed effect model ( Eriksson et al . , 2013 ) to ln-transformed data .", "( 5 ) y=Ai+aij+ ( Bi+bij ) xij+εij , where Ai and Bi are fixed effect intercept and slopes where index i corresponds to different types of data such as SIV CA-RNA or CA-DNA .", "Index j in random effect parameters aij and bij corresponds to different monkeys in case of CA RNA and SIV DNA or in case of reactivation rate to groups of monkeys treated on day 4 or day 27 .", "This model was implemented in R ( v . 3 . 3 . 1 , The R Foundation for Statistical computing ) using standard function nmle for fitting and performing statistical tests .", "Multiple linear regression was performed using both forwards stepwise regression .", "Criteria for inclusion in the model was that the variable had a significance value ( p-value ) of greater than 0 . 05 , and that the regression coefficient was in a biologically meaningful direction ( Table 1 ) .", "The model considers two populations of cells .", "One group is comprised of normal cells , and the other contains highly susceptible cells that are also highly prone to post-treatment reactivation .", "When target cells become infected , some fraction of them , f , become productively infected cells .", "Then ( 1-f ) of them become latently infected cells .", "Highly susceptible cells are infected with a k fold higher infectivity rate than normal cells .", "We assume that reactivation rate is the sum of reactivation from both the normal cells and the highly reactivatable cells , but the highly reactivatable cells contribute a times as much to the reactivation rate .", "The equations are:dTNdt=−βVTNdTSdt=−kβVTSdldt=fβV ( TN+kTS ) −δIIdLNdt= ( 1−f ) βVTN−δLLNdLSdt= ( 1−f ) kβVTS−δLLSdVdt=pI−cV Where TN and TS are normal and susceptible target cells respectively , I are infected cells , LN and LS are normal and susceptible latent cells respectively and V is free virus .", "The parameters of the model are given by: β , the infection rate of normal targets , k the increase in infection rate for susceptible targets , f , the fraction of target cells that become productively infected ( not latent ) , δI , the death rate of productively infected cells , δL , the death rate of latent cells , p , the rate of production of virus and c , the death rate of virus .", "The reactivation rate is determined as:RR∝LN+aLS Parameters of the model .", "TS ( 0 ) =0 . 004TN ( 0 ) ( i . e . Number of highly susceptible cells is initially 0 . 4% of the number of normal cells ) , V0 = 40 , f = 0 . 2 ( i . e . 80% of cells become infected and 20% become latent ) , β = 1 . 4x10-7 , k = 100 , a = 500 .", "TN ( 0 ) = 1000 cells , δI = 1/day , δL=1200/day , c = 20/day , p = 5x105/day ." ] ]

[ "There is currently a need for proxy measures of the HIV rebound competent reservoir ( RCR ) that can predict viral rebound after combined antiretroviral treatment ( cART ) interruption .", "In this study , macaques infected with a barcoded SIVmac239 virus received cART beginning between 4- and 27 days post-infection , leading to the establishment of different levels of viral dissemination and persistence .", "Later treatment initiation led to higher SIV DNA levels maintained during treatment , which was significantly associated with an increased frequency of SIV reactivation and production of progeny capable of causing rebound viremia following treatment interruption .", "However , a 100-fold increase in SIV DNA in PBMCs was associated with only a 2-fold increase in the frequency of reactivation .", "These data suggest that the RCR can be established soon after infection , and that a large fraction of persistent viral DNA that accumulates after this time makes relatively little contribution to viral rebound ." ]

[ "Several drugs are available to control HIV , but they do not completely eliminate the virus from the body .", "Instead , these treatments stop the virus from multiplying , but unless a person is treated very soon after infection , inactive HIV can hide inside cells , and the infection is not completely cleared .", "Once treatment stops , the inactive virus starts to multiply , reaching pre-treatment levels within weeks .", "This means that infected individuals must continue treatment for life , or the virus will return and may cause disease .", "To prevent this , scientists are trying to find a way to eliminate HIV from the body , permanently curing the disease .", "Testing HIV drugs is difficult because there is no simple way to determine if all the inactive virus has been removed .", "One way to test whether a person is cured is to stop treatment , and see if the virus comes back .", "An alternative method is to measure the amount of inactive HIV present in blood cells .", "However , Pinkevych et al . have now shown that levels of inactive HIV in the blood may not be a good predictor of whether HIV levels will rebound after treatment .", "In the experiments , Pinkevych et al . infected macaques with a monkey version of HIV called simian immunodeficiency virus , or SIV for short .", "The virus was genetically modified to have a ‘genetic barcode’ that allowed researchers to track thousands of individual strains of virus simultaneously .", "The macaques were then treated with a combination of drugs starting either 4 or 27 days after infection , and then the treatment was stopped to check how rapidly strains of the virus would re-emerge .", "Although the virus rebounded in both groups , during treatment the inactive virus was often undetectable in the blood from the group treated four days after infection , suggesting that the virus may be hiding elsewhere .", "In the group treated 27 days after infection , more blood cells had inactive virus and reactivation was more frequent .", "However , the amount of inactive virus in the blood did not directly predict the frequency of reactivation: a 100-fold increase in viral levels only led to reactivation being twice as frequent .", "This suggests that the amount of inactive virus in the blood is not a good read-out for whether the virus will come back .", "These experiments suggest that inactive virus hides in cells quickly , since treatment just four days after infection failed to eliminate the virus completely , and that it must hide in cells other than blood cells .", "Evidence from humans suggests something similar may occur in HIV infection .", "Further studies may help identify which cells harbor the inactive virus and contribute to infections re-emerging after treatment stops ." ]

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

[ "neuroscience" ]

[ [ "Neural circuits are the functional units underlying all thoughts and behaviors , but there is as yet no neural circuit in any organism for which we know with precision how the many signaling events among its cells together generate its activity .", "This would require , among other things , understanding how neuromodulators shape activity at chemical and electrical synapses to alter the excitability of specific cells in the circuit to generate regulated , dynamic patterns of circuit responses and a coherent behavioral output .", "One approach to this problem is to analyze small neural circuits typical of invertebrate model organisms , so that both the simplicity of the circuits and the powerful experimental approaches uniquely possible within these systems can provide a penetrating analysis ( Marder , 2012 ) . 10 . 7554/eLife . 21126 . 003Figure 1 . Cell-specific reporters of activity in the C . elegans egg-laying behavior circuit .", "( A ) Schematic of the circuit .", "HSN ( green ) and VC ( blue ) motor neurons synapse onto the vm2 muscle postsynaptic termini ( center of schematic ) .", "The uv1 neuroendocrine cells ( pink ) extend processes ( grey ) along the vulval slit and vm2 postsynaptic terminus .", "( B–E )", "Individual video frames of the GCaMP5:mCherry fluorescence ratio showing active state Ca2+ transients in HSNs ( B ) , VCs ( C ) , and vulval muscles during twitching ( D ) and egg-laying behaviors ( E ) .", "Arrowheads , HSN and VC presynaptic termini; asterisks , cell bodies; scale bar , 10 µm .", "( F ) 30 min recordings of HSN , VC , and vulval muscle activity ( left panel ) , showing distinct active ( yellow ) and inactive ( grey ) egg-laying behavior states , with expanded timescale of one active state at right .", "Arrowheads show egg-laying events .", "( G ) Scatter plots and median HSN , VC , and vulval muscle ( vm ) inter-transient intervals during egg-laying inactive ( – , filled circles ) and active ( + , open circles ) states .", "Asterisks indicate significant differences ( p<0 . 0001 ) .", "( H ) Relationship between Ca2+ transient amplitude and egg release .", "Scatter plots and medians of normalized amplitude with ( +; open circles ) and without ( –; closed circles ) egg release .", "Also shown is the percent of total transients that accompanied egg release .", "( I ) Timing of HSN , VC , and vulval muscle Ca2+ transients and egg release .", "Shown at top is a curve of the median of Ca2+ from HSN ( green ) , VC ( blue ) , and vulval muscles ( orange ) from normalized ∆R/R traces ( with the peak Ca2+ set to 100% ) synchronized to the moment of egg release ( 0 s , arrowhead and dotted line ) .", "Bars indicate 20% change in median GCaMP5/mCherry ratio .", "The timing of the HSN Ca2+ peak is significantly different from that of the VCs and vulval muscles ( p<0 . 0001 ) .", "Shown at bottom is a trace of median vulval muscle size .", "Bar shows a 5% change in median object size based on mCherry fluorescence . DOI: http://dx . doi . org/10 . 7554/eLife . 21126 . 003 C . elegans egg-laying behavior is controlled by a small circuit that offers many experimental advantages for study ( Schafer , 2006 ) .", "This circuit , diagrammed in Figure 1A , consists of two serotonergic Hermaphrodite Specific Neurons ( HSNs ) and six cholinergic Ventral C neurons ( VCs ) , each of which synapse onto a set of vulval muscles whose contraction expels eggs .", "Four neuroendocrine uv1 cells also regulate egg laying ( Jose et al . , 2007 ) .", "Despite its anatomical simplicity , the egg-laying circuit produces a regulated , rhythmic behavior that alternates between quiescent periods of about 20 min during which no egg laying occurs , and active states lasting a few minutes during which ~5 eggs are laid ( Waggoner et al . , 1998 ) .", "Active states appear to result when the HSNs release serotonin that signals through G protein coupled receptors on the vulval muscles to increase their excitability ( Waggoner et al . , 1998; Shyn et al . , 2003; Hapiak et al . , 2009; Emtage et al . , 2012 ) .", "Strong regulation by sensory stimuli is superimposed on the pattern of alternating behavioral states .", "For example , carbon dioxide regulates neuropeptide release from head sensory neurons that signal through receptors on the HSNs to inhibit egg laying ( Ringstad and Horvitz , 2008; Hallem et al . , 2011 ) .", "Worms also halt egg laying in the absence of food and restart the behavior when re-fed ( Daniels et al . , 2000; Dong et al . , 2000 ) .", "C . elegans egg laying has been studied for decades , and dozens of genes have been identified by mutations that either reduce ( Desai et al . , 1988 ) or increase ( Bany et al . , 2003 ) egg laying .", "Some of the identified genes encode ion channels that regulate cell and synaptic electrical excitability ( Elkes et al . , 1997; Johnstone et al . , 1997; Lee et al . , 1997; Weinshenker et al . , 1999; Jospin et al . , 2002; Jose et al . , 2007; Collins and Koelle , 2013 ) .", "Other identified genes encode components of G protein signaling pathways that act in specific cells of the circuit ( Brundage et al . , 1996; Koelle and Horvitz , 1996; Hajdu-Cronin et al . , 1999; Miller et al . , 1999; Williams et al . , 2007; Porter et al . , 2010 ) .", "These results suggest neuromodulators , including serotonin , signal through G proteins to regulate the excitability of specific cells in the circuit to control circuit activity and egg laying .", "The ability to leverage egg-laying mutants to understand the molecular mechanisms that initiate , sustain , and terminate the active state of egg laying has been limited by the tools available to analyze activity in the cells of the egg-laying circuit .", "Ca2+ activity in cells in the circuit was initially optically recorded in immobilized animals , which have limited ability to engage in egg-laying behavior ( Shyn et al . , 2003; Zhang et al . , 2008 ) .", "More recently we developed methods to optically record Ca2+ activity of the vulval muscles in moving animals as they engage in normal egg-laying behavior .", "Using ratiometric imaging to correct for movement and focus artifacts , and recording at 20 frames/sec for periods of up to one hour , we were able to measure activity of the vulval muscles as animals cycled through active and inactive states ( Collins and Koelle , 2013; Li et al . , 2013 ) .", "Our recordings showed that the vulval muscles are excited rhythmically in phase with the body bends of animal locomotion , and we proposed this was due to signaling from the cholinergic motor neurons , including the VCs , that are rhythmically active during locomotion .", "We found that a conserved ERG K+ channel depresses response of the vulval muscles to excitation , thus maintaining the inactive behavior state , and we proposed that serotonin signaling onto vulval muscle receptors enhances vulval muscle excitability to allow the strong muscle contractions and egg release seen in the active state .", "We have now generated tools to carry out Ca2+ recordings in moving animals for each neuron type in the egg-laying circuit .", "Combining these recordings with precise manipulations of the circuit using mutations , optogenetics , and drugs allows us to deduce how specific signals from these cells drive specific features of circuit activity and behavior ." ], [ "We carried out long-term Ca2+ recordings of HSN and VC neuron activity in behaving animals using methods we developed previously for the vulval muscles ( Collins and Koelle , 2013 ) .", "We generated transgenic strains of C . elegans co-expressing the fluorescent Ca2+ reporter GCaMP5 and the Ca2+-independent fluorescent protein mCherry specifically in the HSN or VC neurons , and used ratiometric imaging to record cell activity and egg-laying behavior as animals moved through active and inactive egg-laying states ( Figure 1 ) .", "Because of the large size of the egg-laying synapse , we were able to clearly observe Ca2+ transients localized to the presynaptic terminus in HSN ( Figure 1B; Video 1 ) and VC ( Figure 1C; Video 2 ) .", "For comparison , we also carried out Ca2+ recordings of vulval muscles , and as in our earlier studies of these muscles , were able to clearly distinguish the weak twitching contractions ( Figure 1D; Video 3 ) from the strong contractions that drove egg release ( Figure 1E ) . 10 . 7554/eLife . 21126 . 004Video 1 . Ratio recording of HSN Ca2+ transients during the egg-laying active state . High Ca2+ is indicated in red while low calcium is in blue .", "Large panel is an expanded view showing the freely-moving animal that has been contrast enhanced to make the worm and its laid eggs visible .", "Inset is cropped to a small area containing the HSN , and stabilized to remove movement .", "Text labels indicate when Ca2+ transients and egg release occur . DOI: http://dx . doi . org/10 . 7554/eLife . 21126 . 00410 . 7554/eLife . 21126 . 005Video 2 . Ratio recording of VC Ca2+ transients during the egg-laying active state . High Ca2+ is indicated in red while low calcium is in blue .", "Contrast is enhanced to make the worm visible , although the laid eggs are not easily visible .", "Text labels indicate when vulval muscle ( vm ) twitches or egg-laying contractions occur , which result in small or large displacements of the VC4 and VC5 cell bodies , respectively . DOI: http://dx . doi . org/10 . 7554/eLife . 21126 . 00510 . 7554/eLife . 21126 . 006Video 3 . Ratio recording of vulval muscle Ca2+ transients during the egg-laying active state . High Ca2+ is indicated in red while low calcium is in blue .", "Large panel is an expanded view showing the freely-moving animal that has been contrast enhanced to make the worm and its laid eggs visible .", "Inset is cropped to a small area containing the vulval muscles , and stabilized to remove movement .", "Text labels indicate when Ca2+ transients and egg release occur . DOI: http://dx . doi . org/10 . 7554/eLife . 21126 . 006 Through long-term Ca2+ imaging , we observed a striking increase in rhythmic activity in the cells of the circuit that began prior to the first egg-laying event and persisted beyond the last egg-laying event of each active state ( Figure 1F ) .", "We defined the active state as one minute before the first egg-laying event and one minute after the last egg-laying event .", "We found that HSNs displayed long ( ~4 s at half-maximum amplitude ) Ca2+ transients that were largely consistent in waveform and amplitude , but that varied in occurring either as single events or trains ( Figure 1F ) .", "There was also significant HSN activity outside of the active state , but HSN transients were more frequent during the active state ( Figure 1G ) .", "Active state HSN transients typically occurred in bursts , within which HSN transients occurred every ~20 s ( Figure 1G ) .", "Only 11% of HSN transients were accompanied by an egg release ( Figure 1H ) , but almost every egg-laying event occurred during an HSN transient ( Figure 1F; out of 49 total egg-laying events observed , 48 occurred within an HSN transient ) .", "VC transients were shorter than HSN transients ( ~2 s at half-max ) and variable in amplitude .", "We rarely observed VC transients during the inactive state , but we found a clear induction of transients during the active state ( Video 2; Figure 1F , G ) , and these transients occurred in bursts around egg-laying events with an inter-transient interval of ~10 s , about twice as frequent as seen for the HSNs .", "Although Ca2+ transients were most intense in the VC presynaptic varicosities and axonal processes , they were also visible in all six VC cell bodies , especially those of the VC4 and VC5 neurons that most closely flank the vulva ( Figure 1C and Video 2 ) .", "Vulval muscle twitching and egg-laying contractions caused significant mechanical deformation of the VC neurons .", "34% of VC transients were accompanied by an egg release , a larger fraction than seen for HSN and vulval muscle Ca2+ transients ( p<0 . 0001; Fisher’s exact test ) .", "Every egg-release event was accompanied by a VC transient .", "There was no correlation seen between the magnitudes of VC or HSN transients that did or did not coincide with egg release , while the magnitude of muscle Ca2+ transients that resulted in egg laying were ~four fold larger than the muscle twitch transients that did not release eggs ( Figure 1H ) .", "The close apposition of HSN , VC , and vulval muscle synaptic regions prevented us from simultaneously imaging more than one cell type , but we were able to compare timing of activity in the different cells using the moment of egg release as an objective landmark .", "HSN Ca2+ transients peaked ~2 s prior to egg release ( Figure 1I ) and decayed slowly .", "Shorter VC Ca2+ transients followed HSN activity , peaking ~0 . 1 s before egg release .", "Vulval muscle Ca2+ transients peaked with egg laying .", "We also tracked vulval muscle contraction by measuring the size and fluorescence intensity of the mCherry labeled muscles , and saw that muscle relaxation and decay of vulval muscle Ca2+ immediately followed egg release ( Figure 1I ) .", "Previous work showed that timing of egg-laying events was not rhythmic but followed a Poisson distribution ( Waggoner et al . , 1998 ) .", "However , we found that HSN , VC , and vulval muscles showed rhythmic activity during the egg-laying active state .", "We calculated the power spectra of HSN , VC , and vulval muscle Ca2+ traces , and we observed three activity rhythms in the egg-laying circuit during the active state ( Figure 2A–E ) .", "Rhythms in HSN and VC were similar , peaking at ~50 mHz with smaller peaks observed at ~100 and~140 mHz ( Figure 2B and C ) .", "The 50 mHz rhythm was also clearly evident in the vulval muscles ( Figure 2D ) , but the 100 mHz peak was significantly stronger ( Figure 2E ) .", "These results show that despite egg-laying behavior being aperiodic , circuit activity underlying that behavior is rhythmic . 10 . 7554/eLife . 21126 . 007Figure 2 . HSN , VC , and vulval muscle activity is rhythmic and phased with animal locomotion .", "( A ) Active-state segments , such as the one shown here , were extracted from Ca2+ recordings , and analyzed for rhythmicity by power spectrum analysis .", "Underlying rhythm frequencies and peak inter-transient intervals were thus extracted for HSN ( B ) , VC ( C ) , and vulval muscle ( D ) traces , and the peak of maximum rhythm for each active state is indicated in bold .", "( E ) The average vulval muscle Ca2+ peak interval ( ~10 s ) was significantly different than the ~20 s rhythm observed in HSN ( p<0 . 0003 ) and VC ( p<0 . 0006 ) Ca2+ recordings .", "HSN and VC rhythms were not significantly different from each other ( p>0 . 9999; one-way ANOVA ) .", "( F ) The position of the vulva within a sinusoidal locomotor body bend at the moment a Ca2+ transient peaked was used to assign a body bend ‘phase’ , in units of degrees , with 180° representing ventral relaxation and 0/360° representing ventral contraction .", "Plots show the percent of Ca2+ transients observed in each of eight 45° bins for HSN ( G ) , VC ( H ) , and vulval muscle ( I ) , with data for transients accompanied by an egg-laying event ( ‘egg transient’ ) plotted in different colors from data for transients not accompanied by an egg-laying event ( ‘no egg transient’ ) .", "These plots show data pooled from recordings of 8–11 animals , and Figure 2—figure supplement 1 shows plots for each animal separately .", "The phasing of VC and vulval muscle transients that did not lead to egg laying is significantly different from an equal number of randomly distributed events ( R , at 12 . 5%; p<0 . 0001; Kruskal-Wallis test ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 21126 . 00710 . 7554/eLife . 21126 . 008Figure 2—figure supplement 1 . Relative timing of HSN , VC , and vulval muscle Ca2+ transients during locomotion body bends . The position of the vulva within a sinusoidal locomotor body bend was used to assign a body bend ‘phase’ , in units of degrees , for each Ca2+ transient analyzed in Figure 1 as described in the Materials and methods .", "The timing of each HSN ( A ) , VC ( B ) , or vulval muscle ( C ) Ca2+ transient peak with egg release ( open circles ) or without egg release ( closed circles ) was determined relative to the position of the vulva during the body bends of locomotion .", "Bar and whisker plots indicate the median and quartiles for each transient phase . DOI: http://dx . doi . org/10 . 7554/eLife . 21126 . 008 What is the source of circuit rhythmicity ?", "C . elegans moves with sinusoidal body bends consisting of rhythmic waves of body-wall muscle contraction driven by the Ventral A and B ( VA/VB ) ventral nerve cord motor neurons .", "We previously observed that vulval muscle Ca2+ transients tend to occur when the vulva is at a particular phase of the locomotion body bend ( Collins and Koelle , 2013 ) , suggesting rhythmic activity in the locomotor circuit may be the source of rhythmicity in the egg-laying circuit .", "Using our video recordings of Ca2+ activity in moving animals , we examined whether HSN and VC activity , like vulval muscle activity , are phased with locomotion .", "Thus we determined timing of the peaks of HSN , VC , and vulval muscle Ca2+ transients relative to when the vulva passed through the previous and subsequent most contracted or relaxed state of a body bend ( Figure 2—figure supplement 1 , see Materials and Methods ) .", "The results were plotted on histograms with ventral contraction at 0 and 360° , ventral relaxation at 180° , and intermediate phases of body bends at the angles in between ( Figure 2F–I ) .", "We found that the HSN , VC , and vulval muscle Ca2+ transients occurred when the vulva was at particular phases of the locomotor body bend .", "HSN transients just prior to egg release reached their maximum at ~180° , when the vulva and adjacent body wall muscles were at their most relaxed state ( Figure 2G ) .", "VC and vulval muscle transients that accompanied egg laying occurred later , starting at ~225° ( Figure 2H and I ) .", "HSN transients that did not result in egg laying showed no clear evidence of phasing ( Figure 2G ) .", "In contrast , VC transients and vulval muscle twitch transients that did not lead to egg release were still phased , but delayed by ~45° toward the most ventral contracted state .", "These results confirm our previous observations that vulval muscle twitching and egg-laying activity are phased with locomotion ( Collins and Koelle , 2013 ) .", "These results extend that analysis to show that HSN , VC , and vulval muscle activity during egg laying events are rhythmic , phased with locomotion , and occur earlier in the body bend cycle , when the adjacent body wall muscles are in a more relaxed state .", "To directly test how neurotransmitter signaling from the HSNs regulates egg-laying circuit activity , we used the egl-6 promoter to express Channelrhodopsin-2 in the HSNs ( Emtage et al . , 2012 ) , allowing us to drive neurotransmitter release specifically from the HSNs with blue light .", "We repeated the observation of Emtage et al . ( 2012 ) that optogenetic activation of HSNs stimulates egg-laying behavior ( Video 4 ) , but we activated the HSNs while simultaneously recording behavior and Ca2+ activity in the VCs ( Figure 3A ) or vulval muscles ( Figure 3B ) .", "We found that activation of HSNs resulted in circuit activity reminiscent of a spontaneous active state , including rhythmic Ca2+ activity of both VCs and vulval muscles , and egg-laying events that accompanied a subset of these Ca2+ transients .", "Some differences from a spontaneous active state were notable .", "Almost every VC transient after optogenetic HSN activation was accompanied by an egg-laying event , whereas only one third of VC transients during spontaneous active states were accompanied by egg-laying events .", "These results suggest that the high level of HSN activity after optogenetic activation induces strong coupling of VC and vulval muscle excitation . 10 . 7554/eLife . 21126 . 009Video 4 . Activation of the HSNs using Channelrhodopsin-2 induces the egg-laying active state . Animals were recorded for a total of 90 s with continuous blue light stimulation beginning at 30 s and ending at 60 s , during which five eggs are laid .", "Recording is sped up three-fold . DOI: http://dx . doi . org/10 . 7554/eLife . 21126 . 009 During both spontaneous and optogenetically-induced active states , rhythmic vulval muscle twitches occur far more frequently than do VC transients , suggesting these twitches are excited by something other than the VCs .", "Careful observation shows that the most dramatic Ca2+ changes during vulval muscle twitching occur at the ventral tips of the vm1 muscles ( Figure 1D ) .", "The VCs innervate vm2 muscles but not the vm1 muscles .", "The vm1 muscles are instead innervated by the VB6 and VA7 neurons that also release acetylcholine to drive body wall muscle contraction for locomotion ( White et al . , 1986 ) .", "Consistent with this , vm1 twitching occurs at the same time the adjacent ventral body wall muscles are contracting during locomotion ( Figure 2 ) .", "Increased electrical excitability of the vulval muscles during a spontaneous or optogenetically-induced active state may allow for vm1 excitation by VB6/VA7 with each body bend .", "Our observation that VC activity always accompanies egg release ( Figures 1I and 3A ) is consistent with a proposal that the VCs release acetylcholine that signals through vulval muscle nicotinic receptors to drive egg laying ( Waggoner et al . , 2000; Kim et al . , 2001 ) .", "While VC4 and VC5 make extensive synapses onto the vm2 muscles , VC1-3 and VC6 make extensive , distributed synapses onto the ventral body wall muscles ( White et al . , 1976 ) .", "How VC activity during egg laying might affect body wall muscle contraction has not been examined .", "We optogenetically activated the HSNs to induce active states and recorded animal speed around egg-laying events ( Figure 3C ) .", "We found a clear and significant reduction of locomotion speed beginning ~1 s before egg release , a time when VC activity is increasing ( Figure 1I ) .", "Locomotion speed recovered immediately after egg release ( Figure 3C ) .", "These results are consistent with the hypothesis that acetylcholine released from the VCs slows locomotion during egg laying . 10 . 7554/eLife . 21126 . 010Figure 3 . Optogenetic activation of HSN neurons initiates the egg-laying active state , and optogenetic activation of VC neurons slows locomotion .", "( A ) Animals expressing Channelrhodopsin-2 ( ChR2 ) in the HSNs and GCaMP5/mCherry in the VC neurons were grown in the presence ( +ATR; top , blue ) or absence ( –ATR; bottom , black ) of all-trans retinal .", "489 nm laser light was used to simultaneously stimulate ChR2 activity and excite GCaMP5 fluorescence during the entire recording .", "Arrowheads indicate egg-laying events .", "( B ) The same experiment as ( A ) except that GCaMP5/mCherry were expressed in the vulval muscles rather than VCs .", "( C ) Brief reduction of animal speed during optogenetically-induced egg laying .", "The egg-laying active state in animals expressing ChR2 in the HSN neurons was induced with 30 s exposure to blue light .", "Average speed ( black trace ) about each egg-laying event ( 0 s ) was calculated from the worm centroid position , and the grey area shows the 95% confidence interval .", "Average speed at the moment of egg laying release ( 0 s ) is reduced compared to 1 s before or after ( p<0 . 001 ) .", "See also Video 4 .", "( D ) Activation of the VC neurons slows locomotion but fails to induce egg laying .", "Animals expressing ChR2 in the VCs were grown in the presence ( +ATR; red ) or absence ( –ATR; black/grey ) of all-trans retinal , and worm length ( upper photos and graph ) was determined .", "Micrographs are still images from a representative animal at indicated time points ( see Video 5 ) .", "Inset arrows indicate regions of the head and tail that remain mobile after VC activation .", "Scatter plots of body bends ( E ) and egg laying ( F ) from the same recordings shown in D . There was a significant change in worm length and body bends , but not egg laying , during blue light in animals grown on ATR ( red; p<0 . 0001 ) .", "Positive control animals expressing ChR2 in the HSNs ( green ) showed a significant increase in egg laying during blue light ( p<0 . 0001 ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 21126 . 010 To test directly how VC activity regulates locomotion and egg laying , we used a lin-11 promoter fragment to express Channelrhodopsin-2 specifically in the VCs ( Bany et al . , 2003 ) .", "Optogenetic activation of the VCs was not sufficient to drive egg laying ( Figure 3F ) , but did lead to a rapid shortening of the body ( Video 5 and Figure 3D ) and cessation of locomotion ( Figure 3E ) .", "The shortening phenotype seen in VC ChR2 animals after blue light exposure closely resembles that seen for optogenetic activation of the body wall muscles ( Nagel et al . , 2005 ) , except the head and tail of VC-activated animals were still mobile and active , consistent with the fact that VC neurons innervate only the central body wall muscles ( White et al . , 1976 ) .", "Our results indicate that while VC activity immediately precedes egg-laying events , it is not sufficient to drive egg release .", "Optogenetic VC activation is sufficient to produce a slowing of locomotion as seen during egg-laying events .", "In contrast , HSN activity is sufficient to induce all of the hallmarks of the active state , including rhythmic vulval muscle twitching , VC transients , and the coordinated vulval muscle contractions that produce egg-laying events . 10 . 7554/eLife . 21126 . 011Video 5 . Activation of the VCs using Channelrhodopsin-2 induces animal paralysis and shortening of body length . Animals were recorded for a total of 90 s with continuous blue light stimulation beginning at 30 s and ending at 60 s .", "Recording is sped up 3-fold . DOI: http://dx . doi . org/10 . 7554/eLife . 21126 . 01110 . 7554/eLife . 21126 . 012Video 6 . Ratio recording of uv1 Ca2+ transients during the egg-laying active state . High Ca2+ is indicated in red while low calcium is in blue .", "Large panel is an expanded view showing the freely-moving animal .", "Inset is cropped to a small area containing the uv1s , and stabilized to remove movement .", "Microscope is focused to image the two uv1 cells on the right side of this animal , while the two additional uv1 cells on the left side are out of focus and not visible .", "Text labels indicate when egg releases occur , each of which is followed by a uv1 Ca2+ transient . DOI: http://dx . doi . org/10 . 7554/eLife . 21126 . 012 We next tested whether HSN activity was necessary for circuit activity by recording vulval muscle activity in egl-1 ( dm ) mutants , which lack HSN neurons ( Trent et al . , 1983 ) .", "Animals lacking HSNs have a substantial lengthening of the inactive state ( Waggoner et al . , 1998 ) and are strongly egg-laying defective , accumulating ~50 eggs in the uterus rather than the 12–15 seen in the wild type ( Trent et al . , 1983 ) .", "We expected to find that loss of HSNs led to a dramatic reduction in vulval muscle activity .", "Surprisingly , we found the opposite; animals lacking HSNs had more frequent vulval muscle Ca2+ transients during the inactive state and robust activity during infrequent active states that led to multiple eggs being laid in a short period ( compare Figure 4A and B; quantitation in Figure 4C ) .", "There was a significant reduction in the amplitude of vulval muscle Ca2+ transients during egg laying in egl-1 ( dm ) mutants while the amplitude of twitch Ca2+ transients was unaffected ( Figure 4D ) .", "Vulval muscle activity in egl-1 ( dm ) mutants lacking HSNs resembled the frequent yet uncoordinated vulval muscle activity found in animals missing the vm2 vulval muscle arms onto which the HSNs normally make synapses ( Li et al . , 2013 ) .", "Such activity involves asynchronous contraction of the anterior and posterior vulval muscles , which is ineffective at allowing egg release .", "One quantitative measure of this asynchrony is that overall vulval muscle Ca2+ transient peaks in egl-1 ( dm ) mutants were longer in duration , likely resulting in individual muscle cells beginning their transients at different times ( Figure 4E ) .", "Vulval muscle Ca2+ transients in animals lacking HSNs remained phased with locomotion , including the ~45° advance in phase of vulval muscle activity that results in egg-laying events ( Figure 4—figure supplement 1 ) .", "These results suggest that the HSNs are necessary to coordinate contraction of anterior and posterior vulval muscles , and that signals from other cells besides the HSNs can also induce the egg-laying active state . 10 . 7554/eLife . 21126 . 013Figure 4 . Active states require egg production but not the HSNs .", "( A ) Vulval muscle activity in wild-type animals either untreated or after sterilization with floxuridine ( FUDR ) .", "Left panel shows 30 min recordings with the grey underlined regions expanded at right .", "( B ) Vulval muscle activity in egl-1 ( n986dm ) mutants lacking HSNs .", "( C–D )", "Scatter plots of vulval muscle inter-transient intervals ( C ) and amplitudes ( D ) .", "Line indicates median , error bars indicate quartiles , and asterisks indicate significant differences ( p<0 . 01 ) .", "( E ) Vulval muscle transients are wider in animals lacking HSNs .", "Cumulative distribution plots of Ca2+ transient peak widths; asterisk indicates p<0 . 0001 ( Mann-Whitney test ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 21126 . 01310 . 7554/eLife . 21126 . 014Figure 4—figure supplement 1 . Persistent phasing of rhythmic vulval muscle activity in animals lacking HSNs and after sterilization . Phasing of vulval muscle Ca2+ transients during twitching ( closed circles ) and egg laying ( open circles ) in five untreated wild-type animals ( A ) and five animals sterilized with FUDR ( B ) .", "Bar and whisker plots indicate the median and quartiles for each transient phase .", "( C ) Histograms of pooled data showing phasing of vulval muscle twitches ( red ) and egg-laying transients ( yellow ) in untreated wild-type animals and twitches after FUDR treatment ( blue ) .", "Phasing of vulval muscle Ca2+ transients during twitching ( closed circles ) and egg laying ( open circles ) in seven untreated egl-1 ( n986dm ) mutant animals lacking HSNs ( D ) and eight egl-1 ( n986dm ) animals sterilized with FUDR ( E ) .", "Bar and whisker plots indicate the median and quartiles for each transient phase .", "( F ) Histogram of pooled data showing phasing of vulval muscle twitches ( red ) and egg-laying transients ( yellow ) in untreated egl-1 ( n986dm ) animals and twitches after FUDR treatment ( blue ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 21126 . 014 We hypothesized that accumulating unlaid eggs could stretch the uterus or body , leading to signals responsible for the increased activity of the vulval muscles independent of neurotransmitter signaling from the HSNs .", "To test if egg accumulation contributed to the onset of the active phase , we sterilized animals with FUDR ( Mitchell et al . , 1979 ) , a DNA synthesis inhibitor that blocks germ line cell division and thus egg production .", "FUDR-treated animals had a dramatic reduction of vulval muscle activity and appeared to be stuck in the inactive state , with no egg-laying transients observed ( Figure 4A and B ) .", "The inhibition of vulval muscle activity by FUDR occurred in both wild-type animals and HSN-deficient egl-1 ( dm ) mutants ( Figure 4C and D ) .", "The residual vulval muscle Ca2+ twitch transients in FUDR-treated animals had phasing identical to those seen in the inactive state of untreated animals ( Figure 4—figure supplement 1 ) .", "These results suggest that germ line activity and/or the accumulation of eggs in the uterus is required to initiate the egg-laying active state .", "We previously identified a role for the uv1 neuroendocrine cells in inhibiting egg-laying behavior ( Jose et al . , 2007 ) .", "The four uv1 cells sit at the junction between the uterine lumen and the vulval canal ( Newman and Sternberg , 1996 ) , and as such are positioned to mechanically sense the accumulation of unlaid eggs in the uterus and/or passage of eggs through the vulval canal as they are laid .", "To record uv1 Ca2+ activity , we co-expressed GCaMP5 and mCherry in uv1 from the ocr-2 promoter and made recordings in behaving animals .", "We found that the uv1 cells were mechanically deformed as each egg passed through the vulva , and then showed strong Ca2+ transients after egg release ( Figure 5A–B; Video 6 ) .", "Egg-laying events did not always trigger Ca2+ transients in uv1 , with uv1-silent events usually being the first or last within an active state ( data not shown ) .", "We also observed weaker uv1 Ca2+ transients a few minutes before or after the egg-laying active state ( Figure 5B–D ) .", "Because these were not closely associated with egg-laying events their functional importance remains unclear .", "Typical active state uv1 Ca2+ transients began immediately with or slightly before egg release , coinciding with the initial mechanical deformation of uv1 caused by the egg , with the Ca2+ transient peak occurring ~2 s later ( Figure 5E ) .", "Based on the position of the uv1 cells , their morphological deformations during passage of eggs , and the timing of uv1 Ca2+ transients , our data suggest the uv1 cells are mechanically activated by eggs passing through the vulva during egg laying .", "The uv1 cells make the biogenic amine neurotransmitter tyramine , and tdc-1 mutations that disrupt tyramine biosynthesis lead to increased egg-laying behaviors ( Alkema et al . , 2005 ) .", "We found that exogenous tyramine reduced egg laying in wild-type animals ( Figure 6A ) .", "To identify targets of tyramine inhibition , we tested response to exogenous tyramine in strains mutant for three known C . elegans tyramine receptors .", "Animals lacking lgc-55 , which encodes a tyramine-gated Cl- channel , showed resistance to inhibition by exogenous tyramine ( Figure 6A ) , although tyramine at high levels still had a residual ability to reduce egg laying in lgc-55 mutants ( Figure 6B ) .", "We transformed into C . elegans a fosmid transgene containing the full-length lgc-55 gene fused to GFP coding sequences , and found LGC-55 is expressed in the HSNs , uv1 , and vulval muscles ( Figure 6C and Pirri et al . , 2009 ) .", "Re-expression of LGC-55 specifically in HSN from the tph-1 promoter restored tyramine sensitivity to lgc-55 mutants ( Figure 6D ) .", "We have previously shown that two Cl- extruding transporters , KCC-2 and ABTS-1 , are expressed in the HSNs where they promote the development of inhibitory ligand-gated Cl- channel signaling ( Tanis et al . , 2009; Bellemer et al . , 2011 ) .", "These data suggest that tyramine signaling through LGC-55 would hyperpolarize the HSN and inhibit activity .", "To test this directly , we compared HSN activity in wild-type and lgc-55 mutant animals .", "We observed a significant increase in the frequency of Ca2+ transients in HSNs of lgc-55 mutant animals ( Figure 6E and F ) in both the inactive and active states of egg-laying behavior .", "Mean HSN inter-transient intervals in wild-type animals were 41 ± 5 s in the inactive state and 17 ± 2 s during the active state , while intervals in lgc-55 mutants were reduced to 22 ± 2 s in the inactive state and 13 ± 1 s during the active state .", "Thus , the absence of inhibitory feedback by tyramine signaling onto the HSNs leads to increased activity in both the active and inactive egg-laying behavior states . 10 . 7554/eLife . 21126 . 016Figure 6 . Tyramine inhibits egg laying , in part , through LGC-55 receptors on the HSNs .", "( A ) Exogenous tyramine inhibits egg-laying behavior and requires the LGC-55 receptor .", "Scatter plots and means showing the average number of eggs laid after 30 min by wild type ( grey ) , tdc-1 ( n3419 ) tyramine biosynthetic enzyme mutant ( blue ) , and lgc-55 ( tm2913 ) , ser-2 ( pk1357 ) , and tyra-3 ( ok325 ) tyramine receptor mutant animals ( orange , red , and purple , respectively ) on plates without ( – , closed circles ) or with ( + , open circles ) 15 mM tyramine .", "Error bars indicate 95% confidence intervals , and asterisks indicate significant responses ( p<0 . 01 ) while n . s . is not significant ( p>0 . 05 ) .", "( B ) Dose-response curve of tyramine inhibition on egg laying in wild-type and lgc-55 mutant animals .", "Asterisks indicate significant differences at 10 mM ( p<0 . 0001 ) and 20 mM tyramine ( p=0 . 0164 ) .", "( C ) LGC-55 is expressed in HSN , uv1 , and vulval muscles .", "Expression of LGC-55 tagged with GFP compared to vulval muscle mCherry was visualized using confocal microscopy; scale bar is 10 µm .", "GFP is localized to the HSN and uv1 cell bodies ( arrows ) as well as the ventral tips of the vulval muscles .", "( D ) Re-expression of LGC-55 in HSN restores tyramine inhibition of egg laying .", "Scatter plots of eggs laid by lgc-55 mutants expressing either GFP or lgc-55 in the HSN from the tph-1 promoter .", "Asterisk indicates significant differences in egg laying ( p=0 . 0029 ) .", "( E ) Loss of LGC-55 increases HSN activity .", "Representative Ca2+ ratio traces show HSN activity in wild type ( top , grey ) and lgc-55 mutant animals ( bottom , blue ) during the active state .", "( F ) Scatter plots and medians of HSN inter-transient intervals in wild type and lgc-55 mutant animals during the egg-laying inactive ( – , closed circles ) and active ( + , open circles ) states .", "Asterisks indicate statistically significant differences ( p<0 . 0001 ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 21126 . 016" ], [ "Using a combination of optogenetic , molecular genetic , and Ca2+ imaging approaches , we show an unexpected level of coordination of C . elegans egg-laying circuit activity by the rhythm of locomotion and by egg production .", "Using recordings of cell activity in behaving animals as they transition from the inactive to active egg-laying states , we were able to show that circuit activity is diminished during the inactive state , strongly increased and rhythmic during the active state , and this rhythm is phased with sinusoidal locomotion .", "Sterilization eliminated the active state rhythm , locking animals in the inactive state .", "Our results suggest that activity in the egg-laying circuit , like that of many other modulated motor circuits , responds to rhythmic input from central pattern generators ( Marder et al . , 2015 ) .", "A working model shown in Figure 7 explains how different inputs into the egg-laying circuit are responsible for the inactive state and the active state rhythms observed during egg-laying behavior .", "Active state vulval muscle Ca2+ transients peak rhythmically each time the vulva approaches a ventral body bend , the point at which the cholinergic VB and VA motor neurons release acetylcholine to rhythmically excite both the vm1 muscles and adjacent ventral body wall muscles during locomotion .", "During the inactive state , K+ channels including ERG depress vulval muscle excitability below threshold , explaining why we fail to observe vulval muscle activity with each body bend during this state ( Collins and Koelle , 2013 ) .", "While we observe rhythmic activity in the HSNs in the active phase , the periodicity and phasing of HSN activity does not correlate with that of locomotion .", "Consistent with this , previous work found changes in osmolarity can drive rhythmic HSN activity even in immobilized animals ( Zhang et al . , 2008 ) .", "Thus HSN rhythmic activity is independent of locomotion , although we found HSN Ca2+ transients that happen to fall at a specific phase of locomotor body bends are more likely to be followed by egg release .", "We propose that release of serotonin from the HSNs , along with an unknown signal resulting from mechanical stretch of the uterus by accumulating eggs , together raise vulval muscle electrical excitability to result in the active state during which the vulval muscles undergo rhythmic twitching or egg laying contractions in response to the same cholinergic VA and VB neurons that drive locomotor body bends .", "This model accounts for the phasing of vm1 transients with body bends , although egg-laying events require additional activity in the vm2 muscles that results in the coordinated contraction of all vm muscles that results in egg release .", "We found that vm transients that lead to egg release are on average slightly phase advanced relative to those that do not .", "This phase advance could from the excitatory input onto the vm2 muscles occurring earlier in the body bend , and candidates for this input are the HSNs and VCs , both of which synapse onto the vm2 muscles and have phased activity during egg-laying events .", "The functional purpose of this phase advance may be mechanical .", "Passage of each 25 × 40 µm egg requires contraction of vm1 and vm2 muscles on both the anterior and posterior sides of the vulva ( Li et al . , 2013 ) .", "There may not be sufficient room for vulva to fully open and release egg unless the adjacent body wall muscles are in a contracted state . 10 . 7554/eLife . 21126 . 017Figure 7 . Working model of how circuit connectivity , signaling , and activity contribute to the observed rhythms that accompany the active and inactive egg-laying behavior states . VA and VB motor neuron synapses release acetylcholine ( ACh ) that rhythmically excites the body wall muscles ( bwm ) and vm1 vulval muscles during each locomotion body bend , every ~10 s .", "Eggs are produced from each gonad arm every 10 min , and we propose the accumulation of 2–3 eggs mechanically excites the uterine muscles ( um ) facilitating exit from the inactive state .", "Sustained bursts of HSN command motor neuron activity trigger serotonin release that drives ~2 min active states .", "HSN also makes synapses and gap junctions with the cholinergic VC motor neurons , and VC makes synapses and gap junctions with the vm2 muscles whose contraction allows egg release .", "Passage of eggs mechanically activates the uv1 cells triggering release of tyramine and neuropeptides , which inhibit HSN activity to terminate the active state .", "See text for further details . DOI: http://dx . doi . org/10 . 7554/eLife . 21126 . 017 The HSNs are command neurons that integrate diverse sensory inputs and initiate the ~2 min active state of the egg-laying circuit .", "Optogenetic activation of HSNs recapitulates features of the active state including phased VC and vulval muscle activity and multiple egg-laying events .", "However , stimulation of the HSNs fails to drive tetanic contraction of the vulval muscles as would be predicted for a motor neuron releasing a fast-acting neurotransmitter .", "Instead , HSN makes and releases the neuromodulator serotonin which signals through G protein coupled serotonin receptors to activate the vulval muscles ( Desai et al . , 1988; Shyn et al . , 2003; Carnell et al . , 2005; Hobson et al . , 2006; Hapiak et al . , 2009 ) .", "The HSNs also release serotonin onto the AVF interneurons in the nerve ring to promote a state of locomotion arousal that accompanies the active state ( Hardaker et al . , 2001 ) .", "Animals defective in serotonin signaling have reduced egg laying ( Tanis et al . , 2008 ) , but this defect is not as severe as that of egl-1 ( dm ) animals missing HSNs ( Trent et al . , 1983 ) , suggesting HSN releases additional neurotransmitters .", "We found that optogenetic activation of HSNs leads to rhythmic activity of VC neurons , which are not known to express serotonin receptors , so an unknown signal derived from HSN could directly activate VCs , as depicted in the Figure 7 model .", "How is HSN activity controlled ?", "The HSNs receive little synaptic input near the vulval except from the PLM mechanosensory neurons ( White et al . , 1986 ) .", "The PLMs inhibit HSN activity and egg laying in response to gentle touch ( Zhang et al . , 2008 ) , but recent work indicates the PLM neurons are also deformed during normal patterns of locomotion that could contribute to rhythmic activity seen in HSN during egg laying ( Topalidou et al . , 2012 ) .", "Neuroendocrine signaling also regulates HSN activity .", "flp-10 and flp-17 neuropeptides are released from the CO2-responsive BAG neurons in the head , and these neuropeptides signal through EGL-6 receptor and Gαo to activate IRK channels that inhibit HSN activity and egg laying ( Ringstad and Horvitz , 2008; Hallem et al . , 2011; Emtage et al . , 2012 ) .", "Because loss of EGL-6 or IRK channels do not increase egg laying as much as cell-specific inhibition of Gαo , there must be additional receptors and effectors of Gαo that inhibit HSN activity .", "Gαq signaling opposes Gαo to increase serotonin release from the HSNs ( Tanis et al . , 2008 ) , but the ligands and receptors that activate Gαq have not yet been identified .", "Because we find that HSN Ca2+ transients vary more in frequency than amplitude , we propose that these diverse sensory signals increase or reduce the probability that HSN reaches a threshold potential that can initiate , sustain , or terminate HSN activity and thus the active behavior state of the egg-laying circuit .", "We have also identified an HSN-independent mechanism that induces the active state .", "Mutants lacking HSNs are strongly egg-laying defective , but eventually enter active states with properly phased vulval muscle twitching and egg-laying transients .", "The active state was eliminated in animals sterilized by FUDR , reducing vulval muscle activity and phasing to that seen in the inactive state .", "Recent work has identified the gonad as an important site of regulation for adult-specific behaviors in C . elegans ( Fujiwara et al . , 2016 ) .", "We propose that the accumulation of unlaid eggs in the uterus , perhaps leading to mechanical stretch , provides an essential , permissive signal for entry into the active state .", "Several steps during embryo production and maturation could regulate entry into the active state to result in the ~20 min period of the egg-laying inactive state ( Waggoner et al . , 1998 ) .", "Animals grown on food generate one new oocyte from each of two gonad arms every ~10 min , balancing out the 3–5 eggs laid in the ~2 min active state that occurs every ~20 min , to result in a ~constant number of unlaid eggs in the uterus ( McCarter et al . , 1999 ) .", "New embryos are moved by gonadal sheath contractions into the spermatheca for fertilization and deposition into the uterus .", "Excitable cells drive each of these steps , providing potential sources of regulation by cell signaling and sensory feedback ( Kariya et al . , 2004 ) .", "Eight uterine muscles line the uterine lumen and form gap junctions with the vm2 muscles ( White et al . , 1986 ) , and the addition of unlaid eggs into the uterus could induce a mechanical stretch that cross-excites the vm2 muscles .", "Future work will be required to understand how the accumulation of unlaid eggs promotes the active state .", "Our results strongly imply that acetylcholine released from the VC motor neurons slows locomotion during vulval muscle contraction .", "We found that animal locomotion slows during egg release , and that optogenetic activation of the VC neurons halts movement and results in body wall muscle contraction and animal shortening .", "This function of the VCs is likely via acetylcholine signaling through nicotinic receptors expressed on ventral body wall muscles and neurons that regulate locomotion ( White et al . , 1986; Nagel et al . , 2005 ) .", "The effect of VC activity to slow locomotion and contract body wall muscles may help compress the body and uterus to encourage expulsion of eggs , analogous to the role of body wall muscle contraction in facilitating defecation ( Liu and Thomas , 1994 ) .", "Our results provide more complex evidence as to whether VC acetylcholine also directly triggers egg release .", "We found that VC activity always accompanies egg-laying contractions of the vulval muscles .", "The vulval muscles also express nicotinic acetylcholine receptors , and receptor agonists including levamisole and nicotine stimulate egg laying ( Weinshenker et al . , 1995; Waggoner et al . , 2000 ) .", "However , we found little correlation between the strength of the VC activity and whether an egg is laid ( Figure 1H ) , and that optogenetic activation of the VC neurons fails to drive egg laying .", "Further , VC-defective and acetylcholine-deficient mutants have increased egg-laying behavior ( Bany et al . , 2003; Ringstad and Horvitz , 2008 ) .", "These data together are consistent with a model in which acetylcholine released from the VA/VB motor neurons signal through nicotinic receptors on the vm1 muscles to stimulate rhythmic vulval muscle twitching , which combined with acetylcholine released from the VC neurons to similarly excite the vm2 muscles results in egg laying .", "High levels of VC activity , such as occurs during optogenetic activation , may drive release of additional signals , such as neuropeptides , that inhibit egg-laying circuit activity ( Bany et al . , 2003; Banerjee and Francis , personal communication ) , or by freezing locomotion may feedback to halt the VA/VB activity required to drive vm1 contraction .", "How is VC activity controlled ?", "The VC neurons are unusual in that they are largely presynaptic , only receiving input from each other and the HSNs .", "As noted above , optogenetic activation of HSNs results in VC activity , suggesting a currently unknown signal from HSN directly activates VCs .", "A second possible input onto VCs is suggested by the fact that the VC neurons extend processes devoid of synapses dorsally along the vulval hypodermis that could be mechanosensory ( White et al . , 1986 ) .", "Thus , rhythmic VC activity could be stimulated mechanically by body bends or by a humoral signal released from the locomotor circuit ( Hu et al . , 2011 ) .", "Further , VC activity could be mechanically stimulated by vulval muscle contraction ( Zhang et al . , 2010 ) , particularly as we observed mechanical deformation of VC presynaptic termini during strong twitching and egg-laying contractions .", "In this way , the VCs could act like baroreceptors–mechanically activated by vulval muscle contraction to drive contraction of the body wall muscles that results in animal slowing until the feedback of egg release .", "In this model , the coordination of locomotion and egg laying by tying both behaviors to the same central pattern generator allows productive egg release .", "The uv1 neuroendocrine cells inform the circuit that eggs have been successfully laid .", "Passage of eggs through the vulva mechanically deforms the uv1 cells and triggers a strong Ca2+ transient .", "Our results suggest this activates uv1 to release tyramine that inhibits egg-laying , at least in part via the LGC-55 receptor on the HSN neurons .", "Active states last ~2 min , and inhibitory tyramine release from uv1 could provide a feedback signal that terminates activity in the circuit .", "The four uv1 cells have a unique structural position in the vulva that may explain how they become activated during egg laying ( Newman et al . , 1996 ) .", "uv1 cells extend processes that contact and make adherens junctions with the ventral vulF vulval cells .", "uv1 cells also makes dorsal attachments to utse , a large , H-shaped multinucleate uterine seam cell that attaches the uterus to the lateral epidermis and that functions as a hymen broken by the first egg-laying event .", "The acute mechanical strain on vulF , uv1 , and/or utse during egg passage could mechanically activate ion channels .", "Previous work has shown uv1 expresses several TRPV channels that may be mechanosensory and contribute to this response ( Jose et al . , 2007 ) .", "A dominant-negative OCR-2 mutant expressed in uv1 leads to hyperactive egg-laying behavior phenotype .", "We still observe Ca2+ transients in uv1 after egg-laying events in this ocr-2 ( vs29 ) mutant ( data not shown ) , suggesting other channels contribute to the uv1 activity we see after egg laying .", "Although our results indicate that uv1 releases tyramine to inhibit the HSNs via LGC-55 , this signaling event does not fully explain the ability of uv1 cells to inhibit the egg-laying circuit .", "We found that inhibition of egg laying by exogenous tyramine is reduced but not eliminated in lgc-55 null mutants , suggesting other tyramine receptors help terminate the active state .", "The uv1 cells also make nlp-7 and flp-11 , two neuropeptides that inhibit HSN activity and egg-laying behavior ( Banerjee and Francis , personal communication ) , and the identification of the receptors and signaling pathways through which they inhibit serotonin release will help to more fully understand how activity of the egg-laying circuit is terminated .", "Every neural circuit may require specific signals to initiate activity when its function is required , signals to coordinate the dynamic activity of its various cells , and mechanisms to determine when the circuit has successfully completed its task and signal to turn off activity .", "Many circuits also must respond to specific signals from central pattern generators that coordinate the activity of multiple circuits .", "A great challenge is to reduce these abstract concepts to concrete , specific mechanisms in the case of a real circuit .", "For the C . elegans egg-laying circuit , signals in each of the categories outlined above are now identified , albeit in varying levels of detail and completeness .", "Continuing analysis of the C . elegans egg-laying circuit using recordings of Ca2+ activity in behaving animals , combined with genetic , optogenetic , and pharmacological manipulation , provides a path towards detailed understanding the mechanisms that control activity of this model circuit ." ], [ "Caenorhabditis elegans strains were maintained as hermaphrodites at 20°C on Nematode Growth Medium ( NGM ) agar plates with E . coli OP50 as a source of food as described ( Brenner , 1974 ) .", "All strains are derived from the Bristol N2 wild-type strain , and all assays were performed using age-matched adult hermaphrodites ~24 hr past the late L4 stage using recommended sample sizes ( Chase and Koelle , 2004 ) .", "Animals were treated with 0 . 1 mg/ml floxuridine ( FUDR ) at a late L4 stage when all non-germline cell divisions are complete .", "A list of strains , mutants , and transgenes used in this study can be found in Table 1 . 10 . 7554/eLife . 21126 . 018Table 1 . Strains used in this study . DOI: http://dx . doi . org/10 . 7554/eLife . 21126 . 018StrainFeatureGenotypeFiguresLX1832 Strain for transgene productionlite-1 ( ce314 ) lin-15 ( n765ts ) X1–6 LX1836 HSN ChannelrhodopsinwzIs30 IV;lite-1 ( ce314 ) lin-15 ( n765ts ) X3LX1918 vulval muscle GCaMP5 , mCherrylite-1 ( ce314 ) vsIs164 lin-15 ( n765ts ) X1 , 2LX1932 HSN Channelrhodopsin , vulval muscle GCaMP5 , mCherrywzIs30 IV; lite-1 ( ce314 ) vsIs164 lin-15 ( n765ts ) X3LX1938 No HSNs , vulval muscle GCaMP5 , mCherryegl-1 ( n986dm ) V; lite-1 ( ce314 ) vsIs164 lin-15 ( n765ts ) X4LX1986 uv1 GCaMP5 , mCherryvsIs177; lite-1 ( ce314 ) lin-15 ( n765ts ) X5LX1960 VC GCaMP5 , mCherryvsIs172; lite-1 ( ce314 ) lin-15 ( n765ts ) X1 , 2LX1970 HSN Channelrhodopsin , VC GCaMP5 , mCherrywzIs30 IV; vsIs172; lite-1 ( ce314 ) lin-15 ( n765ts ) X3LX2004 HSN GCaMP5 , mCherrylite-1 ( ce314 ) , vsIs183 lin-15 ( n765ts ) X1 , 2 , 6LX2038 lgc-55 null mutantHSN GCaMP5 , mCherrylgc-55 ( tm2913 ) Vlite-1 ( ce314 ) vsIs183 lin-15 ( n765ts ) X6MIA3 VC ChannelrhodopsinkeyIs3; lite-1 ( ce314 ) lin-15 ( n765ts ) X3MT13113 tdc-1 null mutant;no tyramine biosynthesistdc-1 ( n3419 ) II6N2 Bristol strainwild type6OH313 ser-2 null mutantser-2 ( pk1357 ) X6VC125 tyra-3 null mutanttyra-3 ( ok325 ) X6QW89 lgc-55 null mutantlgc-55 ( tm2913 ) V6LX2096 vulval muscle mCherryvsIs191; lin-15 ( n765ts ) XLX2081 pan-neuronal TagRFPunc-119 ( ed3 ) I; otIs356 LX2137 vulval muscle mCherry , lgc-55::gfp vsIs191; vsEx791 6LX1330 lgc-55 mutant expressing GFP in HSN from tph-1 promoterlgc-55 ( tm2913 ) V; lin-15 ( n765ts ) X vsEx557 6LX1329 lgc-55 mutant expressing LGC-55 in HSN from tph-1 promoterlgc-55 ( thm2913 ) V; lin-15 ( n765ts ) X vsEx558 6 Animals were mounted between glass coverslips and chunked sections of NGM plates for imaging as described ( Collins and Koelle , 2013; Li et al . , 2013 ) .", "The stage and focus were adjusted manually to keep the egg-laying system in view during recording periods .", "Long-term recordings were cropped to 10–30 min with the first egg-laying event at 5 min .", "Three different imaging systems were used to collect recordings , and each gave quantitatively similar results .", "First , single , two-channel confocal slices ( 18 µm thick ) of GCaMP5 and mCherry fluorescence were collected through a 20x Plan Apochromat objective ( 0 . 8NA ) using a Zeiss Axio Observer microscope and recorded at 20 fps at 256 × 256 pixel resolution , 16-bit depth using a 710 Duo LIVE scanner as described previously ( Collins and Koelle , 2013; Li et al . , 2013 ) .", "Second , widefield GCaMP5 and mCherry fluorescence was imaged through a 20x Plan Apochromat objective ( 0 . 8NA ) using a Zeiss Axio Observer microscope , and the GCaMP5 and mCherry fluorescence was separated using a W-View Gemini image splitter at 20 fps at 256 × 256 pixel resolution , 16-bit depth onto a single , 4 × 4 binned Hamamatsu ORCA Flash 4 . 0 V2 sCMOS sensor after excitation with 470 nm and 590 nm LEDs ( Zeiss Colibri . 2 ) .", "Third , single , two-channel confocal slices ( 20 µm thick ) of GCaMP5 and mCherry fluorescence were collected using an inverted Leica TCS SP5 confocal microscope with the resonant scanner running at 8000 Hz and collected at 20 fps at 256 × 256 pixel resolution , 12-bit depth after 2x digital zoom .", "Quantitative ratiometric analysis was performed after importing image sequences into Volocity ( Perkin Elmer ) .", "GCaMP5/mCherry ratios were calculated , and pixels with mCherry fluorescence intensities ~2 standard deviations above background were selected as objects for mean ratio measurement .", "While the Volocity algorithm set with these parameters typically identified the vulval muscles and uv1 cells as a single object , the complex morphology of the HSN and VC neurons often led to the cells being found as two or more objects within each video frame .", "These were rejoined using a custom script in MATLAB ( Mathworks ) .", "The ratio of the joined object was scaled in proportion to the area of each component object to generate an area-adjusted GCaMP5/mCherry ratio .", "The ratio data were then smoothed using a 150 msec ( three timepoint ) rolling average , and the lowest 10% of the GCaMP5/mCherry ratio values over the entire recording period were averaged to establish a ∆R/R baseline .", "Ca2+ transients and their features ( peak amplitude and width at half-maximum ) were identified from ratio traces using the findpeaks algorithm in MATLAB , and peaks were confirmed by visual inspection of GCaMP5/mCherry recordings and ratio traces .", "Egg-laying events were observed by allowing a small amount of bright field light to transmit into the mCherry channel whose gain was increased for egg visualization .", "The egg-laying active state was defined in recordings to include those transients that occurred one minute before and one minute after each egg-laying event .", "Those transients not found within an active state were considered to be in the inactive state .", "Inter-transient intervals were calculated by measuring the elapsed time between successive Ca2+ transient peaks during the inactive and active state .", "Because some inactive states were devoid of detectable activity ( e . g . Figure 1F , VC Ca2+ ) , inter-transient intervals during such periods were operationally defined as the time before the first or after the last transient in the recording .", "Because the recordings were cropped to focus on the egg-laying active state , the inactive state inter-transient intervals reported here should be considered a lower estimate .", "To facilitate comparisons of ∆R/R between different reporters and recording conditions ( Figures 1H and 5D ) , the maximum peak amplitude was normalized to 1 .", "Spectral analysis of active phase Ca2+ traces was performed by Fast Fourier Transform method of estimation using the Signal Analyzer app in the MATLAB Signal Processing toolbox ( R2016b ) .", "Quantitatively similar results were obtained using the Lomb-Scargle periodogram function and using auto-correlation analysis ( data not shown ) .", "The largest amplitude frequency peak between 0 and 250 mHz from each active state periodogram was compared using one-way ANOVA .", "We recorded the position of the vulva during each sinusoidal body bend of locomotion relative to observed HSN , VC , and vulval muscle Ca2+ transients .", "We recorded the timing of each ventral contraction and/or relaxation flanking each Ca2+ transient peak .", "To approximate the phasing of that Ca2+ relative to locomotion , we operationally defined 0° and 360° as when the vulva was positioned when the adjacent body wall muscles were at their most ventrally contracted state , and we defined 180° when the vulva was positioned when the adjacent body wall muscles were at their most relaxed state .", "The relative timing of each Ca2+ transient peak or behavior event was calculated as fraction of time elapsed between the previous and subsequent contraction or relaxation , regardless of whether the animal was engaged in forward or reverse locomotion , as described previously ( Collins and Koelle , 2013 ) .", "For example , if a Ca2+ transient peaked exactly 2 s after the vulva passed through its most ventrally relaxed state ( 180° ) and 2 s prior to the ventral contracted state ( 360° ) , the Ca2+ transient phasing would be halfway between 180° and 360° , or 270° .", "If a Ca2+ transient peaked halfway between ventral contraction ( 0° ) and ventral relaxation ( 180° ) , the Ca2+ transient phasing of that peak would be 90° .", "This method of estimation allows for phasing information to be extracted and compared independent of differences in individual animal speed and locomotion direction .", "Channelrhodopsin-2 ( ChR2 ) expressing strains were grown in the presence or absence of the ChR2 cofactor all-trans retinal ( ATR ) .", "ATR was prepared at 100 mM in 100% ethanol and stored at −20°C .", "To prepare NGM plates for behavior analysis , ATR was diluted to 0 . 4 mM with warmed cultures of OP50 bacteria in B Broth , and 200 µl of culture was seeded onto each 60 mm NGM plate .", "The plates were allowed to grow for 24 hr at 25–37°C , after which late L4 worms were staged onto prepared plates for behavioral assays 24 hr later .", "During Ca2+ imaging experiments , ChR2 was activated with the same light used for GCaMP5 excitation .", "For timed behavior recording experiments on plates , a OTPG_4 TTL Pulse Generator ( Doric Optics ) was used to trigger image capture ( Grasshopper 3 , 4 . 1 Megapixel , USB3 CMOS camera , Point Grey Research ) and shutter opening on a EL6000 metal halide light source generating 8–16 mW/cm2 ( depending on the magnification used ) of ~470 ± 20 nm blue light via a EGFP filter set mounted on a Leica M165FC stereomicroscope .", "Worm size , speed , and body bends were determined from image sequences using Volocity .", "Three adult animals were placed onto each of three 35 mm agar plates prepared with tyramine-HCl ( Sigma-Aldrich , St . Louis , MO ) and acetic acid seeded with OP50 bacteria as described ( Pirri et al . , 2009 ) .", "The average number of eggs laid was counted after 30 min from at least 11 trials for each genotype and tyramine concentration .", "Data were analyzed using Prism 6 ( GraphPad ) .", "Average peak transient rhythm , worm speed , worm length , body bends , or eggs laid were compared using one-way ANOVA with Bonferroni correction for multiple comparisons , and error bars indicate 95% confidence intervals for the mean .", "Ca2+ transient peak amplitudes , widths , and inter-transient intervals were pooled from multiple animals ( n ≥ 4 animals per genotype/condition per experiment ) , compared using the Mann-Whitney test ( for pair-wise comparisons ) or the Kruskal-Wallis test with Dunn’s correction ( for multiple comparisons ) , and error bars indicate quartile intervals for the indicated median .", "p<0 . 05 interactions were deemed significant and are labeled with an asterisk .", "p values for specific interactions are indicated in the Figure legends .", "Sample sizes for behavioral assays followed previous studies ( Waggoner et al . , 1998; Chase and Koelle , 2004; Collins and Koelle , 2013 ) .", "The number of animals analyzed in each experiment is indicated below , with each Ca2+ transient or egg-laying event serving as a biological replicate ." ] ]

[ "Like many behaviors , Caenorhabditis elegans egg laying alternates between inactive and active states .", "To understand how the underlying neural circuit turns the behavior on and off , we optically recorded circuit activity in behaving animals while manipulating circuit function using mutations , optogenetics , and drugs .", "In the active state , the circuit shows rhythmic activity phased with the body bends of locomotion .", "The serotonergic HSN command neurons initiate the active state , but accumulation of unlaid eggs also promotes the active state independent of the HSNs .", "The cholinergic VC motor neurons slow locomotion during egg-laying muscle contraction and egg release .", "The uv1 neuroendocrine cells mechanically sense passage of eggs through the vulva and release tyramine to inhibit egg laying , in part via the LGC-55 tyramine-gated Cl- channel on the HSNs .", "Our results identify discrete signals that entrain or detach the circuit from the locomotion central pattern generator to produce active and inactive states ." ]

[ "It has been said that if the human brain were so simple that we could understand it , we would be so simple that we couldn’t .", "This quote neatly captures the challenge of working out how 80 billion neurons collectively generate our thoughts and behavior .", "Fortunately , the nervous system is also organized into simpler units called circuits .", "Each consists of a relatively small number of neurons , which communicate with one another to control as little as a single behavior .", "These circuits should in principle be simple enough for us to understand , particularly if we study them in nervous systems less complex than our own .", "Despite this , there is currently not a single circuit in any organism in which we can explain how communication between individual neurons generates behavior .", "Collins et al . therefore set out to characterize a simple neural circuit in one of the simplest model organisms: the egg-laying circuit of the worm C . elegans .", "Using mutations , drugs and molecular genetic techniques , Collins et al . systematically altered the activity and signaling of each of the neurons within the egg-laying circuit .", "The experiments revealed that cells called command neurons trigger egg laying by producing signals that switch on the rest of the circuit .", "Once activated , the circuit is able to respond to waves of activity from a second circuit – called the central pattern generator – that also controls the worm’s movement .", "Finally , laying an egg activates a third set of neurons , which release a signal that returns the circuit to its inactive state .", "The use of distinct signals and neurons to activate the circuit , to coordinate its ongoing activity , and to inactivate the circuit when its task is complete also applies to many other neural circuits .", "Now that these signals have been identified in one circuit , it should be possible to build on these findings to better understand how others work ." ]

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

[ "ecology", "physics of living systems" ]

[ [ "Microbial communities feature an immense diversity of organisms , with the typical human gut microbiota and a liter of seawater both containing hundreds of distinct microbial types ( Lloyd-Price et al . , 2016; Ladau et al . , 2013; Weigel and Pfister , 2019 ) .", "These observations appear to clash with a prediction of some resource-competition models , known as the competitive-exclusion principle – namely , that steady-state coexistence is possible for only as many species as resources ( Levin , 1970; Armstrong and McGehee , 1980 ) .", "This conundrum is familiarly known as the ‘paradox of the plankton’ ( Hutchinson , 1961 ) .", "Solving this paradox may provide one key to predicting and controlling outcomes ranging from ecosystem stability to successful cancer treatments in humans ( Ptacnik et al . , 2008; van Elsas et al . , 2012; Taur et al . , 2014; Stein et al . , 2013 ) .", "Chesson , 2000 classified mechanisms that purport to solve this paradox into two broad categories: stabilizing and equalizing .", "Stabilizing mechanisms prevent extinction by allowing species to recover from low populations , whereas equalizing mechanisms slow extinction by minimizing fitness differences between species .", "Many possible solutions of the paradox that rely on stabilizing mechanisms have been offered:", "( i ) interactions between microbes , such as cross-feeding or antibiotic production and degradation ( Goyal and Maslov , 2018; Kelsic et al . , 2015 ) ,", "( ii ) spatial heterogeneity ( Murrell and Law , 2003; Tilman , 1994 ) ,", "( iii ) persistent non-steady-state dynamics ( Hutchinson , 1961 ) , and", "( iv ) predation ( Thingstad , 2000 ) .", "Equalizing mechanisms have been studied through neutral theory , in which all species are assumed to have equal fitness ( Hubbell , 2005 ) , and recent work has proposed resource-competition models that self-organize to a neutral state ( Posfai et al . , 2017 ) .", "Many proposed solutions for the paradox assume a chemostat framework wherein nutrients are continuously supplied and there is a continuous removal of biomass and unused nutrients ( Palmer , 1994 ) .", "However , in nature nutrients are rarely supplied in a constant and continuous fashion .", "In particular , seasonal variation is ubiquitous in ecology , influencing systems ranging from oceanic phytoplankton communities ( Chang , 2003 ) to the microbiota of some human populations ( Smits et al . , 2017 ) .", "How does a variable nutrient supply influence diversity ?", "Existing literature on seasonality focuses on stabilizing mechanisms and generally finds that seasonality either promotes or has little effect on coexistence ( Chesson , 1994 ) .", "But do these conclusions extend to equalizing mechanisms ?", "To address this question , we consider a known resource-competition model that permits high diversity at steady state due to the equalizing effects of metabolic trade-offs , which assume that microbes have a limited enzyme production capacity they must apportion .", "Here , we investigate the equalizing effect of metabolic trade-offs in the context of serial dilution , to reflect a more realistic variable nutrient supply .", "Serial dilution , in which cultures of bacteria are periodically diluted and supplied with fresh nutrients , is well-established as an experimental approach .", "For example , the bacterial populations in the Lenski long-term evolution experiment ( Lenski and Travisano , 1994 ) , experiments on community assembly ( Goldford et al . , 2018 ) , and antibiotic cross-protection ( Yurtsev et al . , 2016 ) were all performed in serial dilution .", "While previous models of serial dilution have characterized competition between small numbers of species with trade-offs in their growth characteristics ( Stewart and Levin , 1973; Smith , 2011 ) , the theoretical understanding of diversity in serial dilution is much less developed than for chemostat-based steady-state growth .", "Here , we show that under serial dilution , metabolic trade-offs can still support high diversity communities , but that this coexistence is now sensitive to environmental conditions .", "Interestingly , seasonality can both increase and decrease diversity in our model , contrasting what has been observed for stabilizing mechanisms .", "In particular , we find a surprising dependence between community diversity and the amount of nutrient provided to the community .", "These changes in diversity are driven by an ‘early-bird’ effect in which species that efficiently consume nutrients that are initially more abundant gain a population advantage early in the batch .", "To our knowledge , this is the first time this effect has been identified as a major influencer of diversity in seasonal ecosystems .", "This dependence between community diversity and the supplied nutrient concentration allowed us to explore an unresolved question in ecology ( Tilman , 1982; Abrams , 1995; Leibold , 1996 ) : what is the relationship between the amount of nutrient supplied and the resulting diversity of the community ?", "Experimental studies of this question have mainly been performed in macroecological contexts ( Mittelbach et al . , 2001; Waide et al . , 1999; Adler et al . , 2011 ) , though recently there has been increased focus on microbial systems ( Bienhold et al . , 2012; Bernstein et al . , 2017 ) .", "In microbial experiments , some evidence has supported the ‘hump-shaped’ unimodal trend predicted by many theories ( Kassen et al . , 2000 ) .", "However , a meta-analysis by Smith , 2007 found no consistent trend across microbial experiments .", "What we observe here is concordant with Smith’s result: even in our highly simplified model , there is no general relationship between nutrient supply and diversity .", "Among the factors we find that influence this relationship are cross-feeding , relative enzyme budgets , differences in enzyme affinities , and differences in nutrient yields .", "That so much variation appears in a simple model suggests that real ecosystems are not likely to display a single universal relationship between nutrient supply and diversity ." ], [ "One can intuit that our serial dilution model at very low nutrient bolus size will mimic a chemostat .", "Adding a small nutrient bolus , letting it be consumed , then removing the additional biomass , and repeating is tantamount to operating a chemostat with a fixed nutrient supply and dilution rate .", "Indeed , the limit c0≪K yields the same steady state as a chemostat .", "Thus , our results for serial dilution include and generalize those obtained for a closely related chemostat model ( Posfai et al . , 2017 ) .", "For completeness , we now briefly describe the chemostat results from Posfai et al . , 2017 .", "In the presence of metabolic trade-offs , the chemostat can support a higher species diversity than prescribed by the competitive exclusion principle as we demonstrate theoretically in Appendix 4 .", "Specifically , if the nutrient supply lies within the convex hull of the strategies on the simplex ( visualized by stretching a rubber band around the outermost strategies , see Figure 1B–C ) , an arbitrarily large number of species can coexist at steady state .", "In the chemostat , such species coexistence is attained when the system organizes such that all nutrient levels are driven towards equality by consumption .", "Dynamically , if one nutrient level is high , the species that consume it increase in population , leading to faster consumption of that nutrient , thus acting to return the nutrients to equal steady-state levels .", "Such a self-organized neutral state is an attractor of the chemostat dynamics ( Posfai et al . , 2017 ) and , correspondingly , of the c0≪K limit of the serial dilution model .", "Note that the coexistence steady state is not a single fixed point , but rather a degenerate manifold of possible solutions ( details in Appendix 4 ) .", "Thus , in the chemostat-limit of the cases shown in Figure 1B and C all the species will coexist .", "Conversely , if the supply lies outside the convex hull , ( e . g . , if we swapped the positions of the leftmost species and the supply in Figure 1B ) the number of surviving species would be strictly less than the number of nutrients , consistent with competitive exclusion .", "To understand the convex-hull rule , note that a state of arbitrarily high coexistence can only occur if the chemostat self-organizes to a ‘neutral’ state in which the nutrient concentrations are all equal , and thus all strategies have the same growth rate .", "This state is achieved if and only if the total enzyme abundances lie along the same vector as the nutrient supply , which is achievable only if the supply lies within the convex hull of the strategies present .", "As in the chemostat model , the serial dilution model can support either coexistence or competitive exclusion .", "However , if one chooses system parameters near the transition between these two states , it requires a very large number of batches for the simulation to reach steady state .", "This is a manifestation of the well-known phenomenon of critical slowing down ( Dakos and Bascompte , 2014 ) .", "Though in principle , critical slowing down is not a simulation artifact and could manifest in similar real-world systems , we expect that a variety of factors outside our modeling framework would preclude observation of this critical behavior .", "We define the serial dilutions ‘steady state’ to be reached when the relative species abundances after the nutrients are depleted ( time tf after starting the batch ) scale with the relative abundances at the beginning of that batch ( time 0 ) , that is , ρσ⁢ ( tf ) =ρ0+c0ρ0⁢ρσ⁢ ( 0 ) .", "We can expand the implicit equation for the steady state to first order in c0/K ( details can be found in Appendix 4 ) , ( 4 ) c0ρ0=∑iασ , i⁢ci⁢ ( 0 ) ∑σ′ασ′ , i⁢ρσ′⁢ ( 0 ) +O⁢ ( c0K ) 2 .", "Dividing both sides by tf and defining , ( 5 ) δ~=c0ρ0⁢tf , si=ci⁢ ( 0 ) tf , we reach the c0/K≪1 steady-state condition for the serial dilution system: ( 6 ) δ~=∑iασ , i⁢si∑σ′ασ′ , i⁢ρσ′⁢ ( 0 ) .", "Averaged over a batch , si is the average rate that nutrient i is supplied , and δ~ is the average rate that all the nutrients are supplied per unit inoculum biomass .", "In analogy to the chemostat model , one can think of si as the rate nutrient i is continuously supplied .", "Moreover , for a chemostat , the parameter δ~ which would be the rate all nutrients are continuously supplied per unit biomass , would need to equal the dilution rate of the chemostat , δ , to maintain steady state .", "A detailed analysis of the effects of larger bolus size , to second order in c0/K , can be found in Appendix 4 .", "In the chemostat limit , increasing the nutrient supply rate simply proportionally increases the steady-state population abundances .", "However , away from this limit we find that the magnitude of the nutrient bolus can qualitatively affect the steady-state outcome of serial dilutions .", "To understand this effect , we first consider a simple case of two nutrients and two species as depicted in Figure 2A .", "The two species will coexist if each species is invasible by the other .", "In our example , we first determine the invasibility of species R ( strategy indicated by red circle ) by species with strategies lying to its left .", "To this end , we choose a nutrient supply and perform model serial dilutions until steady state is reached .", "For a particular finite bolus size , we find that for all supplies within the hatched region an infinitesimal inoculum of any species lying to the left of R will increase more than R during a batch , and therefore can invade R . Similarly , we determine the invasibility of species B ( strategy indicated by blue circle ) by any species with a strategy lying to its right , and find the second hatched region .", "The intersection of these hatched regions for which ( 1 ) B can invade R and ( 2 ) R can invade B is the supply interval of mutual invasibility where these two species will stably coexist .", "The coexistence interval is bounded by the red and blue triangles , and each of these coexistence boundaries is a unique property of its corresponding species .", "We call these species-specific boundaries remapped because they generally lie at different locations on the simplex than the strategies they originated from , with the extent of remapping depending on the nutrient bolus size .", "At a more technical level , the remapped boundary for a given species and bolus size is the nutrient supply for which , over the course of a batch , all nutrients are equally valuable and so , a species with any strategy can neutrally invade and persist .", "This equality of nutrient value is defined in terms of the Monod function integrals for each nutrient ( for details see Appendix 3 ) .", "Since the remapped coexistence boundaries depend on the nutrient bolus size c0 , changing bolus size can qualitatively change the steady-state outcome of serial dilutions .", "Figure 2B–D depicts an example of how c0 affects remapping , and the consequences for species coexistence .", "At low bolus size , c0≪K , corresponding to the chemostat limit , Figure 2B ( left ) shows that all species present achieve steady-state coexistence .", "This follows because the nutrient supply ( black diamond ) lies inside the convex hull .", "When c0 is increased to c0≈K ( Figure 2C ) , the coexistence boundaries are remapped towards the center of the simplex ( dashed arrow ) .", "In this example , the nutrient supply now lies outside the convex hull .", "This results in one winner species ( the dark blue one nearest the supply ) , with all others decreasing exponentially from batch to batch .", "This loss of coexistence with increasing nutrient bolus size is reminiscent of Rosenzweig’s ‘paradox of enrichment’ in predator-prey systems ( Rosenzweig , 1971 ) .", "Strikingly , however , as bolus size is further increased to c0≫K , the coexistence boundaries are remapped back to their original positions , so that the nutrient supply once again lies within the convex hull , and so steady-state coexistence of all species is recovered .", "What causes the remapping of the coexistence boundaries inwards as c0/K→1 ?", "Let us consider a single species growing on two nutrients supplied in the same proportion as its strategy ( i . e . the fraction of Nutrient 1 is equal to ασ , 1 ) .", "At low c0/K , this marks the remapped coexistence boundary and both nutrients will be equally valuable over the course of a batch .", "The balance is achieved because the nutrient with a higher initial abundance is more rapidly exhausted , while the nutrient with lower initial abundance is consumed more slowly and is therefore available for a longer span of time .", "At small c0/K , the more rapid initial consumption of the more abundant nutrient does not influence the consumption rate of the less abundant nutrient because the bolus size is small relative to the initial population size , so the population does not grow substantially during the batch .", "This changes as c0/K increases: the rapid initial consumption of the more abundant nutrient leads to a substantial increase in the total population .", "The remaining low initial abundance nutrient is now consumed more quickly and is less available to an invader with a more balanced enzyme strategy .", "The nutrients are no longer equally valuable on average , and remedying this requires a more equally balanced nutrient bolus .", "Thus , the remapped coexistence boundary moves inwards ( see Appendix 7—figure 3 ) .", "In essence , as c0/K increases it is more difficult for the invader to grow because the resident gains an ‘early-bird’ advantage: its initial growth allows it to more effectively exhaust the nutrients .", "Why does the coexistence boundary of a species map back to its original strategy in the limit of large bolus size , c0≫K ?", "In this limit , the nutrient uptake functions in Equation 1 will be saturated during almost the entire period of a batch .", "Each species will therefore consume nutrients strictly in proportion to its strategy ασ , i .", "For the case of two nutrients ( e . g . , as shown in Figure 1B ) , if there is only a single species present then if the supply lies anywhere to the left of its strategy , at some time during the batch there will be some of Nutrient 2 remaining after the bulk of Nutrient 1 has been consumed .", "Thus a single species can be invaded by any strategy to its left , provided the supply also lies to its left .", "Similarly , a species can be invaded by any strategy to its right if the supply lies to its right .", "This is exactly the condition for the coexistence boundary of a species to coincide with its actual strategy ( details in Appendix 5 ) .", "We have rationalized coexistence in our serial dilution model in terms of mutual invasibility , but have not explicitly stated the condition for an arbitrary number of species to coexist in steady state .", "In the chemostat , all species coexist when the concentrations of all nutrients are equal , implying the same growth rate for all strategies .", "However , for serial dilutions the nutrient concentrations are generally not equal and are not even constant in time .", "Instead , it is the integrated growth contribution of every nutrient that must be equal to allow for arbitrary coexistence .", "In the case of equal enzyme budgets ( ε=0 ) , this condition occurs when the time integrals of the nutrient Monod functions within a batch are all equal , that is , ( 7 ) Ii=∫0∞ciKi+ci⁢𝑑t=const .", "To understand this condition for coexistence beyond competitive exclusion , note that the instantaneous rate of growth of a species σ is ∑iασ , i⁢ci/ ( Ki+ci ) , so that the fold increase of a species during a batch is exp⁡ ( α→σ⋅I→ ) .", "This fold increase will be equal for all species if and only if Equation 7 holds .", "When there are two nutrients , Equation 7 holds at steady state whenever the supply is inside the convex hull of the coexistence boundaries of the species present ( details in Appendix 3 ) .", "For more nutrients , the corresponding condition is that the region of coexistence is bounded by contours that connect the outermost remapped nodes .", "Given a fixed set of species and a choice of initial populations , repeating the growth-dilution batch procedure results in a steady state where the populations at the beginning of a batch do not change from batch to batch .", "The steady-state populations depend on the initial populations , with the set of all possible steady-state populations defining a coexistence manifold .", "As is apparent in Figure 2C , not all strategies are remapped to the same extent .", "In Figure 3A , we plot the remapping of coexistence boundaries as a function of nutrient bolus c0 .", "Note that:", "( i ) the specialists ( 0 , 1 ) and ( 1 , 0 ) and the perfect generalist ( 0 . 5 , 0 . 5 ) are not remapped at all;", "( ii ) remapping is maximal for c0≈K;", "( iii ) there is no remapping in both the c0→0 and c0→∞ limits ( see also Appendix 7—figure 4 ) .", "The extent of remapping also depends on the inoculum size ρ0 as shown in Figure 3B , which demonstrates that remapping is maximal for ρ0≪K and vanishes for ρ0≫K .", "How does nutrient bolus size influence steady-state species diversity ?", "A useful summary statistic for quantifying diversity ( Jost , 2006 ) is the effective number of species me=eS with the Shannon diversity S=-∑σPσ⁢ln⁡Pσ and Pσ=ρσ*⁢ ( 0 ) /ρ0 , with ρσ*⁢ ( 0 ) the steady-state species abundances at the beginning of a batch .", "Diversity as measured by me is shown in Figure 3C for six choices of nutrient bolus composition .", "Notably , if the two nutrients are supplied equally ( top curve , magenta ) , me is independent of c0 and coincides with the maximal possible diversity ( dashed black line ) , namely equal steady-state abundances of all species ( Figure 3D , top ) .", "Conversely , if Nutrient 1 comprises only 5% of supplied nutrient ( Figure 3C , bottom curve , cyan ) , the number of effective species me is lower than maximal even in the chemostat-limit of small bolus sizes c0≪K and drops even further for c0≈K .", "This loss of diversity is due to the dramatically lowered steady-state abundances of strategies that favor Nutrient 1 ( Figure 3D , bottom ) .", "Two different effects underlie this change in community structure .", "The first is the early-bird effect described above: species specializing in more abundant nutrients gain a population advantage that allows them to rapidly consume less abundant nutrients that would otherwise support species with different enzyme specializations .", "The second effect is a well-known property of single nutrient competition and can be viewed as a ‘single-nutrient’ early-bird effect .", "In this case , species that are superior competitors for a nutrient gain an exponential population advantage over inferior competitors , increasing their share of total nutrient beyond the ratio of initial consumption rates .", "Both of these effects increase in strength with larger bolus size because the early-bird advantage increases as growth proceeds .", "The combination of these effects results in the species specialized in consuming the most abundant nutrients consuming a larger fraction of all nutrients .", "However , for very large bolus sizes , saturation of nutrient uptake rates mitigates these two effects , leading to a lack of remapping for c0≫K and diversity returning to its chemostat-limit .", "Though here we focused on the case of two nutrients , these results extend to more nutrients ( for three nutrients see Appendix 7—figure 5 ) .", "In this final Results section , we consider the effects of relaxing some of our simplifying assumptions .", "We first assess the effect or different enzyme affinities , Ki≠K , and different nutrient yields Yi≠Y .", "This is followed by a model that allows cross-feeding of metabolites .", "Finally , we consider population bottlenecks and what happens when the fixed-enzyme-budget constraint is relaxed .", "We show that in all these cases , the dependence on bolus size can be understood as manifestations of the early-bird effect .", "We have thus far made the simplifying assumption that all enzymes have the same substrate affinity , such that Ki≡K .", "However , in nature different nutrients may have drastically different values of K . For example , the methanogen Methanosarcina barkeri has Ki for the consumption of hydrogen and acetate that differ by approximately three orders of magnitude ( Robinson and Tiedje , 1984; Wandrey and Aivasidis , 1983 ) .", "How would such a large difference in the Ki values impact diversity in our serial dilution ecosystem ?", "In Figure 4A we show diversity as a function of bolus size for a system with a large difference in Ki ( K1=10-3 , K2=1 ) .", "Since the symmetry between nutrients is broken by the unequal Ki , we now show the entire range of nutrient proportions , not just the first half .", "In the chemostat limit , the diversity values are similar to those found in the system with equal Ki .", "This makes sense: in a chemostat the nutrients with higher Ki can accumulate to higher levels to compensate for their slow consumption , leaving the steady-state behavior unchanged .", "However , outside the chemostat regime , differences in the Ki have a drastic effect: when the nutrient with the lower Ki is scant in supply , diversity generally increases with increasing c0 , while the opposite occurs when the nutrient with the lower Ki is higher in supply .", "We can understand these Ki-driven shifts in the nutrient-diversity relationship as due to changes in the identity of the early bird .", "In a model with equal Ki , the identity of the early bird is determined by which nutrient is more abundant: if the two nutrients have equal Ki , a species can gain an early-bird advantage by preferentially consuming the more abundant nutrient .", "This changes if one nutrient has a much lower Ki than the other .", "In this case it may be advantageous to preferentially consume the nutrient with the lower Ki , even if it is the less abundant nutrient .", "If the nutrient with the lower Ki is also the more abundant nutrient , this will intensify the early-bird advantage .", "Why does this change in the early bird’s identity change the form of the nutrient-diversity relationship ?", "This change arises from a clash between optimal feeding behavior in the chemostat and seasonal regimes .", "In the chemostat , it is advantageous to focus on the most abundant nutrient , regardless of the value of Ki .", "Thus , in the chemostat limit , species focusing on the more abundant nutrient have an advantage .", "In the case of equal Ki ( or Ki favoring the more abundant nutrient ) , this advantage is intensified by the early-bird effect , increasing the biomass of already abundant species and lowering diversity .", "By contrast , if the low abundance nutrient has a low Ki , the early-bird effect will have the opposite effect on diversity .", "Now the early-bird effect benefits species that were disadvantaged in the chemostat limit , leading to more equal abundances and higher diversity .", "This shift in abundances is shown in Figure 4B .", "The change in the identity of the early bird can also explain more complex relationships between diversity and bolus size ( see Appendix 7—figure 6 ) .", "In addition to unequal enzyme affinities , it is possible for different nutrients to have different yields , Yi .", "In Figure 4C we show the relationship between bolus and diversity for a system with Y1=10 and Y2=1 .", "As expected , at low c0 the diversity is similar to that in the case of equal Yi .", "As c0/K increases , the diversity decreases initially and the symmetry-related bolus-composition cases ( e . g . [0 . 2 , 0 . 8] and [0 . 8 , 0 . 2] ) eventually diverge , with one’s diversity rising and the other’s continuing to fall .", "This behavior is explainable by the same logic as in the variable Ki case: diversity rises or falls depending on whether the early-bird species was also favored in the chemostat limit .", "However , unlike the case of variable Ki , the diversity curves do not eventually return to the chemostat limit .", "Regardless of which nutrient the Yi favor , the diversity eventually begins decreasing monotonically as c0/K increases .", "This difference between the variable Ki and variable Yi cases can be understood by considering what occurs when both nutrients are saturating .", "In the variable Ki case , saturating nutrients are equal in value , implying a return to the chemostat limit as the early-bird effect weakens .", "In contrast , for variable Yi , there remains a difference in the value of the two nutrients in the saturated regime , meaning that the early-bird effect will grow stronger and the early bird will take over the population .", "The beginning of this takeover can be seen at high bolus sizes in Figure 4D .", "Note that for both variable Ki and variable Yi , these trends are also reflected in the remapping ( see Appendix 7—figure 7 ) .", "Despite the large variation in relationships between diversity and bolus size , these phenomena can all be understood as consequences of the early-bird effect .", "As the model becomes more complex there are additional factors to consider in determining which nutrient will provide an early-bird advantage , but the fundamental mechanism of exploiting early growth advantages remains .", "It is possible to extend Equations 2 and 3 beyond a single trophic layer , allowing for consumption of metabolic byproducts .", "This is a form of cross-feeding , which has generally been found to promote diversity ( Goyal and Maslov , 2018 ) and stable community structure ( Goldford et al . , 2018 ) .", "Here , cross-feeding is introduced through the byproduct matrix Γi , i′σ , which converts the consumption of nutrient i′ to production of nutrient i such that , ( 8 ) d⁢cid⁢t=-∑σρσ⁢ ( jσ , i-∑i′Γi , i′σ⁢jσ , i′ ) .", "In this framework , nutrient i′ is converted to nutrient i at no extra enzymatic cost , meaning that nutrient i is simply a byproduct whenever nutrient i′ is consumed for growth ( it would be straightforward to modify this framework so that nutrient conversion can be carried out independently from growth ) .", "We focus on the simplest case: initially supplying only Nutrient 2 , with Nutrient 1 solely derived as a metabolic byproduct via Γi , i′σ= ( 0Γ00 ) for all species .", "When Γ=1 , upon consumption Nutrient 2 is perfectly converted to Nutrient 1 , leading to an equal total supply of the two nutrients .", "More generally , ∫0∞∑σρσ⁢jσ , 1⁢d⁢t=Γ⁢c2⁢ ( 0 ) which allows a direct comparison between the unitrophic and bitrophic regimes: starting with c2⁢ ( 0 ) results in ( Γ+1 ) ⁢c2⁢ ( 0 ) total nutrient , and hence the Nutrient 1 fraction is Γ1+Γ of the total .", "How does cross-feeding influence diversity in our serial dilution model ?", "In Figure 5A we compare bitrophic diversity for six values of Γ to their unitrophic equivalents ( in Figure 3C ) .", "We note that:", "( i ) bitrophy still supports diversity greater than the competitive-exclusion limit;", "( ii ) in the chemostat regime , c0≪K , the unitrophic and bitrophic schemes have identical values of me , and these drop as c0→K;", "( iii ) but for bitrophy the me does not recover for c0≫K;", "( iv ) even when the total supply of both nutrients is equal ( Γ=1 ) , bitrophy leads to lower than maximal me outside the chemostat limit .", "These features are clarified in Figure 5B , which shows steady-state species abundances for Γ values leading to a total Nutrient 1 supply fraction of 0 . 5 and 0 . 05 , and highlights the lower diversity for bitrophy compared to unitrophy for large nutrient bolus size .", "This difference is due to an early-bird effect: the species consuming supplied nutrient early in the batch can build a sizable population before the competing species that rely on its byproduct .", "The early-bird population then outcompetes the others for byproduct consumption .", "As such , this effect increases with c0/ρ0 .", "The effect also becomes stronger at low c0/K ( with constant c0/ρ0 ) , since this allows the early-bird species more time to grow before the byproduct accumulates to high enough levels to be significantly consumed ( Appendix 7—figure 8 ) .", "Note that this effect is dependent on metabolite byproducts being also consumed by their producer .", "If the species in each trophic layer are single-nutrient specialists , then changes in c0/K have no impact on community diversity .", "The behavior of the model with cross-feeding shows that the early-bird effect extends beyond simple metabolic trade-offs .", "More broadly , when species compete for multiple resources that are supplied in batches , a species’ survival depends on more than its ability to efficiently consume nutrients .", "An early-bird species , being more specialized in consuming the nutrients that are initially more abundant , gains a population advantage early in the batch .", "This population advantage may allow the early-bird species to out-compete other species even when consuming nutrients it is not specialized to consume .", "Despite its consumption inefficiencies , through sheer numbers the early-bird species can consume more of the remaining nutrients than its more specialized competitors .", "So far we have considered deterministic dynamics , which is appropriate for large populations .", "In natural settings , however , there are often small semi-isolated communities .", "For these communities , fluctuations can play an important role .", "In particular , population bottlenecks can lead to large demographic changes ( Abel et al . , 2015 ) .", "In our model , how does the nutrient supply affect diversity in such communities ?", "To address this question , we applied discrete sampling of a finite population when diluting from one batch to the next ( see Appendix 1 ) .", "With this protocol , an ‘extinction’ occurs when sampling yields zero individuals of a species .", "For a long enough series of dilutions such extinctions would ultimately lead to near-complete loss of diversity .", "For small real-world populations , however , diversity may be maintained by migration .", "To model such migration we augmented the population at each dilution with a ‘spike-in’ from a global pool of species , in the spirit of MacArthur’s theory of island biogeography ( MacArthur and Wilson , 2001 ) .", "Specifically , in the spike-in procedure , to prevent extinctions caused by sampling fluctuations , every new batch is inoculated with a small number of the original , founder species .", "In Figure 6A we show results of spike-in serial dilutions for a population bottleneck of 1008 cells .", "95% of these cells are sampled from the previous batch , while 5% are sampled from a global pool , with equal abundances of 21 equally spaced strategies ( cf . Figure 3A ) .", "The resulting me vs . c0 curves have maximal me for all six nutrient fractions in the regime c0≪K where the 5% spike-in dominates sampling noise .", "As expected , for a balanced nutrient supply at any c0 , all species have the same average abundance ( Figure 6B top ) .", "By contrast , when Nutrient 1’s fraction is low ( Figure 6A cyan and 6B bottom ) , increasing c0 increases the abundance gaps between the species , reflecting the uneven competition for Nutrient 2 .", "Overall , the spike-in protocol leads to higher diversity at low c0 than the deterministic case ( starting from equal species abundances but with no spike-in , Figure 3C ) .", "For large c0 , the me vs . c0 curves for these two protocols are indistinguishable .", "The only noticeable difference is that the spike-in maintains a higher level of the least competitive strains , but since these abundances are still low , this difference in not reflected in the me values ." ], [ "Natural ecosystems experience variations in the timing and magnitude of nutrient supply , and the impact of these variations on species diversity is not fully understood ( Smith , 2011; Smith , 2007 ) .", "To explore the impact of variable nutrient supply , we modeled resource competition in a serial dilution framework and analyzed the model’s steady states .", "We found that variable nutrient supply still allows for the high diversity seen in the continuous supply ( ‘chemostat’ ) version of the model .", "Indeed , the serial dilution steady state mimics that of a chemostat when the amount of nutrients supplied in each batch is small .", "Surprisingly , however , supplying the nutrients as a bolus led to a dependence of diversity on the amount of supplied nutrients .", "In contrast to existing literature on seasonality , we find that environmental fluctuations can both weaken and strengthen coexistence in this model .", "This occurs as the result of an ‘early-bird’ effect associated with supplying nutrients as large seasonal boluses instead of continuously .", "Some species can capitalize on rapid initial growth on an abundant nutrient to reach a large population size , which then allows them to deplete the remaining nutrients at the expense of their competitors .", "This early-bird effect can both restrict and expand the range of environments in which communities can self-organize to a neutral state .", "We show that even when metabolic trade-offs are combined with stabilizing mechanisms , the impact of the early-bird effect remains .", "For example , in the case of cross-feeding , the community diversity falls as a function of c0/K due to the early-bird advantages gained by species at higher trophic levels .", "While the idea of species gaining early advantages has been explored , such as in the literature on founder effects and speciation ( Barton and Charlesworth , 1984; Brown , , 1957 ) , to the best of our knowledge this is the first demonstration of the influence of the early-bird effect on the diversity of seasonal ecosystems .", "We believe that this effect will occur in a variety of such ecosystems , as its only fundamental requirement is competition for multiple nutrients that are supplied in a time-dependent manner .", "Interestingly , while the early-bird effect plays a large role in our model , it is not the only bolus-dependent effect that influences diversity .", "We also observe another effect that can be viewed as a ‘single-nutrient’ version of the early-bird effect .", "This effect arises from a well-studied property of competition: as growth proceeds , a superior competitor for a nutrient gains an exponential advantage over inferior competitors for that nutrient .", "Like the early-bird effect , this shifts the system’s biomass towards species more specialized in initially abundant nutrients , particularly for large but non-saturating nutrient bolus sizes .", "This single-nutrient effect can co-occur with the early-bird effect , for example in competition for an abundant nutrient between two early-bird species .", "The form of seasonality we explore in this manuscript , where mixed boluses are supplied periodically , is only one possible form of seasonal nutrient supply .", "The impact of the early-bird effect and single-nutrient competition will likely differ between different forms of seasonality .", "For example , we show in Appendix 7—figure 1 that supplying cycles of single nutrient boluses that approach an equal distribution of nutrients results in lower diversity than supplying mixed equal nutrient boluses .", "While this form of seasonality differs from the one we characterized , we can still understand the loss of diversity as arising from the single-nutrient competition effect initially observed in our mixed-bolus models .", "We expect the principles gleaned from our models to be of use in understanding diversity in a variety of seasonal ecosystems .", "Finding a general relation between the amount of nutrient supplied to a community and its diversity is a long-standing goal of theoretical ecology ( Tilman , 1982; Abrams , 1995; Leibold , 1996 ) .", "We found that in our model the form of the nutrient-diversity relation ( NDR ) can change based on model details .", "The model has two regimes: a low diversity and a high diversity regime .", "The former satisfies competitive exclusion ( no more species coexisting than resources ) , whereas the latter exceeds competitive exclusion and occurs when the nutrient supply lies within the convex hull of the remapped metabolic strategies present ( Posfai et al . , 2017 ) .", "At the bifurcation point between the two regimes , we observe critical slowing down in that the number of dilutions required to reach steady state diverges .", "In the high diversity regime , the NDR can take several forms , resulting from the interplay of the early-bird effect and other mechanisms .", "Even with a single trophic layer , the NDR can be U-shaped , hump-shaped , monotonically decreasing , or have multiple peaks .", "These trends can then be further modified by the addition of more trophic layers , differences in enzyme budgets , etc .", "Experimental studies that characterize the NDRs of microbial ecosystems have reached similarly variable conclusions .", "For example , one work studying bacterial communities in Arctic deep-sea sediments found an increasing trend between energy input and richness ( Bienhold et al . , 2012 ) , while a study on photosynthetic microbial mats found a negative relationship between energy input and richness ( Bernstein et al . , 2017 ) .", "A meta-analysis of aquatic microbial ecosystems found examples of both monotonic and non-monotonic NDRs , with no single form dominating ( Smith , 2007 ) .", "Our theoretical results , together with these experimental findings , indicate that there may be no single universal NDR in microbial ecosystems .", "This conclusion suggests that the best approach for characterizing the NDR of a given ecosystem is not to apply a one-size-fits-all theory , but to analyze the role of different factors such as cross-feeding , trade-offs , and immigration in determining that particular ecosystem’s NDR .", "While we have focused on microbial systems , the absence of a universal NDR is consistent with results from recent work in plants ( Adler et al . , 2011 ) .", "We found that the stringency of metabolic trade-offs has a large impact on community diversity .", "We imposed a metabolic enzyme budget on each species to reflect the reality that microbial cells have a finite capacity to synthesize proteins and must carefully apportion their proteome ( Basan et al . , 2015 ) .", "However , while it is true that microbes have limited biosynthetic capacity , it is unclear how strict are the resulting trade-offs .", "For this reason , we characterized versions of the model with both exact and inexact trade-offs .", "Our results show that the form of an ecosystem’s NDR can depend on the stringency of metabolic trade-offs .", "This finding is not exclusive to the serial dilution model .", "The stringency of trade-offs was also important in the original chemostat setting: in a birth-death-immigration framework , small violations of the enzyme budget still allowed for high levels of coexistence , but large violations disrupted coexistence ( Posfai et al . , 2017 ) .", "These results suggest that an experimental characterization of the stringency of metabolic trade-offs among microbes would provide a valuable ecological parameter .", "Note that metabolic trade-offs are only one of the many types of trade-offs microbes are subject to; other types of trade-offs , such as constraints between biomass yield and growth rate ( Wortel et al . , 2018 ) , may also shape a community’s NDR .", "In constructing a model , we made a number of assumptions about the way in which microbes consume and utilize nutrients .", "Some of these assumptions do not apply to all microbial communities , and the impact of relaxing these assumptions can affect the NDR .", "For example , we mostly focused on communities where all nutrients are equally valuable ( i . e . Yi=Yj⁢∀i , j ) .", "However , biomass yields can vary between nutrients and between species , which we explored in Figure 4C–D .", "Notably , unequal yields create differences between nutrients even in the saturating regime ( c0≫K ) , leading to a departure from the chemostat limit at large nutrient boluses .", "Coexistence in the serial dilution model is robust to varying yield , as long as all species have the same yield on a given nutrient .", "The scenario where species have different biomass yields on the same nutrient is conceptually similar to the case of inexact trade-offs , since some species will have a strict advantage over others .", "Thus , it is likely that these unequal yields between species will lead to a reduction in community diversity .", "However , varying the yield in this manner also allows for the inclusion of new trade-offs that may impact diversity , such as the aforementioned trade-off between yield and growth rate ( Wortel et al . , 2018 ) .", "We also explored the effects of unequal Monod constants for different nutrients ( cf . Figure 4A–B ) .", "We found that if a low-abundance nutrient also has a low Ki , the early-bird effect favors species that were disadvantaged in the chemostat limit , thus reversing the equal-Ki NDR and leading to hump-shaped NDR curves .", "Indeed , large differences in Ki values can lead to a multi-peaked NDR as shown in Appendix 7—figure 6 .", "Our model assumes that all nutrients are substitutable ( i . e . only one of the multiple nutrients is required for growth ) .", "In real ecosystems , microbes can require multiple complementary nutrients to grow , e . g . sources of carbon , nitrogen , and phosphorus .", "In cases where one class of complementary nutrient is strongly limiting , a model with both complementary and substitutable resources would essentially reduce to the current model of only substitutable resources .", "This case is likely the more common one , e . g . as many soils are carbon limited ( Aldén et al . , 2001; Demoling et al . , 2007 ) .", "However , in cases where no single nutrient is strongly limiting , the presence of complementary nutrients would possibly lead to different NDRs , which will be an interesting direction for future study .", "Our modeling predictions , e . g . the convex hull condition and the changes in diversity due to the early-bird effect , are in principle testable .", "To connect our modeling assumptions to real microbial systems , we compare our growth model of substitutable and simultaneous nutrient consumption to previously published experimental data from Escherichia coli growing in batch and chemostat conditions .", "We find that our modeling assumptions are consistent with both datasets and outline potential future experiments to test the model’s multispecies predictions , detailed in Appendix 6 .", "As is apparent in Appendix 6—figure 1 , the growth dynamics of E . coli at low nutrient levels is well described by our modeling framework .", "The experiments we compared were performed with the same strain of E . coli , meaning that inclusion of different microbes would be needed to test the multispecies predictions .", "To determine the strategies of other microbes , including other strains of E . coli , the most practical approach would likely be batch culturing .", "Once strains with different strategies have been identified , nutrient-diversity relationships could then be obtained by competing strains in serial dilution culture and measuring the community diversity ( e . g . via fluorescent tags or by 16S rRNA sequencing ) as a function of the total concentration of multiple , substitutable nutrients provided at the start of each batch ." ] ]

[ "Microbial communities feature an immense diversity of species and this diversity is linked to outcomes ranging from ecosystem stability to medical prognoses .", "Yet the mechanisms underlying microbial diversity are under debate .", "While simple resource-competition models don't allow for coexistence of a large number of species , it was recently shown that metabolic trade-offs can allow unlimited diversity .", "Does this diversity persist with more realistic , intermittent nutrient supply ?", "Here , we demonstrate theoretically that in serial dilution culture , metabolic trade-offs allow for high diversity .", "When a small amount of nutrient is supplied to each batch , the serial dilution dynamics mimic a chemostat-like steady state .", "If more nutrient is supplied , community diversity shifts due to an 'early-bird' effect .", "The interplay of this effect with different environmental factors and diversity-supporting mechanisms leads to a variety of relationships between nutrient supply and diversity , suggesting that real ecosystems may not obey a universal nutrient-diversity relationship ." ]

[ "In most environments , organisms compete for limited resources .", "The number and relative abundance of species that an ecosystem can host is referred to as ‘species diversity’ .", "The competitive-exclusion principle is a hypothesis which proposes that , in an ecosystem , competition for resources results in decreased diversity: only species best equipped to consume the available resources thrive , while their less successful competitors die off .", "However , many natural ecosystems foster a wide array of species despite offering relatively few resources .", "Researchers have proposed many competing theories to explain how this paradox can emerge , but they have mainly focused on ecosystems where nutrients are steadily supplied .", "By contrast , less is known about the way species diversity is maintained when nutrients are only intermittently available , for example in ecosystems that have seasons .", "To address this question , Erez , Lopez et al . modeled communities of bacteria in which nutrients were repeatedly added and then used up .", "Depending on conditions , a variety of relationships between the amount of nutrient supplied and community diversity could emerge , suggesting that ecosystems do not follow a simple , universal rule that dictates species diversity .", "In particular , the resulting communities displayed a higher diversity of microbes than the limit imposed by the competitive-exclusion principle .", "Further observations allowed Erez , Lopez et al . to suggest guiding principles for when diversity in ecosystems will be maintained or lost .", "In this framework , ‘early-bird’ species , which rapidly use a subset of the available nutrients , grow to dominate the ecosystem .", "Even though ‘late-bird’ species are more effective at consuming the remaining resources , they cannot compete with the increased sheer numbers of the ‘early-birds’ , leading to a ‘rich-get-richer’ phenomenon .", "Oceanic plankton , arctic permafrost and many other threatened , resource-poor ecosystems across the world can dramatically influence our daily lives .", "Closer to home , shifts in the microbe communities that live on the surface of the human body and in the digestive system are linked to poor health .", "Understanding how species diversity emerges and changes will help to protect our external and internal environments ." ]

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

[ "neuroscience" ]

[ [ "One of the most remarkable characteristics of a human hand is its ability to gather a rich variety of sensory information about the external world .", "In particular , tactile information conveyed by the four classes of low-threshold mechanoreceptor afferent units in the fingertips ( Johnson et al . , 2000; Johnson , 2001; Vallbo and Johansson , 1984a; Abraira and Ginty , 2013 ) is fundamental for manipulation activities ( Edin et al . , 1992; Edin and Johansson , 1995 ) .", "In addition , the biological sensors innervating the glabrous skin can provide complex types of information such as the onset of the contact with an object , the level of performed grasping force , and discrimination of textural features ( Weber et al . , 2013; Darian-Smith et al . , 1980a ) .", "The possibility of providing natural and rich sensory information to hand prosthesis users represents a major achievement .", "In fact , even though a significant research in biology , medicine and engineering has produced artificial hands that progressively approached the performance of a “natural hand” , it is a shared belief that providing a sense of touch is still the missing milestone ( Kwok , 2013 ) .", "This achievement will allow the “symbiosis” between the user and interface to become more adaptive ( to changing tasks and situations ) , more robust ( beyond interfering stimuli ) , more effective ( learn from the past to anticipate or predict the future ) , and more natural ( rapidly becoming part of the body scheme ) .", "Therefore , the restoration of sensory perception is the crucial step to achieve in the development of next generation of artificial limbs and hand prostheses , in particular .", "Thus far , promising results have been recently achieved to restore information about the touch of objects ( Tan et al . , 2014 ) and also the level of produced grasping force ( Raspopovic , 2014 ) .", "The restoration of ability to judge textural features represents the next significant step towards the re-establishment of close-to-natural sensory skills of a natural hand .", "Here , we sought to achieve this goal via an integrated approach to mimic natural coding using a neuromorphic , real-time , mechano-neuro-transduction ( Spigler et al . , 2012 ) ( MNT ) process through a sensorized artificial finger ( Oddo et al . , 2011a ) that integrates a Micro Electro-Mechanical System ( MEMS ) tactile sensor ( Beccai et al . , 2005 ) .", "In this framework , the temporal coding of tactile information was based on the use of a biologically plausible neural model ( Izhikevich , 2003 ) , which has shown promise as a versatile and computationally efficient framework for reproducing a wide range of phenomenological neural responses to stimuli .", "In this study , the MNT process was first tested in intact subjects by delivering electrical stimulation to their sensory peripheral nerve fibers during microstimulation via tungsten needle microelectrodes ( Vallbo et al . , 1984b; Torebjörk et al . , 1987 ) .", "Furthermore , the effects of natural mechanical stimulation at the fingertip and of the MNT-based electrical stimulation were qualitatively compared in terms of electrophysiological signals elicited in the contralateral sensory cortex of the subjects .", "Moreover , we wanted to evaluate whether the results achieved during needle microstimulation could be translated into experiments , which would be related to the real-time and longer-term use of hand prostheses .", "In fact , percutaneous needle microstimulation of peripheral nerves cannot be used in amputees as part of an effective long-term assistive device because a stable needle-to-fibers spatial relationship cannot be maintained in a moving limb .", "Therefore , its use can only be limited to a single experimental session lasting a few hours at most ( Vallbo et al . , 2004 ) .", "Instead , implantable neural interfaces , such as Transverse Intrafascicular Multichannel Electrodes ( TIMEs ) ( Boretius et al . , 2010 ) , are surgically positioned and firmly stabilized to adhere to nerve fascicles and do not require an arm rest compared with needle interfaces .", "Therefore , implantable neural interfaces are suitable for long-term implants .", "In this study , we developed a novel computational model to investigate the similarities between neural effects , such as in afferent nerve recruitment curves , achieved using microstimulation with percutaneous needles and with TIME neural interfaces .", "This comparison allowed verification of the possibility of extending the results achieved using percutaneous needle stimulation to TIMEs .", "The MNT process was tested in acute conditions in four intact subjects and was validated in a TIMEs implanted subject with a transradial amputation .", "For the first time in human hand neuroprosthetics , these integrated approaches allowed to show that discrimination of textural features can be reliably provided to users in different experimental conditions using peripheral intraneural electrical stimulation .", "The range of tested tactile stimuli in the current work is on the order of millimeters ( 0 . 5–3 . 0 mm ) .", "Thus , these stimuli pertain to the lower boundary ( towards the fine region ) of stimuli that are typically classified as coarse ( Weber et al . , 2013 ) ." ], [ "The MNT process translates surface coarseness into the injection of current pulses into the nerve .", "It qualitatively mimics the neuronal activity recorded during human microneurographic experiments ( Oddo et al . , 2011b ) .", "The MNT approach was initially tested in four intact volunteers using percutaneous electrical microstimulation of the median nerve ( Vallbo et al . , 1984b; Torebjörk and Ochoa , 1980 ) ( Figure 1a , Figure 2 ) .", "The participants - without visual or acoustic cues about the stimuli - were asked to discriminate surface pairs ( Figure 1b ) that differed in the Spatial Period ( SP ) of alternating ridges and grooves ( gratings ) , i . e . , in the distance between consecutive ridges separated by grooves ( defined in Figure 2a ) , which was a constant quantity in each half grating ( as shown in Figure 1b ) . 10 . 7554/eLife . 09148 . 003Figure 1 . Experimental setup and performance metrics .", "( a ) Sensorized artificial finger and tactile stimulation platform .", "( b ) Tactile stimuli that were used in the three-alternative forced-choice ( 3AFC ) psychophysical protocol and the raster plot of spike trains that were generated in all sessions with one subject by the artificial finger while the gratings were slid .", "( c ) Setup of percutaneous electrical microstimulation ( left ) and implanted intrafascicular stimulation ( right ) of the median nerve , and discrimination performance during all experimental sessions involving four intact subjects and one transradial upper limb amputee . Source data of the spike trains that were transduced by the artificial finger while the gratings were indented and slid over have been deposited in Dryad ( Oddo et al . , 2016 ) .", "Such spikes were used to trigger the neural stimulator in all the experimental sessions with DAS amputee ( raster plot depicted in Figure 1b ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 09148 . 00310 . 7554/eLife . 09148 . 004Figure 2 . Mechano-neuro-transduction process .", "( a ) MEMS sensor with 4 transducing piezoresistors implanted at the base of a cross-shaped structure ( sensor piezoresistive outputs Sx+ and Sx- are represented in blue and in green , respectively ) , grating with ridges and grooves that alternate with spatial period SP , and sensorized fingertip , which is in contact with tactile stimulus .", "( b ) The sequence of presentation of surface pair to the artificial finger .", "( c ) Example of implementation of the Izhikevich model for real-time conversion of MEMS sensor data into a sequence of artificial neural spikes .", "The blue and green traces show raw sensor outputs from the pair of opposing channels depicted in panel a .", "The red trace shows the input to the Izhikevich artificial neuron , which results from the application of Equation 1 and 2 ( described in the Materials and methods section ) .", "The black lines depict the spikes that are generated when the membrane potential of the Izhikevich artificial neuron reaches the threshold ( Equation 5 in the Materials and methods section ) and , thus , the neural stimulator is triggered .", "( d–e )", "Implementation of the Izhikevich model with a close-up view during the sliding motion over the first and second halves of the grating .", "The traces and color-coding are shown in panel c . DOI: http://dx . doi . org/10 . 7554/eLife . 09148 . 004 Via percutaneous electrical neural microstimulation , they reported mechanical sensation pertaining to the palmar side of the first four fingers of the hand .", "Microstimulation allowed users to reach discrimination ability above 77% ( 107/138 , Figure 1c , Figure 3a ) during a three-alternative forced-choice ( 3AFC ) psychophysical procedure ( Perez et al . , 2010; Gibson and Craig , 2005 ) mediated by the artificial touch system , which is based on the use of a MEMS sensor embedded into a human-sized robotic fingertip ( Video 1 ) .", "Confidence analyses indicated that percutaneous electrical microstimulation successfully induced percepts that were used to assess the coarseness of textured surfaces ( Figure 3b ) .", "The capability to discriminate between the two sides of the surface pairs was correlated with the difference between their spatial periods ( Figure 3c ) . 10 . 7554/eLife . 09148 . 005Figure 3 . Responses of intact subjects during the 3AFC psychophysical protocol with percutaneous electrical microstimulation of the median nerve . Each column reports results of the analyses on individual subject basis .", "( a ) Each panel displays the confusion matrix of behavioral responses relative to the four intact subjects with microstimulation .", "The titles indicate correct/total responses ( percentage ) .", "( b ) Vertical bars display correct responses that are associated with each stimulus .", "The vertical solid lines over each bar indicate the 95% confidence intervals ( Clopper Pearson exact interval ) per stimulus .", "The dashed horizontal line indicates chance level ( 1/3 ) .", "( c ) The fraction of trials a pair of stimuli is perceived as different as a function of difference in spatial period ( ΔSP ) between the two stimulus halves , and logistic fit ( dashed line ) .", "The title reports the R2 associated with the fit , i . e . , the fraction of data variance explained by the logistic function , and the significance of the Pearson correlation between data points and the fit . DOI: http://dx . doi . org/10 . 7554/eLife . 09148 . 00510 . 7554/eLife . 09148 . 006Video 1 . An example of the 3AFC psychophysical experiment with implanted intrafascicular stimulation of DAS amputee ( as illustrated in Figure 1 ) .", "The video includes an interview with DAS amputee subject reporting the percepts immediately after one experimental session . DOI: http://dx . doi . org/10 . 7554/eLife . 09148 . 006 As described hereafter , the comparison between the EEG activity that was evoked by the natural mechanical tactile stimulation of the real fingertip in the right hand and the one evoked by the substitutive electrical stimulation showed no significant differences in source topography , response timing , and clustering of cortical connections between the two stimulation modalities .", "Event-related potentials ( Figure 4a ) after substitutive electrical ( n = 4 , estimated power 0 . 75 , Figure 4—figure supplement 2 ) and natural mechanical stimulation ( n = 4 , estimated power 0 . 79 , Figure 4—figure supplement 3 ) conditions did not reveal any statistical difference ( Montecarlo statistics with cluster correction for multiple comparisons ) .", "Furthermore , a network graph analysis approach ( Vecchio et al . , 2015a ) revealed a lateralized EEG frequency modulation that was evoked both by electrical and mechanical stimuli ( Figure 4b ) .", "Indeed , the primary sensorimotor areas in the hemisphere contralateral to the stimulus presented a significant reduction ( 3-way ANOVA followed by Duncan’s multiple range test , F ( 1 , 6 ) = 6 . 48 , p<0 . 05 , comparison to the ipsilateral hemisphere ) in the clustering coefficient following the incoming sensory stimulus , regardless of its tactile or substitutive nature ( Figure 4c ) .", "Moreover , the generator sources of short-latency components of Somatosensory Evoked Potentials ( SEPs ) that were elicited by the substitutive electrical stimulation were localized at the Postcentral Gyrus ( Brodmann Areas 2 and 3 ) , which was consistent with a physiological tactile activation of the primary somatosensory cortex ( Figure 5 ) , as previously described using a hand area functional source separation method ( Di Pino et al . , 2012 ) . 10 . 7554/eLife . 09148 . 007Figure 4 . Cortical response to mechanical and electrical stimulation using a surface with 1 . 5 mm SP .", "( a ) Grand average event related potentials ( ERPs ) of all subjects ( n = 4 ) for both substitutive neuromorphic electrical ( red ) and natural mechanical tactile ( blue ) stimulation , ranging from -1500 to 3000 ms with respect to the stimulus onset .", "Each channel was normalized for the standard deviation of the prestimulus .", "( b ) eLORETA connectivity maps for delta , theta , alpha , low beta and high beta bands .", "Each tract ( red for electrical and blue for mechanical stimulations ) among the 7 sensorimotor regions of interest ( Brodmann Areas BA 1–7 ) reports the connectivity value higher than the cut-off threshold ( functional coupling >0 . 3 ) .", "( c ) Clustering modulation ( percentage of variation during stimulation with respect to baseline ) in the left and right hemispheres with electrical and mechanical stimulations .", "A significant reduction in clustering modulation across all frequencies occurred in the hemisphere contralateral to the stimulation ( p<0 . 05 , comparison to the right hemisphere , Duncan test after ANOVA ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 09148 . 00710 . 7554/eLife . 09148 . 008Figure 4—figure supplement 1 . Grand average event related potentials ( ERPs ) of all subjects ( n = 4 ) at the FC1 electrode for both the substitutive neuromorphic electrical ( red ) and natural mechanical tactile ( black ) stimulation , in the -150 to 350 ms window with respect to stimulus onset , with confidence interval bars . The horizontal black bar indicates the time window ( 210–240 ms ) when the evoked potential reached significance compared with the prestimulus voltage ( 2 Standard Deviations from the mean prestimulus voltage ) , both after the electrical microstimulation and after the mechanical stimulation . DOI: http://dx . doi . org/10 . 7554/eLife . 09148 . 00810 . 7554/eLife . 09148 . 009Figure 4—figure supplement 2 . Sample size computation based on the effect size of the prestimulus and the evoked activity within the significant time-window for the electrical microstimulation . DOI: http://dx . doi . org/10 . 7554/eLife . 09148 . 00910 . 7554/eLife . 09148 . 010Figure 4—figure supplement 3 . Sample size computation based on the effect size of the prestimulus and the evoked activity within the significant time-window for the mechanical stimulation . DOI: http://dx . doi . org/10 . 7554/eLife . 09148 . 01010 . 7554/eLife . 09148 . 011Figure 4—figure supplement 4 . Sample size computation based on the prestimulus effect sizes preceding the electrical microstimulation and the mechanical stimulation . DOI: http://dx . doi . org/10 . 7554/eLife . 09148 . 01110 . 7554/eLife . 09148 . 012Figure 4—figure supplement 5 . Sample size computation based on the effect size of the stimulus voltages ( ERPs within the significant time-window after the electrical microstimulation , and after the mechanical stimulation ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 09148 . 01210 . 7554/eLife . 09148 . 013Figure 5 . Cortical localization of a 1 Hz electrical microstimulation sensory evoked potential .", "( a ) Butterfly plot of SEPs for all 64 channels of one subject ( M4 ) .", "All traces are aligned to the electrical stimulus delivery .", "On top , the topographic representation of amplitude distribution at different time lags ( ms ) .", "( b ) Position of the associated equivalent dipole , which corresponds to P27 peak , superimposed on the individual horizontal , coronal and sagittal structural MRI planes of the subject . DOI: http://dx . doi . org/10 . 7554/eLife . 09148 . 013 The possibility to translate results from needle microstimulation to TIMEs was investigated by developing a novel hybrid model ( Raspopovic et al . , 2011 ) of the median nerve ( Figure 6 ) with both a microneedle and TIME interfaces inserted inside the nerve trunk ( Figure 7 , Figure 8 ) .", "The model , which takes into account realistic anatomical ( Jabaley et al . , 1980 ) and neurophysiological ( Vallbo et al . , 1984 ) data , indicated that the stimulated portion of axonal population ( Figure 7 ) , and , therefore , the type of sensation , together with the stimulation threshold ( statistically non-different: p>0 . 05 , Kruskal-Wallis test ) were similar for the two interfaces .", "The range of electric charge necessary for recruitment in both cases was comparable ( Figure 7 , Figure 8 ) .", "These findings provided evidence that implanted intraneural electrodes could achieve results that were comparable to needle percutaneous microstimulation and justified the transition from the acute to implantable interface to pursue a long-term use . 10 . 7554/eLife . 09148 . 014Figure 6 . Representation of the physical design of electrodes for the hybrid model .", "( a ) Implementation for the TIME electrode .", "( b ) Implementation for the microneedle .", "( c ) Different locations for stimulating active site and tip , that were used in the model to compare the TIME interface versus the microstimulation needle .", "Red dot represents the evaluated intrafascicular positioning of the TIME active site and the end of microstimulation needle tip .", "Yellow dot represents close-to-fascicle location , where the end of microstimulation needle tip and the active site of TIME interface were placed .", "Blue dot represents an example position with a shielding fascicle .", "The X marker represents the targeted fascicle , where the fiber activation was calculated for different locations of microneedle and TIME for 9 different populations that emulated biologically inherent uncertainty in the placement and extension of fibers innervating a specific hand district .", "This procedure was performed analogously for medium and small fascicles and confirmed the results . DOI: http://dx . doi . org/10 . 7554/eLife . 09148 . 01410 . 7554/eLife . 09148 . 015Figure 6—figure supplement 1 . Finite element model development for the human median nerve , starting from histological pictures and resulting in the solution of voltage distribution within the nerve . DOI: http://dx . doi . org/10 . 7554/eLife . 09148 . 01510 . 7554/eLife . 09148 . 016Figure 7 . Comparison of needle microstimulation and TIME stimulation using hybrid FEM/Neuron models .", "( a , b ) , Recruitment curves of sensory axon populations resulting from different positions of the active sites for the needle microstimulation and TIME stimulation ( mean +/- S . E . M . of percentage of recruited fibers ) .", "R4/L4 is the pair of active sites within the target fascicle; R5/L5 is close to it , and R6/L6 is shielded with respect to it .", "For each position of the stimulating tip/active site , nine different axonal populations were computed .", "The fiber was considered active when the spike travelled until the last node of Ranvier that was implemented .", "Figure insets represent voltage distributions for different positions of active sites , as calculated using the FEM solver .", "These results are representative for several electrode insertion configurations .", "The same computations were performed for medium and small fascicles and confirmed the results . DOI: http://dx . doi . org/10 . 7554/eLife . 09148 . 01610 . 7554/eLife . 09148 . 017Figure 8 . Fiber recruitment as a function of injected charge with microstimulation and the TIME implant .", "( a ) Recruitment results obtained for placement of the microneedle tip ( insulated and non-insulated tip ) and the TIME active site ( L4 and R4 ) within the fascicle of interest .", "The recruitment curves are the mean +/- S . E . M . for the 9 implemented populations in 4 different FEM configurations .", "The results for the microstimulation needle and TIME interface are similar , which supports the translation from percutaneous towards intraneural stimulation .", "The 2 cases of microneedle tip exposure yielded comparable outcomes .", "( b ) Voltage distribution induced by the close active site and positioning of 9 different fiber populations that were implemented to emulate the inherent anatomical uncertainty of sensory axon locations ( see Figure 8—figure supplement 1 for the correspondence between each color of the fibers illustrated in the panel and the specific implemented population ) .", "( c ) The colored dots indicate another randomized positioning of 9 different fiber populations implemented in the simulations of the hybrid electrical-biophysical model of the median nerve , with active site inside of fascicle . DOI: http://dx . doi . org/10 . 7554/eLife . 09148 . 01710 . 7554/eLife . 09148 . 018Figure 8—figure supplement 1 . Representation of the 9 neural populations within large fascicles that were implemented in the hybrid electrical-biophysical model simulations to compare needle microstimulation and stimulation via implanted TIME interface . DOI: http://dx . doi . org/10 . 7554/eLife . 09148 . 018 The 3AFC psychophysical protocol used during microstimulation experiments was also evaluated during sessions with subject DAS , a transradial amputee that was implanted with TIMEs ( Raspopovic , 2014 ) .", "The sensations elicited by temporal modulation of spiking electrical stimulation of the median nerve through a single implanted electrode were reported by DAS as pertaining to the palmar side of the index finger of the missing hand and as providing a realistic representation of the mechanical alternation between ridges and gaps of the experimented gratings ( Video 1 ) .", "DAS showed to be able to comply with the entire 3AFC psychophysical protocol via neural stimulation , without any visual input or guidance by the experimenter , and without interruption in each session , and in 77/80 trials ( Figure 1c , Figure 9a ) of trials he was able to correctly identify whether the two sides of the surface had the same SP or which one had a larger SP ( Figure 10 ) .", "This performance was significant ( p<0 . 05 confidence , Clopper Pearson exact interval ) for each presented single surface ( Figure 9b ) .", "These results indicate that the discrimination ability obtained using TIME stimulation was higher than the one with microstimulation ( Figure 1c , Figure 3 ) .", "This was attributed to the prolonged use/training of the implanted neural interface before the present study ( Raspopovic , 2014 ) . 10 . 7554/eLife . 09148 . 019Figure 9 . Subject behavior and analysis based on the stimulus spatial period ( SP ) , inter-burst interval ( IBI ) and average firing rate ( AFR ) in the session with DAS amputee .", "( a ) Confusion matrix of the responses given by DAS subject .", "( b ) Vertical bars display the correct responses , which are associated with each stimulus .", "The vertical solid lines over each bar indicate the 95% confidence intervals ( Clopper Pearson exact interval ) per stimulus .", "The dashed horizontal line indicates chance level ( 1/3 ) .", "( c ) Fraction of trials for which a pair of stimuli is perceived as different as a function of the difference in spatial period ( ΔSP ) between the two stimulus halves , and logistic fit ( dashed line ) .", "The title reports the R2 associated with the fit , i . e . , the fraction of data variance explained by the logistic function , and the significance of the Pearson correlation between data points and the fit .", "( d , e )", "Comparison between IBI-based and AFR-based discrimination .", "Difference between IBIs ( ΔIBI , panel", "d ) and difference between AFRs ( ΔAFR , panel", "e ) measured in the spike patterns elicited by the two sides of each stimulus and plotted as a function of the difference between grating SPs ( ΔSP ) .", "Error bars indicate the interquartile range .", "The title reports the fraction of explained variance . DOI: http://dx . doi . org/10 . 7554/eLife . 09148 . 01910 . 7554/eLife . 09148 . 020Figure 10 . Temporal coding of the spatial features of the experimented tactile stimuli . In all panels the spatial structure ( scale: 2 mm ) of the grating is superimposed over a sample temporal pattern ( scale: 200 ms ) of the spike train that was obtained as a result of transduction with the artificial finger .", "The ratio between the spatial and temporal scales turns into the stimulus sliding velocity ( 10 mm/s ) .", "The instantaneous firing rate ( scale: 50 spikes/s ) is shown together with the average firing rate during the stimulus sliding motion .", "( a ) ( above ) Stimulus ∆0 . 0+ is characterized by thw presentation of two half surfaces in the SP1 - SP2 order ( first: 1 . 5 mm , second: 1 . 5 mm ) ; ( below ) stimulus ∆0 . 0- , is characterized by the presentation of two half surfaces in the SP2 – SP1 order ( first: 1 . 5 mm , second: 1 . 5 mm ) .", "For stimulus ∆0 . 0 , the two half surfaces have the same coarseness .", "Therefore , ∆0 . 0+ and ∆0 . 0- result in spiking patterns with a common temporal structure .", "( b ) , ( above ) Stimulus ∆1 . 0+ is characterized by the presentation of two half surfaces in the SP1 - SP2 order ( first: 2 . 0 mm , second: 1 . 0 mm ) ; ( below ) stimulus ∆1 . 0- is characterized by the presentation of two half surfaces in the SP2 – SP1 order ( first: 1 . 0 mm , second: 2 . 0 mm ) .", "( c ) ( above ) Stimulus ∆2 . 0+ is characterized by the presentation of two half surfaces in the SP1 - SP2 order ( first: 3 . 0 mm , second: 1 . 0 mm ) ; ( below ) stimulus ∆2 . 0- is characterized by the presentation of two half surfaces in the SP2 – SP1 order ( first: 1 . 0 mm , second: 3 . 0 mm ) .", "( d ) ( above ) Stimulus ∆2 . 5+ is characterized by the presentation of two half surfaces in the SP1 - SP2 order ( first: 3 . 0 mm , second: 0 . 5 mm ) ; ( below ) stimulus ∆2 . 5- is characterized by the presentation of two half surfaces in the SP2 – SP1 order ( first: 0 . 5 mm , second: 3 . 0 mm ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 09148 . 02010 . 7554/eLife . 09148 . 021Figure 10—figure supplement 1 . Spatial modulation index that was calculated from the artificial-touch spike patterns from our study ( left ) compared to neurophysiological data shown by Phillips and Johnson for SA1 units ( right , adapted from a previous study ( Phillips and Johnson , 1981 ) .", "The depicted spatial modulations have the same monotonic trend versus the period of grating .", "The spatial modulation of our spike trains is globally higher in comparison with the neurophysiological data that was previously shown ( Phillips and Johnson , 1981 ) for surfaces with the same spatial period ( highlighted by a red box in the figure ) .", "We hypothesize that this difference exists because our surfaces had a constant ridge width of 0 . 25 mm , whereas , the surfaces in the study performed by Phillips and Johnson were square-wave gratings ( i . e . , ridge width was equal to groove width and was equal to half the spatial period ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 09148 . 021 The behavioral performance as a function of stimulus in subject DAS can be fit exactly ( R = 1 ) using a logistic function of the difference of SPs between the two halves of each surface ( Figure 9c ) .", "To understand how the very good surface discrimination , which was shown both by intact subjects with microstimulation and by DAS with the TIME implant , was achieved ( Figure 1c ) , we tested which neural features correlated with the difference of SPs .", "During the conditions of constant sliding velocity ( 10 mm/s ) and regulated load force ( 400 mN ) , the periodic contact between the sensor and the texture ridges generated regular patterns in the sensor output .", "The spiking response was given by a sequence of bursts of reliable duration , time-locked to the alternation of ridges and grooves during the sliding motion of gratings ( Figure 10 ) .", "This suggested that the Inter Burst Interval ( IBI , illustrated in Figure 10a ) , given by the temporal distance between the onset of consecutives bursts , might characterize the response to each texture .", "Indeed , the MNT process led to linear correlation between the stimulus SP of each texture and a highly specific IBI ( ANOVA test , Tukey Kramer correction for multiple comparisons , p<0 . 00001 ) reproducing neurophysiological findings in non-human ( Darian-Smith et al . , 1980a; Phillips and Johnson , 1981 ) and human ( Oddo et al . , 2011b ) primate studies with similar gratings .", "Consequently , the difference in IBI between the MNT output that was elicited by the two sides of each stimulus correlated almost perfectly ( R2 = 0 . 997 ) with the difference in SP ( Figure 9d ) .", "Additionally , the behavioral responses of DAS were perfectly ( R = 1 ) fit by a logistic function of the difference in IBI .", "The abovementioned results show that DAS behavioral responses may be based on the temporal structure of stimulation .", "However , they do not rule out the possibility that the responses were based simply on the count of the total number of spikes injected during each stimulus .", "Then , we tested whether a rate code not taking into account the temporal structure of the response was able to lead to comparable discrimination of stimuli as the one observed behaviorally .", "A commonly accepted definition of a rate code is the count of spikes within the time frame of encoding window ( Theunissen and Miller , 1995; Panzeri et al . , 2010 ) which in our case is assumed to be the 2 s presentation of each stimulus .", "Then , the sensor response to each stimulus , based on which we performed the IBI-based analysis , was also quantified by measuring the overall average firing rate ( AFR ) , i . e . , the ratio between the total number of spikes emitted during the sliding and the ( fixed ) 2 s sliding duration .", "We found that the AFRs generated in response to different SPs were often similar because a larger IBI was associated with a more intense bursting activity ( Figure 10 ) .", "In particular , the AFRs of responses to 0 . 5 , 1 . 0 and 1 . 5 mm were not significantly different , and the AFRs of responses to 2 . 0 mm and 3 . 0 mm were not significantly different ( p<0 . 05 ANOVA test with multiple comparisons ) .", "Consequently , the difference ΔAFR in AFR between the two sides of a stimulus correlated very weakly with the difference between their SP ( R2 = 0 . 066 for ΔAFR vs R2 = 0 . 997 for ΔIBI , compare Figure 9e and Figure 9d ) .", "Note that no logistic function of the difference in AFR was able to fit DAS behavioral responses ( R2 = 0 . 04 , p = 0 . 8 ) , which was a significant contrast with the perfect fit achieved with ΔIBI .", "Therefore , because the total spike count was not able to encode differences between stimuli to a degree compatible with behavioral responses , we concluded that the rate code was not responsible for the subject perception of the presented textures .", "This further strengthens the hypothesis that the subject exploited the temporal structure of the response to discriminate the stimuli .", "Figure 10 shows the instantaneous firing rate ( Lánský et al . , 2004 ) of different responses computed as the inverse of the inter-spike interval .", "It is apparent that the temporal evolution of the instantaneous firing rate matches the alternation of ridges and grooves .", "Indeed , it is already known that , when sliding occurs at fixed velocity , the temporal structure of the sensor output is a linear transformation of the spatial structure of the texture ( Oddo et al . , 2011b; 2011c ) , at least for regular textures such as those considered here .", "Therefore , this is a case of temporal coding of the stimulus ( Theunissen and Miller , 1995; Borst and Theunissen , 1999 ) : the temporal evolution of the spiking response reflects the time evolution of the stimulus rather than internal dynamics of stimulus encoding .", "Because the spatial structure is highly regular and the textures primarily differ for a single spatial variable ( the SP ) , it is possible to discriminate between them using the corresponding single temporal variable ( the IBI ) .", "Then , this may be the feature of the injected spike trains that allows subjects to precisely decode the presented stimuli ." ], [ "Recent neuroprosthetic studies showed that pressure sensation can be elicited by injecting a train of pulses with a fixed shape and different frequencies via intrafascicular ( Dhillon and Horch , 2005 ) or cuff ( Tan et al . , 2014; Ortiz-Catalan et al . , 2014 ) interfaces .", "Additionally , reconstruction of tactile sensation of force levels and object shape was shown using multi-electrode stimulation via pulses with current amplitude linearly increasing with sensor outputs ( Raspopovic , 2014 ) .", "In contrast , this work focused on eliciting textural features by injecting a biomimetic ( Saal and Bensmaia , 2015 ) temporal structure of pulse trains with a fixed current amplitude in each experimental session .", "Discrimination of textural features is a remarkable skill of our somatosensory system , which is used in everyday activities to interact with a peri-personal space .", "The subjects that use hand prostheses will significantly benefit from the restoration of this sensory function , which represents the next desirable feature after recent achievements ( Tan et al . , 2014; Raspopovic , 2014; Ortiz-Catalan et al . , 2014 ) .", "The results of this study show that the texture discrimination skills can be artificially provided to users using both needle microstimulation in intact subjects and implantable intraneural interfaces in a transradial amputee .", "Both in needle and TIME-based experiments , the subjects were able to use our ‘artificial’ feedback to perform a 3AFC psychophysical test with very good and comparable performance .", "The similarity between the needle and TIME-based stimulations was predicted by our hybrid FEM-biophysical model , which supported the transition from acute preparation towards chronically implanted electrodes .", "In addition , this result opens up interesting scientific and clinical opportunities .", "In the future , needle microstimulation can be used during translational experiments to test different types of peripheral neural coding strategies , which - if successful in this prototypal situation - would be implemented using implantable neural interfaces in disabled patients .", "Furthermore , model predictions were confirmed by the quasi-perfect discrimination ( 96% overall stimuli , see Figure 1c , and see Figure 9a , b for the analysis on a per-stimulus basis ) that was achieved by DAS amputee with TIME stimulation .", "The results of the present study showed that a temporal neuronal coding of spatial structure ( Weber et al . , 2013 ) can successfully elicit tactile cues in case of coarse surfaces that were explored under a passive- dynamic-touch protocol with a constant sliding velocity and a regulated load force , thus , involving controlled motion of the tactile stimulus while the fingertip was not moving ( Yoshioka et al . , 2001 ) .", "In such passive-touch framework there is a lack of voluntary movement .", "This allows decoupling of cutaneous information ( which was our interest in this study ) from the kinaesthetic afferent sensory feedback and efference copy associated with movement dynamics .", "Thus , it was possible to compare the EEG activities evoked by the substitutive neuromorphic electrical stimulation versus the natural mechanical tactile stimulation .", "No significant differences in topography or frequency modulation clustering were shown by the EEG signals in the two cases .", "The behavioral results showed that injecting the sensor output via a single stimulation channel was sufficient to induce accurate responses ( particularly from DAS ) in the designed experimental protocol with locked indentation force and tangential velocity of stimulus pairs .", "Additionally , these experiments allowed us to investigate how the subjects were able to achieve these results in this specific case .", "In fact , the relationship between IBI and behavioral performance seems to indicate that responses of subject DAS were based on temporal structures of the injected stimuli ( which , due to the linearity of our MNT process in mapping the geometry of stimuli , correspond to temporal modulation in the spiking activity captured by the IBI for regular textures ) , rather than on their average firing rate alone .", "IBI was previously shown to be given by the ratio between the surface spatial period SP and the sliding velocity V in human ( Oddo et al . , 2011b ) and animal ( Weber et al . , 2013; Darian-Smith et al . , 1980a ) neurophysiological investigations and in artificial touch ( Oddo et al . , 2011c ) studies .", "Because in our protocol the sliding velocity V was fixed ( 10 mm/s ) , the IBI was proportional to the SP , and so were their differences ( ∆IBI and ∆SP ) between the two sides of the stimuli ( Figure 9d ) .", "Our results indicate that a temporal single-channel MNT-based intraneural stimulation allows gathering of textural features of medium-coarse surfaces with constant and slow sliding velocity .", "However , for more complex texture discriminating tasks it is plausible that integration of spatiotemporal information from neighboring receptors distributed along the fingertip ( Johansson and Flanagan , 2009; Jörntell et al . , 2014 ) and , thus , multi-channel stimulation , combined with amplitude modulation ( Raspopovic , 2014 ) , would be needed in less restrictive conditions that involve velocity or force variations and everyday life stimuli ( Weber et al . , 2013; Dépeault et al . , 2008; Rongala et al . , 2015 ) .", "The promising results obtained with microstimulation in four intact subjects , combined with robust translational indications from the hybrid model and an excellent outcome from one amputee , prompt the idea that neuromorphic stimulation could be a natural and effective tool for eliciting texture discrimination abilities via hand prostheses .", "Neuroprosthetic research ( Tan et al . , 2014; Raspopovic , 2014; Dhillon and Horch , 2005; Rossini et al . , 2010 ) can in parallel contribute to the evaluation of open neuroscientific hypotheses about tactile perception ( Weber et al . , 2013; Yoshioka et al . , 2001; Johansson and Flanagan , 2009; Hollins and Risner , 2000 ) for the advancement of understanding of human somatosensory physiology ." ], [ "The core element of the artificial fingertip was a Micro Electro Mechanical System ( MEMS ) sensor with 4 transducing piezoresistors implanted at the base of a cross-shaped structure ( Beccai et al . , 2005 ) ( Figure 3 ) .", "The MEMS was packaged with polymeric compliant material ( Dragon Skin , Smooth-On , USA ) .", "Sensor data were sampled at 380 Hz per channel by a 24-bit Analog to Digital Converter ( ADS1258 , Texas Instruments , USA ) integrated in the fingertip , and acquired via SPI by a Field Programmable Gate Array ( Cyclone II FPGA , Altera , USA ) .", "The FPGA streamed the acquired information via Ethernet to a PC for real-time implementation of an artificial mechanoreceptor model emulating the tactile coding recorded during human microneurography sessions carried out with surfaces and experimental apparatus as those of current study ( Oddo et al . , 2011b; 2011d ) .", "To this aim , MEMS sensor signals were converted into sequences of neural spikes via a real-time C++ implementation of the Izhikevich spiking neuron ( Izhikevich , 2003 ) .", "Such model was originally conceived to emulate neuron-to-neuron signaling , whereas in this modified implementation the input is haptic rather than synaptic .", "As detailed in the following sections , these transduced spike trains were injected in the median nerve as train of pulses of fixed width and amplitude through microstimulation electrode in the case of intact subjects and through TIME interface in the case of DAS amputee .", "We did not model the fine details of the complex spatio-temporal mechanical interaction between physical stimuli , soft tissues and receptors ( Hayward et al . , 2014 ) , but we injected as input current in the neuron model a signal generated from the outputs of sensors integrated in the artificial fingertip , as follows .", "Sensor piezoresistive outputs ( Sx+ and Sx- , represented in blue and in green in Figure 2c ) belonging to opposite tethers of the cross-shaped structure were subtracted ( Equation 1 ) to obtain a component , proximal to distal on the biomimetic fingertip , highly correlated to the frictional shear stress ( Oddo et al . , 2007 ) arising along the direction of the stimulus sliding motion .", "This component was half-rectified and amplified ( Equation 2 , as represented in red in Figure 2c ) in order to inject it as input current in the spiking neuron model ( Ix in Equation 3 ) .", "Equations 3 and 4 describe the subthreshold evolution of the membrane potential v and the recovery variable u in the implemented artificial neuron model ( Spigler et al . , 2012; Izhikevich , 2003 ) .", "Whenever the membrane potential v reached the threshold level , a spike was triggered , v was set to a reset value c and u was increased of a fixed value d ( Equation 5 ) .", "The spike was broadcasted by TCP communication to successive services and graphical user interface ( Labview , NI , USA ) .", "The computed binary response constituted the output of the neuromorphic artificial touch system , which triggered via USB the current stimulator ( STG4008 , Multichannel System , Germany ) each time that a spike template had to be delivered .", "The coefficients of the model ( Spigler et al . , 2012; Izhikevich , 2003 ) were tuned so to achieve a phasic firing with respect to the moving ridges of surfaces ( Gardner and Palmer , 1989 ) .", "The MNT process emulated to some extent the neural representation of stimulus spatial patterning observed in previous non-human ( Weber et al . , 2013; Darian-Smith et al . , 1980a ) and human ( Oddo et al . , 2011b ) primate recordings of SA1 afferents associated to Merkel mechanoreceptors , a functionally relevant class of tactile units able to encode medium-coarse textures ( Weber et al . , 2013; Phillips and Johnson , 1981; Yoshioka et al . , 2001; Darian-Smith et al . , 1980b ) such as the surfaces evaluated in the present study .", "The MNT process coded the temporal period of the surface , a feature scaling linearly with spatial period of the grating and inversely with the stimulus tangential velocity .", "The resulting firing ( Figure 2 ) was characterized by biomimetic ( Sathian et al . , 1989 ) bursts of multiple spikes per ridge with coarser surfaces ( see 3 . 0 mm and 2 . 0 mm SP in Figure 10 ) , by triplets or duplets for surfaces with medium coarseness ( see 1 . 5 mm and 1 . 0 mm SP in Figure 10 ) , down to a single spike per ridge with finer surfaces ( see 0 . 5 mm SP in Figure 10 ) , thus implementing a neural code of stimulus geometry based on the modulation of discharge rather than on the mean discharge frequency , resembling SA1 electrophysiological recordings under similar tactile stimulation conditions ( Darian-Smith et al . , 1980a; Phillips and Johnson , 1981 ) ( Figure 10—figure supplement 1 ) .", "Furthermore , the instantaneous firing rate of the implemented artificial mechanoreceptor model achieves a modulation up to tens of spikes/s ( see red traces in Figure 10 ) , coherently with typical values reported in the literature for SA1 units ( Johnson , 2001; Phillips and Johnson , 1981 ) .", "( 1 ) Sx=Sx+−Sx− ( 2 ) Ix=KSx , x≥00 , x<0 ( 3 ) dvdt=Av2+Bv+C−u+IxRCm ( 4 ) dudt=a ( bv−u ) ( 5 ) if ( v≥vth ) , thenv←cu←u+d The following parameters were used: K = 15 , 000; A = 0 . 04/sV; B = 5/s; C = 140 V/s; Cm =1F; R = 1C; a = 0 . 02; b = 0 . 2/s; c = -65 mV; d = 8 mV; vth = 30 mV .", "Four intact subjects ( 2 males and 2 females , 25–26 years old ) underwent the experiments of texture discrimination with tactile feedback elicited by a stimulation injected through two microneurographic electrodes ( FHC UNP40GAS , shaped as needles ) .", "The enrolment was subjected to signing a written Informed Consent , approved by the Campus Bio-Medico University Ethics Committee , where this set of experiments took place .", "A neurologist trained in microneurography performed the procedure ( Hagbarth and Vallbo , 1967 ) , consisting of two phases of electrical stimulation , superficial and percutaneous , necessary to identify the correct site for electrodes insertion .", "The reference electrode was positioned just through the skin and the active one into the nerve .", "Initially , we stimulated the skin of the right arm of the subject in an area centred 2–3 cm proximal and medial to the elbow .", "A 1 Hz cathodic biphasic balanced square current was delivered to the subject .", "Pulse duration was 0 . 2 ms , while amplitude was changed in the range 1–10 mA .", "The nerve area was identified whenever a hand muscle twitch occurred in correspondence of 1–2 mA amplitude .", "Reference and active electrodes were then inserted through subject’s skin , after cleaning .", "A cathodic biphasic balanced square wave was injected through the needles with a frequency of 1 Hz .", "Pulse duration was 0 . 2 ms , while the current changed within the 1–100 μA range .", "The neurologist moved slightly the active electrode towards the inner of the arm seeking for a reported sensation or a muscular twitch over/of the hand .", "If one of those conditions occurred with amplitude of the injected current between 1–10 μA , the electrode impaled the nerve fiber .", "The neurologist then moved the needle in the area of insertion until the subject reported a distinct tactile sensation over one of the first four fingers .", "As final evaluation of proper placement of the electrodes , the electroneurogram ( ENG ) was visualized and acoustically identified: afferent ENG from the median nerve had to be discriminated over the background nerve activity as some mechanical stimuli were exerted over the fingers .", "Acquisition was performed by means of a system comprising a single channel amplifier ( 15LT Bipolar , Grass Instrument , USA ) and a 16-bit data acquisition board ( PCI-6251 , NI , USA ) installed on a personal computer running a custom interface ( Labview , NI , USA ) .", "The subject was instructed to avoid any voluntary movement that could alter the needle placement once the nerve search procedure was completed .", "An experimenter-guided session of microstimulation was performed in order to identify appropriate parameters to use in the upcoming 3AFC psychophysical protocol: 500 ms trains of cathodic biphasic balanced squares were delivered while changing frequency , pulse duration ( Ts ) and amplitude ( As ) starting from 10 Hz , 5 μs and 2 μA , respectively .", "The tuning of stimulation parameters was operated heuristically among the subjects while performing pilot trials , such as:", "a ) subject connected to the stimulator while the experimented touched manually the artificial fingertip ,", "b ) subject exploring the gratings with the non-stimulated hand and then", "c ) perceiving the same surfaces via the artificial fingertip .", "An additional experimental session of recording somatosensory evoked potentials ( SEP ) ( Kunesch et al . , 1995 ) at 1 Hz stimulation rate was performed with subjects M3 and M4 before starting the 3AFC psychophysical experimental protocol: the spike template was injected in the nerve through microstimulation .", "Stimulation was delivered as square pulses lasting 200 μs at 1 Hz with a number of repetitions ( 180 for M3 and 700 for M4 ) allowing a robust Independent Component Analysis ( Artoni et al . , 2014 ) on EEG data ( as detailed in the specific session ) .", "In order to allow a better comparison among subjects , stimulation amplitude was individually tailored and chosen as an amplitude that was clearly perceived by the subject , without producing any discomfort ( between 5 and 10 μA ) .", "Pilot trials of the 3AFC psychophysical test with neuromorphic sensory feedback were then repeated for variable duration among subjects just before starting the actual experimental protocol .", "Participant DAS had suffered a traumatic transradial left arm amputation 10 years before the experiments .", "He was selected from a group of 31 persons with hand amputation because of the stump characteristics and his psychophysical abilities .", "All procedures were approved by the Institutional Ethics Committees of Policlinic A . Gemelli at Catholic University , where the surgery was performed , IRCCS S . Raffaele Pisana ( Rome ) , where the experiments were performed , the Ethics Committee of Campus Bio-Medico University and the Italian Ministry of Health .", "TIME neural interfaces were implanted under general anaesthesia .", "After superficial disinfection of the medial aspect of the left upper limb , placed extrarotated , the skin was cut along the medial edge of the biceps muscle for about 15 centimetres , from few centimetres below the axilla to about 6 cm above the elbow .", "The ulnar and the medial nerves were exposed along their course , through careful smooth dissection of the dermal and hypodermic structures , fascial bands , and muscles .", "Following an epineurial microdissection , performed under a surgical microscope ( Zeiss , Pentero ) to visualize the fascicles , two TIME electrodes were inserted into the nerve trunk of each nerve ( median and ulnar , only the former of which was used for electrical stimulation in the present work ) .", "Electrodes were pulled inside the nerve , until the embedded active contacts reached the targeted location , close in contact with the nerve fascicles .", "Cable segments were placed in subcutaneous pockets , while four holes were made in close proximity of the skin incision , two medially and two laterally for the electrode emission ( Raspopovic , 2014; Di Pino et al . , 2014 ) .", "The electrodes were removed after 30 days , in accordance with EU guidelines .", "At the time of removal however , the TIME electrodes were still performing extremely well , and did not cause any discomfort to the subject .", "The follow-up of the clinical condition of the participant almost 2 years after the end of the protocol did not reveal any subjective or objective side effects .", "Upon connection of the neural interface implanted in the median nerve with the current stimulator , an experimenter-guided session of intraneural stimulation was performed in order to identify appropriate parameters to use in the upcoming 3AFC psychophysical protocol , also capitalizing on the information gathered during the previous days of experimental activities with DAS .", "Pulse duration ( Ts ) and amplitude ( As ) were initially set to 100 μs and 100 μA , respectively .", "The tuning of stimulation parameters was operated heuristically in 33 min while performing pilot trials , such as:", "a ) DAS connected to the stimulator while he ( with the intact hand , to have a sort of self-touch experience ) or the experimenter touched manually the artificial fingertip ,", "b ) DAS exploring the gratings with the intact hand and then", "c ) perceiving the same surfaces via the artificial fingertip .", "Pilot trials of the 3AFC psychophysical test with neuromorphic sensory feedback were then repeated for 8 min with the selected parameters ( As = 160 μA , Ts = 100 μs ) just before starting the experimental protocol .", "The protocol structure was essentially the same during microstimulation of intact subjects , and stimulation through implanted TIMEs in the amputee .", "The artificial fingertip underwent mechanical stimulation with surface pairs that were presented using a mechatronic platform ( Oddo et al . , 2011d ) designed to implement standardized human and artificial passive-touch ( Jones and Smith , 2014 ) experimental protocols .", "The tactile stimuli were gratings , fabricated with 3D printing of plastic material ( Project HD 3000 , 3D Systems ) , consisting of a sequence of alternating ridges and grooves with spatial period ( SP ) from a minimum of 0 . 5 mm to a maximum of 3 . 0 mm depending on the stimulus and on the half portion of the surface under test ( Figure 1b , Figure 2 , Figure 10 ) .", "The sequence of presented stimuli was randomized within sessions of 16 trials composed of 4 presentations of the 4 surfaces ∆0 . 0 , ∆1 . 0 , ∆2 . 0 and ∆2 . 5 .", "In each session , the 4 trials per each stimulus were composed of 2 trials with presentation of the two surface halves in the SP1-SP2 order ( labeled +: ∆0 . 0+ , ∆1 . 0+ , ∆2 . 0+ and ∆2 . 5+ ) , and 2 trials with reversed SP2-SP1 order ( labeled 2: ∆0 . 0- , ∆1 . 0- , ∆2 . 0- and ∆2 . 5- ) .", "The first half-surface was indented at 400 mN on the artificial fingertip and , after 4 s of indentation without tangential movement , it was slid at 10 mm/s for 2 s under regulated load force .", "The sliding motion was followed by 2 s of indentation at 400 mN without stimulus movement , and then the surface was detached .", "The same sequence was applied to the second half-surface , starting 3 s after the end of presentation of the first half ( see Figure 2 and Video 1 for the representation of the whole stimulation sequence ) .", "At the end of the presentation of the surface pair , the subject stated whether the first half-surface was perceived as having coarser , finer or same spatial coarseness in comparison to the second half .", "During the experiment the subject received no feedback about the correctness of the responses .", "For the intact subjects who participated in the microstimulation sessions , the gratings were also presented mechanically , via the tactile stimulation platform ( Oddo et al . , 2011d ) , directly on the finger ( fixed to the platform via a finger holder glued to the nail ) that was identified as source of the sensory perception elicited with microstimulation .", "During the experiment the subjects received no feedback about the correctness of the responses .", "The 95% confidence intervals for performance estimate from individual subjects responses were computed with exact Clopper Pearson method ( binofit function in Matlab ) and compared against chance level ( 1/3 ) to assess performance significance .", "Parameters for logistic fit of performance as a function of stimulus features were estimated with generalized linear regression ( glmfit function in Matlab ) , then the optimal logistic fit function was generated ( glmval function in Matlab ) and its accuracy evaluated as the squared correlation coefficient between data and fit .", "In order to investigate the neural correlates of natural and substitutive texture discrimination , EEG signals were recorded in all microstimulation sessions using a 64 channel EEG device ( SD LTM Express , Micromed S . p . A . , Italy ) with a 2 kHz sampling rate .", "The montage was in accordance with the 5% 10/20 system ( Oostenveld and Praamstra , 2001 ) .", "Careful scalp preparation granted electrodes impedance below 5 kΩ in at least 90% of derivations , as measured at the experiment onset .", "Data were analyzed by Matlab scripts based on the EEGLAB toolbox ( Delorme and Makeig , 2004 ) .", "EEG signals were processed via independent component analysis ( ICA ) filtering to remove non-neural sources and artifacts .", "In order to optimize the dipolarity of the independent components ( IC ) extracted ( Artoni et al . , 2014 ) and to maximize the amount of data fed to the algorithm ( while maintaining a sufficient density of information ) the EEG continuous data were epoched .", "Data were high-pass filtered using a zero-phase 1 Hz , 24th order , Chebyshev type II filter and low-pass filtered using a zero-phase , 45 Hz , 71th order , Chebyshev type II filter to remove slow drifts and high-frequency noise respectively , then resampled at 256 Hz .", "Channels with prolonged prominent artifacts ( by visual inspection ) or with probability more than five times the standard deviation from the mean across all channels were removed ( in the end the remaining numbers of channels were 21 , 40 , 59 , 61 respectively for the four subjects ) , then a common average reference was used for the remaining channels .", "Epochs containing high-amplitude artefactual potentials , high-frequency muscle noise and other irregular artifacts , as identified by visual inspection , were removed and remaining data were submitted to AMICA ( Palmer et al . , 2007 ) , a generalization of the Infomax algorithm ( Makeig et al . , 1996 ) to multiple mixture approaches ( Lee et al . , 1999 ) under the hypothesis that the ICs are spatially static ( general stationarity , e . g . recording environment ) .", "ICA decompositions were performed separately on each subject over all conditions .", "Stereotyped artifacts such as eye movements , eye blinks and muscle tension were removed by ICA .", "The ICA decomposition was then saved and reapplied to data , pre-processed using the same described procedure , but high-passed using a 0 . 5 Hz , 94th order , Chebyshev type II filter but using the pre-computed weights .", "This procedure allowed to efficiently remove artifacts while retaining the low-frequency EEG information .", "Regarding source localization , for subject M4 a realistic head model was obtained by means of the NFT toolbox ( Acar and Makeig , 2010 ) using a T1-weighted magnetic resonance ( MR ) image of the subject and a 4-layer model accounting for scalp ( σ = 0 . 33 ) , skull ( σ = 0 . 0132 ) , brain tissue ( σ = 0 . 33 ) , and cerebrospinal fluid ( σ = 1 . 79 ) respectively .", "Electrodes positions on the head were co-registered with an optoelectronic neuronavigation system SoftAxic ( E . M . S . srl , Italy ) and aligned with the Montreal Neurological Institute MNI , Canada .", "The Finite Element Method ( FEM ) was used for the numerical solution of the forward problem with Boundary Element Method ( BEM ) meshes as boundaries .", "Given the potentials distribution across the scalp , the position of a best-fitting single equivalent current dipole ( inverse problem ) was determined using the ( Dipfit toolbox of EEGLAB ) ( Oostenveld and Oostendorp , 2002; Delorme et al . , 2012 ) .", "EEG recording time-locked with 1 Hz intraneural median nerve microstimulation was exploited to evidence the early somatosensory potentials , the components more strictly due to the stimulation .", "SEP data epochs were selected from 20 ms before to 100 ms after each stimulation onset .", "Noisy epochs were rejected by careful visual inspection .", "Similarly to the continuous data , the criteria for epoch removal were the presence of high amplitude artifacts ( e . g . , Jaw clenching ) .", "Source localization was then performed on the most reliable short-latency potential of cortical origin , namely the P27 peak ( Figure 5 ) .", "Event-related potentials ( ERPs ) were time-locked to the onset of either the electrical microstimulation or of the sliding phase of the mechanical stimulation ( Figure 4 , Figure 4—figure supplement 1 ) .", "ERPs were normalized for the standard deviation of the prestimulus ( 1000 ms ) .", "ERP’s statistical significance between conditions ( electrical microstimulation vs . mechanical stimulation ) was assessed using a Montecarlo statistics with cluster correction for multiple comparisons ( triangulation and maxsum as clustered statistics ) ( Maris and Oostenveld , 2007 ) , adapted from the FieldTrip toolbox ( Oostenveld et al . , 2011 ) .", "Statistical power of ERPs comparisons between stimulation conditions was computed for dependent groups ( GPower , Duesseldorf , Germany ) ( Figure 4—figure supplements 2–5 ) .", "EEG functional connectivity analysis was performed using the eLORETA exact low-resolution electromagnetic tomography ( Vecchio et al . , 2015; Pascual-Marqui , 2011; Vecchio et al . , 2014a; 2014b; 2015b ) software .", "To obtain a topographic view of the sensorimotor network , brain connectivity was computed with sLORETA/eLORETA software in 7 regions , positioning the center in Brodmann Areas ( BAs: 1–7 ) separately on the left and right hemispheres .", "For each subject and for each hemisphere , among the eLORETA current density time series of the 7 Regions of Interest ( ROIs ) , intracortical Lagged Linear Coherence , extracted using a sphere with 19 mm of radius ( Pascual-Marqui , 2011; 2007 ) , was computed between all possible pairs of the ROIs for each of the five independent EEG frequency bands: delta ( 2–4 Hz ) , theta ( 4–8 Hz ) , alpha ( 8–13 Hz ) , low beta ( 13–20 Hz ) and high beta ( 20–30 Hz ) .", "A weighted network was built based on the connectivity between ROIs .", "The nodes of the network were ROIs , and the edges of the network were weighed by the lagged linear coherence values .", "The vertices of the network were the estimated cortical sources in the BAs , and the edges were weighted by the Lagged Linear value within each pair of vertices .", "The measure considered here was the clustering ( C ) that characterizes the tendency of the nearest neighbors of a node to be interconnected .", "The mean clustering coefficient was computed for all nodes of the graph and was then averaged to estimate the tendency of network elements to form local segregated clusters .", "Finally , to obtain individual normalized relative measures , the values of each mean clustering coefficient were divided by the mean values obtained by their average in all bands of each subject .", "Statistical analysis of percent clustering modulation with respect to baseline period was performed with Statistica v . 7 software ( StatSoft Inc . , USA ) .", "Greenhouse and Geisser correction was used for the protection against the violation of the sphericity assumption in the repeated measure ANOVA .", "Besides , statistical significance was determined by 3-way ANOVA followed by Duncan’s multiple range test .", "ANOVA was performed between three factors: stimulation mode ( mechanical and electrical; independent variable ) , hemisphere ( left and right ) , and band ( delta , theta , alpha , low beta , high beta ) .", "Hybrid models ( Raspopovic et al . , 2011; McIntyre et al . , 2002 ) account for the anisotropy of extracellular conductivity during the calculation of the electrical field induced by the injection of the electrical current into the tissue , present in real nerves , and for the nonlinear response of cells to the extracellular stimulation .", "Those two aspects were addressed by means of a finite element method ( FEM ) to solve the voltage distribution generated by injected currents , and by calculating the neuronal dynamics to estimate the axonal response to the electrical stimulation .", "The volume conductor model implemented via multiphysics FEM ( COMSOL Multiphysics , Sweden ) can solve Poisson’s equation provided by proper boundary conditions .", "To do so , an anatomically shaped geometrical model of the median nerve ( Jabaley et al . , 1980 ) was generated by image segmentation ( ImageJ , USA ) .", "Coordinates of the image segmentation were exported ( livelink COMSOL-Matlab ) to edit a 3D nerve model ( procedure illustrated in Figure 6—figure supplement 1 ) .", "Perineurium had a thickness of 3% of the diameter of the fascicles ( Grinberg et al . , 2008 ) , and coordinates were interpolated .", "The nerve diameter had a maximum of 4 . 6 mm and minimum of 2 . 3 mm , as obtained from unpublished histological data provided by Dr . Xavier Navarro and from post-mortem human cadavers dissections at Universitat Autonoma de Barcelona , Spain .", "A cylinder , representing the nerve’s outer space , filled with saline , enclosed the nerve .", "Boundary optimal dimensions of cylinder were found to be 69 mm for the diameter and 140 mm for the height , using convergence calculations ( Raspopovic et al . , 2011 ) .", "Electrical ground was therefore fixed in this cylindrical boundary of the structure .", "On the biophysical side , MRG model ( McIntyre et al . , 2002 ) was used to model the nerve tactile sensory fibers .", "This model represents the nonlinear modified Hodgkin-Huxley Equations for the active compartment of the axons ( the nodes of Ranvier ) and a detailed realistic representation of the myelinated tracts .", "The model ( available in NEURON model repository ) is capable of reproducing several experimental aspects of cells dynamics ( McIntyre et al . , 2002 ) .", "Then , a fiber with 21 segments ( Raspopovic et al . , 2011; McIntyre et al . , 2002 ) of nodes of Ranvier was built and extracellular stimulation procedure used to excite the cell .", "For a fiber of diameter D , a model had internodal spacing L = 100D .", "A fiber was considered recruited when a generated action potential travelled along its whole length ( i . e . , reached the last node of Ranvier ) .", "The total recruitment was calculated as the portion of the fibers activated for the specific charge injected with respect to the total number of fibers implemented .", "Furthermore , nodal length was fixed at 1 μm and nodal diameter scaled from a previous study ( McIntyre et al . , 2002 ) .", "A list of plausible assumptions had to be taken , during the model construction .", "The sensory axon populations were constructed ( NEURON , USA ) by using a probabilistic distribution of fibers diameters ( Vallbo and Johansson , 1984 ) for different tactile units of the human hand , resulting in two Gaussian distributions which differentiate nociceptive fibers from fibers responsible for pressure/touch sensation , and the latter was used .", "One of the assumptions constraining the model was that the stimulation of different fiber types , such as nociceptive fibers , was not induced , at used current range .", "A total amount of 100 modeled fibers for each fascicle were placed randomly in the specific target fascicle , for several placements ( Figure 8 ) , as explained in continuation .", "As the fiber organization within different fascicles in the nerve is unknown , we assumed that fibers within one fascicle innervate the same hand area ( Jabaley et al . , 1980 ) ( i . e . , the fibers in one fascicle are innervating a single finger tip and not many of them ) .", "Moreover , since there is an inherent anatomical uncertainty in placement and extension of fibers innervating a specific hand district , for every position of stimulating electrode/needle we implemented 9 populations having different extensions and centroids for the same large fascicle ( i . e . , spanning from having the whole fascicle uniformly populated , to the case of very concentrated population where the fibers are almost touching each-other , Figure 8 and Figure 8—figure supplement 1 ) .", "Then the analysis has been performed for the range of significant fascicle sizes , defined as median size representatives of three groups: small ( 1 population implemented ) , medium ( 5 populations implemented ) and large ( 9 populations ) .", "Different device placements were also studied: within , close , or far from fascicle , finally resulting in n = 90 simulations for TIME and n = 45 simulations cases for microneedle ( this is because for every position of needle tip there are 2 corresponding positions of TIME: left and right ) .", "The microstimulation needle ( FHC UNP40GAS ) was replicated as a cylinder with a cone-like ending ( Figure 6 ) : the cylinder had 3 mm length , and cone ( tip ) with semi-angle 12 . 78° and shank diameter 250 μm .", "The whole structure was insulated and had electrical conductivity of 6 . 67 10–14 S/m and the tip was non-insulated with a conductivity of 1 . 89 107 S/m .", "Since in the case of microstimulation needle the tip was un-insulated by the neurologist , imminently before the insertion , the precise un-insulated cone dimension is not known .", "In order to account for this uncertainty , two models were implemented and the results analyzed for both ( Figure 8 ) .", "The TIME electrode was built as a rectangular structure where seven circled active sites were placed in each side of the structure ( Figure 6 ) .", "Conductivity of the polyimide substrate was set to 6 . 67 10–14 S/m .", "The radius for the active sites was 40 μm and the thickness 300 nm .", "The overall structure had 4 mm length , 0 . 35 mm width and 20 μm thickness .", "The electrical conductivity values used for the FEM were [0 . 0826i 0 . 0826j 0 . 571k] S/m for the endoneurium ( Raspopovic et al . , 2011; McIntyre et al . , 2002 ) , 880 μS/m for perineurium , 0 . 0826 S/m for the epineurium ( Raspopovic et al . , 2011; McIntyre et al . , 2002 ) , 2 S/m for saline .", "Conductivity of perineurium was recalculated from a previous study ( Weerasuriya et al . , 1984 ) taking into account the thickness of the perineurium as 3% of diameter of the fascicle and the difference of temperature between frogs and humans .", "Although TIME electrode was implanted in vivo and no saline placed outside the nerve , this value was used as in previous studies ( Raspopovic et al . , 2011; McIntyre et al . , 2002 ) .", "The similarity between needle and TIME is estimated by comparison of charges necessary for 10% of recruitment of fibers , for respectively 45 and 90 populations computed , by means of Kruskal-Wallis test , with significance level fixed at 0 . 05 ." ] ]

[ "Restoration of touch after hand amputation is a desirable feature of ideal prostheses .", "Here , we show that texture discrimination can be artificially provided in human subjects by implementing a neuromorphic real-time mechano-neuro-transduction ( MNT ) , which emulates to some extent the firing dynamics of SA1 cutaneous afferents .", "The MNT process was used to modulate the temporal pattern of electrical spikes delivered to the human median nerve via percutaneous microstimulation in four intact subjects and via implanted intrafascicular stimulation in one transradial amputee .", "Both approaches allowed the subjects to reliably discriminate spatial coarseness of surfaces as confirmed also by a hybrid neural model of the median nerve .", "Moreover , MNT-evoked EEG activity showed physiologically plausible responses that were superimposable in time and topography to the ones elicited by a natural mechanical tactile stimulation .", "These findings can open up novel opportunities for sensory restoration in the next generation of neuro-prosthetic hands ." ]

[ "Our hands provide us with a wide variety of information about our surroundings , enabling us to detect pain , temperature and pressure .", "Our sense of touch also allows us to interact with objects by feeling their texture and solidity .", "However , completely reproducing a sense of touch in artificial or prosthetic hands has proven challenging .", "While commercial prostheses can mimic the range of movements of natural limbs , even the latest experimental prostheses have only a limited ability to ‘feel’ the objects being manipulated .", "Oddo , Raspopovic et al . have now brought this ability a step closer by exploiting an artificial fingertip and appropriate neural interfaces through which different textures can be identified .", "The initial experiments were performed in four healthy volunteers with intact limbs .", "Oddo , Raspopovic et al . connected the artificial fingertip to the volunteers via an electrode inserted into a nerve in the arm .", "When moved over a rough surface , sensors in the fingertip produced patterns of electrical pulses that stimulated the nerve , causing the volunteers to feel like they were touching the surface .", "The volunteers were even able to tell the difference between the different surface textures the artificial fingertip moved across .", "The temporary electrodes used in this group of volunteers are unsuitable for use with prosthetic limbs because they can easily be knocked out of position .", "Therefore , in a further experiment involving a volunteer who had undergone an arm amputation a number of years previously , Oddo , Raspopovic et al . tested an implanted electrode array that could , in principle , remain in place long-term .", "This volunteer could also identify the different textures the artificial fingertip touched , with a slightly higher degree of accuracy than the previous group of intact volunteers .", "Further studies are now required to explore the potential of this approach in larger groups of volunteers ." ]

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

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

[ [ "Superfast muscles ( SFMs ) are the fastest known synchronous muscle phenotypes in vertebrates ( Rome , 2006 ) .", "Their ability to repetitively contract and relax fast enough to produce work at cycling rates over around 90 Hz and up to 250 Hz sets them apart from other muscles by almost two orders of magnitude and allows the execution of central motor commands with millisecond temporal precision ( Rome , 2006; Rome et al . , 1988; Elemans et al . , 2004 , 2008 , 2011 ) .", "The phenotype is defined by its mechanical performance and therefore establishing a muscle to be superfast requires quantification of its performance-profile ( Rome , 2006 ) .", "Although once considered an extremely rare muscle phenotype , SFMs have now been established in most major vertebrate lineages ( mammals [Elemans et al . , 2011] , birds [Elemans et al . , 2004; Elemans et al . , 2008] , reptiles [Rome et al . , 1996] , ray-finned fish [Rome et al . , 1996] ) .", "SFMs in the bat larynx control the rapid , high frequency calls used by laryngeally echolocating bats to detect , orient to and track prey ( Elemans et al . , 2011 ) .", "SFMs in the songbird syrinx control the precisely timed , rapidly produced acoustic elements and frequency sweeps of bird vocalizations ( Elemans et al . , 2004 , 2008 ) used for territorial defense and mate attraction ( Collins , 2004 ) .", "SFMs in the Oyster toadfish swimbladder and rattlesnake tailshaker both set the fundamental frequency of the produced sound by rhythmically contracting the swimbladder and shaking tail rattles respectively ( Rome et al . , 1996 ) .", "Other muscles have been suggested to be of the superfast phenotype , such as some extraocular or limb muscles ( Fuxjager et al . , 2016 ) , but non-isometric mechanical tests are lacking to classify them as such .", "Taken together , SFMs seem a commonly occurring muscle phenotype that interestingly so far has been established only in motor systems involving sound production and control .", "Force modulation in vertebrate skeletal muscles is precisely timed via the highly conserved excitation-contraction coupling ( ECC ) pathway that consists of several sequential steps whose individual kinetics affect the maximally attainable force modulation speed .", "In brief , firing of a motor neuron first triggers the release of intracellular calcium ions ( Ca2+ ) stored in the sarcoplasmic reticulum ( SR ) .", "Subsequent binding of Ca2+ to troponin in sarcomeres triggers the exposure of binding sites for the motor protein myosin along actin filaments , allowing the cyclical binding and unbinding of myosin motor head domains to actin , forming actomyosin crossbridges that generate force .", "Finally , force decreases when SR Ca2+-ATPases ( SERCA ) pump Ca2+ back into the SR , consecutively lowering the cytoplasmic free Ca2+ concentration ( [Ca2+]i ) and returning thin filament inhibition thus preventing further crossbridge formation .", "Extensive study of SFMs in the oyster toadfish ( Opsanus tao ) swimbladder has demonstrated that no single step in the ECC pathway is rate-limiting , but that multiple hallmark traits have adapted to allow superfast cycling rates .", "In swimbladder SFMs , a far greater proportion of cellular volume is dedicated to SR compared to locomotory muscles , which increases the number and density of SERCA pumps ( Rome et al . , 1999 ) .", "Furthermore small , ribbon-like myofibrils ( Appelt et al . , 1991 ) greatly reduce diffusion distances for Ca2+ .", "Together , these adaptations lead to the shortest [Ca2+]i transient times observed in any muscle ( Rome et al . , 1996 ) .", "Additionally , the rate of actomyosin crossbridge detachment in SFM of the toadfish swimbladder is extremely fast , which is necessary to ensure a rapid force drop after the return of actin filament inhibition ( Rome et al . , 1999 ) .", "Whether these hallmark ECC pathway adaptations are shared with avian ( songbird syrinx ) and mammalian ( bat larynx ) SFMs are currently unknown .", "In vertebrates , kinetic tuning of the actomyosin crossbridge cycle for different energetic and biomechanical requirements is accomplished largely via the differential expression of specific myosin heavy chain ( MYH ) genes each with unique kinetic properties ( Bottinelli et al . , 1991 ) .", "Because of its thoroughly characterized phylogeny ( Desjardins et al . , 2002 ) and a demonstrated role in force generation ( Schiaffino and Reggiani , 2011 ) the MYH gene family is well suited to shed light onto the evolutionary origin and timing of SFM phenotypes .", "However , MYH characterization and expression are currently not known for any SFM .", "Bat and songbird SFM are excellent models to", "( i ) determine whether SFMs share a common evolutionary origin and to", "( ii ) improve our understanding of muscle function , because sequenced genomes ( Warren et al . , 2010 ) and identified neural substrates for quantifiable , learned vocalizations exist in some of these taxa ( Fee and Scharff , 2010; Brainard and Doupe , 2013; Moss and Sinha , 2003 ) .", "Interestingly , functional trade-offs associated with the above ECC adaptations do appear to be common to all SFMs .", "The fast actomyosin detachment rate of SFM in the toadfish swimbladder , in the absence of a commensurate increase in the rate of crossbridge formation , reduces the proportion of myosin motors actively bound to actin at a given time , and thus force and power production ( Rome et al . , 1999 ) .", "Furthermore , the increased volumetric allocation for required SR and mitochondria leave less space for contractile machinery , further reducing volume-specific force and power ( Rome and Lindstedt , 1998 ) .", "Consequentially , the SFMs in swimbladder and tailshaker trade force for speed , constraining movement to very low masses at low efficiency ( Rome et al . , 1999; Rome and Lindstedt , 1998; Young and Rome , 2001 ) .", "SFMs in bat , songbird and toadfish , which produce work up to similar cycling limits ( around 200 Hz ) , develop very similar low force ( 10–20 kN/m2 ) ( Rome , 2006; Elemans et al . , 2004 , 2008 , 2011; Rome et al . , 1996 ) .", "Because of the bias for the SFM phenotype to have evolved in sound production systems we asked whether this apparent functional convergence in SFM force profiles is ( 1 ) the result of selective pressures common to motor control of sound production and modulation , or ( 2 ) due to constraints inherent to the otherwise conserved architecture of synchronous vertebrate sarcomeric muscle , or ( 3 ) both ?", "Here , we test if SFMs share a common evolutionary origin by characterizing MYH gene expression in the SFM of zebra finch ( Taeniopygia guttata ) syrinx and Daubenton’s bat ( Myotis daubentonii ) larynx .", "The employment of evolutionarily and ontologically distinct MYH genes suggests separate evolutionary origins of SFM .", "Furthermore , by quantifying cellular morphometry and [Ca2+]i signal transduction in all known SFM , we show that all SFMs share hallmark adaptations in the ECC pathway and have identical intracellular calcium dynamics at their operating temperatures in vivo .", "Our data suggest that SFMs converge at a maximum speed allowed by fundamental constraints in vertebrate synchronous muscle architecture .", "This implies that motor control of complex acoustic communication is fundamentally limited by synchronous vertebral muscle architecture ." ], [ "To test the hypothesis that SFMs in the bat larynx and songbird syrinx share a common evolutionary origin ( Figure 1 ) , we used sequenced genomes of zebra finch ( Warren et al . , 2010 ) and little brown bat ( Myotis lucifigus ) , an echolocating bat in the same genus as Daubenton’s bat , to identify the dominant MYH genes expressed in their SFMs .", "Among known MYH genes , orthologs of human MYH13 are prime candidates for superfast motor performance ( Bloemink et al . , 2013 ) .", "In humans and other mammals , MYH13 and five other MYH genes ( MYH8 , MYH4 , MYH1 , MYH2 , MYH3 ) are linked head-to-tail in what is known as the fast/developmental cluster ( Desjardins et al . , 2002 ) , which arose from multiple gene duplication events , the first believed to have occurred in an early tetrapod ( Ikeda et al . , 2007 ) .", "Here , we identified the orthologous fast/developmental MYH gene clusters in syntenic regions of zebra finch and little brown bat genomes ( Figure 2a , b ) .", "To place putative superfast bird and bat MYH genes in the context of well-characterized vertebrate fast myosin evolution , we additionally used human MYH13 and the slow/cardiac MYH7 motor domain amino acid sequence to identify predicted MYH genes by BLASTp in the large flying fox ( Pteropus vampyrus ) ( Kersey et al . , 2016 ) , chicken ( Gallus gallus ) ( International Chicken Genome Sequencing Consortium , 2004 ) , Burmese python ( Python molurus ) ( Castoe et al . , 2013 ) , clawed frog ( Xenopus tropicalis ) ( Nasipak and Kelley , 2008 ) , and torafugu puffer fish ( Takifugu rubripes ) ( Aparicio et al . , 2002 ) .", "Phylogenetic analysis of myosin rod amino acid sequence revealed orthologs of MYH13 in syntenic genomic regions of all mammalian and avian species included in our analysis corroborating previous studies ( Desjardins et al . , 2002; Nasipak and Kelley , 2008 ) .", "We also found further lineage-specific expansions of fast/developmental clusters ( Figure 2b ) , though gene convergence events complicate precise phylogenetic reconstruction of more recently diverging genes ( Desjardins et al . , 2002 ) .", "Some ray-finned fishes , including torafugu , only have a single MYH gene in duplicated genomic regions syntenic to the tetrapod fast/developmental cluster ( Ikeda et al . , 2007 ) , supporting the view that the duplication event at this locus , which gave rise to an ancestral MYH13 postdated the tetrapod/ray finned fish last common ancestor .", "Interestingly , laryngeal muscles of the frog , which power vocalizations in the range of 70 Hz , express a MYH gene ( MyHC-101d ) that does not appear to have descended from the ancestral fast/developmental locus ( Nasipak and Kelley , 2008 ) .", "The python possesses an ortholog of MYH3 ( LOC103063479 ) at the expected 5’ end of the cluster , but the presence of a 3’ flanking MYH13 ortholog is unknown due to the presence of a scaffold end .", "To test the hypothesis that MYH13 orthologs are expressed in bat laryngeal and zebra finch syringeal SFMs , we first used a panel of MYH13-specific commercial and custom-made antibodies ( See Materials and methods ) .", "Surprisingly , we did not find immunoreactivity against MYH13 in bat laryngeal SFM ( Figure 3a ) .", "However , a fast myosin antibody ( See Materials and methods ) , which binds gene products of MYH 1 , 2 , and 4 , showed immunoreactivity .", "In agreement with that , sequenced amplicons of qPCR analysis revealed that bat SFM laryngeal muscle is highly enriched for the mammal-lineage-specific MYH4 transcript , which codes for a locomotory myosin also known as MyHC-2B ( Figure 3b ) .", "In male zebra finch syrinx SFM , multiple attempts at immunostaining were unsuccessful ( see Materials and methods ) , but sequenced amplicons of qPCR analysis unambiguously showed that it was highly enriched for the MYH13 ortholog transcript ( Figure 4a ) .", "Only one other myosin gene ( H0Z747 ) was expressed and we could not distinguish whether this myosin was expressed in different cells , as suggested by observation of fast myosin antibody staining in starlings ( Uchida et al . , 2010 ) and zebra finches ( Figure 4—figure supplement 1 ) , or co-expressed in the same cells .", "Extraocular muscle expressed MYH13 at the same level as syringeal SFM ( p=0 . 06 , Kruskal Wallis ) , and also four additional MYH genes from the fast/developmental cluster ( Figure 4a ) .", "To identify possible species-specific adaptations to MYH4 in the echolocating bat , and to MYH13 in the zebra finch that could affect crossbridge kinetics , we aligned predicted motor domain amino acid sequence from these genes to their orthologs in the genomes of human , large flying fox and chicken , respectively , focusing on two hypervariable regions identified as potential modifiers of crossbridge kinetics ( Figure 2c , d ) .", "Loop one is situated near the nucleotide binding pocket , and its flexibility is thought to influence the rate of ADP dissociation , and thus is a potentially critical region for the rapid crossbridge detachment seen in SFM ( Ikeda et al . , 2007 ) .", "The only non-synonymous sequence difference between little brown bat and human MYH4 in loop 1 , an insertion of a glycine at position 214 , is also shared with the large flying fox .", "Although human MYH13 is known to possess rapid ADP dissociation ( Bloemink et al . , 2013 ) , the zebra finch ortholog differs from those of both human and chicken by the substitution of a proline at position 210 .", "Loop two potentially affects maximum shortening velocity via influence on actin-myosin binding ( Lorenz and Holmes , 2010 ) .", "Both zebra finch MYH13 and bat MYH4 possess amino acid substitutions in loop two and in the case of the finch the insertion of a glycine at position 643 not shared by chicken , flying fox , or human orthologs .", "To further explore the relationship between MYH gene expression and muscle speed , we next investigated properties of songbird SFM in three groups of zebra finches that differ with respect to song production: juvenile males during the song learning phase singing juvenile , variable song , adult males singing adult , invariable song , and females that do not sing .", "All three groups produce different types of mostly unlearned calls .", "Female syringeal SFMs have two-fold slower twitch kinetics than male syringeal SFMs ( Elemans et al . , 2008 ) , and expressed more than five times less MYH13 compared to male syringeal SFMs ( Figure 4b ) .", "Juvenile male songbirds develop their vocal behavior by sensory-guided motor practice in a process bearing many parallels to human speech acquisition ( Fee and Scharff , 2010; Brainard and Doupe , 2013 ) .", "Over the 75-days long sensorimotor phase young zebra finches produce vocalizations that slowly change in acoustical parameters ( Tchernichovski et al . , 2001 ) .", "During this period , the variability of acoustic output decreases and accuracy increases , which is attributed to increasingly precise timing of vocal motor pathway activity ( Ölveczky et al . , 2011 ) .", "To see whether these vocal changes are associated with changes in SFM performance , we first quantified MYH expression in syringeal SFM over song ontogeny .", "Of all MYH genes , only MYH13 changed significantly with age ( p=0 . 02 , KW-test ) and was significantly upregulated progressively ( p<0 . 05 , Tukey-Kramer post hoc ) in juvenile male syringeal SFM ( Figure 4c ) .", "Second , we found that muscle twitch duration significantly decreased progressively ( p<0 . 05 , Wilcoxon-signed rank tests ) during the sensorimotor period of vocal learning ( Figure 4d ) .", "Taken together , these results suggest that in zebra finch syrinx SFM levels of MYH13 expression are negatively associated with twitch duration and thus positively with muscle speed .", "Because the above results established that SFMs do not share a common evolutionary origin , we next investigated whether SFM share adaptive mechanisms to ECC pathway traits .", "We first examined whether cellular adaptations for rapid calcium transients are present across all known SFMs .", "Representative transmission electron-microscopy cross-sections of SFMs in zebra finch syrinx , bat larynx , toadfish swimbladder and rattlesnake tailshaker showed strikingly more SR compared to intraspecific skeletal muscles ( Figure 5a ) .", "Based on transmission electron-microscopy images , we quantified volumetric percentages of sarcoplasmatic reticulum , mitochondria and myofibrils in all four SFM and intraspecific skeletal muscles ( Figure 5b ) .", "Zebra finch syrinx SFMs contained 15 ± 1 , 22 ± 2 and 50 ± 2% of SR , mitochondria and myofibrils , respectively ( N = 3 ) and transmission electron-microscopy images resembled those of dove ( Elemans et al . , 2006 ) and oilbird ( Suthers and Hector , 1985 ) syrinx muscles .", "Bat larynx SFMs contained 24 ± 2 , 31 ± 4 and 34 ± 2% of SR , mitochondria and myofibrils , respectively ( N = 2 ) ( Figure 5b ) .", "SR volume was ≥15% and SR per myofibril ratio was ≥30% for all SFMs and significantly higher ( p<0 . 05 , KW test ) compared to intraspecific skeletal muscles in zebra finch , bat and rattlesnake .", "Thus the morphometric cellular adaptation of larger SR volume to increase signal transduction ( Rome , 2006; Rome and Lindstedt , 1998 ) is a hallmark adaptation present in all SFM .", "We investigated whether [Ca2+]i transients in songbird syrinx and bat larynx SFM attain similar extreme kinetics as toadfish swimbladder and rattlesnake tailshaker SFM ( Rome et al . , 1996 ) .", "We measured force and real-time [Ca2+]i dynamics in zebra finch syrinx and toadfish swimbladder SFM as a function of temperature ( Figure 6 ) .", "At their respective operating temperatures of 39°C and 25°C , the full width at half maximum ( FWHM ) values of calcium transients for zebra finch syrinx and toadfish swimbladder SFMs did not differ significantly ( p=0 . 54 , one-tailed paired t-test ) and measured 1 . 98 ± 0 . 65 ms ( N = 4 ) and 1 . 91 ± 0 . 69 ms ( N = 2 ) , respectively .", "The FWHM[Ca2+]i in rattle snake tailshaker SFM was 1 . 5 ms at 35°C ( Rome et al . , 1996 ) .", "Taken together , the FWHM[Ca2+]i values for SFMs in zebra finch , toadfish , and rattlesnake converged to 1 . 5–2 . 0 ms at their operating temperatures ( Figure 6b ) .", "In toadfish SFMs , the magnitude of [Ca2+]i transients and ATP-usage reduces from the first to consecutive twitches that make up the vast majority of the boatwhistle call ( Harwood et al . , 2011 ) .", "If the FWHM of [Ca2+]i transients also decreases from first to consecutive twitches , our above comparison would underestimate the speed of the toadfish SFM transient .", "Therefore , we quantified [Ca2+]i kinematics as a function of twitch number in toadfish and zebra finch SFM at operational temperatures .", "While the magnitude of the [Ca2+]i transient decreased from first to consecutive twitches confirming earlier work on toadfish SFM ( Harwood et al . , 2011 ) , the FWHM of the [Ca2+]i transients did not change as a function of twitch number ( Figure 6—figure supplement 1 ) .", "In zebra finch SFMs , there was no significant magnitude ( KW , p>0 . 05 ) or FWHM decrease ( KW , p>0 . 05 ) of [Ca2+]i transients as a function of twitch number ( Figure 6—figure supplement 1 ) .", "Because of limited tissue availability of bat laryngeal SFMs , we used an in vitro assay to quantify calcium uptake and release rates of homogenized SR vesicles as a proxy for whole cell behavior ( see Materials and methods ) .", "This technique yields comparable values for SFMs in zebra finch , toadfish and bat , which were significantly elevated ( p<0 . 01 , ANOVA ) 5–10 times compared to intraspecific skeletal muscle and also compared to the comparable rat fast twitch m .", "extensor digitorum longus ( Figure 6c , Supplementary file 1A ) .", "These data thus suggest that bat larynx SFMs have equally fast calcium dynamics as zebra finch syrinx and toadfish swimbladder SFMs .", "This in turn implies that all established SFMs have similar speeds of calcium dynamics at their operating temperatures in vivo .", "In conclusion , all SFM phenotypes demonstrate hallmark cellular adaptations and functionally convergent calcium transduction dynamics consistent with superfast cycling rates ." ], [ "We identified the MYH genes that power superfast motor performance in songbird syrinx and bat larynx muscle: zebra finch syrinx SFMs predominantly express the avian ortholog of MYH13 , which encodes superfast MyHC-sf ( aka extraocular MyHC-eo ) , while Daubenton’s bat larynx SFMs express an ortholog of the mammalian locomotory MYH4 ( MyHC-2b ) .", "Our analysis is consistent with earlier findings ( Desjardins et al . , 2002; Ikeda et al . , 2007 ) that both MYH13 and MYH4 were the result of gene duplication events that occurred in tetrapods , and would , therefore , have no ortholog in the ray-finned fish , the taxon to which the toadfish belongs .", "Which MYH gene is expressed in the rattlesnake tailshaker SFM remains unknown , but no MYH13 ortholog was found in the Burmese python , which could mean that the gene was lost , or the genome is incomplete and the fast/developmental cluster was truncated by the end of a scaffold .", "Since extremely fast crossbridge kinetics are necessary , though not sufficient , for superfast performance ( Rome , 2006 ) , and these are set largely by MYH gene expression in vertebrates ( Bottinelli et al . , 1991 ) , the parsimonious conclusion is that the SFM phenotype found in the sound-producing organs of bats , songbirds , and toadfish descends from different evolutionary events .", "We further show that all SFMs share hallmark ECC pathway adaptations to speed up signal transduction .", "First , we establish that all SFMs converge upon identical speed of in vivo calcium dynamics , which can be explained by increased SR per myofibril volume ratio .", "Additional mechanisms to increase the speed of [Ca2+]i transients are also possible and include ( 1 ) adaptations that affect the function of ryanodine receptors or the SERCA pumps ( Nelson et al . , 2016 ) , ( 2 ) faster Ca2+ detachment from troponin due to differential expression of Troponin C genes , or ( 3 ) the employment of calcium-sequestering proteins such as parvalbumin ( Rome , 2006 ) that have already been shown to play an important role in toadfish ( Harwood et al . , 2011; Tikunov and Rome , 2009 ) .", "The potential contribution of these mechanisms needs further investigation across all SFMs .", "Second , actomyosin crossbridge cycling with a higher than normal detachment rate also appears to be a hallmark of SFM , as evidenced by consistently low developed isometric tensions in all cases ( Rome , 2006; Elemans et al . , 2004 , 2008 , 2011 ) .", "The expression of a MYH13 ortholog in zebra finch syrinx SFM is consistent with the human protein’s superfast kinetics ( Bloemink et al . , 2013 ) , but if and how the observed predicted protein sequence differences between zebra finch and human MYH13 orthologs affect crossbridge kinetics remains unknown .", "Similarly , further investigation is required to understand how the normally much slower and higher force locomotory MYH4 realizes superfast crossbridge kinetics in bats .", "The insertion of a second glycine in loop one may be a potential mechanism to increase crossbridge cycling by altering mechanical flexibility of the loop .", "However , this insertion is shared by the large flying fox that does not produce calls at high repetition rates , which casts doubt on the insertion being a SFM phenotype specific adaptation .", "Importantly , superfast crossbridge kinetics may also be the result of adaptations independent of MYH genes , so that potential modifiers of crossbridge kinetics , such as myosin light chain genes and phosphorylation state ( Sweeney and Stull , 1990 ) , also warrant further study .", "Both mechanistic as well as functional characteristics are convergent in SFMs .", "Previous observations show that SFMs from diverse taxa have remarkably similar maximum performance characteristics ( force and cycling frequency ) ( Rome , 2006; Elemans et al . , 2004 , 2008 , 2011; Rome et al . , 1996; Moon et al . , 2002 .", "Here , we demonstrate that the similarity extends to the speed of signal transduction itself within the ECC pathway ( calcium release and reuptake ) , and to mechanisms of attaining those speeds ( volume and distribution of SR ) .", "Because we establish SFM’s divergent origin , we propose that these similarities must be attributed to convergent evolution .", "There is good evidence for selective pressures on high cycling speeds in the motor systems in which SFMs have evolved: female canaries prefer faster trill rates ( Drăgănoiu et al . , 2002 ) , echolocating bats could produce much higher call rates before introducing call-echo ambiguity to their sensory system ( Elemans et al . , 2011 ) and in toadfish call fundamental frequency , which is set 1:1 by muscle speed ( Elemans et al . , 2014 ) , positively correlates with male fitness ( Amorim et al . , 2010 ) .", "Thus faster muscles would be expected to evolve .", "What mechanisms set the maximum speed to power motion in vertebrates ?", "In toadfish swimbladder SFMs , a trade-off between force-generating capacity and speed is well-established and results from two constraints related to", "( i ) crossbridge dynamics and", "( ii ) increased ECC pathway requirements ( Rome , 2006 ) .", "The consequence of fast relaxation rate is that a lower proportion of myosin motors is bound to actin at a given time reducing the developed force and , by extension , the specific force of the myofibril as a whole ( Rome et al . , 1999 ) .", "While detachment rates of intact toadfish SFM fibers ( Rome et al . , 1999 ) and isolated MYH13 ( Bloemink et al . , 2013 ) are very high , attachment rates approximate that of locomotory muscle , and are likely limited by diffusion of the unbound myosin head ( Rome et al . , 1999 ) .", "Furthermore , the necessity for very rapid ECC signal transduction requires more cell volume dedicated to increased demands on calcium dynamics and ATP production , limiting space for myofibrils , further diminishing force generation per unit volume of muscle ( Rome and Lindstedt , 1998 ) .", "Importantly , additional SR and mitochondria are likely to affect the muscle cell’s viscoelastic properties .", "The ultimate limit to how much force can be traded for speed , while the muscle still maintains the ability to do external work , is dictated by the minimum force required to overcome unavoidable viscous losses associated with shortening .", "Taken together , these observations support the view that SFMs converge at a maximum speed allowed by fundamental constraints in vertebrate synchronous muscle architecture .", "Interestingly , only arthropods have pushed the envelope of force/speed tradeoffs further than vertebrate SFMs .", "In insect asynchronous flight muscle force cycling is uncoupled from calcium cycling , minimizing force loss and allowing for high power production at similar cycling speeds to SFM ( Syme and Josephson , 2002 ) .", "However , this increase in power at high speeds is at the great cost of sacrificing precise temporal control of contraction ( Syme and Josephson , 2002 ) .", "The cicada’s synchronous tymbal muscles cycle at over 500 Hz ( Josephson and Young , 1985 ) , but it is unknown if mass reduction or adaptations to the ECC account for this extreme performance .", "In the much larger vertebrates , the low forces developed by SFMs restrict them to low-mass systems as found in sound production and modulation where precise control is essential .", "Myoblast lineage and postnatal motor activation patterns and exercise can play crucial roles in the development of muscle groups or allotypes ( Spencer and Porter , 2006 ) and can alter myosin expression in craniofacial ( Rhee et al . , 2004; Brueckner et al . , 1999 ) and body ( axial ) muscle ( Pette and Vrbová , 1985 ) .", "Our discovery of MYH13 ortholog expression in avian syrinx SFM corroborates earlier developmental studies ( Noden et al . , 1999; Noden and Francis-West , 2006 ) and places syringeal myogenic precursor cells in the craniofacial muscle group , because this muscle group can uniquely express MYH genes , such as MYH13 , 15 and 16 , that have never have been found in axial somatic muscle cells .", "In juvenile songbirds , MYH13 expression was progressively upregulated during the sensorimotor period of vocal learning and positively associated with muscle speed ( Figure 4c , d ) .", "This observation raises the interesting questions whether MYH13 upregulation and increase of muscle speed in songbird syrinx SFMs is causally related and due to a set developmental program , hormonal influences , muscle exercise in the form of increased neural stimulation , or some combination of these factors .", "Interestingly , the SFM in the larynx of juvenile bats may also increase speed during the first 30 days after birth as suggested by an increase in frequency modulation speed of echolocation calls ( Moss et al . , 1997 ) .", "In other motor systems , neural stimulation can drive MYH expression patterns: neural activity associated with optokinetic and vestibulo-ocular reflexes stimulates particularly MYH13 expression in rat extraocular muscle ( Brueckner et al . , 1999; Moncman et al . , 2011 ) , and transnervation from cranial nerve X with XII increases speed of laryngeal muscles in dogs ( Paniello et al . , 2001 ) .", "Because in extraocular muscle MYH13 is found close to the neuromuscular junction ( Briggs and Schachat , 2002 ) , a possible mechanism could be that both electrical and chemical activation of the motor neuron directly stimulate MYH13 production linked to actetylcholine receptors ( Rubinstein et al . , 2004; Sanes and Lichtman , 2001 ) .", "We speculate that vocal muscle training can be associated with optimizing SFM function .", "Reversely , our data suggest that , next to neural constraints , SFMs can peripherally constrain the precision to execute skilled motor sequences during birdsong and echolocation call ontogeny .", "To conclude , the non-orthology of the dominant myosin heavy chains expressed in SFMs of birds , mammals , and fish indicates that SFMs likely evolved independently in each lineage .", "However , these independent SFM each employs similar qualitative and quantitative adaptations to ECC , and arrive at near identical maximum performance .", "Taken together , these results suggest that SFMs operate at the maximum speed allowed by vertebrate synchronous muscle architecture .", "In each case , SFM evolution involved the same specific set of constraints ( high cycling speeds and synchronous control ) and allowances ( minimal force and power ) , particular to motor systems associated with sound production .", "Motor-driven acoustic modulation rates in complex communication are thus fundamentally limited by muscle architecture ." ], [ "All procedures were carried out in accordance with the Danish Animal Experiments Inspectorate ( Copenhagen , Denmark ) .", "Songbird: All bird data presented were collected in zebra finches ( Taeniopygia guttata ( Vieillot , 1817 ) .", "All adult birds ( older than 100 days ) were kept in group aviaries at the University of Southern Denmark , Odense , Denmark , and juvenile zebra finches used for muscle kinetics measurements were raised in separate cages together with both parents and siblings at 13 hr light:11 hr dark photoperiod and given water and food ad libitum .", "The juvenile male zebra finches used for MYH mRNA quantification came from the long-term breeding colony at the Freie Universität Berlin , Germany .", "These animals were kept on a 14 hr light:10 hr dark photoperiod and given water and food ad libitum .", "We dissected the syringeal muscle tracheobronchialis ventralis ( VTB ) on ice immediately after isoflurane euthanasia ( Elemans et al . , 2008 ) .", "Extraocular ( m . rectus and m . oblique; EOM ) , flight ( m . pectoralis; PEC ) and leg ( m . tibialis anterior; TA ) muscles were collected as reference tissues .", "Toadfish: Adult male oyster toadfish ( Opsanus tau , Linnaeus 1766 ) were obtained from the Marine Biological Laboratories ( MBL , Woods Hole , MA , USA ) and housed individually in 80 × 40×40 cm seawater tanks on constant flow-through of fresh seawater at the Fjord and Bælt field station , Kerteminde , Denmark .", "Toadfish were euthanized by a blow on the head and double pithing .", "Superfast swimbladder muscle was isolated bilaterally from the midsection of the swimbladder .", "Bats: Efforts were focused on Daubenton’s bat ( Myotis daubentoni ) where superfast behavior of the anterior portion of the laryngeal muscle m .", "cricothyroideus anterior ( ACTM ) was previously established ( Elemans et al . , 2011 ) .", "We obtained permission ( Licenses SNS-3446–00001 and NST-3446–00001 ) to capture four individuals of Myotis daubentoni from the Skov- og Naturstyrelsen Inspectorate ( Denmark ) .", "After isoflurane euthanasia , the ACTM muscle was isolated and extraocular and leg ( m . tibialis anterior , TA ) muscles were collected as reference tissues .", "Rats: Adult male Sprague Dawley rats ( Rattus norvegicus ) were purchased from the Institute of Biomedicine , Odense University Hospital .", "The rats were housed in cages with a 12 hr:12 hr light:dark cycle and provided unrestricted access to water and food .", "The rats were killed by a blow on the head followed by cervical dislocation .", "M . soleus ( SOL ) and m .", "extensor digitorum longus ( EDL ) were collected .", "Fast/developmental myosin heavy chain gene clusters were identified in the genomes of Myotis lucifigus and Taeniopygia guttata by BLASTP using ENSEMBL genome resources ( http://useast . ensembl . org/Myotis_lucifugus/Info/Index and http://useast . ensembl . org/Taeniopygia_guttata/Info/Index , RRID:SCR_006773 ) with the rod domain of human MYH3 ( conserved motor domain/rod junction proline 839 to C terminus ) as query sequence .", "Seven Ensembl genebuild annotated predicted proteins were clustered on forward strand of the zebra finch chromosome 18 .", "Six predicted proteins in bat were similarly clustered on the reverse strand of scaffold GL430049 ( Supplementary file 1B ) .", "The Ensemble annotation for little brown bat MYH13 was missing exons 1 through 12 .", "Fgenesh gene-finder ( hidden markov model based gene structure prediction - http://linux1 . softberry . com/berry . phtml ? topic=fgenesh&group=programs&subgroup=gfind , RRID:SCR_011928 ) was used to partially reconstruct the full-length gene , including loop 1 and loop two subdomains , from flanking genomic sequence ( Figure 2 ) .", "TBLASTN using the same query sequence returned no additional MYH domains in these genomic regions .", "Fast/developmental cluster and cardiac orthologs from flying fox , chicken , and clawed frog genomes were identified by BLAST using ensemble genome resources , and NCBI genome , with human MYH3 and MYH7 ( conserved motor domain/rod junction proline 839 to C terminus ) as query sequence .", "Torafugu sequences were obtained directly from ( Ikeda et al . , 2007 ) .", "2D dot-plot analysis of genomic DNA vs . human MYH3 protein sequence was used to identify clawed frog genes described in ( Nasipak and Kelley , 2008 ) , which had been named using scaffold names from an earlier assembly ( Supplementary file 1B ) .", "Fgenesh gene finder was used to reconstruct MyHC-101d sequence from genomic sequence on scaffold 172772 . 1 .", "Predicted protein sequences were downloaded and analyzed using Macvector 14 . 5 software ( Macvector Inc . Apex , NC ) .", "Human MYH sequences were obtained from the NCBI protein database ( Supplementary file 1B ) .", "To remove the influence of insertions/deletions , multiple protein sequence alignments of human sarcomeric MYHs , along with non-muscle MYH9 , were performed using Clustal W ( Thompson et al . , 1994 ) in Macvector on rod domains from the conserved proline at the motor domain/rod junction ( 839 in MYH3 ) to the C terminus .", "To reconstruct the phylogeny of fast/developmental cluster MYH genes from zebra finch , little brown bat , chicken , large flying fox , Burmese python , clawed frog , torafugu and human genomes ( Warren et al . , 2010; Kersey et al . , 2016; International Chicken Genome Sequencing Consortium , 2004; Castoe et al . , 2013; Nasipak and Kelley , 2008; Aparicio et al . , 2002 ) , alignments were performed on 556-residue regions of the rod domain starting with the conserved alanine ( 1331 in human MYH3 ) .", "Here full-length rods were not aligned due to a region of unknown sequence in bat MYH13 .", "Maximum likelihood trees were generated and branch lengths calculated in Macvector using the Neigbor Joining method ( Saitou and Nei , 1987 ) rooted by the non-muscle MYH9 ( Figure 2a , not shown ) .", "Bootstrap analysis from 1000 replicates was used to evaluate internal branches .", "For the purposes of comparing relevant hypervariable regions within the motor domain , Clustal W alignments were performed on N-terminal motor domain predicted protein sequence , terminating at the conserved most-c-terminal proline ( 839 in human MYH3 ) from human MYH13 , little brown bat and flying fox MYH4; chicken and zebra finch MYH13 .", "The three-dimensional homology model in Figure 2c was generated for human MYH3 protein , with scallop myosin head structures ( ProteinData Bank code 1kk8 ) as a template , using the SWISS-MODEL ( RRID:SCR_013032 ) server ( Arnold et al . , 2006 ) as in ( Bloemink et al . , 2013 ) .", "Muscle tissue was rapidly dissected and frozen in liquid nitrogen after sacrifice .", "Immunofluorescence staining was carried out on 5 µm thick frozen sections .", "After initial washing with PBS three times for 5 min , sections were incubated for 20 min in a 1% solution of Triton X-100 ( Mannheim , Germany ) in 0 . 01 M PBS ( Roche , Mannheim , Germany ) and rinsed in PBS three times .", "The sections were then incubated in 10% normal goat serum ( Life technologies , MD , USA ) for 15 min , and were thereafter incubated with a mouse monoclonal anti-myosin ( Skeletal , fast ) antibody ( clone MY-32 ( RRID:AB_784724 ) , M4276 ( RRID:AB_477190 ) , Sigma-Aldrich , MO , USA ) , diluted 1:100; a rabbit polyclonal myosin 13/Extraocular myosin ( Hinge region ) ( MP4571 ( RRID:AB_2713977 ) , ECM Biosciences , KY , USA ) , diluted 1:50; and a rat monoclonal anti-Laminin 3 alpha antibody ( 4H8-2 , ab11576 ( RRID:AB_298180 ) , Abcam , MA , USA ) , diluted 1:500 in PBS with BSA for 60 min at 37°C .", "The skeletal fast antibody MY-32 binds to MYH1 , 2 and 4 .", "To our knowledge no existing antibody can reliably distinguish between these myosins .", "After incubation with antiserum and PBS wash , a new incubation in 10% normal goat serum followed , after which the sections were incubated in goat anti-mouse IgG1 , Human ads-TRITC ( 1070–03 , SouthernBiotech , AL , USA ) , donkey anti-rabbit IgG-TRITC ( 711-025-152 , Jackson ImmunoResearch , PA , USA ) and goat anti-Rat IgG Alexa 488 ( ab150157 , Abcam , MA , USA ) diluted 1:300 for 30 min at 37°C .", "The sections were thereafter washed in PBS and then mounted in Vectashield Mounting Medium ( H-1500 ) with DAPI ( Vector Laboratories , CA , USA ) .", "Images were taken using Leica DM6000 at University of Pennsylvania Microscopy Core Facility .", "We explored six antibodies to identify MYH13 in zebra finch , but all attempts were unsuccessful .", "Summarizing , we tried to raise one poly- and one monoclonal antibody against a peptide derived from ortholog-specific loop 2 sequence , tested three commercially available antibodies ( of which one was used successfully in bats above ) , and tested one antibody used successfully in rabbits ( Rhee and Hoh , 2008 ) .", "All these antibodies were tested by experienced workers and were either unsuccessful or lacked positive identification ( i . e . absence of evidence ) .", "Muscle tissues were rapidly dissected and frozen in liquid nitrogen after sacrifice .", "Tissues were homogenized in Trizol using an Ultra-turrax homogenizer and total RNA was extracted according to manufacturer´s instructions ( Invitrogen ) .", "Reverse transcription was performed by incubating 1 µg of total RNA with 0 . 25 µg of random hexamers ( Amersham Pharmacia Biotech ) and 0 . 9 mM dNTPs at 65°C for 5 min , followed by incubation with 1x First Strand Buffer ( Invitrogen ) , 10 mM DTT and 200 units of Moloney murine leukemia virus reverse transcriptase ( Life Technologies ) at 37°C for 1 hr .", "For more details see ( Osinalde et al . , 2016 ) .", "Quantitative PCR was performed using 20 µl reactions containing SYBR Green JumpStart Taq ReadyMix ( Sigma-Aldrich ) , 5 µl of diluted cDNA , 0 . 2 µl of reference dye and 150 nM of each primer .", "Reaction mixtures were preheated at 95°C for 2 min followed by 40 cycles of melting at 95°C for 15 s , annealing at 60°C for 45 s , and elongation at 72°C for 45 s on a Mx3000P qPCR System ( Agilent Technologies , Santa Clara , CA ) .", "The expression levels of targets genes were measured in technical triplicates and normalized to GAPDH ( in birds [Feng et al . , 2010] ) or HPRT ( in bats ) gene expression .", "In bat TA and extraocular muscle tissues no reference gene ( GAPDH , HPRT , tubulin ) could be amplified and MYH expression in these tissues could thus not be normalized .", "The sequences of the primers used are provided in the Supplementary file 1C , D .", "Amplicons were sequenced ( Eurofins Genomics , Germany ) after all experiments to confirm identity .", "We only included genes where we had a positive control in the tissues sampled .", "Data for zebra finches were normalized to the maximum value of all muscles per gene , after which mean ± SE was calculated .", "Because of the lower amount of muscle tissue present in females compared to males ( Wade and Buhlman , 2000 ) , we pooled the tissue of three females prior to tissue homogenization .", "To compare across juvenile male birds , we collected syringeal tissue of three individuals of the ages 25 , 50 , 75 and 100 DPH .", "The left syringeal muscle VTB and left TA was dissected in three adult male zebra finches .", "The left laryngeal muscle ACTM and left TA was dissected in two adult Daubenton’s bats .", "Tissues were fixed with a 2 . 5% glutaraldehyde in 0 . 1 M sodium cacodylate buffer ( pH 7 . 3 ) for 24 hr and subsequently rinsed four times in 0 . 1 M sodium cacodylate buffer .", "Following rinsing , muscles were post-fixed with 1% osmium tetroxide ( OsO4 ) and 1 . 5% potassium ferrocyanide ( K4Fe ( CN ) 6 ) in 0 . 1 M sodium cacodylate buffer for 90 min at 4°C .", "After post-fixation , muscles were rinsed twice in 0 . 1 M sodium cacodylate buffer at 4°C , dehydrated through a graded series of alcohol at 4–20oC , infiltrated with graded mixtures of propylene oxide and Epon at 20°C , and embedded in 100% Epon at 30°C .", "Ultra-thin ( 60 nm ) sections were cut ( Leica Ultracut UCT ultramicrotome , Leica Microsystems , Brønshoj , Denmark ) at three depths ( separated by 150 µm ) and contrasted with uranyl acetate and lead citrate .", "Sections were examined and photographed at 40 , 000x magnification in a pre-calibrated transmission electron microscope ( Philips EM 208 and a Megaview III FW camera or JEOL-1400 microscope ( JEOL , Nieuw Vennep , The Netherlands ) and a Quemesa camera ( EMSIS GmbH , Münster , Germany ) ) .", "We quantified volumetric percentage estimates ( VPE ) of sarcoplasmatic reticulum ( VPESR ) , mitochondria ( VPEmt ) and myofibrils ( VPEmf ) .", "The VPESR and VPEmito and VPEmyo were determined by point counting using grid sizes of 135 , 135 and 300 nm , respectively ( Nielsen et al . , 2010 ) .", "For zebra finch SFM the VPESR and VPEmyo were based on 168 images obtained from 14 fibres in 2 animals , and the VPEmito was based on 288 images obtained from 24 fibres in 3 animals ( 4 , 10 , 10 ) .", "For zebra finch TA all the estimates were based on 61 images obtained from 5 fibres in 2 animals .", "For bat CT all the estimates were based on 80 images obtained from 7 fibres in 2 animals , and for bat TA all the estimates were based on 48 images from 4 fibres in 2 animals .", "The estimated coefficient of error for ratio estimators were 0 . 05 , 0 . 03 and 0 . 08 for the estimates of SR , myofibrils and mitochondria , respectively .", "The rest volume consisted of myoplasm , lipids , nuclei and glycogen particles .", "SR per myofibril ratio was defined as VPESRVPEmf*100% ) .", "TEM images of the Toadfish white muscle body ( as prepared in Felder and Franzini-Armstrong , 2002 ) for intraspecific comparison to toadfish swimbladder SFM were kindly provided by Dr . C . Franzini-Armstrong .", "Other values were taken from the literature: Crotalus atrox tailshaker and dorsal mid-body muscle ( Schaeffer et al . , 1996 ) ; and Opsanus tau swimbladder muscle ( Appelt et al . , 1991 ) and teleost white muscle ( Johnston , 1983 ) .", "Meaningful statistical comparison of the toadfish data could not be performed on published mean values in toadfish .", "Force development curves of zebra finch syringeal VTB muscle were measured at 50 ( n = 4 ) , 75 ( n = 4 ) and 100 DPH ( n = 4 ) at 39°C at optimal stimulus amplitude and muscle length as described previously ( Elemans et al . , 2008 , 2011 ) .", "Real-time force development and intracellular calcium concentration kinetics were measured in four adult male zebra finches and two adult male toadfish on a custom-built microscope setup .", "Briefly , the muscle was suspended between a force transducer ( model 400A , Aurora Scientific , Aurora , ON , Canada ) and micromanipulator submerged in temperature controlled ( accuracy ±0 . 1°C ) Ringers solution .", "This muscle chamber was mounted on an inverted microscope ( Nikon eTI , DFA , Glostrup , Denmark ) .", "First , force development curves of single and multiple stimuli ( 100 ms , 100 Hz trains ) were measured at a range of temperatures ( 10–39°C ) at optimal stimulus amplitude and muscle length .", "Stimulus trains were measured at 25°C and 39°C for toadfish and zebra finch respectively .", "Second , we flushed the sample with 20 µM BTS in Ringers for 20 min to inhibit actomyosin interaction and avoid movement artifacts for the subsequent calcium measurements ( Cheung et al . , 2002 ) .", "Third , to image intracellular calcium , we used a high-affinity calcium dye ( Hollingworth et al . , 2009 ) ( Mag-Fluo-4 , AM stock , ThermoFisher scientific , Slangerup Denmark , # F14201 ) in DMSO , diluted to 2 µM in ringer’s solution ) and incubated the samples for 30 min at 21°C after which they were rinsed three times with indicator-free medium .", "A 20x air objective was used to image the sample using a Metal Halide lamp as illumination source and a FITC filter cube ( Nikon , DFA , Glostrup , Denmark ) to isolate the calcium dye signal .", "We imaged a small portion of the sample to increase the signal to noise ratio .", "The resulting signal was passed through a 535 ± 15 nm filter ( AHF , Germany ) detected by a photo multiplier tube ( Model R928 , Hamamatsu , Denmark ) and amplified ( model SR560 , Stanford Research Systems , Sunnyvale , CA ) .", "The initial temperate series was now repeated at identical stimulation settings .", "Force , stimulus , and calcium signals were digitized ( National Instruments USB6259 ) , aligned on the stimulus onset and analyzed in Matlab ( RRID:SCR_001622 ) .", "We performed these measurements in zebra finch syringeal VTB muscle ( N = 5 ) and toadfish swimbladder muscle ( N = 2 ) .", "The bat laryngeal CT muscle’s force data presented in Figure 6b was collected as part of a previous study ( Elemans et al . , 2011 ) .", "Because of limited tissue availability of bat muscle and the rapid experimental rundown of mammalian muscles in vitro at relevant physiological temperatures , we could not optimize the real-time calcium imaging technique for bats and used an alternative technique to measure free calcium uptake and release by isolated SR vesicles in muscle homogenate ( Nielsen et al . , 2007 ) .", "Briefly , the SR vesicle oxalate mediated , calcium uptake and release was measured fluorometrically ( Ratiomaster RCM , Photon Technology International , Brunswick , NJ ) at 37°C using a fluorescent calcium indicator ( indo-1 ) .", "The [Ca2+] release and uptake curves were fitted using exponential equations as previously described ( Nielsen et al . , 2007 ) .", "No sample sizes were computed before the experiments .", "A technical replicate is a replicate of the measurement on the same preparation , and a biological replicate is an individual .", "Data are presented as mean values ± 1 SE .", "MYH13 mRNA levels in juvenile birds were compared using Kruskal-Wallis test , followed by Dunn’s posthoc test .", "Juvenile twitch times were compared using Kruskal-Wallis test , followed by Wilcoxon signed rank tests .", "SR volume and SR per myofibril ratio were compared between each SFM and the intraspecific skeletal muscle using a Kruskal-Wallis test .", "Statistical test results for the rattlesnake TEM data were reported in Schaeffer et al . , 1996 .", "dF/Fr peak magnitude and FWHM values were compared between binned twitch number ( 1 , 2–4 and 5–10 ) using a Kruskal-Wallis test .", "SR vesicle [Ca2+] uptake and release data were compared between SFM and intraspecific reference muscle by one-way analysis of variance ( ANOVA ) .", "The number of technical and biological replicates are listed above with each essay .", "No outliers or data were excluded ." ] ]

[ "Superfast muscles ( SFMs ) are extremely fast synchronous muscles capable of contraction rates up to 250 Hz , enabling precise motor execution at the millisecond time scale .", "SFM phenotypes have been discovered in most major vertebrate lineages , but it remains unknown whether all SFMs share excitation-contraction coupling pathway adaptations for speed , and if SFMs arose once , or from independent evolutionary events .", "Here , we demonstrate that to achieve rapid actomyosin crossbridge kinetics bat and songbird SFM express myosin heavy chain genes that are evolutionarily and ontologically distinct .", "Furthermore , we show that all known SFMs share multiple functional adaptations that minimize excitation-contraction coupling transduction times .", "Our results suggest that SFM evolved independently in sound-producing organs in ray-finned fish , birds , and mammals , and that SFM phenotypes operate at a maximum operational speed set by fundamental constraints in synchronous muscle .", "Consequentially , these constraints set a fundamental limit to the maximum speed of fine motor control ." ]

[ "Across animals , different muscle types have evolved to perform vastly different tasks at different speeds .", "For example , tortoise leg muscles move slowly over several seconds , while the flight muscles of a hummingbird move quickly dozens of times per second .", "The speed record holders among vertebrates are the so-called superfast muscles , which can move up to 250 times per second .", "Superfast muscles power the alarming rattle of rattlesnakes , courtship calls in fish , rapid echolocation calls in bats and the elaborate vocal gymnastics of songbirds .", "Thus these extreme muscles are all around us and are always involved in sound production .", "Did superfast muscles evolve from a common ancestor ?", "And how do different superfast muscles achieve their extreme behavior ?", "To answer these questions , Mead et al . studied the systems known to limit contraction speed in all currently known superfast muscles found in rattlesnakes , toadfish , bats and songbirds .", "This revealed that all the muscles share certain specific adaptations that allow superfast contractions .", "Furthermore , the three fastest examples – toadfish , songbird and bat – have nearly identical maximum speeds .", "Although this appears to support the idea that the adaptations all evolved from a shared ancestor , Mead et al . found evidence that suggests otherwise .", "Each of the three superfast muscles are powered by a different motor protein , which argues strongly in favor of the muscles evolving independently .", "The existence of such similar mechanisms and performance in independently evolved muscles raises the possibility that the fastest contraction rates measured by Mead et al . represent a maximum speed limit for all vertebrate muscles .", "Any technical failure in a racecar most likely will slow it down , while the same failure in a robustly engineered family car may not be so noticeable .", "Similarly in superfast muscle many cellular and molecular systems need to perform maximally .", "Therefore by understanding how these extreme muscles work , we also gain a better understanding of how normal muscles contract ." ]

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

[ "chromosomes and gene expression", "genetics and genomics" ]

[ [ "Complex tissues require the coordinated activity of a wide array of specialized cell types .", "It has been proposed that cellular diversity arises in the course of evolution through a ‘division of labor’ process , in which a multifunctional ancestral cell type gives rise to descendant cell types with divergent and novel functions ( Arendt et al . , 2009; Brunet and King , 2017 ) .", "Such descendants are often referred to as ‘sister’ cell types , and typically share a range of morphological , functional , and transcriptional features while at the same time displaying key differences ( Arendt , 2003; Arendt et al . , 2016 ) .", "A canonical example of sister cell types are mammalian retinal photoreceptors and bipolar cells ( BCs ) ( Arendt , 2008; Lamb , 2013 ) .", "In a typical vertebrate retina , photoreceptors synapse onto bipolar cells , which , in turn , synapse onto retinal ganglion cells that send their axons to the brain .", "Bipolar cells therefore constitute the central interneuronal cell class in the vertebrate retina .", "In mice , an array of 15 distinct bipolar cell types , broadly categorized as ON and OFF based on their response to light onset/offset , serve as a scaffold upon which the complex information-processing circuitry of the retina is built ( Euler et al . , 2014 ) .", "In this paper , we refer to photoreceptors and bipolar cells as cell ‘classes’ , since they each comprise multiple distinct cell types .", "During retinal development , photoreceptors and bipolar cells arise from the same population of OTX2-expressing progenitor cells ( Koike et al . , 2007; Wang et al . , 2014; Emerson et al . , 2013 ) , share a similar elongate morphology ( Lamb , 2013 ) , and possess the molecular machinery required for ribbon synapse formation , a structure not found in any other retinal cell class ( tom Dieck and Brandstätter , 2006 ) .", "In some vertebrate species , a subset of bipolar cells exhibit additional photoreceptor-like features , including localization of their cell bodies in the outer nuclear layer and the presence of an inner segment-like structure known as Landolt’s club , which extends from the dendrite to the outer limiting membrane and contains a 9+0 cilium ( Tauchi , 1990; Hendrickson , 1966; Locket , 1970; Quesada and Génis-Gálvez , 1985 ) .", "These ‘transitional’ cell types point to the evolutionary origin of bipolar cells from photoreceptors ( Arendt , 2008; Lamb , 2013 ) .", "Both shared and divergent features of sister cell types are mediated by the transcriptional regulatory networks that govern gene expression in each cell type .", "In vertebrates , photoreceptors and bipolar cells express the paired-type homeodomain ( HD ) TFs CRX and OTX2 , which are master regulators of gene expression in both cell classes ( Omori et al . , 2011; Nishida et al . , 2003; Hennig et al . , 2008; Corbo et al . , 2010 ) .", "A third paired-type HD TF , VSX2 , is expressed specifically in bipolar cells and is required for bipolar fate ( Livne-Bar et al . , 2006; Liu et al . , 1994 ) .", "Paired-type homeodomains recognize a core ‘TAAT’ motif , with additional specificity conferred by amino acids in positions 47 , 50 , and 54 of the homeodomain ( Treisman et al . , 1989; Noyes et al . , 2008; Berger et al . , 2008 ) In particular , a lysine at position 50 ( K50 , as found in CRX and OTX2 ) favors recognition of TAATCC , whereas a glutamine ( Q50 , as found in VSX2 ) favors recognition of TAATTA/G .", "Thus , substitution of a single amino acid in the HD toggles the TF’s binding preference for the nucleotides 3’ of the TAAT core ( Treisman et al . , 1989 ) .", "Various bHLH TFs , which recognize E-box motifs ( CANNTG ) , also play important roles in photoreceptor and bipolar cell gene expression programs ( Tomita et al . , 2000; Akagi et al . , 2004 ) .", "For example , NEUROD1 is required for photoreceptor survival , and BHLHE22 and BHLHE23 are required for development of OFF cone bipolar cells and rod bipolar cells , respectively ( Bramblett et al . , 2004; Feng et al . , 2006; Huang et al . , 2014; Pennesi et al . , 2003 ) .", "Our lab has previously shown that the cis-regulatory elements ( CREs; i . e . , enhancers and promoters ) of mouse rods and cones are strongly enriched for K50 HD motifs as well as moderately enriched for Q50 HD and E-box motifs ( Hughes et al . , 2017 ) .", "In addition , we recently used a massively parallel reporter assay to analyze the activity of thousands of photoreceptor enhancers identified by CRX ChIP-seq and found that both K50 HD and E-box motifs are positively correlated with enhancer activity in photoreceptors while Q50 HD motifs have a weakly negative correlation with enhancer activity ( Hughes et al . , 2018 ) .", "In contrast , studies of individual reporters have shown that Q50 HD motifs mediate weak activation of expression via RAX , and that RAX can either enhance or suppress the transactivation activity of CRX , depending upon RAX expression levels ( Irie et al . , 2015; Kimura et al . , 2000 ) .", "Thus , Q50 HD motifs appear to have both positive and negative effects of photoreceptor enhancer activity , depending on context .", "In contrast , Q50 motifs in bipolar cells appear to be strongly repressive when bound by VSX2 , which has been proposed to inhibit the expression of photoreceptor genes in bipolar cells ( Livne-Bar et al . , 2006; Dorval et al . , 2006; Clark et al . , 2008 ) .", "The opposing effects on transcriptional activity mediated by K50 and Q50 motifs suggest that even subtle changes in HD binding sites may mediate major differences in gene expression .", "Indeed , a recent study in Drosophila showed that single base pair substitutions that interconvert Q50 and K50 half-sites within dimeric motifs are sufficient to switch the specificity of opsin expression within photoreceptor sub-types ( Rister et al . , 2015 ) .", "Photoreceptors and bipolar cells offer an attractive model system in which to examine the mechanisms of cis-regulatory divergence in evolution and development , but the cis-regulatory landscape of bipolar cells is currently unknown .", "To elucidate the cis-regulatory grammar of bipolar cells we isolated specific bipolar cell populations from mouse retina and obtained profiles of open chromatin and gene expression .", "By comparing these datasets to matching data from mouse rod and cone photoreceptors we identified differential enrichment of Q50 motifs in photoreceptor-specific enhancers and a corresponding enrichment of E-boxes in bipolar-specific enhancers .", "We propose that the differential partitioning of Q50 motifs in photoreceptor and bipolar enhancers was a key evolutionary innovation contributing to transcriptomic divergence between the two cell classes ." ], [ "To obtain cell class-specific transcriptome profiles of mouse bipolar cells , we used fluorescence-activated cell sorting ( FACS ) to purify bipolar cell populations from adult mice .", "We first isolated all bipolar cells using Otx2-GFP mice .", "This line harbors a GFP cassette knocked into the endogenous Otx2 locus ( Fossat et al . , 2007 ) .", "Adult Otx2-GFP mice display high-level GFP in bipolar cells and low-level expression in photoreceptors ( Figure 1B ) .", "To purify ON and OFF bipolar cells separately , we crossed Otx2-GFP mice with a Grm6-YFP line , in which YFP is driven by the Grm6 promoter and expressed only in ON bipolar cells ( Morgan et al . , 2011 ) .", "In the double transgenic mice ( Otx2-GFP+; Grm6-YFP+ ) , ON bipolar cells co-express GFP and YFP and can be separated from OFF bipolar cells based on fluorescence intensity ( Figure 1B ) .", "We subjected OFF bipolar cells to a second round of sorting to maximize purity from the adjacent weakly fluorescent photoreceptor population .", "Purity of bipolar cell populations was confirmed by RT-qPCR which showed enrichment of ON- and OFF-specific genes in the appropriate populations and depletion of markers for other retinal cell classes as compared to whole retina ( Figure 1C and Figure 1—figure supplement 2 ) .", "We then used RNA-seq to profile gene expression in purified populations of bipolar cells , obtaining highly reproducible profiles across biological replicates ( Figure 1—figure supplement 1 ) .", "To define the transcriptional differences between photoreceptor and bipolar cells we compared RNA-seq data from bipolar cells to similar data from wild-type rods and Nrl-/- photoreceptors previously generated in our lab ( Hughes et al . , 2017 ) .", "We used Nrl-/- photoreceptors as a surrogate for blue cones ( i . e . , Opn1sw-expressing cones ) since mouse photoreceptors lacking Nrl transdifferentiate into blue cones during development ( Daniele et al . , 2005; Nikonov et al . , 2005 ) .", "We identified a total of 5259 genes with at least a two-fold difference in expression between bipolar cells and either rods or blue cones ( FDR < 0 . 05 ) ( Supplementary file 5 ) .", "Despite the large number of differentially expressed genes , published single-cell RNA-seq profiles indicate that the bipolar cell transcriptome is more similar to that of photoreceptors than to that of any other retinal cell class ( Macosko et al . , 2015 ) .", "To evaluate the functional differences between photoreceptor and bipolar cell gene expression programs we compared the top ~ 30% most differentially expressed genes in each cell class ( 832 bipolar cell , 818 photoreceptor ) using the gene ontology ( GO ) analysis tool , PANTHER ( Thomas et al . , 2003 ) .", "Top bipolar-enriched GO terms were typical of many neuronal cell types and related to aspects of synaptic transmission , while photoreceptor-enriched GO terms mainly related to light-sensing ( Supplementary file 6 ) .", "Next , we compared the list of differentially expressed genes to a database of mouse TFs ( AnimalTFDB3 . 0; Hu et al . , 2019 ) , which revealed that 394 of the differentially expressed genes encode putative transcriptional regulators ( Supplementary file 5 ) .", "These include TFs known to be responsible for controlling gene expression in rods ( Nrl , Nr2e3 , Neurod1 ) , cones ( Thrb ) , bipolar cells ( Vsx2 , Neurod4 ) , or both cell classes ( Crx ) .", "Nearly one-third ( 176 ) of differentially expressed TFs are members of the zinc finger ( ZF ) family , many of which are more highly expressed in rods compared to bipolar cells but not in blue cone compared to bipolar cells .", "Conversely , of the top 10% differentially expressed TFs , the majority ( 25 of 35 ) are more highly expressed in bipolar cells compared to either rods or cones , and of these , most ( 16 of 25 ) are classified as HD , ZF or bHLH .", "Thus , the transcriptomes of bipolar cells and photoreceptors are significantly divergent , despite various functional and morphological similarities between the two cell classes .", "In contrast , comparison of the transcriptomes of ON and OFF bipolar cells identified only 680 genes that were differentially expressed by at least two-fold ( 317 ON- and 363 OFF-enriched at FDR < 0 . 05; Supplementary file 5 ) .", "This figure is less than half of the number of differentially expressed genes identified between rods and blue cones ( 1 , 471 ) , indicating that the transcriptomes of the two categories of bipolar cells are quite similar .", "Of note , a recent study by Shekhar et al . ( 2016 ) , described single-cell expression profiles for bipolar cell types using Drop-seq .", "To compare our results with this study , we pooled data from Shekhar et al . across inferred cell types to generate ‘pseudo-bulk’ ON , OFF , and pan bipolar cell gene expression profiles .", "We found that expression estimates from bulk and pooled pseudo-bulk single-cell RNA-seq are well correlated ( Pearson correlation coefficients range from 0 . 82 to 0 . 85 , Figure 2—figure supplement 1A–C ) .", "Similarly , pairwise comparisons showed that bulk ON , OFF , and pan bipolar cell populations were most strongly correlated with their pseudo-bulk counterparts , suggesting that the two approaches yield similar cell type-specific expression profiles ( Figure 2—figure supplement 1D ) .", "Shekhar et al . also identified an ON bipolar cell type ( BC5D ) that expresses low levels of Grm6 and could thus have been incorrectly sorted into our OFF population .", "To evaluate this possibility , we examined RNA-seq data for Lrrtm1 , a gene specific to BC5D bipolar cells ( Shekhar et al . , 2016 ) .", "We observed low read counts for Lrrtm1 in both ON and OFF bipolar populations , suggesting that BC5D cells were not sorted into either group .", "To investigate further , we repeated the FACS experiments , this time also collecting cells exhibiting fluorescence levels in between those of the ON and OFF populations ( termed YFP-low ) .", "We found that the YFP-low population expressed both the ON BC marker Isl1 and the BC5D marker Lrrtm1 , while the ON and OFF populations did not express Lrrtm1 ( Figure 1—figure supplement 2 ) .", "These results indicate that low levels of Grm6-YFP expression caused BC5D cells to be excluded from both the ON and OFF bipolar populations .", "Given the rarity of BC5D cells , which constituted 2 . 3% of cells identified as bipolar cells by Shekhar et al . , we believe their absence does not materially impact our analysis .", "To gain insight into gene expression among individual bipolar cell types , we compared the list of genes differentially expressed between ON and OFF bipolar cells with the data of Shekhar et al . Overall , we found a strong correlation between the results of bulk ON and OFF bipolar cell expression profiling and single-cell transcriptome analysis ( Figure 2; Figure 2—figure supplement 1E and Figure 2—source data 1 ) .", "Additionally , 50 of the 680 differentially expressed genes found in our bulk analysis were not present in the Drop-seq data ( Supplementary file 5 ) .", "These 50 genes are generally expressed at low levels , even in the cell population in which they are enriched .", "This finding suggests that bulk RNA-seq of purified populations can provide information complementary to that obtained by single-cell profiling .", "Taken together , these data indicate that despite their sister cell type relationship , photoreceptor and bipolar cells have markedly distinct transcriptional profiles , while ON and OFF bipolar cells are more similar at the transcriptome level than rods and cones .", "To compare chromatin accessibility between photoreceptor and bipolar cells , we used ATAC-seq ( Assay for Transposase-Accessible Chromatin by sequencing ) to generate open chromatin profiles from FACS-purified bipolar cells ( Buenrostro et al . , 2013 ) .", "Similar to our RNA-seq data , ATAC-seq generated highly reproducible profiles across biological replicates ( Pearson correlation 0 . 95–1 . 00 , Figure 1—figure supplement 1 ) .", "For the purpose of our bioinformatic analyses , we define ‘promoters’ as those ATAC-seq peaks that occur between 1000 bp upstream and 100 bp downstream of a transcription start site ( TSS ) ; we refer to ATAC-seq peaks outside of this range as ‘enhancers’ or ‘TSS-distal’ elements .", "To compare chromatin accessibility across tissues , we combined ATAC-seq peaks from bipolar cells with previously generated ATAC-seq data from purified mouse rods , blue cones , and ‘green’ cones ( i . e . , Opn1mw-expressing cones ) as well as DNase-seq data from whole retina , brain , heart , and liver to obtain a list of > 345 , 000 open chromatin regions ( Hughes et al . , 2017; ENCODE Project Consortium , 2012 ) .", "Hierarchical clustering of chromatin accessibility profiles at enhancers showed that photoreceptors , bipolar cells , and whole retina cluster separately from other tissues ( Figure 3A ) .", "Thus , the sister cell type relationship between photoreceptors and bipolar cells is reflected by the similarity of genome-wide patterns of enhancer chromatin accessibility .", "We found that whole retina DNase-seq clustered with bipolar cell samples ( Figure 3A ) .", "This finding was unexpected given that rod photoreceptors constitute ~ 80% of all cells in the mouse retina ( Figure 1A ) ( Jeon et al . , 1998 ) and indicates that the open chromatin profile of a complex tissue is not necessarily dominated by the most abundant cell type .", "This is in line with our previous comparison of genome-wide patterns of chromatin accessibility in rods , cones and whole retina , which showed that more than half of the whole retina peaks did not overlap with photoreceptor peaks , suggesting that they derive from non-photoreceptor retinal cell types ( Hughes et al . , 2017 ) .", "This finding may be a consequence of the distinctive pattern of global chromatin closure that we previously described in rod photoreceptors ( Hughes et al . , 2017 ) .", "We next set out to examine patterns of chromatin accessibility within the retinal cell types in greater detail .", "We combined TSS-distal ATAC-seq peaks from bipolar cells and photoreceptors with DNase-seq peaks from whole retina to create a list of 99 , 684 retinal open chromatin regions .", "Clustering these regions based on chromatin accessibility across retinal and non-retinal cell types offers a broad view of cell class- and cell type-specific regions of open chromatin ( Figure 3B ) .", "We found a large subset of bipolar-enriched peaks , many of which were also accessible in whole retina and brain ( Figure 3B , green box ) .", "Conversely , a smaller subset of peaks showed selective accessibility in photoreceptors , with lower levels of accessibility in bipolar cells , and even less in other cell types ( Figure 3B , red box ) .", "While pan BCs and ON-BCs showed nearly identical open chromatin profiles , OFF-BC open chromatin patterns were somewhat divergent , with slightly more accessibility in the photoreceptor-enriched subset ( red box ) and less in the bipolar-enriched subset ( green box ) compared to ON-BC .", "Overall , the sum of photoreceptor and bipolar cell ATAC-seq peaks accounted for 83 percent of whole-retina DNase-seq peaks , with the remainder presumably deriving from other inner retinal cell classes .", "These data suggest that relatively few regions of open chromatin are truly photoreceptor-specific , and that regions enriched in bipolar cells are more likely to be accessible in other tissues , particularly in brain .", "For instance , compared to photoreceptors , a greater proportion of bipolar cell peaks corresponded to DNase-seq peaks in brain ( 47% vs 37% ) .", "Finally , direct comparison of ATAC-seq peaks from photoreceptor and bipolar cells identified 55 , 402 differentially accessible regions ( FDR < 0 . 05 ) , 75% of which are more accessible in bipolar cells ( Supplementary file 7 ) .", "While we observed significant differences in global chromatin accessibility between photoreceptors and bipolar cells , the open chromatin profiles of ON and OFF bipolar cells were largely similar , consistent with the high degree of similarity between their transcriptomes .", "Specifically , only 4263 peaks were differentially accessible between ON and OFF bipolar cells .", "Of note , 79% ( 3359 ) of these differential peaks were more accessible in ON bipolar cells .", "When examining loci surrounding genes expressed in both ON and OFF bipolar cells , we found that ON and OFF subclasses typically had similar open chromatin profiles ( e . g . , Vsx2 , Figure 3C ) .", "In contrast , some ON- or OFF-specific genes exhibit cell subclass-specific patterns of chromatin accessibility ( e . g . , Isl1 and Esam , Figure 3D–E ) .", "We found an overall correlation between accessibility and associated gene expression ( Figure 5—figure supplement 1 ) but did not see this pattern at all gene loci ( e . g . , Grik1 , Figure 3F ) .", "Having compared global accessibility between photoreceptor and bipolar cells , we next sought to compare them in terms of ‘cis-regulatory grammar’ , which we define as the number , affinity , spacing and orientation of TF binding sites within the open chromatin regions of a given cell type or class .", "To begin , we assessed all 319 TF binding site motifs from the HOMER database for enrichment within bipolar cell open chromatin regions ( Heinz et al . , 2010 ) .", "The most highly enriched motifs within enhancers corresponded to CTCF , K50 HD , E-box , nuclear receptor , and MADS box motifs ( Supplementary file 8 ) .", "All of these motifs were previously shown to be among the most enriched motifs in photoreceptor ATAC-seq peaks as well ( Hughes et al . , 2017 ) .", "The similarity in the patterns of TF binding site enrichment between photoreceptors and bipolar cells can be better understood in the context of known patterns of TF expression in these cell classes .", "Specifically , the K50 HD TFs OTX2 and CRX are master regulators of gene expression programs in photoreceptor and bipolar cells .", "Likewise , bHLH TFs ( which recognize E-box motifs ) play roles in fate specification and maintenance of both photoreceptor and bipolar cell gene expression programs , as described in the Introduction .", "Finally , enrichment of ZF motifs recognized by CTCF in both cell classes is in line with reports that CTCF motifs often lie in ubiquitously accessible chromatin regions , where CTCF recruitment is thought to mediate interactions between promoters and enhancers , among other functions ( Splinter et al . , 2006; Rao et al . , 2014; Song et al . , 2011 ) .", "Despite the overall similarity between the cis-regulatory grammars of bipolar cells and photoreceptors , there are notable quantitative differences in motif enrichment between the two cell classes .", "To systematically identify these differences , we compared the proportion of peaks containing each of the 319 motifs between pan-bipolar cells and each photoreceptor cell type ( Supplementary file 8 ) .", "The most differentially enriched motifs corresponded to those with the highest enrichment in each cell class and are summarized in Figure", "4 . Although both photoreceptors and bipolar cells showed marked enrichment for K50 HD motifs , these motifs were more enriched in photoreceptors ( Figure 4 ) .", "Conversely , E-box motifs were more enriched in bipolar cells than photoreceptors .", "The most striking difference in TF binding site enrichment between the two cell classes was the enrichment of both monomeric and dimeric Q50 HD motifs in photoreceptor open chromatin regions and their lack of enrichment in bipolar regions ( Figure 4 ) .", "The most well-characterized Q50 HD TF expressed in photoreceptors is RAX , which is required for cone gene expression and survival ( Irie et al . , 2015 ) .", "In contrast , bipolar cells express multiple Q50 HD TFs ( VSX2 , VSX1 , ISL1 , LXH3 , LHX4 , AND SEBOX ) ( Shekhar et al . , 2016 ) .", "VSX2 is required for bipolar cell development and is expressed in all mouse bipolar cell types throughout development and into adulthood ( Livne-Bar et al . , 2006; Liu et al . , 1994 ) .", "The paradoxical absence of Q50 HD motif enrichment in bipolar open chromatin regions despite the presence of multiple Q50 HD TFs in this cell class may be explained by the observation that VSX2 acts as a repressor of photoreceptor CREs ( Dorval et al . , 2006 ) .", "The lack of Q50 motif enrichment in bipolar cells could be due to selective repression of photoreceptor-specific open chromatin regions by VSX2 , which , in turn , prevents ectopic expression of photoreceptor genes in bipolar cells , a possibility that we will return to in the final section of the Results .", "To further compare the cis-regulatory grammars of bipolar cells and photoreceptors , we examined TF binding site co-occurrence and spacing within each cell class .", "For this analysis we compared a combined list of enhancer regions from rod and cones to that of bipolar cells .", "As expected , motif pairs enriched in specific peak sets tended to include motifs that showed the highest individual enrichment in the same cell type .", "Likewise , differentially enriched motif pairs included one or more differentially enriched motifs , such as K50 and Q50 HD motifs in photoreceptors and bHLH motifs in bipolar cells ( Figure 4—figure supplement 1A–B ) .", "Of note , while we identified specific motif pairs enriched in each cell type , the frequencies of these pairs are consistent with an independent model ( i . e . , the number of occurrences of motif pairs is approximately what would be expected given the number of occurrences of each individual motif and assuming a random distribution of motifs across peaks ) .", "As with chromatin accessibility , enrichment of motif pairs is highly similar between ON and OFF bipolar cells , with nominally differentially enriched pairs showing similar proportions ( Figure 4—figure supplement 1C ) .", "Finally , to investigate preferences in spacing and orientation between pairs of motifs , we plotted the density of highly enriched motifs ( those depicted in Figure 4 ) centered on regions flanking K50 and Q50 HD motifs in each peak set .", "As described previously for photoreceptors , spacing and orientation preferences in bipolar open chromatin regions were minimal ( Figure 4—figure supplement 2 ) ( Hughes et al . , 2017 ) .", "Thus , the primary differences in the cis-regulatory grammar of the two cell classes appears to be the degree of HD and E-box motif enrichment .", "We next sought to determine the extent to which photoreceptor- and bipolar-enriched open chromatin regions correlate with cell type-specific gene expression .", "To this end , we assigned each of the 55 , 402 regions identified as differentially accessible between photoreceptor and bipolar cells to a candidate target gene based on proximity to the nearest transcription start site and compared mean RNA-seq expression values for the assigned genes .", "As described in previous studies , we observed a modest correlation between enhancer accessibility and gene expression , and a more robust correlation between promoter accessibility and gene expression ( Figure 5—figure supplement 1 ) ( Hughes et al . , 2017; Pastor et al . , 2014; Ampuja et al . , 2017; de la Torre-Ubieta et al . , 2018 ) .", "Our analysis of global chromatin accessibility suggested that many of the differentially accessible peaks were also open in other tissues , especially those enriched in bipolar cells compared to photoreceptors ( Figure 3E ) .", "To gain a better understanding of the cell type-specific open chromatin regions that drive gene expression differences between these two cell classes , we refined our analysis to exclude peaks shared with non-retinal cell types .", "We identified 8435 enhancer regions which are accessible either in photoreceptors or bipolar cells , but not accessible in brain , liver or splenic B cells ( Figure 5A ) .", "This set includes 1291 regions that are open in both photoreceptors and bipolar cells , and 7144 regions that are differentially accessible between the two cell classes ( Figure 5A ) .", "We found that ~ 46% ( 3 , 270 ) of the differentially accessible peaks were more open in bipolar cells .", "Thus , most of the bipolar cell-enriched regions identified in the previous section were also accessible in one or more non-retinal tissues .", "As with the enhancer regions from the unfiltered list , assigning genes to this more retina-specific set of differentially accessible regions also shows a correlation between accessibility and gene expression ( Figure 5B ) .", "To visualize this association and identify the peaks that underly it , we plotted all 8435 peaks according to fold-change differences in accessibility and gene expression between bipolar cells and rods ( Figure 5C ) and between bipolar cells and blue cones ( Figure 5—figure supplement 1D ) .", "We then selected for further analysis those peaks that exhibited correlated accessibility and gene expression in photoreceptors ( highlighted in red or blue in Figure 5C and Figure 5—figure supplement 1D; n = 901 ) or bipolar cells ( highlighted in green in Figure 5C and Figure 5—figure supplement 1D; n = 833 ) .", "These differentially enriched peaks represent strong candidates for CREs that mediate the gene expression differences between the two cell classes .", "Indeed , the photoreceptor peak set contains known enhancers responsible for driving cell type-specific expression of Rhodopsin ( Rho ) and components of the rod-specific phototransduction cascade ( Corbo et al . , 2010; Nie et al . , 1996 ) , while the bipolar peak set contains a known enhancer that drives Vsx2 expression in bipolar cells ( Kim et al . , 2008 ) .", "To gain insight into the possible biological functions of these peaks we used GREAT to assign biological annotations based on nearby genes ( McLean et al . , 2010 ) .", "As was found with the unfiltered datasets , photoreceptor peaks are linked with genes associated with light sensation , whereas bipolar peaks are linked to genes involved in more generic neuronal functions ( Figure 5—figure supplement 2 ) .", "Next , we asked whether the patterns of TF binding site enrichment observed with aggregate sets of ATAC-seq peaks from each cell class would be preserved within the retina-specific peak sets associated with correlated gene expression .", "We compared photoreceptor-enriched regions , bipolar-enriched regions , and regions that share accessibility between the two cell classes which were either specific to the retina ( Figure 5A , ‘shared retina’ n = 1 , 291 ) , or unfiltered ( ‘shared all’ , n = 47 , 550 ) .", "We found that K50 HD motifs were enriched in both shared and cell class-selective regions , but to a lesser extent in regions specifically enriched in bipolar cells .", "The bipolar-selective regions were markedly enriched for E-box motifs but completely lacked enrichment for Q50 HD motifs ( Figure 5D ) .", "Conversely , photoreceptor-selective regions were enriched for Q50 HD motifs , but lacked E-box motif enrichment .", "Peaks that were shared between the two cell classes showed an intermediate pattern of motif enrichment , highlighting the roles of both HD and bHLH TFs in regulating the gene expression programs of each class .", "Of note , CTCF enrichment was absent in all but the unfiltered peak set , suggesting that the CTCF enrichment observed in Figure 4 is attributable to ubiquitously accessible peaks .", "Taken together , these findings suggest that differential enrichment of Q50 HD and E-box motifs are the key features that distinguish the cis-regulatory grammars of photoreceptors and bipolar cells .", "Given the critical roles of HD TFs in the regulation of photoreceptor and bipolar gene expression , we further investigated the role of K50 and Q50 motifs in the cis-regulatory region upstream of Gnb3 .", "Gnb3 encodes the β subunit of a heterotrimeric G-protein required for cone phototransduction as well as ON bipolar cell function ( Dhingra et al . , 2012 ) .", "Gnb3 is expressed in rods , cones , and bipolar cells during early postnatal retinal development in the mouse .", "Selective repression of Gnb3 in rods by the nuclear receptor TF NR2E3 results in a cone + bipolar pattern after postnatal day 10 ( Haider et al . , 2009; Corbo and Cepko , 2005 ) .", "We focused on an 820 bp region around the TSS of Gnb3 which drives robust expression in rods , cones , and bipolar cells when electroporated into early postnatal mouse retina .", "This region lacks Q50 motifs but contains five K50 HD motifs of varying affinity which occur in two clusters , one immediately upstream of the TSS ( −65 bp ) and the other more distally ( −350 bp ) ( Figure 6A ) .", "To evaluate the role of these five K50 motifs in mediating photoreceptor and bipolar expression , we engineered reporter constructs in which each of the five motifs was individually inactivated by mutating the TAAT core to TGGT .", "We then introduced wild-type and mutant reporters into mouse retinal explants via electroporation and compared expression levels after 8 days .", "Mutations in K50 motifs 2 , 4 and 5 resulted in coordinate loss of expression in both photoreceptor and bipolar cells , indicating that these motifs are required for reporter expression in both cell classes ( Figure 6B , D ) .", "Conversely , mutations in site 1 or 3 had no effect on expression in either cell class ( Figure 6B , D ) .", "Binding site affinity did not correlate with expression , as site 3 has a higher predicted affinity than sites 4 or", "5 . Thus , the Gnb3 promoter region contains both essential and nonessential K50 motifs , underscoring the critical role for these shared motifs in both photoreceptors and bipolar cells .", "Next , we sought to determine the effect of introducing Q50 motifs into the Gnb3 promoter region .", "For these experiments , reporters were introduced into newborn mouse retinas via in vivo electroporation and harvested for histologic analysis after 20 days .", "First , we electroporated identical wild-type sequences driving both DsRed and GFP to confirm that essentially all photoreceptors and bipolar cells received both constructs ( Figure 6C ) .", "Next , we compared the expression of a Gnb3 promoter containing mutations in K50 motifs 1 and 3 to that of a wild-type promoter , confirming that elimination of both of these sites has no effect on expression in either photoreceptors or bipolar cells ( Figure 6C , D and Figure 6—source data 1 ) .", "To test the effect of introducing Q50 motifs into the Gnb3 promoter region , we replaced K50 motifs 1 and 3 with a Q50 motif ( TAATTA ) , both individually and in combination .", "Whereas introduction of a Q50 motif into site one had no apparent effect , replacement of site three caused a selective decrease in bipolar expression with no change in photoreceptor expression .", "When we introduced Q50 motifs into both sites , reporter expression was markedly reduced in bipolar cells , with no effect on photoreceptor expression ( Figure 6C , D and Figure 6—source data 1 ) .", "These data , along with previous reports of VSX2-mediated repression of photoreceptor-specific promoters and enhancers , suggest that Q50 motifs play an important role in mediating repression of photoreceptor genes in bipolar cells ." ], [ "In this study we generated open chromatin maps and transcriptome profiles of mouse bipolar cells , including FACS-purified ON and OFF bipolar cell populations , and compared them to analogous data from rod and cone photoreceptors .", "We found that photoreceptors and bipolar cells differ in the expression of thousands of genes and yet have very similar cis-regulatory grammars .", "The key cis-regulatory differences that distinguish the two cell classes are the preferential enrichment of Q50 HD motifs in open chromatin regions associated with photoreceptor-specific gene expression and a corresponding enrichment of E-box motifs in chromatin associated with bipolar-specific expression .", "The cellular features and transcriptional mechanisms shared by photoreceptors and bipolar cells have prompted speculation that these two sister cell types arose from a single ancestral photoreceptor cell type via a process of progressive cellular divergence ( Arendt , 2008; Lamb , 2013 ) .", "We propose that the elimination of Q50 motifs from bipolar-specific CREs likely played a key role in differentiating the bipolar transcriptome from that of photoreceptors during early stages of vertebrate retinal evolution .", "Alternatively , given the multiplicity of K50 HD binding sites within both photoreceptor and bipolar cell regulatory elements , a subset of photoreceptor-specific elements may have emerged through the simple conversion of K50 ( TAATCC ) to Q50 ( TAATTA/G ) sites .", "Prior studies of individual photoreceptor CREs showed a role for the Q50 HD TF , VSX2 , in selectively repressing photoreceptor genes in bipolar cells ( Livne-Bar et al . , 2006; Dorval et al . , 2006 ) .", "Our results generalize this conclusion , suggesting that VSX2 plays a genome-wide role in silencing photoreceptor gene expression in bipolar cells .", "A similar role for VSX2 has recently been described in the spinal cord , where closely related progenitor cells give rise to either motor neurons or V2a interneurons ( Clovis et al . , 2016 ) .", "VSX2 promotes V2a identity by directly repressing the motor neuron gene expression program and by competing for Q50 sites at motor neuron enhancers .", "Thus , in both retina and spinal cord , expression of VSX2 promotes interneuron fate at the expense of the alternative neuronal ( photoreceptor or motor neuron ) cell type .", "These parallels suggest that transcriptional repression by cell type-specific TFs such as VSX2 represent a common mechanism for differentiating the gene expression programs of two closely related cell types .", "In addition to differential enrichment of Q50 sites , we also observed enrichment of E-box motifs in regions associated with bipolar-specific expression ( Figure 5C ) .", "The lack of corresponding enrichment in regions associated with photoreceptor-specific expression suggests that bHLH TFs also play an important role in distinguishing the gene expression programs of photoreceptor and bipolar cells .", "It is important to note that this finding does not contradict known roles for bHLH TFs in photoreceptors , as E-box motifs are also enriched within the complete set of photoreceptor ATAC-seq peaks ( Figure 4 ) , as well as in the subset that exhibits shared accessibility with bipolar cells ( Figure 5C ) .", "The role of bHLH TFs in establishing the cellular identity of both classes is further demonstrated by multiple loss-of-function studies ( Tomita et al . , 2000; Bramblett et al . , 2004; Feng et al . , 2006; Huang et al . , 2014; Pennesi et al . , 2003 ) .", "In contrast to the differences between photoreceptors and bipolar cells , ON and OFF bipolar cell populations displayed striking similarities in their cis-regulatory landscape and gene expression profiles .", "In a paper that appeared after our profiling studies had been performed , Shekhar et al . ( 2016 ) documented the single-cell expression profiles of individual bipolar cell types and showed that the transcriptomes of ON and OFF cone bipolar cells are more similar to each other than to that of rod bipolar cells ( RBCs ) .", "This finding implies that the ON bipolar population analyzed in the present study represents a grouping of distinct bipolar cell types ( RBC and cone ON BC ) , which should be separated for a more informative comparison .", "Despite this drawback , our analysis indicates that there are relatively few differences in chromatin accessibility between bipolar cell types .", "We estimate that RBCs constitute nearly 60% of the cells in our ON population ( RBCs compose 56% of ON bipolar cells identified in Shekhar et al . , 2016 , and ~ 57% of those identified as ON BCs by Wässle et al . , 2009 ) .", "Thus , much of the signal in our ON bipolar RNA-seq and ATAC-seq data likely derives from RBCs .", "It remains to be determined whether open chromatin profiling of individual bipolar subtypes will reveal additional differences in their epigenomic landscapes beyond those reported here .", "We found an imperfect correlation between chromatin accessibility and gene expression in bipolar cells .", "For example , ATAC-seq peaks upstream of Grik1 are open in ON bipolar cells despite an absence of Grik1 expression in this cell type ( Figure 3F ) .", "We observed similar instances of discordance between chromatin accessibility and gene expression in our previous analysis of rod and cone photoreceptors ( Hughes et al . , 2017 ) , and other groups have documented this discrepancy in other cell types ( de la Torre-Ubieta et al . , 2018; Lara-Astiaso et al . , 2014; Starks et al . , 2019 ) .", "Presumably , transcriptional activity in these instances requires expression of additional cell type-specific factors ( Heinz et al . , 2015 ) , perhaps most clearly demonstrated in developmental contexts , wherein accessibility is frequently established prior to the onset of transciption ( Lara-Astiaso et al . , 2014 ) .", "Support for the idea that bipolar cells diverged from photoreceptors via progressive partitioning of cellular function is provided by the existence of cell types in the retinas of non-mammalian vertebrates with features intermediate between those of mammalian photoreceptors and bipolar cells .", "In some turtle species ~ 30% of the cell bodies in the photoreceptor layer ( the outer nuclear layer ) belong to bipolar cells , not photoreceptors ( Tauchi , 1990 ) .", "These so-called ‘displaced bipolar cells’ possess an inner segment-like process that extends to the outer limiting membrane and contains abundant mitochondria and even a sensory-type ( ‘9 + 0’ ) cilium ( Figure 7A , B ) .", "Thus , displaced bipolar cells closely resemble typical photoreceptors except that they lack an outer segment , possess dendrites in the outer plexiform layer , and synapse directly onto retinal ganglion cells .", "Another intermediate type of bipolar cell occurs in nearly all non-mammalian vertebrate classes and even in some mammalian species ( Hendrickson , 1966; Locket , 1970; Quesada and Génis-Gálvez , 1985; Young and Vaney , 1990 ) .", "This bipolar type has a nucleus localized to the inner nuclear layer , but retains an inner segment-like structure ( Landolt’s club ) , which extends from the cell’s dendritic arbor to the outer limiting membrane and contains abundant mitochondria and a sensory-type cilium ( Figure 7A ) ( Hendrickson , 1966; Locket , 1970; Quesada and Génis-Gálvez , 1985 ) .", "We suggest that displaced bipolar cells and those with a Landolt’s club represent ‘transitional forms’ on the evolutionary path from photoreceptor to typical bipolar cell .", "The existence of these transitional forms suggests that bipolar cells may have evolved via the stepwise repression of discrete gene modules required for the development of individual cellular features , or ‘apomeres’ , that are specific to photoreceptors ( Arendt et al . , 2016 ) .", "If this evolutionary model is correct , then how can we account for the co-existence of ‘transitional’ bipolar cell types and ‘conventional’ bipolar cells in a single retina ?", "One testable hypothesis is that VSX2 may be expressed at lower levels in transitional bipolar cell types , thereby permitting expression of additional photoreceptor gene modules and their corresponding apomeres .", "Alternatively , it is possible that additional activating Q50 HD TFs are expressed in transitional bipolar types , and these TFs can overcome VSX2-mediated repression of selected photoreceptor gene modules .", "Indeed , we and others have found that multiple Q50 HD TFs are expressed in subsets of mouse bipolar cells ( Shekhar et al . , 2016 ) .", "In addition , there is evidence that transitional bipolar cell types with Landolt’s club may exist in the mouse ( Rowan and Cepko , 2004 ) .", "Thus , individual bipolar cell types may control the number of photoreceptor apomeres they express by modulating the balance of activating and repressing Q50 HD TFs in their nuclei .", "The evolutionary divergence of bipolar cells from photoreceptors likely required coordinated changes in both cis-regulatory grammar and HD TF expression .", "The Q50 HD TF , RAX , is expressed in developing vertebrate rods and cones and is required for normal activation of photoreceptor gene expression in mice ( Irie et al . , 2015; Rodgers et al . , 2018 ) .", "The expression of a RAX homolog in the photoreceptors of the tadpole larva of the protochordate , Ciona intestinalis , suggests a primordial role for this Q50 HD TF in activating photoreceptor gene expression in chordates ( Cao et al . , 2019 ) .", "These data suggest that both K50 and Q50 motifs were present in the CREs of the ancestral vertebrate photoreceptor prior to the evolutionary emergence of bipolar cells , and that both K50 ( OTX2 and CRX ) and Q50 ( RAX ) HD TFs were required for gene activation in that ancestral cell type ( Figure 7C ) .", "In this context , the emergent expression of a repressive Q50 HD TF ( VSX2 ) in a primordial bipolar cell would have permitted selective repression of CREs containing Q50 motifs , allowing the cis-regulatory landscape of the two nascent cell types to begin to diverge .", "Maintaining expression of selected ‘photoreceptor’ genes in bipolar cells ( e . g . , Gnb3 ) would then have required the elimination of Q50 motifs from the cis-regulatory regions of those genes .", "These evolutionary considerations suggest that the modern vertebrate retina arose from an ancestral retina in which photoreceptors directly synapse onto projection neurons ( i . e . , ganglion cells ) without an intervening layer of interneurons ( left side of Figure 7C ) .", "Two lines of evidence suggest that such a retina may have existed .", "First , the retina of the hagfish , the most primitive extant vertebrate , reportedly has photoreceptors that directly synapse onto projection neurons ( i . e . , ganglion cells ) ( Lamb , 2013; Locket and Jørgensen , 1998 ) .", "Second , some vertebrate species ( including reptiles , amphibians , and larval lamprey ) have an unpaired , median ‘parietal eye’ developmentally related to the pineal gland , which contains photoreceptors that directly synapse onto ganglion cells ( Eakin , 1973; Dodt , 1973; Mano and Fukada , 2007; Solessio and Engbretson , 1993 ) .", "It is possible that the parietal eye evolved from the midline ‘eye’ of a protochordate ancestor , akin to the present-day ascidian larva .", "The simple lateral eyes of a hagfish-like vertebrate ancestor may then have emerged via co-option of the gene networks required for parietal eye development .", "Subsequently , bipolar cells may have arisen in the lateral eyes of early vertebrates via subtle changes in cis-regulatory grammar and TF expression , paving the way for the emergence of the sophisticated interneuronal circuitry found in present-day vertebrate retinas ." ], [ "All animal experiments were carried out in accordance with the regulations of the IACUC at Washington University in St . Louis .", "Retinal dissociation and FACS were carried out using Otx2-GFP or Otx2-GFP; Grm6-YFP mice .", "Otx2-GFP mice were heterozygous for a GFP cassette inserted at the C-terminus of the endogenous Otx2 locus ( Fossat et al . , 2007 ) .", "The Grm6-YFP line harbors a YFP transgene driven by the Grm6 promoter ( Morgan et al . , 2011 ) .", "All electroporation experiments were carried out in CD-1 mice .", "Following dissection , retinas of mice aged 6–8 weeks , or 3 months ( for one biological replicate of Figure 1—figure supplement 2 ) were dissociated with papain as described previously ( Trimarchi et al . , 2007 ) .", "Briefly , two retinas were incubated in 400 µl of calcium/magnesium free Hanks’ Balanced Salt Solution ( HBSS ) ( Thermo Fisher ) containing 0 . 65 mg papain ( Worthington Biochem ) for 10 min at 37°C .", "Cells were then washed in a DMEM ( Thermo Fisher ) solution containing 100 units DNase1 ( Roche ) and incubated an additional 5 min at 37°C .", "Cells were then resuspended in 600 µl of sorting buffer ( 2 . 5 mM EDTA , 25 mM HEPES , 1% BSA in HBSS ) and used directly for sorting .", "Cells were sorted on a FACS Aria-II ( BD biosciences ) with gates based on forward scatter , side scatter , and GFP fluorescence .", "OFF bipolar cell populations ( Otx2-GFP+;Grm6-YFP- ) were immediately sorted a second time to increase purity .", "An 820 bp region encompassing part of the Gnb3 5’ UTR and upstream sequence was amplified from mouse genomic DNA .", "Site-directed mutagenesis by overlap extension was used to modify K50 sites ( Ho et al . , 1989 ) .", "The resultant PCR products were digested and ligated into GFP or DsRed reporter vectors derived from pCAGGS ( Hsiau et al . , 2007 ) .", "After verification by sequencing , plasmid DNA was resuspended in PBS at a concentration of ~ 6–7 µg/µl prior to injection .", "All primers are listed in Supplementary file", "2 . In vivo subretinal injection and electroporation of newborn CD1 mice was performed as previously described ( Matsuda and Cepko , 2004 ) .", "Briefly , mice were first anesthetized on ice .", "A 30-gauge needle was then used to incise the eyelid and puncture the sclera , and a Hamilton syringe with a 33-gauge blunt-tipped needle was used to inject the DNA into the subretinal space .", "Tweezer electrodes placed across the head were then used to electroporate with five square pulses of 80 volts and 50 millisecond duration at 950 millisecond intervals .", "Explant electroporation was carried out as described previously ( Hsiau et al . , 2007 ) , except that the electroporation chamber contained a solution of 0 . 5 µg/µl DNA in PBS .", "Eyes were removed at P21 , punctured with a 26-gauge needle , and incubated in 4% paraformaldehyde for 5 min before dissection to remove the cornea .", "The lens was removed , and eyes were then incubated for an additional 45 min in 4% paraformaldehyde .", "Eye cups were next washed in PBS and incubated overnight at 4°C in 30% sucrose-PBS .", "The following day , eye cups were incubated in a 1:1 mixture of OCT compound ( Sakura ) and sucrose-PBS before being flash frozen in OCT and stored at −80°C .", "Retinal sections of 14 µm were cut using a cryostat ( Leica CryoCut 1800 ) , mounted on Superfrost Plus slides ( Fisher ) , and stored at −20°C .", "Prior to placement of cover slips , slides were washed with PBS to remove OCT .", "The sections were then stained with DAPI , and coverslips were mounted using Vectashield mounting medium ( Vectorlabs ) .", "Retinal sections were imaged on a Zeiss 880 laser-scanning confocal microscopy in the Washington University Center for Cellular Imaging ( WUCCI ) Core .", "Transposition and library preparation from sorted cell populations were performed as previously described ( Buenrostro et al . , 2015 ) .", "Briefly , 30 , 000–100 , 000 sorted cells were pelleted at 500 G and washed twice in ice-cold PBS before lysis .", "Transposition reactions were incubated at 37°C for 30 min and purified using a Qiagen MiniElute PCR Purification kit .", "Libraries were amplified with Phusion High-Fidelity DNA Polymerase ( NEB ) .", "Cycle number was calibrated by a parallel qPCR reaction .", "Gel electrophoresis was used to assess library quality , and final libraries were quantified using KAPA Library Quantification Kit ( KAPA Biosystems ) .", "Equimolar concentrations of each library were pooled and run on an Illumina HiSeq2500 to obtain 50 bp paired-end reads .", "ATAC-seq and RNA-seq reads from bipolar cell populations were processed in an identical manner to those previously obtained from rod and cone photoreceptor cells ( Hughes et al . , 2017 ) .", "ATAC-seq reads were aligned to the GRCm38/mm10 mouse genome assembly using Bowtie2 ( v2 . 3 . 5 ) with a max fragment size of 2000 ( Langmead and Salzberg , 2012 ) .", "Alignments were filtered using SAMtools ( v1 . 9 ) ( Li et al . , 2009 ) , PCR duplicates were removed using Picard ( v2 . 19 . 0 ) ( https://broadinstitute . github . io/picard/ ) , and nucleosome-free reads were selected by removing alignments with an insertion size greater than 150 bp .", "Peaks were called using MACS2 ( v2 . 1 . 1 ) ( Zhang et al . , 2008 ) and annotated with HOMER ( v4 . 8 ) ( Heinz et al . , 2010 ) .", "DNase-seq datasets generated by ENCODE ( ENCODE Project Consortium , 2012 ) were downloaded as FASTQ files from https://www . encodeproject . org/ and processed in the same manner as ATAC-seq data .", "RNA-seq reads were aligned to the GRCm38/mm10 using STAR ( v2 . 7 . 0d ) ( Dobin et al . , 2013 ) , with an index prepared for 50 base-pair reads and the RefSeq gene model , and read counts were calculated using HTSeq ( v1 . 9 ) ( Anders et al . , 2015 ) .", "All datasets are listed in Supplementary file", "3 . Single-cell data from Shekhar et al . were downloaded from the Gene Expression Omnibus ( GSE81904 ) and processed as described by the authors to yield a digital expression matrix of normalized counts for each gene for each cell as well as cluster assignments for each cell .", "‘Pseudo-bulk’ expression estimates were then generated by taking the weighted average of counts for each gene across cells belonging to clusters that constituted bulk populations of interest ( RBC , BC5A , BC5C , BC5D , BC6 , BC7 , and BC8/BC9 for ON BCs; BC1A , BC1B , BC2 , BC3A , BC3B , BC4 for OFF BCs; and these clusters combined for pan BCs ) .", "Finally , pseudo-bulk expression estimates were re-scaled to match bulk expression estimates .", "Motif enrichment , co-enrichment , and spacing analyses for ATAC-seq and DNase-seq datasets were performed as described previously using HOMER ( v4 . 8 ) ( Hughes et al . , 2017; Heinz et al . , 2010 ) .", "Differential motif enrichment was determined using a test of equal proportions ( R stats v3 . 5 . 3 ) to compare each motif between pan BC and rods , blue cones or green cones .", "The top motifs across the three comparisons were manually filtered for redundancy and are shown in Figure", "4 . Motif co-occurrence analysis was performed using a list of 66 non-redundant motifs ( Hughes et al . , 2017 ) to which the motif for LHX3 ( representing a Q50 HD motif ) was manually added .", "For purposes of the analysis , peaks from rod , green cones and blue cones were merged to obtain a ‘photoreceptor’ peak list , while those from pan- , ON- and OFF-bipolar cells were merged to create a ‘bipolar cell’ peak list .", "Enrichment for co-occurrence was calculated by taking the log2 ( observed pairs +1/expected pairs+1 ) .", "Expected frequency of individual pairs was estimated from the counts for each motif within the pair ( motif one count × motif two count ÷ number of total peaks ) .", "Differential enrichment between tissues was calculated with a Fisher’s exact test .", "Motif spacing was analyzed for the top enriched peaks shown in Figure", "4 . The same set of peaks for co-occurrence were centered on individual K50 or Q50 motifs , and the density of flanking secondary motifs was plotted on either strand .", "DESeq2 ( v1 . 14 . 1 ) ( Love et al . , 2014 ) was used to test for differential expression or differential accessibility using a log2 fold-change threshold of 1 and an FDR of 0 . 05 .", "For comparison of ATAC-seq data with DNase-seq data from non-retinal tissues ( Figure 5 ) , photoreceptors were collapsed into a single level .", "Differentially expressed genes are listed in Supplementary file 5 , and differentially accessible regions are listed in Supplementary file 7 .", "For each comparison ( i . e . ON versus OFF , pan BC versus rod ) , gene expression stemming from low-level contamination of bipolar cell populations with either rod or cone photoreceptors was filtered out .", "When comparing pan BC to photoreceptor populations , potential contaminating genes from the alternate photoreceptor type ( i . e . rod genes identified as enriched in pan BC versus blue cone ) were identified as those highly expressed ( >16 fold ) and specific to rod compared to blue cone , and also more highly expressed in rod compared to pan BC ( at least four-fold ) .", "In comparing ON and OFF bipolar cells , genes enriched in each bipolar cell population were filtered for those which were also identified as highly specific to either photoreceptor population compared to the enriched bipolar cell type .", "For example , genes increased in OFF- compared to ON-bipolar cells were filtered for genes that were also highly enriched ( >16 fold ) in rods and blue cones compared to OFF bipolar cells .", "In total , 38 genes were filtered from those identified as enriched in pan BC compared to photoreceptors , and 39 genes were filtered from the ON versus OFF comparison ( 12 from the ON-enriched , 27 from the OFF-enriched ) .", "These genes include those known to be expressed at very high levels in either rod or cone photoreceptors ( Corbo et al . , 2007 ) .", "Sorted bipolar cell populations were resuspended in 500 µl TRIzol reagent ( Invitrogen ) , and RNA was isolated according to manufacturer’s instructions .", "Prior to sequencing , RNA quality was analyzed using an Agilent Bioanalyzer .", "cDNA was prepared using the SMARTer Ultra Low RNA kit for Illumina Sequencing-HV ( Clontech ) per manufacturer’s instructions .", "cDNA was fragmented using a Covaris E210 sonicator using duty cycle 10 , intensity 5 , cycles/burst 200 , time 180 s .", "cDNA was blunt-ended , an ‘A’ base added to the 3′ ends , and Illumina sequencing adapters were ligated to the ends of the cDNAs .", "Ligated fragments were amplified for 12 cycles using primers incorporating unique index tags .", "Replicate libraries from each bipolar cell population were pooled in equimolar ratios and sequenced on an Illumina HiSeq 3000 ( single-end 50 bp reads ) .", "For qPCR , RNA samples were treated with TURBO DNase ( Invitrogen ) and cDNA was synthesized with SuperScript IV ( Invitrogen ) and oligo ( dT ) primers according to manufacturer’s instructions .", "For Figure 1C , expression was normalized to the average of reference genes Gapdh , Sdha , Hprt , and Pgk .", "For Figure 1—figure supplement 2 , expression was normalized to Gapdh alone .", "Primers for Grm6 , Gnat1 , Lhx1 , Pax6 , Rlbp1 , Slc17a6 , Vsx2 , Grik1 , Tacr3 , Isl1 , and Lrrtm1 are listed in Supplementary file 2 ." ] ]

[ "Multicellular organisms evolved via repeated functional divergence of transcriptionally related sister cell types , but the mechanisms underlying sister cell type divergence are not well understood .", "Here , we study a canonical pair of sister cell types , retinal photoreceptors and bipolar cells , to identify the key cis-regulatory features that distinguish them .", "By comparing open chromatin maps and transcriptomic profiles , we found that while photoreceptor and bipolar cells have divergent transcriptomes , they share remarkably similar cis-regulatory grammars , marked by enrichment of K50 homeodomain binding sites .", "However , cell class-specific enhancers are distinguished by enrichment of E-box motifs in bipolar cells , and Q50 homeodomain motifs in photoreceptors .", "We show that converting K50 motifs to Q50 motifs represses reporter expression in bipolar cells , while photoreceptor expression is maintained .", "These findings suggest that partitioning of Q50 motifs within cell type-specific cis-regulatory elements was a critical step in the evolutionary divergence of the bipolar transcriptome from that of photoreceptors ." ]

[ "Humans see the world through a light-sensitive tissue at the back of the eye called the retina , which is made up of three layers that each contain specific cell types .", "The layers form a circuit , with light-sensing photoreceptor cells in the outermost layer connected to bipolar cells in the middle layer , which connect to the brain via specialized cells in the innermost layer .", "Photoreceptors and bipolar cells share similar characteristics and are thought to be ‘sister cells’ which evolved from a common ancestral cell type .", "However , it is not well understood how these two cells types diverged during evolution .", "Every cell type has a specific role , which is largely determined by the set of genes that it switches on or off .", "Specialized regions of DNA , called enhancers , determine whether a gene is turned on or off in a particular cell type .", "In each cell , DNA strands are bundled together with proteins into a coiled structure known as chromatin .", "In some cells , a particular enhancer may be ‘shut down’ and rendered inactive on account of being tightly packed within chromatin .", "Whilst in other cells , the same enhancer may be ‘open’ and ready for action .", "For a given cell type , which genes are turned on is determined , in part , by which enhancers are open .", "One way to distinguish between cells is by examining how their chromatin is packaged to see which enhancers are open .", "Researchers have previously characterized the chromatin structure of photoreceptor cells , but the structure of chromatin in bipolar cells , and how it compares to that of photoreceptors , remained unknown .", "Now , Murphy et al . have examined the chromatin profile of bipolar cells from the mouse retina in order to gain a better understanding of how these two cell types may be evolutionarily related .", "The analysis revealed that although bipolar and photoreceptor cells switch on different sets of genes , the enhancers open in each cell type are very similar .", "Despite this similarity , Murphy et al . were able to detect subtle differences in short sequences of DNA , known as motifs , present in bipolar and photoreceptor enhancers .", "Further experiments showed that one of these motifs may be responsible for turning photoreceptor genes off in bipolar cells .", "This motif therefore appears to play a critical role in distinguishing photoreceptors from bipolar cells .", "This comparison of photoreceptor and bipolar cells has provided a possible mechanism whereby photoreceptor and bipolar cells diverged in evolution from a single common ancestral cell type .", "This insight may help explain how complex organisms with many cell types may have evolved from a single-cell ancestor long ago ." ]

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

[ "epidemiology and global health", "microbiology and infectious disease" ]

[ [ "Artemisinin combination therapy ( ACT ) , the frontline treatment for P . falciparum infection , has played a major part in reducing the number of deaths due to malaria over the past decade ( World Health Organization , 2014b ) .", "However the increasing prevalence of artemisinin resistant P . falciparum across large parts of Southeast Asia threatens to destabilise malaria control worldwide ( Dondorp et al . , 2009; Hien et al . , 2012; Phyo et al . , 2012; Kyaw et al . , 2013; Ashley et al . , 2014; World Health Organization , 2014a ) .", "One of the main contemporary challenges in global health is to prevent artemisinin resistance from becoming established in Africa , where the consequences for childhood mortality could be disastrous ( Dondorp and Ringwald , 2013 ) .", "Understanding the epidemiological and evolutionary driving forces behind artemisinin resistance is essential to develop effective strategies to stop it spreading .", "At the molecular level , artemisinin resistance is caused by mutations in a kelch protein encoded by PF3D7_1343700 on P . falciparum chromosome 13 , referred to here as kelch13 .", "More specifically , non-synonymous mutations in the kelch13 propeller and BTB-POZ domains ( KPBD ) result in reduced sensitivity of P . falciparum to artemisinin , as demonstrated by multiple lines of evidence including laboratory studies of artificially acquired resistance , genetic association studies of natural resistance and allelic replacement experiments ( Ariey et al . , 2014; Ghorbal et al . , 2014; Miotto et al . , 2015; Straimer et al . , 2015; Takala-Harrison et al . , 2015 ) .", "Parasites with KBPD mutations tend to grow more slowly in the early part of the erythrocytic cycle , and have an enhanced unfolded protein response , both of which might act to protect against oxidative damage caused by artemisinin ( Dogovski et al . , 2015; Mok et al . , 2015 ) .", "It has also recently been shown that the PI3K protein is the target of artemisinin action , and that kelch13 binds to PI3K to mark it for degradation; by affecting this binding , KBPD mutations result in high levels of PI3K which counteract the effect of artemisinin ( Mbengue et al . , 2015 ) .", "A striking characteristic of the current distribution of artemisinin resistance is that it is caused by multiple independent KPBD mutations emerging in different locations , i . e . it does not originate from a single mutational event .", "More than 20 KPBD SNPs have been associated with delayed parasite clearance during artemisinin treatment and there are several documented instances of the same allele arising independently in different locations ( Ashley et al . , 2014; Miotto et al . , 2015; Takala-Harrison et al . , 2015 ) .", "These are classic features of a soft selective sweep which , according to evolutionary theory , is most likely to arise in populations where the selected alleles are already present as standing genetic variations or have been repeatedly introduced by de novo mutations ( Hermisson and Pennings , 2005; Pennings and Hermisson , 2006; Messer and Petrov , 2013 ) .", "There is ongoing debate among evolutionary biologists about how commonly soft selective sweeps occur in nature ( Jensen , 2014; Garud et al . , 2015 ) but they have clearly played a role in previous forms of antimalarial drug resistance ( Nair et al . , 2007; Salgueiro et al . , 2010 ) and the current epidemic of artemisinin resistance is the most extreme example of a soft selective sweep thus far observed in eukaryotes .", "This creates a practical problem in monitoring the global spread of resistance .", "Artemisinin resistance can be measured directly , by following the rate of parasite clearance in patients ( Flegg et al . , 2011 ) or by testing parasite isolates in vitro ( Witkowski et al . , 2013 ) , but these phenotypic assays are resource intensive and impractical for large-scale screening in resource-poor settings .", "Genetic approaches are therefore preferable for practical implementation of large-scale surveillance , but the soft selective sweep of artemisinin resistance produces much more heterogeneous genetic signatures than previous global waves of chloroquine and pyrimethamine resistance , where hard selective sweeps were the dominant mode of spread .", "Thus there is considerable uncertainty about the epidemiological significance of the growing number of non-synonymous KPBD mutations reported in Africa ( Ashley et al . , 2014; Hopkins Sibley , 2015; Kamau et al . , 2015; Taylor et al . , 2015 ) .", "Previous studies in Africa have not identified variants known to cause resistance in Southeast Asia ( Kamau et al . , 2015; Taylor et al . , 2015 ) .", "At the same time , there are documented instances of African parasites that can be rapidly cleared by artemisinin treatment , although they carry KPBD mutations ( Ashley et al . , 2014 ) .", "In the absence of comprehensive phenotypic data , it is not known which of these mutations , if any , are markers of resistance .", "This is a limitation of conventional molecular epidemiology , which tracks specific mutations and haplotypes and is poorly equipped to monitor soft selective sweeps where new mutations are continually arising on different haplotypic backgrounds , making it difficult to keep track of their phenotypic effects and evolutionary trajectories .", "Here we explore how genomic data might help overcome these practical obstacles to monitoring the current epidemic of artemisinin resistance .", "This analysis includes genome sequencing data for 3 , 411 clinical samples of P . falciparum obtained from 43 locations in 23 countries .", "This large dataset was generated by the MalariaGEN Plasmodium falciparum Community Project , ( www . malariagen . net/projects/parasite/pf ) , a collaborative study in which multiple research groups working on different scientific questions are sharing genome variation data to generate an integrated view of polymorphism in the global parasite population .", "Data resources arising from the present analysis , including genotype calls and sample metadata , can be obtained at www . malariagen . net/resource/16 .", "The MalariaGEN Plasmodium falciparum Community Project also developed a user-friendly web application , with interactive tools for exploring and querying the latest version of the data: www . malariagen . net/apps/pf ." ], [ "Paired-end sequence reads were generated using the Illumina platform and aligned to the P . falciparum 3D7 reference genome , applying a series of quality control filters as previously described ( see Materials and methods ) ( Manske et al . , 2012; Miotto et al . , 2013 ) .", "The initial alignments identified 4 , 305 , 957 potential SNPs , which after quality control filtering produced a set of 935 , 601 exonic SNPs that could be genotyped with high confidence in the majority of samples , and that form the basis for the current analysis .", "As summarised in Table 1 , the dataset comprised 1 , 648 samples from Africa and 1 , 599 samples from Southeast Asia , allowing us to compare these two groups directly without the need for sample size corrections .", "We identified a total of 155 SNPs in the kelch13 gene , of which 128 were seen in Africa and 62 in Southeast Asia ( Table 2 ) .", "Studies in Southeast Asia have found that artemisinin resistance is associated with non-synonymous polymorphisms in the KPBD .", "Out of a total of 46 non-synonymous SNPs in these domains we found a similar number in Africa ( n = 26 ) and Southeast Asia ( n = 34 ) , with 14 seen in both places ( Table 3 ) .", "Seven of those observed in Africa have previously been associated with artemisinin resistance in Southeast Asia , including C580Y , the most common allele in resistant parasites ( Miotto et al . , 2015 ) . 10 . 7554/eLife . 08714 . 003Table 1 . Count of samples analysed in this study . DOI: http://dx . doi . org/10 . 7554/eLife . 08714 . 003RegionCodeSample countsCountryCodeSample countsWest AfricaWAF957Burkina FasoBF56CameroonCM134GhanaGH478Gambia , TheGM73GuineaGN124MaliML87NigeriaNG5East AfricaEAF412KenyaKE52MadagascarMG18MalawiMW262TanzaniaTZ68UgandaUG12Central AfricaCAF279D .", "R . CongoCD279South AmericaSAM27ColombiaCO16PeruPE11South AsiaSAS75BangladeshBD75West Southeast AsiaWSEA497MyanmarMM111ThailandTH386East Southeast AsiaESEA1102CambodiaKH762LaosLA120VietnamVN220OceaniaOCE62Indonesia ( Papua ) ID17Papua New GuineaPG4510 . 7554/eLife . 08714 . 004Table 2 . Worldwide distribution of kelch13 mutations .", "Number of kelch13 propeller and BTB-POZ domain ( KPBD ) mutations present ( not necessarily exclusively ) in 5 populations ( AFR = Africa , SEA = Southeast Asia , SAS = South Asia , OCE = Oceania , SAM = South America ) .", "Sample size is reported for each population . DOI: http://dx . doi . org/10 . 7554/eLife . 08714 . 004AFR ( N = 1 , 648 ) SEA ( N = 1 , 599 ) SAS ( N = 75 ) OCE ( N = 62 ) SAM ( N = 27 ) Non-synonymousKPBD2634110Upstream region4216211SynonymousKPBD389100Upstream region22310010 . 7554/eLife . 08714 . 005Table 3 . List of non-synonymous KPBD mutations .", "Non-synonymous mutations found in the kelch13 propeller and BTB-POZ domains ( KPBD ) and their position on chromosome Pf3D7_13_v3 .", "For each mutation is reported where it has been observed and in how many samples .", "Where known , we reported if the mutation has been previously observed in patients with a prolonged parasite clearance half-life ( >5 hr ) by Miotto et al . 2014 and/or Ashley et al 2014 .", "Sample size is reported for each population . DOI: http://dx . doi . org/10 . 7554/eLife . 08714 . 005MutationGenomic coordinatesAFR ( N = 1 , 648 ) SEA ( N = 1 , 599 ) SAS ( N = 75 ) OCE ( N = 62 ) SAM ( N = 27 ) Observed in ART-R samples ?", "D353Y1725941-4---YesF395Y1725814-1---NoI416V172575211---I416M17257501----K438N1725684-1---NoP441L1725676-27---YesP443S1725671-1---F446I1725662-7---YesG449A1725652-7---YesS459L172562222---A481V1725556-4---YesS485N1725544-1---Y493H1725521176---YesV520I17254401----S522C172543421---YesP527H172541815---C532S17254041----V534L17253982----N537I172538811---NoG538V1725385-19---YesR539T1725382-63---YesI543T1725370-34---YesP553L1725340224---YesA557S17253291----R561H1725316124---YesV568G1725295-6---YesT573S17252802----P574L1725277-12---YesR575K1725274-3---A578S172526618----NoC580Y17252592423-1-YesD584V1725247-3---YesV589I17252332----T593S17252211----E612D17251621----Q613E172516151---Q613L17251601----NoF614L1725158-1---NoY630F172510921---V637I17250892----P667A1724999-2---P667L1724998-21--F673I1724981-3---YesA675V1724974118---YesA676S172497223---H719N172484318---Yes We asked whether kelch13 polymorphisms seen in Africa had emerged independently , as opposed to migrating from Southeast Asia .", "We started by grouping samples according to genome-wide genetic similarity , based on a neighbour-joining ( NJ ) tree ( Figure 1A ) .", "African and Southeast Asian samples formed two well-separated and distinct clusters , suggesting that gene flow between the two regions is very modest or negligible , a view supported by an alternative analysis using principal coordinate analysis ( Figure 1B ) .", "None of the African parasites carrying kelch13 mutations grouped with the Southeast Asian population , supporting the idea that these mutations are indigenous . 10 . 7554/eLife . 08714 . 006Figure 1 . Differentiation between African and Asian genomes .", "( A ) Neighbour-joining tree showing population structure across all sampling sites , with sample branches coloured according to country groupings ( Table 1 ) .", "Branches with large open tip circle indicate the sample is a kelch13 mutant , while those with a small black symbol are mixed infections ( i . e . mixture of wild-type and mutant parasites or two mutant parasites with different mutations ) .", "Branches without tip symbols are kelch13 wild type .", "African kelch13 mutants are , at a genomic level , similar to other African samples .", "( B ) Plot of second principal component ( PC2 ) against the first ( PC1 ) , computed from a principal coordinate analysis ( PCoA ) of all samples in the present study , based on the same pairwise genetic distance matrix used for the tree of Figure 1A .", "PC1 clearly separates African samples from those collected in SEA , while PC2 is mainly driven by extreme population structure in ESEA . DOI: http://dx . doi . org/10 . 7554/eLife . 08714 . 006 We considered that recombination between African and imported parasites could result in the transfer of DNA segments of Asian origin into genomes that otherwise appear to be local , and therefore not be easily detectable through genome-wide analysis .", "Thus , we analyzed the regions immediately flanking kelch13 in both directions , using a probabilistic method to reconstruct the most likely geographical origin of haplotypes observed in African kelch13 mutants ( Figure 2 ) .", "The vast majority of kelch13 mutants showed no evidence that flanking regions may have been imported , which indicates that most mutations observed in Africa do not have a common origin with those in Asia , and are likely to have emerged independently on a variety of haplotypes ( Figure 2—figure supplement 1 ) . 10 . 7554/eLife . 08714 . 007Figure 2 . Local origin of African kelch13 mutations .", "( A ) Chromosome painting ( see Materials and methods ) of the 52 African kelch13 mutants across the two 250 kbp flanking regions on each side of the kelch13 gene .", "Each genome chunk is coloured according to the aggregated posterior probabilities that it originated in the African ( red ) or SEA ( blue ) population , according to the scale shown .", "( B ) Detail of the flanking regions over a span of approximately 15 kbp , using the same colour scheme .", "The country of origin is indicated on the left , followed by the proportion of African chunks identified; the kelch13 mutation carried by the sample is shown on the right .", "Samples are sorted by the proportion of Asian chunks in this window , and the same order was applied to Panel A . Only five samples ( lower region of the panel ) show strong probability of Asian origin of the chunks closest to kelch13 . DOI: http://dx . doi . org/10 . 7554/eLife . 08714 . 00710 . 7554/eLife . 08714 . 008Figure 2—figure supplement 1 . Diversity within kelch13-flanking haplotypes in African mutants . Using the same SNPs utilized in the analysis shown in Figure 2B , we estimated diversity in a 1 kbp sliding window around kelch13 ( highlighted in grey ) , by computing the mean SNP heterozygosity in a random set of 56 African kelch13 wild type genomes .", "The blue shaded area reports 95% of the distribution of the values over 1000 iterations .", "We also computed the heterozygosity estimate for the set of 56 African kelch13 mutants ( red line ) , and found it not to be significantly different from those in wild-type parasites , suggesting that no common flanking haplotype is shared among these mutants . DOI: http://dx . doi . org/10 . 7554/eLife . 08714 . 00810 . 7554/eLife . 08714 . 009Figure 2—figure supplement 2 . Origin of flanking haplotypes in selected African kelch13 mutants . Here , chromosome painting is applied to the 15 kbp region around the kelch13 gene ( as in Figure 2B ) for five samples for which kelch13 was flanked by chunks with >50% probability of Asian origin .", "Here , the SEA population was divided into two groups: kelch13 wild type ( blue ) and mutants ( yellow ) , to estimate the likelihood that the haplotype originated in SEA kelch13 mutants .", "Strong evidence of origin from SEA mutants was only found in two samples: Y483H mutant from Ghana ( top row ) , and a C580Y mutant from Cameroon ( bottom row ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 08714 . 009 Only 5 samples out of 56 were assigned a significant probability of Southeast Asian origin in at least one flanking sequence , and only two of these samples carried haplotypes similar to those in SEA mutants ( Figure 2—figure supplement 2 ) .", "Both in the NJ tree and in the PCA , these two African kelch13 mutants do not cluster with the bulk of African samples , but occupy a somewhat isolated position ( Figure 1 ) .", "We note that they exhibit unusually high levels of heterozygosity ( FWS < 0 . 4 ) , with mixed calls randomly distributed across the genome , consistent with a mix of African and SEA parasites .", "Continuous genetic monitoring of the parasite population will determine whether these are indeed just isolated cases , or they constitute very early evidence of gene flow between the two regions .", "Since these samples passed through a number of different laboratories , we cannot absolutely rule out the possibility that these mixtures could be the result of biological contamination during sample preparation and processing .", "Historical demographic changes such as population expansions and bottlenecks ( Tanabe et al . , 2010 ) , and epidemiological and environmental factors ( Prugnolle et al . , 2010 ) are highly influential forces that shaped the allele frequency spectra of P . falciparum populations across the globe ( Nielsen et al . , 2009; Manske et al . , 2012 ) .", "In order to properly contextualize the numbers and frequencies of kelch13 mutations , it is therefore important to characterize genomic variation patterns in different geographical regions .", "One of the most striking features of this dataset is the high number of rare variations in the high-quality SNP list .", "At more than half of all polymorphic sites , the minor allele was only carried by a single sample ( referred to as singletons , n = 330 , 783 or 35% ) or by two samples ( doubletons , n = 214 , 179 or 23% ) , often in heterozygous calls .", "By contrast , only 13 , 383 polymorphisms ( 1 . 4% ) had a minor allele in ≥5% of samples .", "Rare alleles , however , are not evenly distributed geographically .", "There is a large excess of polymorphisms with minor allele frequency ( MAF ) below 0 . 1% in Africa ( 71% of all SNPs , vs . 17% in SEA ) , while numbers in the two regions are similar for SNPs with MAF>1% ( 2% of all SNPs , Figure 3a ) .", "Rare variations in Africa are not confined to a limited set of highly variable genes , but evenly distributed across the genome , as attested by the distribution of variants across all genes: SNP density in Africa ( median = 67 SNPs/kbp , interquartile range = 51–84 ) is approximately 3 . 9 times higher than in SEA ( median = 17 , IQR = 13–22 , p < 10–16 , Figure 3b ) .", "Very similar ratios are estimated in both non-synonymous ( Africa median SNP density = 43 , IQR = 27–58; SEA median = 11 , IQR = 7–15 ) and synonymous variants ( Africa median SNP density=25 , IQR = 20–30 , SEA median = 6 , IQR = 4–8 ) .", "Accordingly , we found virtually identical distributions of the ratio of non-synonymous to synonymous mutations ( N/S ratio ) in the two regions ( Figure 3c ) .", "This suggests that the huge disparity in SNP density between the two regions is more likely to be the result of different demographic histories and epidemiological characteristics , such as changes in effective population size ( Joy et al . , 2003 ) , rather than the product of different selective constraints . 10 . 7554/eLife . 08714 . 010Figure 3 . Number and density of variants in Africa and Southeast Asia .", "( a ) Allele frequency spectrum for Africa ( red ) and SEA ( blue ) .", "Polymorphisms were binned by their minor allele frequency ( MAF ) , and the counts in each bin were plotted against frequency , shown on a logarithmic scale .", "Although the number of high-frequency variations is consistent between the two regions , samples from Africa possess an excess of low-frequency variations .", "( b ) SNP density per gene: for each gene , the number of variants found in the two regions is normalized by the length of the coding region ( in kbp ) .", "African samples have on average 3 . 9 times more mutations than parasites from SEA .", "( c ) Non-synonymous/synonymous ratio per gene: ratios of non-synonymous to synonymous mutations found per gene are similar in the two regions .", "To reduce artifacts due to small numbers , only genes with at least 10 SNP were considered in both analyses . DOI: http://dx . doi . org/10 . 7554/eLife . 08714 . 01010 . 7554/eLife . 08714 . 011Figure 3—figure supplement 1 . Site frequency spectrum for non-synonymous mutations in the KPBD . Allele frequency spectra for Africa ( red ) and SEA ( blue ) of non-synonymous mutations in the KPBD .", "Polymorphisms were binned by their minor allele frequency ( MAF ) , shown on a logarithmic scale , and the counts in each bin reported .", "The two populations exhibit dramatically different behaviours with an abundance of medium and high frequency variations in SEA contrasted by an extreme paucity in Africa . DOI: http://dx . doi . org/10 . 7554/eLife . 08714 . 011 In summary , we observe many more rare variants in Africa than in SEA; however we expect N/S ratios to be similar in these two regions in genes that are not subjected to selective pressures .", "The density of kelch13 synonymous variations in the two continents is roughly consistent with that observed in the rest of the genome ( Africa: 28 SNPs/kbp , SEA: 6; Figure 4a ) , which is expected since synonymous changes are less likely to be affected by selection .", "The excess of African non-synonymous mutations in the upstream region is also consistent with expectations ( Africa: 44 SNPs/kbp , SEA: 16 ) .", "In contrast , non-synonymous polymorphisms in the KPBD show a reversal of this relationship: SEA parasites possess about 30% more polymorphisms than African ones ( 34 in SEA vs . 26 in AFR ) .", "In addition , all but two non-synonymous changes in the KPBD are observed in Africa at very low frequency ( singletons and doubletons ) , while in SEA more than half of the changes are observed in >2 samples ( Table 4 and Figure 3—figure supplement 1 ) . 10 . 7554/eLife . 08714 . 012Figure 4 . Structure of kelch13 , positioning of mutations in Africa and Southeast Asia , and sequence conservation .", "( a ) The amino acid positions of kelch13 polymorphisms observed in Africa ( red ) and SEA ( blue ) are shown .", "Coloured rectangles describe the extents of the resistance domains ( BTB/POZ: aa . 349–448; kelch propeller: aa . 443–721 ) and upstream region , with the locations of non-synonymous changes indicated above , and that of synonymous changes below .", "Short lines represent singleton/doubleton mutations , while longer lines represent more frequent mutations .", "( b ) Conservation score across amino acid residues of kelch13 , derived by applying a CCF53P62 matrix on alignments of the P . falciparum gene coding sequence with its homologues in six other Plasmodium species for which high-quality sequence data were available: P . reichenowi , P . vivax , P . knowlesi , P . yoelii , P . berghei , and P . chabaudi ( see Materials and methods ) .", "Although the region below position 200 is less conserved , particularly in rodent species ( P . yoelii , berghei , and chabaudi ) , there is remarkably high conservation across all species over the rest of the gene , which includes the KPBD . DOI: http://dx . doi . org/10 . 7554/eLife . 08714 . 01210 . 7554/eLife . 08714 . 013Table 4 . Frequency of the non-synonymous KPBD mutations .", "Counts of non-synonymous mutations in the conserved propeller and BTB-POZ domains of kelch13 are shown for each geographical region , stratified by the number of samples in which they are observed .", "Sample size for each population is reported . DOI: http://dx . doi . org/10 . 7554/eLife . 08714 . 013AFR ( N = 1 , 648 ) SEA ( N = 1 , 599 ) SAS ( N = 75 ) OCE ( N = 62 ) SAM ( N = 27 ) 1–2 samples24131103–5 samples17000>5 samples114000 At a first approximation , these observations are consistent with a high number of non-synonymous changes that have risen in frequency in SEA parasites because of their association with artemisinin resistance , and a low number of mostly rare alleles in Africa , where artemisinin has been introduced more recently and resistance is yet to be reported .", "Although the function of kelch13 is as yet unclear , an alignment of its homologous gene sequences in eight Plasmodium species shows that the propeller and BTB-POZ domains are part of a highly conserved region ( Figure 4b ) , suggesting a crucial role in parasite fitness .", "A reconstruction of ancestral alleles from this alignment suggests that P . falciparum accumulated only five conservative amino acid changes in the kelch13 propeller domain since diverging from other species 55 Myr ago ( Escalante and Ayala , 1995 ) ( Table 5 ) .", "Given this extreme level of conservation , non-synonymous polymorphisms may appear surprisingly numerous in the present dataset , both in SEA ( n = 34 ) and in Africa ( n = 26 ) .", "Such elevated numbers may be produced by selection processes; alternatively , they may be present in a large neutrally-evolving population , in which low-frequency variations continually emerge , but can only be detected for a brief span of time before they are removed by genetic drift and/or purifying selection .", "The question is , then , whether neutral evolution can account for the pattern of kelch13 mutations observed here . 10 . 7554/eLife . 08714 . 014Table 5 . Kelch13 propeller domain mutations in different Plasmodium species .", "Here we report amino acid allele differences in a multiple sequence alignments of kelch13 homologues for seven species of Plasmodium parasites for which high-quality sequence data were available: P . falciparum ( Pf ) , P . reichenowi ( Pr ) , P . vivax ( Pv ) , P . knowlesi ( Pk ) , P . yoelii ( Py ) , P . berghei ( Pb ) , and P . chabaudi ( Pc ) .", "The species formed three groups by similarity: Laverania ( Pf , Pr ) , primate Plasmodia ( Pv , Pk ) and rodent Plasmodia ( Py , Pb and Pc ) .", "An allele shared by all members of two different groups was identified as a putative ancestral allele .", "The table shows , for each position where at least one species exhibits a difference from the others: the amino acid position in the Pf kelch13 sequence; the putative ancestral amino acid allele; the alleles in the various species ( columns with heading listing multiple species show mutations common to those species ) ; and a substitution score of the mutation , based on a CCF53P62 substitution matrix ( see Materials and methods ) .", "All substitution scores are ≥0 , denoting conservative substitutions . DOI: http://dx . doi . org/10 . 7554/eLife . 08714 . 014Pf PositionAncestral AllelePf , PrPv , PkPkPy , Pb , PcPy , PbPbPcSubstitution Score434F------Y2447C----S--1448I-M-----2517TV------0519FY------2520V---L---0534V---I---2550S--C----1566V---I---2568V-I-----2578A-S-----0584D--E----1590I----V--2593T---A---0605DE------1613Q----N-K0648D---E---1666V---I---2676A---T---0691DE------1708IL------0711S-----P-0723I---V---2 To answer this question , we compared patterns of kelch13 mutations to those in the rest of the P . falciparum genome Supplementary file 1 .", "Since fewer non-synonymous mutations are expected in more conserved genes , we applied genomic calibration , i . e . we stratified these analyses by evolutionary conservation .", "Each gene was assigned a conservation score determined from a sequence alignment of the P . falciparum gene with its P . chabaudi homologue , using a substitution matrix corrected for the AT bias in the Pf genome ( Brick and Pizzi , 2008 ) .", "P . chabaudi was chosen since it was the member of the group most differentiated from P . falciparum ( rodent plasmodia ) with the most complete reference sequence .", "A genome-wide non-linear negative correlation between gene conservation and N/S ratio is clearly observable; this trend is almost identical in the two populations ( Figure 5a ) .", "Although kelch13 did not diverge significantly from this relationship in Africa ( P = 0 . 2 ) , its N/S ratio in SEA was the highest observed at its level of conservation , far exceeding the expected ratio ( P<0 . 001 ) .", "Accordingly , kelch13 showed the most significant difference in N/S ratios between Africa and SEA genome-wide ( 3 . 7-fold , P = 2 × 10-4 by Fisher’s exact test , Figure 5b and Supplementary file 1 ) , even compared to other well-known drug resistance genes ( Figure 5—figure supplement 1 ) .", "Such unusually high N/S ratio in SEA parasites is mainly due to an excess of high frequency non-synonymous variations ( Figure 5—figure supplement 2 ) , suggesting that multiple independent origins of artemisinin resistance ( Miotto et al . , 2015; Takala-Harrison et al . , 2015 ) have produced an unusually large number of common non-synonymous mutations . 10 . 7554/eLife . 08714 . 015Figure 5 . Genome-wide analysis of N/S ratio .", "( a ) For each gene with more than 2 synonymous or non-synonymous SNPs , the N/S ratio in Africa ( red points ) and in SEA ( blue points ) are plotted against the conservation score of the gene coding sequence .", "The kelch13 gene values are represented by larger circles .", "For each region , a solid line show median values , while dotted lines delimit 95% of the genes at varying levels of conservation .", "This plot is truncated on the y-axis to show more clearly the bulk of the distribution; the full range is shown in Figure 5—figure supplement 2 .", "( b ) Histogram showing the distribution of the ratio of N/S ratios in SEA and Africa , for all genes with ≥5 synonymous and ≥5 non-synonymous SNPs on each region .", "An arrow shows the placement of kelch13 . DOI: http://dx . doi . org/10 . 7554/eLife . 08714 . 01510 . 7554/eLife . 08714 . 016Figure 5—figure supplement 1 . N/S ratio of well-known drug resistance genes . This figure shows the same data as Figure 5a , extended by mapping genes known to have undergone selection for antimalarial resistance .", "For each gene , we show circles marking the N/S ration in Africa ( red letter ) and SEA ( blue letter ) , paired by a vertical line annotated with a p-value ( by Fisher’s exact test ) that the two ratios are significantly different .", "The genes shown are kelch13 ( denoted by the letter ‘K’ ) , pfcrt ( chloroquine resistance , ‘C’ ) , pfdhfr ( pyrimethamine resistance , ‘R’ ) , pfdhps ( sulfadoxine resistance , ‘S’ ) , and pfmdr1 ( resistance to chloroquine and other drugs , ‘M’ ) .", "These results are best analyzed in the context of the underlying selective sweeps .", "For example , the small difference between ratios for the pfmdr1 gene can be explained by the association of the drug resistance phenotype with gene amplification , rather than with non-synonymous changes .", "Also , the extremely high N/S ratio for pfcrt in SEA is largely due to a paucity of synonymous SNPs , caused by the near-fixation of a very small number of haplotypes in the SEA region , yielding a p-value comparable to that of kelch13 . DOI: http://dx . doi . org/10 . 7554/eLife . 08714 . 01610 . 7554/eLife . 08714 . 017Figure 5—figure supplement 2 . Genome-wide analysis of high-frequency SNP density .", "( a ) Plot of the density of frequent ( present in 3 or more samples ) non-synonymous mutations in Africa ( red ) and SEA ( blue ) against conservation , for all genes with more than 2 synonymous or non-synonymous SNPs , showing that kelch13 ( larger circle ) is consistent with other similarly conserved genes in Africa , but has excess non-synonymous polymorphisms in SEA .", "( b ) An analogous plot for synonymous mutations also shows that kelch13 follows the normal trend among genes in Africa , but has far fewer SNPs than expected in SEA . DOI: http://dx . doi . org/10 . 7554/eLife . 08714 . 017 From this analysis we conclude that the high prevalence of kelch13 non-synonymous variants in SEA is not explainable by neutral evolution , but is consistent with selection of artemisinin-resistance alleles .", "In Africa , on the other hand , the observed non-synonymous changes appear to constitute a “physiological” level of variation consistent with a population rich in low-frequency alleles .", "The different kelch13 mutation repertoires in Africa and in SEA raise the question of whether these sets of mutations have different structural and functional properties .", "While there is high conservation across the whole of the propeller domain , it is unlikely that all possible amino acid changes have the same functional relevance or that they all carry the same fitness cost for parasites .", "Although direct measures of functional relevance are not yet available , and the exact function of kelch13 is hitherto unknown , we can make statistical comparisons of some properties of the observed changes , in at least two respects .", "First , assuming that kelch13 function is conserved across Plasmodium species , we can assess the strength of evolutionary constraints at any given position by examining whether amino acid substitutions between species are conservative or radical .", "Second , given that kelch proteins have been shown in other organisms to play an adapter role , with key binding sites defined by the arrangement of hydrophobic β-strands in the propeller domain ( Adams et al . , 2000 ) , we can assess changes in hydrophobicity caused by the observed mutations , which may be informative of their functional importance .", "Detailed mapping against the secondary structure of the propeller domain suggests that the polymorphisms found in SEA parasites occur in different blades , preferentially at positions proximal to the first and second β-strand of the propeller’s blades ( Figure 6 ) .", "This may indicate that these two strands play a role in defining the binding interface to the PI3K protein involved in artemisinin resistance ( Mbengue et al . , 2015 ) , but this needs to be confirmed by in-depth structural analyses . 10 . 7554/eLife . 08714 . 018Figure 6 . Structure of the kelch13 propeller domain , showing the position of mutations in Southeast Asia and Africa . The sequence of the kelch13 propeller domain ( amino acids 443–726 ) is organized according to its 6-blade tertiary structure , with the four β-strands characterizing each blade highlighted in colour .", "Polymorphisms observed in SEA ( top panel ) and Africa ( bottom panel ) are shown by circles above the mutated position .", "Small circles indicate very rare mutations ( singletons and doubletons ) , while larger circles are used for more frequent mutations . DOI: http://dx . doi . org/10 . 7554/eLife . 08714 . 01810 . 7554/eLife . 08714 . 019Figure 6—figure supplement 1 . Characterization of kelch13 mutations in Africa and Southeast Asia . In these plots , all kelch13 amino acids in the propeller domains , downstream of position 430 , are plotted against their Kyte-Doolittle hydrophobicity score ( KD , gray ) .", "Circle symbols in plot", "( a ) derived amino acid alleles in P . falciparum , coloured according to the conservation score against the ancestral allele ( Table 5 ) , derived from the CFF53P62 substitution matrix; lower values indicate more radical substitutions .", "The remaining plots use the same colouring scheme to show polymorphisms observed in ≥3 samples in Africa", "( b ) or in SEA", "( c ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 08714 . 019 We characterized changes in the propeller domain by a conservation score derived from a substitution matrix specific to AT-rich genomes ( Brick and Pizzi , 2008 ) , and assigned a hydrophobicity score to each site , estimated from the Kyte-Doolittle ( KD ) hydropathicity score ( Kyte and Doolittle , 1982 ) .", "The five putative derived alleles that have become established in the P . falciparum kelch13 propeller domain since its divergence from other Plasmodia are all conservative changes at hydrophilic sites ( Figure 6—figure supplement 1a ) .", "Common mutations in Africa have characteristics broadly consistent with this conservative history of change ( Figure 6—figure supplement 1b and Table 6 ) .", "Polymorphisms in SEA parasites , on the other hand , show a pattern of changes that are more radical than those in Africa ( P = 10–3 ) and more commonly found at hydrophobic sites ( Figure 6—figure supplement 1c and Table 6 ) . 10 . 7554/eLife . 08714 . 020Table 6 . Conservation score of KPBD mutations . The table shows , for each non-synonymous KPBD mutation observed in the dataset , the number of samples carrying the mutation in Africa ( AFR ) , in Southeast Asia ( SEA ) , and a substitution score of the mutation , based on a CCF53P62 substitution matrix; lower values indicate more radical substitutions .", "Mutations observed in Africa tend to have higher conservation score , whereas in SEA mutations tend to be more radical . DOI: http://dx . doi . org/10 . 7554/eLife . 08714 . 020MutationAFRSEACFF53P62Q613E512Y630F212V637I202V589I202T573S202Y493H1762I416V112T593S102V520I102I416M102F395Y012S522C211E612D101C532S101R575K031A578S1800A676S230V534L200R561H1240A675V1180H719N180P527H150A557S100R539T0630I543T0340G449A070F446I070A481V040F673I030P667A020F614L010S485N010P443S010C580Y2423-2D353Y04-2K438N01-2P553L224-3P441L027-3G538V019-3P574L012-3V568G06-3P667L02-3S459L22-4N537I11-4Q613L10-4D584V03-5 Taken together , the above results suggest that the propeller domain has long been under very strong evolutionary constraints , and that the number and nature of the changes observed in African parasites is consistent with these constraints , once we discard the abundant rare alleles expected in such a large population .", "In contrast , mutations in SEA parasites are not only far more numerous than expected , but they produce radical changes that are likely to be important determinants of the binding properties of the kelch13 protein , consistent with recent findings that binding of kelch13 to the PI3K protein is a critical factor in P . falciparum response to artemisinin ( Mbengue et al . , 2015 ) .", "A recent study has shown that resistance-causing KPBD mutations are significantly more likely to arise in parasites with a particular genetic background ( Miotto et al . , 2015 ) .", "This predisposing genetic background is marked by specific SNP alleles of the genes encoding ferredoxin ( fd ) , apicoplast ribosomal protein S10 ( arps10 ) , multidrug resistance protein 2 ( mdr2 ) and chloroquine resistance transporter ( crt ) .", "Here we extend this analysis , confirming that this particular combination of variants is extremely common in the parts of Southeast Asia where artemisinin resistance is known to be established , and is absent from Africa and other regions sampled here ( Table 7 ) . 10 . 7554/eLife . 08714 . 021Table 7 . Frequency of the genetic background alleles across the world .", "Frequency of the four genetic background alleles identified in Miotto et al . ( 2015 ) for each geographical region .", "For each SNP , we show mutation name; chromosome number; nucleotide position; and frequencies of the mutant allele in the various populations . DOI: http://dx . doi . org/10 . 7554/eLife . 08714 . 021MutationChrPosAFRSASSEAPNGSAMarps10-V127M1424810700 . 0%0 . 0%59 . 4%0 . 0%0 . 0%fd-D193Y137483950 . 1%2 . 2%62 . 8%23 . 9%0 . 0%mdr2-T484I1419562250 . 1%5 . 7%64 . 2%0 . 4%0 . 0%crt-N326S74053620 . 8%28 . 2%68 . 6%0 . 1%0 . 0%" ], [ "This study demonstrates the value of genomic data in characterising the current epidemic of artemisinin resistance , which is problematic for conventional molecular epidemiology since new resistance-causing mutations are continually emerging on different haplotypic backgrounds .", "A key problem is to define the geographical origin of KBPD mutations , and we show that this can be solved by using genomic epidemiological data to analyse ancestral relationships between samples , thereby demonstrating that the KPBD mutations observed in Africa are of local origin .", "Another important question is whether KPBD mutations are under positive selection in Africa , which is difficult to determine by standard haplotype-based methods because so many independent mutations are involved .", "This question is further complicated by marked geographical variation in normal levels of genetic diversity , i . e . there are many more rare variants in Africa than Southeast Asia , most likely due to the larger population size and other demographic factors ( Manske et al . , 2012 ) .", "Here we address this question by comparing kelch13 against other genes in the same samples , a process that we refer to as genomic calibration .", "We show that for most genes the ratio of non-synonymous to synonymous mutations is relatively constant across geographical regions , despite geographic differences in genetic diversity , and that this ratio is correlated with the level of sequence conservation across different Plasmodium species .", "When calibrated against other genes with the same level of cross-species conservation , allowing for geographical differences in the overall level of genetic diversity , kelch13 shows a marked excess of non-synonymous substitutions in Southeast Asia , but appears normal in Africa .", "Moreover , KPBD mutations causing radical amino acid changes at highly conserved positions are found at relatively high frequency in Southeast Asia but remain at very low frequency in Africa .", "Taken together , these findings indicate that non-synonymous KBPD mutations are undergoing strong evolutionary selection in Southeast Asia , whereas those seen in Africa have originated locally and most likely reflect normal variation .", "These findings have practical implications for the prevention of artemisinin resistance in Africa , where there is evidently a deep reservoir of low frequency genetic variations that could potentially allow resistance to emerge rapidly as the levels of selective pressure increase .", "In most parts of Africa , the selective pressure of artemisinin is probably relatively low at present , for several reasons .", "Artemisinin has been widely used in Southeast Asia for over two decades , whereas its usage in Africa is more recent , and it is estimated that only 20% of infected African children currently have access to frontline treatment ACT medication ( World Health Organization , 2014b ) .", "Another factor is that people living in regions of high malaria endemicity acquire partial immunity , resulting in asymptomatic infection , so that there is a large reservoir of parasites in Africa that are not exposed to antimalarial drugs because asymptomatic individuals do not seek treatment ( Hastings , 2003 ) .", "The situation could change dramatically as malaria control efforts are intensified , and it will be vital to monitor the effects of major interventions on the emergence of resistance , particularly in African countries that have already achieved relatively low levels of malaria transmission .", "A key question for the future is whether parasite populations in certain locations possess genetic features that predispose to the emergence of artemisinin resistance , as suggested by the strong association of certain fd , arps10 , mdr2 and crt alleles with resistance-causing KPBD mutations in Southeast Asia ( Miotto et al . , 2015 ) .", "Another concern is that growing resistance to ACT partner drugs , now emerging in Southeast Asia ( Saunders et al . , 2014 ) may spread to Africa , or evolve independently there , and lead to increased selective pressure for artemisinin resistance there .", "The global spread of resistance to chloroquine and sulfadoxine-pyrimethamine was dominated by hard sweeps of specific haplotypes originating in Southeast Asia , although it is clear that there were also localised emergences ( Mita et al . , 2009 ) .", "Although we still know relatively little about the functional properties of different KPBD mutations , it is clear that some are more successful than others , e . g . the C580Y allele has emerged at multiple locations in Southeast Asia and Africa , and a specific C580Y haplotype is approaching fixation in large parts of Western Cambodia ( Miotto et al . , 2015 ) .", "The high level of sequence conservation of KPBD across Plasmodium species indicates that mutations in these domains incur fitness costs , and this is supported by the observation that although there have been multiple independent origins of resistance-causing kelch13 mutations , individual kelch13 mutations appear to have limited spread , implying that there is a substantial fitness cost in the absence of sustained drug pressure .", "The danger is that these fitness costs may be compensated by other genetic variants , either in kelch13 or elsewhere in the genome , and that , as a result of this continuing evolutionary process , parasites in Southeast Asia will progressively acquire higher levels of artemisinin resistance ( World Health Organization , 2014a ) coupled with strong biological fitness and the ability to propagate across a wide range of vector species .", "Under these circumstances , the current soft sweep of artemisinin resistance could give way to a pervasive hard sweep with potentially disastrous consequences .", "These findings demonstrate the utility of applying genomic epidemiology to identify features of parasite demography and evolution that affect how drug resistance spreads .", "Future strategies to combat resistance will require better understanding of the evolutionary consequences of malaria control interventions , e . g . how the selective advantage of a resistance allele is counterbalanced by its fitness cost under different control regimes and in different geographical settings .", "It is now possible to approach this problem prospectively , by conducting systematic spatiotemporal sampling and genome sequencing of the parasite population as an integral part of public health interventions to prevent resistance spreading ." ], [ "All samples in this study were derived from blood samples obtained from patients with P . falciparum malaria , collected with informed consent from the patient or a parent or guardian .", "At each location , sample collection was approved by the appropriate local and institutional ethics committees .", "The following local and institutional committees that gave ethical approval for the partner studies: Comité d'Éthique , Ministère de la Santé , Bobo-Dioulasso , Burkina Faso; Navrongo Health Research Centre Institutional Review Board , Navrongo , Ghana; Kintampo Health Research Centre Institutional Ethics Committee , Kintampo , Ghana; Noguchi Memorial Institute for Medical Research Institutional Review Committee , University of Ghana , Legon , Ghana; Ghana Health Service Ethical Review Committee , Accra , Ghana; Gambia Government/MRC Joint Ethics Committee , Banjul , The Gambia; Comité d'Ethique National Pour la Recherche en Santé , Guinea; Ethics Committee of Faculté de Médecine , de Pharmacie et d'Odonto-Stomatologie , University of Bamako , Bamako , Mali; Ethical Review Committee , University of Ilorin Teaching Hospital , Ilorin , Nigeria; Institutional Review Board , Faculty of Health Sciences , University of Buea , Cameroon; Comité d’Ethique , Ecole de Santé Publique , Université de Kinshasa , Ministère de l’Enseignement Superieur , Universitaire et Recherche Scientifique , D . R . Congo; Comité National d'Ethique auprès du Ministère de la Santé Publique , Madagascar; Institutional Review Committee , Med Biotech Laboratories , Kampala , Uganda and Uganda National Council for Sciences and Technology ( UNCST ) ; College of Medicine Research Ethics Committee , University of Malawi , Blantyre , Malawi; KEMRI National Ethical Review Committee , Kenya; Medical Research Coordinating Committee of the National Institute for Medical Research , Tanzania; Ethical Review Committee , Bangladesh Medical Research Council , Bangladesh; Ethics Committee of the International Centre for Diarrheal Disease Research , Bangladesh; Institutional Ethical Review Committee , Department of Medical Research ( Lower Myanmar ) ; Ministry of Health , Government of The Republic of the Union of Myanmar; National Ethics Committee for Health Research , Ministry of Health , Phnom Penh , Cambodia; Ministry of Health National Ethics Committee For Health Research , Laos; Ethics Committee , Faculty of Tropical Medicine , Mahidol University , Bangkok , Thailand; Ethical Committee , Hospital for Tropical Diseases , Ho Chi Minh City , Vietnam; Eijkman Institute Research Ethics Commission , Jakarta , Indonesia; Institutional Review Board , Papua New Guinea Institute of Medical Research , Goroka , Papua New Guinea; Institutional Review Board , International Center for Medical Research and Training , Cali , Colombia; Institutional Review Board , Universidad Nacional de la Amazonia Peruana , Iquitos , Peru; Human Research Ethics Committee of NT Department of Health and Families and Menzies School of Health Research , Darwin , Australia; Institutional Review Board , New York University Medical School , NY , USA; Institutional Review Board , National Institute of Allergy and Infectious Diseases , Bethesda , MD , USA; Institutional Review Board , Walter Reed Army Institute of Research , Washington DC , USA; Ethics Review Committee , World Health Organization , Geneva , Switzerland; Ethics Committee of the Faculty of Medicine , Heidelberg , Germany; Ethics Committee of the Medical University of Vienna; Ethics Committee , London School of Hygiene and Tropical Medicine , London , UK; Oxford Tropical Research Ethics Committee , Oxford , UK .", "DNA was extracted directly from blood samples taken from patients at admission time , after leukocyte depletion to minimize human DNA .", "Leukocyte depletion was achieved by CF11 filtration in most samples ( Venkatesan et al . , 2012 ) , or alternatively by Lymphoprep density gradient centrifugation ( Axis‐Shield , Dundee , UK ) followed by Plasmodipur filtration ( Euro‐Diagnostica , Malmö , Sweden ) ( Auburn et al . , 2011 ) or by Plasmodipur filtration alone .", "Genomic DNA was extracted using the QIAamp DNA Blood Midi or Maxi Kit ( Qiagen , Hilden , Germany ) , and quantities of human and Plasmodium DNA were determined by fluorescence analysis using a Qubit instrument ( Invitrogen , Carlsbad , California ) and multi-species quantitative PCR ( Q‐PCR ) using the Roche Lightcycler 480 II system , as described previously ( Manske et al . , 2012 ) .", "Samples with >50 ng DNA and <80% human DNA contamination were selected for sequencing on the Illumina HiSeq platform following the manufacturer’s standard protocols ( Bentley et al . , 2008 ) .", "Paired‐end sequencing reads of length 200–300 bp were obtained , generating approximately 1 Gbp of read data per sample .", "All short read sequence data have been deposited in the European Nucleotide Archive ( http://www . ebi . ac . uk/ena/data/search/ ? query=plasmodium ) , and metadata will be released at the time of publication .", "Polymorphism discovery , quality control and sample genotyping followed a process described elsewhere ( Manske et al . , 2012 ) .", "Short sequence reads from 3 , 411 P . falciparum samples included in the MalariaGEN Plasmodium falciparum Community Project ( http://www . malariagen . net/projects/parasite/pf ) were aligned against the P . falciparum 3D7 reference sequence V3 ( ftp://ftp . sanger . ac . uk/pub/pathogens/Plasmodium/falciparum/3D7/3D7 . latest_version/version3/ ) , using the bwa program ( Li and Durbin , 2009 ) ( http://bio-bwa . sourceforge . net/ ) as previously described ( Manske et al . , 2012 ) , to identify an initial global set of 4 , 305 , 957 potential SNPs .", "This list was then used to guide stringent re-alignment using the SNP-o-matic algorithm ( Manske and Kwiatkowski , 2009 ) , to reduce misalignment errors .", "The stringent alignments were then examined by a series of quality filters , with the aim of removing alignment artefacts and their sources .", "In particular , the following were removed:", "a ) non-coding SNPs;", "b ) SNPs where polymorphisms have extremely low support ( <10 reads in one sample ) ;", "c ) SNPs with more than two alleles , with the exception of loci known to be important for drug resistance , which were manually verified for artifacts;", "d ) SNPs where coverage across samples is lower than the 25th percentile and higher than the 95th percentile of coverage in coding SNPs ( these thresholds were determined from artifact analysis ) ;", "e ) SNPs located in regions of relatively low uniqueness ( Manske et al . , 2012 ) ;", "f ) SNPs where heterozygosity levels were found to be inconsistent with the heterozygosity distribution at the SNP’s allele frequency; and", "g ) SNPs where genotype could not be established in at least 70% of the samples .", "These analyses produced a final list of 935 , 601 high-quality SNPs in the 14 chromosomes of the nuclear genome , whose genotypes were used for analysis in this study .", "All samples were genotyped at each high-quality SNP by a single allele , based on the number of reads observed for the two alleles at that position in the sample .", "At positions with fewer than 5 reads , the genotype was undetermined ( no call was made ) .", "At all other positions , the sample was determined to be heterozygous if both alleles were each observed in more than 2 reads; otherwise , the sample was called as homozygous for the allele observed in the majority of reads .", "For the purposes of estimating allele frequencies and genetic distances , a within-sample allele frequency ( fw ) was also assigned to each valid call .", "For heterozygous calls , fw was estimated as ratio of non-reference read count to reference read count; homozygous calls were assigned fw = 0 when called with the reference allele , and fw = 1 when called with the non-reference allele .", "The genotype of kelch13 was derived from read counts at non-synonymous SNP in the KPBD using a procedure described previously ( Miotto et al . , 2015 ) For a given population P , we estimated the non-reference allele frequency ( NRAF ) at a given SNP as the mean of the within-sample allele frequency ( fw ) for all samples in P which have a valid genotype at that SNP .", "The minor allele frequency ( MAF ) at is the computed as min ( NRAF , ( 1 – NRAF ) ) .", "To investigate the global population structure , we started by computing an NxN pairwise distance matrix , where N is the number of samples .", "Each cell of the matrix contained an estimate of genetic distance between the relevant pair of samples , obtained by summing the pairwise distance , estimated from within-sample allele frequency ( fw ) , at each SNP in the 100 kbp window considered .", "When comparing a pair of samples sA and sB at a single SNP i where a genotype could be called in each sample , with within-sample allele frequencies fA and fB respectively , the distance dAB was estimated as dAB = fA ( 1- fB ) + fB ( 1- fA ) .", "The genome-wide distance DAB between the two samples is then calculated as DAB=αnAB∑iwidAB where nAB is the number of SNPs where both samples could be genotyped , wi is an LD weighting factor ( see below ) and α is a scaling constant , equal to 70% of the number of coding positions in the genome ( since our genotyping covers approximately 70% of the coding genome ) .", "The exact value of α is uninfluential towards the analyses conducted in this study .", "The LD weighting factor , which corrects for the cumulative contribution of physically linked polymorphisms , was computed at each SNP i with MAF ≥ 0 . 1 in our sample set , by considering a window of m SNPs ( j = 0 . . m ) centred at", "i . For each j , we computed the squared correlation coefficient r2ij between SNPs i and", "j . Ignoring positions j where where r2ij < 0 . 1 , the weighting wi was computed by wi=11+∑j rij2 A neighbour-joining tree was then produced using the nj implementation in the R ape package .", "Principal coordinate analysis ( PCoA ) was performed using the same pairwise distance matrices using the Classical Multidimensional Scaling ( MDS ) method ( Gower , 1966 ) PCoA is a computationally efficient variant of principal component analysis ( PCA ) in which a pairwise distance matrix is used as input , rather than a table of genotypes .", "The matrix was supplied as input to the MDS algorithm , using the R language cmdscale implementation .", "We analyzed homologous protein sequences of kelch13 genes for seven Plasmodium species for which high-quality sequence data were available: P . falciparum , P . reichenowi , P . vivax , P . knowlesi , P . yoelii , P . berghei , and P . chabaudi .", "The sequences were retrieved from the OrthoMCL cluster ORTHOMCL894 in GeneDB ( http://www . genedb . org/ ) , and a multiple alignment was obtained using ClustalW ( Larkin et al . , 2007 ) at default settings .", "In turn , we considered each pair alignment of P . falciparum with one of the remaining species , assigning a substitution score to each kelch13 amino acid position , derived from the CCF53P62 substitution matrix .", "Although this matrix was chosen due to its suitability for AT-rich codon biases ( Brick and Pizzi , 2008 ) , we found that use of the more commonly used BLOSUM62 matrix ( Henikoff and Henikoff , 1992 ) did not have a significant effect on the results .", "Finally , each amino acid position was assigned a conservation score for the pair alignment , equal to the mean of the substitution scores in a 9-residue window centered at that position .", "To reconstruct putative ancestral and derived alleles in the propeller domain , we catalogued all polymorphic positions in the multiple sequence alignment .", "We organized the seven species into three groups by similarity: Laverania ( P . falciparum , P . reichenowi ) , primate Plasmodia ( P . vivax , P . knowlesi ) and rodent Plasmodia ( P . yoelii , P . berghei , and P . chabaudi ) , and observed that at each position , only one of the groups presented an allele different from that in the remaining groups .", "This group-specific allele was labelled as a putative derived allele , and the alternative allele as ancestral .", "( see Table 5 ) .", "Rodent species were found to carry the highest number of derived alleles , and therefore deemed to be good comparators for genome-wide conservation scoring .", "P . chabaudi was selected as a representative species in this group , and used for subsequent comparative analyses .", "We estimated a gene conservation score for every P . falciparum gene for which a P . chabaudi orthologue sequence could be obtained from PlasmoDB ( http://www . plasmodb . org/ ) .", "The details of the method are described elsewhere ( Gardner et al . , 2011 ) .", "Briefly , alignments of orthologous protein sequences were performed using ClustalW ( Larkin et al . , 2007 ) at default settings , and each amino acid position was assigned a CCF53P62 substitution score ( see above ) .", "The gene conservation score assigned was equal to the mean substitution score for all amino acid positions in the gene .", "Each amino acid position in the kelch13 was assigned a hydrophobicity score , estimated by computing the mean of the Kyte-Doolittle index in a 14-residue window centered at the position , using the protein sequence translated from the 3D7 reference sequence .", "To reconstruct the probable origin of kelch13 flanking haplotypes in African mutants , we used chromosome painting ( Lawson et al . , 2012 ) , a method that compares haplotypes in a sample to those in the remaining samples , and estimates probabilities that a genome fragment originates each population , by identifying individuals that share the same haplotype .", "For all African and SEA samples , we applied chromosome painting across the 250 kbp flanking regions on each side of the kelch13 gene .", "For each sample , haplotypes surrounding genome loci ( chunks ) were assigned posterior copying probabilities with respect to all remaining samples ( unrestricted painting ) .", "We aggregated these probabilities according to the geographical origin of the donor samples , assigning to each chunk a probability that it originates from each of the populations .", "For each sample , we then estimated the expected fraction of chunks copied from each population .", "In this analysis , we assumed a mutation rate per base per generation of 3 . 9-10 ( Claessens et al . , 2014 ) and a uniform recombination map .", "Since the two populations differ substantially in effective recombination rates ( Mu et al . , 2005 ) , we assumed a conservative recombination rate of 30 kbp/cM .", "Effective population size was initially set to 10 , 000 and optimized by 10 iterations of the expectation-maximization procedure ( Lawson et al . , 2012 ) .", "To account for the presence of heterozygous genotypes due to mixed infections , we modified the matrix of emission probabilities by introducing a novel parameter ( ε , set to 10-8 ) to represent the probability of emitting a mixed call .", "We repeated the analysis varying this parameter set , to assess the effects of misspecification , and results were found to be very similar qualitatively ( data not shown ) .", "Illumina sequence reads have been submitted to the European Nucleotide Archive with study accessions ERP000190 ( www . ebi . ac . uk/ena/data/view/ERP000190 ) and ERP000197 ( www . ebi . ac . uk/ena/data/view/ERP000197 ) .", "ENA accession numbers and metadata for the samples used in this paper can obtained via the MalariaGEN website , which also provides access to genotype calls on individual samples ( www . malariagen . net/resource/16 ) .", "Further details of all SNPs reported in this dataset including their genome coverage , mapping quality and allele frequencies in different populations , together with tools for querying the data , can be explored at www . malariagen . net/apps/pf ." ] ]

[ "The current epidemic of artemisinin resistant Plasmodium falciparum in Southeast Asia is the result of a soft selective sweep involving at least 20 independent kelch13 mutations .", "In a large global survey , we find that kelch13 mutations which cause resistance in Southeast Asia are present at low frequency in Africa .", "We show that African kelch13 mutations have originated locally , and that kelch13 shows a normal variation pattern relative to other genes in Africa , whereas in Southeast Asia there is a great excess of non-synonymous mutations , many of which cause radical amino-acid changes .", "Thus , kelch13 is not currently undergoing strong selection in Africa , despite a deep reservoir of variations that could potentially allow resistance to emerge rapidly .", "The practical implications are that public health surveillance for artemisinin resistance should not rely on kelch13 data alone , and interventions to prevent resistance must account for local evolutionary conditions , shown by genomic epidemiology to differ greatly between geographical regions ." ]

[ "Malaria is an infectious disease caused by a microscopic parasite called Plasmodium , which is transferred between humans by mosquitos .", "One species of malaria parasite called Plasmodium falciparum can cause particularly severe and life-threatening forms of the disease .", "Currently , the most widely used treatment for P . falciparum infections is artemisinin combination therapy , a treatment that combines the drug artemisinin ( or a closely related molecule ) with another antimalarial drug .", "However , resistance to artemisinin has started to spread throughout Southeast Asia .", "Artemisinin resistance is caused by mutations in a parasite gene called kelch13 , and researchers have identified over 20 different mutations in P . falciparum that confer artemisinin resistance .", "The diversity of mutations involved , and the fact that the same mutation can arise independently in different locations , make it difficult to track the spread of resistance using conventional molecular marker approaches .", "Here , Amato , Miotto et al . sequenced the entire genomes of more than 3 , 000 clinical samples of P . falciparum from Southeast Asia and Africa , collected as part of a global network of research groups called the MalariaGEN Plasmodium falciparum Community Project .", "Amato , Miotto et al . found that African parasites had independently acquired many of the same kelch13 mutations that are known to cause resistance to artemisinin in Southeast Asia .", "However the kelch13 mutations seen in Africa remained at low levels in the parasite population , and appeared to be under much less pressure for evolutionary selection than those found in Southeast Asia .", "These findings demonstrate that the emergence and spread of resistance to antimalarial drugs does not depend solely on the mutational process , but also on other factors that influence whether the mutations will spread in the population .", "Understanding how this is affected by different patterns of drug treatments and other environmental conditions will be important in developing more effective strategies for combating malaria ." ]

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

[ "neuroscience" ]

[ [ "Accurate control of grip force is essential in the manipulation of objects in everyday life .", "Knowledge of how gripping force is encoded in the brain would facilitate the design and control of brain machine interfaces ( BMI ) driving neuroprosthetics to help physically impaired patients .", "However , the results of studies aimed to decode force based on cortical neural activity are still far from consistent and satisfactory , and no BMI user has yet achieved manipulation of the force generated by a robotic hand ( Velliste et al . , 2008; Collinger et al . , 2013 ) , or the control of the simulated grasp force used for a virtual object ( Bensmaia and Miller , 2014 ) .", "Motor areas of the basal ganglia have long been associated with the scaling of motor vigour , measured in terms of the amplitude and speed of a movement or gripping force , although this is certainly not likely to be their exclusive function ( DeLong and Wichmann , 2010 ) .", "Neuronal recordings in monkeys and imaging studies in healthy humans have suggested that the basal ganglia play an important role in the control of the scaling of motor responses ( DeLong et al . , 1984; Turner and Anderson , 1997; Spraker et al . , 2007; Vaillancourt et al . , 2007 ) .", "Direct recordings from basal ganglia targets in patients suggest that changes in frequency specific activities in the local field potential ( LFP ) contribute to the selection of effort or force levels for voluntary movements .", "For example , the power over the gamma band ( 60–80 Hz ) in the LFP in the globus pallidus correlates with the movement amplitude and velocity of the contralateral hand of patients with cranial dystonia ( Brücke et al . , 2012 ) .", "Similar correlations have been noted in patients with Parkinson’s disease between movement speed and the power in the gamma band in the LFP picked up from the STN ( Joundi et al . , 2012 ) .", "Our previous studies also showed that suppression in the beta band ( 13–30 Hz ) and power increase in the gamma band of the STN LFP may correlate with forces or efforts made over the lower and higher effort ranges , respectively , in a manner independent from the effector that was activated ( Tan et al . , 2013 , 2015 ) .", "These results suggest that the signals from basal ganglia may serve as a central signal indexing motor effort , which in turn modulates force in manual grips .", "However , most previous studies are based on static linear correlations and averaged data; the dynamic relationship between activities of different frequencies in the basal ganglia LFP and generated force , and whether this relationship can be used to decode gripping force based on basal ganglia LFP signals has not been investigated on a trial by trial basis .", "The aim of the current study was to decode gripping force profiles from LFPs recorded in the STN .", "We hypothesized that beta and gamma band activities will be the most informative features in predicting the force profile generated by the contralateral hand , and that a simple first order linear dynamic model is sufficient to capture the relationship between STN LFP features and generated force .", "Our results suggest that reciprocal changes in synchronised oscillatory population activity in different frequency ranges provide potential control signals for the motor plant , the action of which can be modelled as a first order linear dynamical system .", "At the same time our results raise the possibility of using the LFP signal recorded from deep brain structures to provide stable and high-performance control signals for BMI driven neuroprosthetic grasping in paralysed patients ." ], [ "Our main interest was the initialisation , development and the average force during the ‘holding phase’ of each grip; therefore we focused on the period of time from one second before the cue to 2 . 8 s after the cue ( before force releasing ) for decoding .", "The force trajectory of each individual trial of each subject was normalized against the average maximal force that subject achieved in their maximal effort trials .", "We hypothesised that the relationship between LFP features and generated grip force ( the transfer function ) could be captured by a first order linear dynamic model ( Equation 1 ) : ( 1 ) Force=LFP* KpTp∙s+1e−Td∙s Where Kp is the steady state proportional gain , Tp is the time constant of the first order system which is a measure of how fast the force output responds to brain signal changes , and Td is the time delay between the brain control signal ( LFP ) and the measured force which describes the latency between the timing of brain signal changes and force output onset .", "Different models with different assumptions about what is the effective force control feature from the STNr ( STN region ) LFP and how different features are combined to encode force were tested ( see Table 2 and details in Materials and methods ) . 10 . 7554/eLife . 19089 . 006Table 2 . Model details . Models 1–3 use activity change in the beta band ( β ) and gamma bands ( γ ) from the STN LFP as model inputs .", "Models 4 and 5 take into account extra information about the low frequency activity change ( α ) .", "Models 6–8 only use the activity change from a single frequency band ( α , β and γ , respectively ) as model input .", "Tp and Td are the time constant and time delay of the first order linear dynamic model , respectively . DOI: http://dx . doi . org/10 . 7554/eLife . 19089 . 006Model IDModel equation No .", "of free parameters1Force= ( γ−β ) * KpTp∙s+1e−Td∙s32Force= ( Kp1∙γ+Kp2∙β ) * 1Tp∙s+1e−Td∙s43Force=γ* Kp1Tp1∙s+1e−Td1∙s+ β* Kp2Tp2∙s+1e−Td2∙s64Force= ( Kp1∙γ+Kp2∙β+ Kp3∙α ) * 1Tp∙s+1e−Td∙s55Force=γ* Kp1Tp1∙s+1e−Td1∙s+ β* Kp2Tp2∙s+1e−Td2∙s+ α* Kp3Tp3∙s+1e−Td3∙s96Force=α* KpTp∙s+1e−Td∙s37Force=β* KpTp∙s+1e−Td∙s38Force=γ* KpTp∙s+1e−Td∙s3 Cross-validation , within each STN/contralateral hand as well as across different patient groups , was used to evaluate the generalisability and decoding performance of the proposed models for predicting gripping force ( see details in Materials and methods ) .", "Within each STN/contralateral hand , after the model parameters had been fitted based on training data , several key variables were quantified to evaluate the performance of the model in predicting force in another set of test data recorded from the same hand: ( 1 ) the correlation coefficient between the predicted force and measured force ( WithinTrialR ) , which can be used to evaluate the prediction accuracy of the force development and force trajectory within each individual trial .", "( 2 ) The root mean square error , which quantifies the average distance between the prediction and actual measurements normalized by the maximal measured value ( nRMSE ) .", "( 3 ) The correlation coefficient between the average predicted force at the holding phase ( over 1–2 s after cue onset , when the gripping force was relatively stable ) and the measured force at the holding phase across different trials ( StableFrcR ) .", "This was used to evaluate the prediction accuracy of the stable target force across different effort levels .", "( 4 ) The difference between the timing of the predicted force onset and the timing of the measured force onset ( DifRT ) .", "The performances of different models in predicting force averaged across multiple trials using different STN features ( see Materials and methods for details ) were first evaluated .", "To achieve this , gripping force and LFPs measured from the contralateral STNr were grouped into low effort trials ( with self-reported effort <= 5 , trial number = 15±1 ) and high effort trials ( with self-reported effort > 5 , trial number = 14±1 ) .", "Average STNr LFP features and force trajectories were calculated for each effort condition .", "The STNr LFP features and force for one effort condition were used to estimate the model parameters ( model fitting ) , and the models were then used to predict force for the other effort condition ( model testing ) .", "The within-trial correlation coefficients between the predicted force and measured force ( WithinR ) and the nRMSE of the predicted force from different models during model testing are shown in Figure 2A and B . There was no significant difference consistent across the STNs between the three models using beta and gamma ERS as model inputs ( Models 1–3 ) in the predictive performance in terms of either WithinR or nRMSE .", "These three models with both beta and gamma activity as inputs performed better than models in which extra information about alpha activity was also included ( Models 4 , 5 ) , or models with activities from a single frequency band as model input only ( Models 6–8 ) .", "Figure 2C shows the BIC values combining the force prediction for low effort and high effort for all models .", "One-way repeated ANOVA identified a significant effect of models in the BIC ( F ( 7 , 56 ) =11 . 777 , p<0 . 001 ) .", "Paired t-tests showed that Model 2 was significantly better , in terms of BIC , than Models 4–8 after FDR multiple comparison correction: ( t ( 8 ) =−3 . 63 , p=0 . 007 compared with Model 4; t ( 8 ) =−4 . 950 , p=0 . 001 compared with Model 5; t ( 8 ) =−9 . 347 , p<0 . 001 compared with Model 6; t ( 8 ) =−5 . 757 , p<0 . 001 compared with Model 7; t ( 8 ) =−3 . 675 , p=0 . 007 compared with Model 8 ) . 10 . 7554/eLife . 19089 . 007Figure 2 . Force prediction performances of different models evaluated in terms of within-trial correlation ( A ) , RMSE ( B ) and BIC ( C ) .", "The filled dots and shaded bars show the median and range across all STNs; the open circles and stars show the data for each individual STN .", "The red dots and bars show performance in predicting high effort forces , while using data from low effort trials for model fitting; the blue dots and bars show performance in predicting low effort forces , while using data from high effort trials for model fitting .", "( C ) The total BIC values combing the force predictions for low effort and high effort for all tested models .", "The filled dots and shaded bars show the median and range across all STNs; the stars show the data for each individual STN ( some overlap ) .", "Models 1–3 use beta and gamma ERS as model inputs; Models 4–5 use activities from all three frequency bands ( alpha , beta and gamma ) as model inputs; Models 6–8 use activities from a single frequency band ( alpha , beta and gamma , respectively ) as model inputs . DOI: http://dx . doi . org/10 . 7554/eLife . 19089 . 00710 . 7554/eLife . 19089 . 008Figure 2—source data 1 . The excel data file related to Figures 2 and 6 . It contains data about median WithinR and the normalized RMSE when different models were used for force prediction , as well as the average power modulations at the beta band and gamma band for each STN that was recorded in the main paradigm and used for the analysis . DOI: http://dx . doi . org/10 . 7554/eLife . 19089 . 008 Figure 3 shows how Model 2 with beta and gamma having different linear gains can be used to predict the force generated by the contralateral hand for individual STNs .", "In Figure 3A , data averaged across low effort trials ( with self-reported effort <= 5 ) were used for the estimation of model parameters ( model fitting ) .", "The predictive performance of the model was evaluated on the data averaged across high effort trials for the same STN ( with self-reported effort > 5 , Figure 3B ) .", "Figure 3C shows the fitting and predictive performance of the model on data from the other 8 STNr electrodes in which consistent movement-related power modulations in both the beta and the gamma band were observed .", "The correlation coefficient between the predicted force and the measured force across all STNs was between 0 . 902 and 0 . 987 with a median value of 0 . 952 for high effort force ( data during low effort used for model parameter estimation ) and between 0 . 921 and 0 . 992 with a median value of 0 . 969 for low effort force ( data during high effort used for model parameter estimation ) .", "The nRMSE of the prediction force was between 5 . 60% and 28 . 07% with median value of 16 . 5% for high effort levels and between 10 . 5% and 23 . 8% with median value of 16 . 4% for low effort levels .", "The differences in reaction times ( DifRT ) between the predicted force and the measured force were 11 ± 27 ms for high effort levels and −26 ± 31 ms for low effort levels .", "These were not significantly different from zero ( t = 1 . 25 , p=0 . 247 for high effort levels and t = 1 . 03 , p=0 . 333 for low effort levels , one-sampled t-test compared to zero ) .", "The estimated Td ranged between 0 and 264 ms for low effort trials and ranged from 0 and 277 ms for high effort trials across STNs .", "Together , the results demonstrated that a simple first order linear dynamic model with beta and gamma as inputs with different linear gains can be used to describe the relationship between STNr activity change and measured force; and that , in addition , the model with parameters derived from one set of data can be used to predict force exerted at other effort levels in the same subject . 10 . 7554/eLife . 19089 . 009Figure 3 . Fitting and predicting performance of the model for predicting force averaged across multiple trials .", "( A ) The fitted model based on data from low effort trials for one exemplar STN and the contralateral hand .", "( B ) The fitted model was used to predict the average force for high effort trials for the same STN and contralateral hand .", "( C ) The fitted ( dashed lines ) and predicted force were compared against the measured force for the other 8 STNs in which consistent movement-related modulations in both beta and gamma bands were observed .", "Predicted force traces for high effort trials were derived from the model fitted to data from low effort trials and vice versa .", "Time 0 indicates the onset of the cue to start a grip in all plots . DOI: http://dx . doi . org/10 . 7554/eLife . 19089 . 00910 . 7554/eLife . 19089 . 010Figure 3—source data 1 . The Matlab data file containing source data related to Figure 3 . The variable names are self-explanatory .", "‘DataPlotC . Axisi’ contain data for the ith axis in PlotC .", "Column 1–7 in this variable corresponds to time , predicted force for a low effort , predicted force for high effort , fitted the force for a high effort , fitted the force for a low effort , measured the force for a high effort and measured the force for a low effort , respectively . DOI: http://dx . doi . org/10 . 7554/eLife . 19089 . 010 Figure 4 shows the performance of the model with beta and gamma power change in the STNr LFP as inputs ( Model", "2 ) in predicting contralateral gripping force in individual trials for one exemplar STN .", "For half of all the individual trials ( n = 21 ) recorded from this STN and contralateral hand , the within-trial correlation coefficient between the predicted force and measured force was equal or larger than 0 . 78 .", "The actual measured stable force during the holding phase and the predicted stable force were then quantified for each individual trial ( Figure 4D ) .", "Figure 4D shows that the stable force during the holding phase varied from zero to 100% of the maximal voluntary force across all the trials .", "Linear correlation was applied to the actual measured stable force during the holding phase and the predicted stable force of all individual trials .", "This predicted stable force correlated with the measured stable force ( n = 21 , r = 0 . 815 , p<0 . 001 ) , suggesting that the STNr LFP in conjunction with the dynamic model can predict both the force trajectory in individual trials and the stable force achieved across trials with different effort .", "The line of best fit between the predicted stable force and measured stable force was y = 0 . 96x + 2 . 4 .", "The regression gradient close to unity suggests that the prediction matches well with the measurement with no systematic overestimation or underestimation .", "During one trial for this patient , there is a force increase predicted from the STNr LFP , but there is no actual measured force change in the dynamometer ( indicated by ** in Figure 4B ) .", "This may be due to changes in the STNr LFP signal following the cue without the actual force change being registered in the dynamometer , as might arise when movements of the limb did not involve the dynamometer . 10 . 7554/eLife . 19089 . 011Figure 4 . Predicting force profile of individual grips based on beta and gamma activities from STN LFP ( one exemplar subject ) .", "( A ) Time-evolving power spectrum of the bipolar STN LFP channel used for decoding force .", "( B ) The predicted force ( in red ) compared with the measured force ( in black ) .", "** indicates the trial where STN LFP predicted increased force but with no measured force from the dynamometer .", "Grips are concatenated in A and B . ( C ) Distribution of the within-trial correlation coefficient ( WithinR ) between predicted force and measured force , with the dashed blue line the median value of the WithinR for all trials .", "( D ) Scatter plot between the predicted stable force ( average force during the second of holding phase ) and measured stable force for all tested trials .", "The correlation coefficient between the predicted and measured stable force across trials was 0 . 815 for this subject .", "The regression slope of 0 . 96 , which is close to 1 , shows that there is no systematic under-estimation .", "The black lines show the regression line and 95% confidence interval . DOI: http://dx . doi . org/10 . 7554/eLife . 19089 . 01110 . 7554/eLife . 19089 . 012Figure 4—source data 1 . The Matlab data file containing source data related to Figure 4 . The variable ‘DataPlotA’ is a data structure containing fields about the movement-related power changes over time ( ‘ERS’ ) for one exemplar subject .", "‘DataPlotB’ is a data structure containing fields about measured force ( ‘MeasForce’ ) and predicted force based on Model 2 ( ‘EstForce’ ) .", "‘DataPlotD’ is a data structure containing measured stable force and predicted stable force for each individual trial and the linear regression between them . DOI: http://dx . doi . org/10 . 7554/eLife . 19089 . 012 Considering all the STNs in which significant movement-related modulations were observed in both beta and gamma bands ( N = 9 ) , the correlation between the predicted stable force and measured stable force across trials ranged from 0 . 448 to 0 . 913 for the contralateral hand when Model 2 was used , with the regression gradient between the predicted stable force and measured stable force ranging from 0 . 91 to 1 . 12 ( not significantly different from unity across STNs based on one-sampled T-test , p=0 . 431 ) .", "There was no significant difference between the three models which took into account both beta and gamma activities ( F ( 2 , 16 ) =0 . 207 , p=0 . 815 , Figure 5A ) .", "In addition , the distribution of the within-trial correlation coefficients across all trials was not significantly different between Model 2 and Model 1 ( ks-test p=0 . 2850 ) , Model 1 and Model 3 ( ks-test p=0 . 9584 ) or Model 2 and Model 3 ( ks-test p=0 . 9228 ) .", "For convenience therefore , Model 2 was taken as representative of Models 1–3 .", "Considering all the individual trials from the 9 STNr , the median value for the within-trial correlation coefficient was 0 . 732 for Model 2 ( Figure 5B and C ) .", "There were around 13% of trials during which the correlation between the measured force and the predicted force were negative , suggesting a failure in predicting force trajectory . 10 . 7554/eLife . 19089 . 013Figure 5 . STN LFP features predict gripping force profile generated by the contralateral hand .", "( A ) The correlation coefficients between the measured stable force and the predicted stable force were higher for the force generated by the contralateral hand than that by the ipsilateral hand .", "There was no significant difference when different models based on both beta and gamma activities from STN LFP were used .", "The dots and bars show the median value and the range of values for different STNs .", "** indicate a significant difference in the prediction performance when the LFPs from the ipsilateral STN was used for decoding .", "( B ) The histogram of the within-trial correlation coefficients between predicted force and measured force ( WithinR ) for the contralateral hand considering all the trials and all the STNs .", "( C ) Cumulative distribution function ( CDF ) of the WithinR for the force generated by the contralateral hand ( solid lines ) and the ipsilateral hand ( dashed lines ) .", "The CDF indicates the probability that WinthinR has a value less than or equal to a certain value on the x-axis .", "Data presented in this figure are for all the STNs in which significant modulations were observed in both the beta band and gamma band . DOI: http://dx . doi . org/10 . 7554/eLife . 19089 . 01310 . 7554/eLife . 19089 . 014Figure 5—source data 1 . The Matlab data file containing source data related to Figure 5 . The variable ‘WithinR_Test_Contr’ contains data for the WithinR for each individual trial when Models 1–5 were used to predict force generated by the contralateral hand , for the 9 STNs in the main paradigm that are included in the main analysis .", "‘WithinR_Test_Ipsi’ contains the WithinR when the models were used to predict force generated by the ipsilateral hand .", "‘StbR_Test_Contr’ and ‘StbR_Test_Ipsi’ contain the correlation coefficients between the predicted stable force and measured stable force for the 9 STNs when different models were used . DOI: http://dx . doi . org/10 . 7554/eLife . 19089 . 014 The predictive performance of the STNr LFP for the force generated by the ipsilateral hand was also evaluated .", "The cross-trial correlations between the predicted stable force and measured stable were significantly lower for the ipsilateral hand than for the contralateral hand ( F ( 2 , 16 ) =10 . 629 , p=0 . 012 ) .", "The distributions of WithinR for ipsilateral force prediction were significantly different from that for the contralateral force prediction ( ks-test p<0 . 001 no matter whether Model 1 , Model 2 or Model 3 was used ) , indicating that the STNr LFP was more informative in predicting force generated by the contralateral than the ipsilateral hand .", "However , the degree of lateralisation could have been underestimated due to the potential presence of mirror movements , even though we asked patients to avoid these and in half those recordings used for decoding we also had bilateral upper limb EMG recordings .", "Figure 6 shows how the performance of force prediction based on the STNr LFP changes with movement related reactivity in the gamma band and beta band .", "Here we considered all the STNr with significant movement-related modulations , whether either in one or other , or both frequency bands of interest ( N = 12 ) .", "The median values of the within-trial correlation coefficients and stable force correlation coefficients increased with the average movement related synchronisation in the gamma band , with an exponential model y=a*e−bx+k explaining 75 . 7% ( p<0 . 001 ) of the variance in WithinR and 88 . 1% ( p<0 . 001 ) of the variance in StableR across STNs .", "There was also a trend for better prediction of force with increasing movement related beta desynchronization , but linear regression fitting was not significant for either WithinR or StableR .", "If we assume that movement-related synchronisation in gamma activity upon gripping is a good proxy for proximity to the motor region of the STN ( and perhaps the upper limb representation within it ) then these findings suggest that the electrode has to be very near to this region if recorded activity is to have a decent prospect of force prediction .", "This assumption was borne out by the fact that the movement-related synchronisation in gamma activity dropped by 71 ± 7 . 7% from the bipolar channel showing the most movement-related modulation to the average modulation in the remaining two bipolar channels .", "This drop was not so acute , 59 ± 7 . 9% , for movement related modulation in the beta band , which also only showed a trend for better prediction of force as movement related modulation in this band increased .", "Finally , there was a lack of significant correlation between the accuracy of the prediction of force and baseline beta ( Spearman r = 0 . 468 , p=0 . 13 for WithinR; Spearman r = 0 . 363 , p=0 . 25 for StableR ) or gamma activity ( Spearman r = 0 . 281 , p=0 . 37 for WithinR; Spearman r = 0 . 273 , p=0 . 39 for StableR ) measured during rest across subjects , suggesting that it is the reactivity of the power changes during gripping that is important for prediction , perhaps because it is more specific for the corresponding motor representation . 10 . 7554/eLife . 19089 . 015Figure 6 . Factors affecting the gripping force prediction performance . The median values of the WithinR ( A ) and stable force correlation coefficients ( B ) increased with the average movement- related modulation in the gamma band .", "Each dot is the data for one STN and the blue line shows the exponential fit of the data ( y=a∙e−bx+k , p<0 . 001 for the fitting ) .", "The median values of the WithinR ( C ) and stable force correlation coefficients ( D ) show a trend of increasing with average movement related desynchronization in the beta band .", "The blue lines show a linear fitting , but the fits were not significant .", "In this Figure , we consider all the STNr with significant movement-related modulations , whether either in one or other , or both frequency bands of interest ( N = 12 ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 19089 . 015 Finally , the first order dynamic model with beta and gamma power changes as inputs ( Model", "2 ) was used to predict the gripping force from individual trials in an independent patient group that performed a different , but related , paradigm .", "Ten patients were recorded in this study and were asked to grip as fast and as strongly as they could in each trial in response to an external cue .", "Twenty trials were collected for each hand when the patients were on their normal dopaminergic medication .", "Details of patient information and experimental paradigm were previously reported ( Anzak et al . , 2012 ) .", "The average movement related power change in the STN LFP activities and results of force prediction in this patient group are shown in Figure 7 .", "The median correlation coefficient between predicted force and measured force for individual trials ( median WithinR ) ranged between 0 . 3848 and 0 . 9421 for the 20 STNs and contralateral hand ( Figure 7B ) .", "Considering all the 397 individual trials across all STNs , more than 50% of the trials had WithinR more than 0 . 7859 ( Figure 7C and E ) .", "We also found that incorporating alpha activity ( Model", "4 ) improved the force prediction accuracy for maximal effort gripping in this patient group .", "When Model 4 was used , the median WithinR ranged between 0 . 6731 and 0 . 9625 across all STNs , and more than 50% of all individual trials had WithinR more than 0 . 8699 .", "BIC analysis showed that Model 4 ( BIC = 2116 . 2±135 . 7 ) was significantly better than Model 2 ( BIC = 1873 . 0±110 . 3 ) for this patient group performing maximal effort gripping ( ∆BIC = 243 . 3±95 . 9 , t ( 19 ) =2 . 5369 , p=0 . 020 comparing the BIC values , Figure 7D ) . 10 . 7554/eLife . 19089 . 016Figure 7 . Validation of the models for force prediction on an independent patient group during maximal effort gripping .", "( A ) Average power change in the STN LFP activity associated with the gripping movement .", "The power change is relative to the average over a 1 s period pre-cue .", "Time 0 is the timing of cue onset .", "( B ) Median WithinR for individual STN and contralateral hands , * indicates data for each individual STN and contralateral hand .", "( C ) Histogram and ( E ) cumulative distribution function ( CDF ) of the WithinR for all the 397 individual trials across all the 20 STNs .", "( D ) BIC analysis showed that Model 4 considering alpha , beta and gamma power changes significantly improved force prediction compared to Model 2 during maximal effort gripping .", "* indicates p<0 . 05 using a paired t-test . DOI: http://dx . doi . org/10 . 7554/eLife . 19089 . 01610 . 7554/eLife . 19089 . 017Figure 7—source data 1 . The Matlab data file containing source data related to Figure 7 ( data from an independent patient group on a maximal effort gripping paradigm ) .", "The variable ‘ERS_AllSTN’ contains the average movement related power changes from 1 to 90 Hz in the LFP signal from each STN recorded .", "‘WithinR_IndividualSTN’ contains data of WithinR for each individual trial recorded from each individual STN when Models 1–5 that are tested on this paradigm .", "‘WithinR_Median_AllSTN’ contains data for the median WithinR from each STN for the five models that are tested .", "( Data for Models 2 and 4 are presented in Figure 7 ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 19089 . 017" ], [ "When gripping was performed over a range of efforts we found that most information about the gripping force profile was contained in the beta and gamma bands in the STN LFP .", "The rich information in the gamma band was consistent with prior studies of grasp decoding using ECoG from human motor cortex ( Flint et al . , 2012 , 2014; Pistohl et al . , 2012 ) .", "Changes in beta activity also contributed to the prediction of the force profile and inclusion of beta activity improved the encoding accuracy .", "This is consistent with our previous finding that beta desynchronization can encode gripping force , especially at the low effort levels ( Tan et al . , 2013 , 2015 ) .", "It is unlikely that the predictive power of activities in the beta and gamma band was related to contamination by movement artefacts as we used bipolar LFPs for the decoding in which any common artefact is removed through common mode rejection .", "In addition , we observed increased activity in the low frequency ( delta and alpha bands ) , but features extracted from these frequency bands deteriorated the decoding of force during our core paradigm , even though separate parameters in a first order linear model were estimated for the low frequency activities .", "This contrasts with previous research which showed that the local motor potential and delta activity ( 0–4 Hz ) in the LFP from the hand area of the primary motor cortex contains information about muscle activity ( Flint et al . , 2012 ) and pinching force ( Flint et al . , 2014 ) .", "Our first order linear dynamic model was cross validated in an independent patient cohort in a paradigm in which patients were asked to grip as fast and hard as they could .", "In this case , though , the additional incorporation of theta/alpha activity further improved the accuracy and reliability of the force prediction .", "This may relate to the focus on maximal voluntary contractions and their execution as fast as possible , placing additional attentional demands on the participants .", "Oscillatory activity in the theta/alpha range may be involved in attentional mechanisms ( for review see Palva and Palva , 2007 ) .", "In particular , alpha activity ( 7–13 Hz ) in the subthalamic nucleus of patients with Parkinson’s disease is coherent with parieto-temporal cortical activity in a circuit that has been proposed to subserve attentional functions ( Hirschmann et al . , 2011; Litvak et al . , 2011 ) .", "Another consideration is the potential for the predictive power of low frequency activities to be related to contamination by movement artefacts secondary to movement overflow during maximal contractions .", "Taken as a whole our results suggest that reciprocal changes in synchronised oscillatory activity in the STN can potentially provide control signals for the motor plant .", "In line with this is the impaired effort scaling in subjects lacking beta and gamma related motor reactivity in the STN LFP .", "We speculate that the motor plant may behave as a first order linear dynamical system translating a basal ganglia effort signal to force for a given effector .", "The present analyses allow quantitative assessment of the importance of such a simple transfer function model of basal ganglia-motor function in terms of its remarkable ability to predict both the pattern with which force is developed and the static force achieved on a single trial basis .", "Note that although the prediction is being used here in terms of its statistical meaning of accounting for the variance in a second signal , it also satisfies the physiological implications of this term in that changes in the STN LFP preceded changes in the measured force by about 200–300 ms . Our quantitative approach also allowed us to demonstrate that effort encoding is relatively lateralised in the basal ganglia .", "Patients with Parkinson’s disease have been shown to have attenuated power modulation in both the beta band ( Doyle et al . , 2005; Devos and Defebvre , 2006; Androulidakis et al . , 2007; Anzak et al . , 2012 ) and the gamma band ( Androulidakis et al . , 2007 ) during movement initialization and when a constant force is meant to be sustained ( Tan et al . , 2013 ) when off dopaminergic medication .", "Such a diminished range of power modulation in the beta and gamma bands during movement may restrict the dynamic range of the coding of force in gripping in Parkinson’s disease .", "This may impair the force generation , and thereby cause bradykinesia , hypokinesia and weak movements when off compared to when on medication , presuming that the relationship between the STN force-encoding signal and generated force remains fixed across drug states ( Figure 8 ) .", "This is consistent with previous observations that untreated PD patients produce normal muscle activation patterns , but that muscle activity is not adequately scaled to produce the required force ( Berardelli et al . , 1986; Turner and Desmurget , 2010 ) , PD patients are weaker off dopaminergic medication or DBS ( Corcos et al . , 1996; Alberts et al . , 2004 ) and that PD patients show an increased probability of selecting slow movement speeds ( Mazzoni et al . , 2007 ) .", "On the other hand , if the range of the maximal force to be achieved is to be kept similar to normal when off medication , the scale between the STN force-encoding signal and gripping force will be steeper when untreated ( as indicated by Figure 8B ) .", "This will lead to abnormally high grip forces in Parkinsonian patients , as observed when they are asked to lift and hold an object ( Fellows et al . , 1998 ) .", "Increased force scaling can also lead to the more coarse control of force and difficulty in finely tuning generated force .", "This is consistent with abnormally increased gripping force observed in precision grasping movements in Parkinson’s disease ( Wenzelburger et al . , 2002 ) . 10 . 7554/eLife . 19089 . 018Figure 8 . Implications of reduced movement related modulation in beta and gamma band activity in STN LFP with reduced dopaminergic input .", "( A ) The range of forces that can be generated will be reduced if the scale between the STN encoding signal and the force is to remain the same .", "This will lead to unscaled , bradykinetic force generation .", "( B ) The scale between the STN encoding signal and the force will be increased if the range of force that can be generated is to be kept similar .", "This will lead to abnormally high force generation and more coarse force control . DOI: http://dx . doi . org/10 . 7554/eLife . 19089 . 018 Our findings suggest that the STN LFP could provide a high-performance control signal for BMI driven neuroprosthetic grasping in paralysed patients , leveraging advances in surgery for deep brain stimulation which has now become a relatively safe procedure ( Larson , 2014 ) .", "Surgical subcortical targets , such as the STN and globus pallidus ( GPi ) , are involved in motor planning and execution .", "Activities from STN and GPi have been shown to correlate with movement parameters such as movement amplitude and speed ( Brücke et al . , 2012; Joundi et al . , 2012 ) , and are also modulated by movement intention ( Kühn et al . , 2006 ) .", "Basal ganglia output has been theorized to regulate movement gain in healthy motor control , and can contain important information about the motor vigour ( Shadmehr and Krakauer , 2008; Turner and Desmurget , 2010 ) .", "Recordings of STN LFP signals have been shown to be stable over months using an implanted amplifier ( Quinn et al . , 2015; Neumann et al . , 2016 ) , and the signals are similar at re-operation several years later ( Giannicola et al . , 2012 ) .", "It is also possible that the predictive potential of the STN LFP might be further improved in patients without Parkinson’s disease , as dopamine increases the reactivity of beta and gamma activities around the time of movement ( Doyle et al . , 2005; Androulidakis et al . , 2007 ) .", "Moreover the reliability of predictions was dependent on the relative scale of movement-related power changes in the gamma band which could be improved by refinements in electrode design and targeting , perhaps using this reactivity to select the optimum implantation target .", "Therefore , LFP signals recorded from the basal ganglia have the potential to provide stable and high-performance BMI input signals for the control of neuroprosthetic devices in paralysed patients , complementary or alternative to spikes or ECoG signals recorded from the cortex .", "In this regard , it is important to note that some LFP reactivity is retained when movements are imagined rather than actioned ( Kühn et al . , 2006 ) , suggesting that peripheral afferents may not be necessary for the beta suppression and gamma increases tracked here .", "Nevertheless , several critical issues need to be addressed before neuroprosthetic control by STN LFPs can be considered for implementation in BMIs .", "Any application is predicated on the assumption that the spectral reactivity demonstrated in the STN of treated PD patients is more-or-less preserved in chronically paralysed patients .", "This remains to be proven .", "Although our results suggest that basal ganglia LFP changes might potentially be useful in force control , the selection of movements or effectors is also important in BMI control .", "Whether and to which extent movements involving different body parts , such the lower as opposed to the upper limb , can be decoded from the STN LFP remains unaddressed , although microelectrode recordings suggest some spatial segregation in activity related to different limbs in the human STN ( Rodriguez-Oroz et al . , 2001; Theodosopoulos et al . , 2003 ) .", "In addition , we have not demonstrated the specificity of STN LFPs for the decoding of force .", "Finally , plasticity of cortical control signals is likely to play a significant role in the maximisation of the performance of BMIs ( Carmena et al . , 2003; Ganguly and Carmena , 2009 ) , and yet whether subcortical signals can adapt over time remains to be seen .", "There are some limitations in the current study which need to be acknowledged .", "First and foremost , we were unable to predict the force in about half of our patients .", "There may be many reasons for this , including disease related impairments despite dopaminergic therapy , post-operative stun effects and failure to pick up LFP activity from the ‘motor’ STN due to electrode targeting error .", "Confirmation that some electrode contacts were in or touching the STN was given by the surgical team at each centre upon review of pre- and post-operative imaging blinded to the electrophysiological results .", "According to this standard seven out of the nine electrodes affording force prediction were on target , with the other two electrodes in the posterolateral tail of STN .", "This suggests that significant movement-related spectral modulation in the beta and gamma bands , -the basis for selecting out this group in the first place , might be a good electrophysiological marker of proximity to the STN , and perhaps to the region of the STN involved in the motor representation of the hand in particular .", "Noteworthy in this regard , the spatial gradient of gamma reactivity was more acute than that of beta reactivity .", "In contrast , six out of the 12 electrodes that did not allow reasonable force prediction were identified as neither being in nor touching the STN and these subsequently revised on four sides or left inactive on two sides .", "A stun effect or disease related impairment might help account for the lack of joint beta and gamma reactivity in the remaining patients in this group , although two of these still had significant modulation in the beta band and one in the gamma band .", "Such factors are also likely to have contributed the variation in prediction performance between subjects in whom some force prediction was possible .", "Amongst these , predicted force based on the STN LFP features was also noisier than the measured force .", "This could be caused by dynamic fluctuations and short-time-scale events in the STN LFP oscillatory activity ( Feingold et al . , 2015 ) .", "It could be improved by incorporating a time history of the LFP signals which is equivalent to filtering the control input from the brain , and by incorporating filtering algorithms in the predicted force ( the plant output of the model ) in the time domain , such as the Kalman filter , or by convolving the output with a static nonlinearity ( Fagg et al . , 2009; Flint et al . , 2014 ) .", "Alternatively the relatively noisy predictions might in part reflect involuntary dyskinetic movement that did not impinge on grip force and yet may have been parameterised in the LFP control signal .", "Second , in a minority of trials grip forces were predicted based on the STN LFP but there was no measured force .", "This might represent a movement intention with consequent LFP change without movement execution , or voluntary movements that were not captured by the force dynamometer .", "Likewise these factors might have modified baseline LFPs in some trials leading to the negative force predicted in rare trials , unless predictions were clamped so as not to fall below zero .", "Third , the force prediction presented here focuses on the rest , force initialization , development and stable force holding phase of a grip , but does not include the force release or termination phase .", "Finally , despite the observation that the first order linear model provides a good approximation of the relationship between STN LFP features and gripping force , the model may be an over simplification .", "Moreover , the results do not settle the discussion as to which circuits predominantly account for the selection of motor scaling , which can equally be attributed to cortical function ( DeLong and Wichmann , 2010 ) .", "Despite these limitations , our findings do suggest that signals elaborated in and/or transmitted through the basal ganglia , and the STN in particular , carry information about motor scaling .", "We have also shown that features in the STN LFP combined with a simple dynamic model can be used to reliably predict the gripping force profile of the contralateral hand , even in individual grips .", "We propose that the LFPs from deep brain structures such as the STN could potentially provide stable and high-performance BMI input signals , complementary or alternative to neuronal spikes or ECoG signals recorded from the cortex .", "The recording stability and rich information content in the STN LFP about movement intentions and parameters make it an interesting signal with respect to BMI control ." ], [ "In the main paradigm , eleven patients with idiopathic Parkinson’s Disease ( mean disease duration 11 . 3 years , mean age 61 . 3 years , range 49–73 years; seven males ) provided informed consent to take part in this study , which was approved by the local ethics committees .", "Patients underwent bilateral implantation of DBS electrodes into the STN , as a prelude to therapeutic high frequency stimulation for advanced idiopathic PD with motor fluctuations and/or dyskinesia .", "Techniques to target and implant electrodes in the STN have previously been described ( Foltynie and Hariz , 2010 ) .", "Microelectrode recordings were not made during surgery .", "The permanent quadripolar macroelectrode used was model 3389 ( Medtronic Neurologic Division , Minneapolis , MN , USA ) featuring four platinum-iridium cylindrical surfaces .", "Its contacts are numbered 0 , 1 , 2 , and 3 , with 0 being the most caudal and contact three being the most cranial .", "Localisation was supported intra-operatively by the effects of direct stimulation and by immediate post-operative stereotactic imaging .", "Nonetheless , in acknowledgement of the fact that not all electrode contacts could be expected to lie in the STN per se , we term the area sampled by the electrode contact the STN region ( STNr ) .", "DBS electrode extension cables were externalized through the scalp to enable recordings prior to connection to a subcutaneous DBS pacemaker , implanted in a second operative procedure up to seven days later .", "One out of the eleven patients ( case 2 ) had only one electrode externalised for testing , thus we could record from 21 STN regions ( STNr ) .", "Clinical details of the patients are given in Table 1 .", "The patients showed 53 . 4 ± 6 . 0% ( p<0 . 001 ) improvement in the motor section of the Unified Parkinson’s Disease Rating Scale ( UPDRS ) on treatment with levodopa , indicating good responsiveness to this drug .", "Subjects were seated in a comfortable chair with their shoulders adducted and their elbows flexed at about 90° .", "Subjects were first asked to grip the dynamometer with maximal effort three times , with each trial lasting for 3 s .", "Then they were presented with a series of imperative visual cues ( red light-emitting diode illuminated for 3", "s ) , separated by 11–13 s , and instructed to ‘choose an effort level from the scale provided and then to squeeze the force dynamometer at this chosen effort level when the light comes on and maintain this squeeze for the duration of the light’ .", "Subjects were provided with the Rated Perceived Exertion Scale with 11 levels ranging from zero to 10 ( Borg , 1998 ) printed on a piece of A4 paper .", "They were asked to try and randomise their selection of effort levels , so that the levels were varied from trial to trial , and all levels were represented .", "The subjects reported the effort level verbally after each grip .", "The mean number ( ± SEM ) of trials per hand per subject was 31 ± 2 grips , with a mean number of trials per level per subject of 3 ( ±1 ) .", "In particular , 2–3 trials were self-rated as maximal effort and 2–3 trials self-rated as minimal effort in each subject .", "Patients were asked to grip following illumination of the LED , but were not requested to respond as quickly as possible .", "There was no other feedback provided to the patients related to the generated force .", "Recordings were made when the patients were ON their usual dopaminergic medication , 3–6 days postoperatively , while electrodes were externalized and before implantation of the pulse generator .", "Grip force was measured one hand at a time using an isometric dynamometer with standard Jamar design , and its handle set in the second of the five discrete grip diameter adjustments possible ( G200; Biometrics Ltd , Gwent , UK ) .", "The order in which left and right hands were tested was counterbalanced across subjects .", "Monopolar LFPs recorded with a TMSi porti ( TMS international , Netherlands ) and its respective software .", "A common average reference was used for the monopolar recordings and these were low and high pass filtered at 0 . 5 and 500 Hz , respectively .", "Bipolar signals were derived offline by subtracting the monopolar recordings between neighbouring contacts on each electrode .", "The force was only a low pass filtered at 200 Hz .", "EMG signals from the first dorsal interosseous ( FDI ) of the activated hand were recorded in all patients , and the EMG signals from the extensor digitorum communis or extensor carpi ulnaris of both lower arms were recorded in six out of the 11 patients .", "LFP , EMG and force measurements were initially sampled at 2048 Hz .", "The effort level the subject reported verbally after each grip was logged manually and then used to label each individual trial .", "Each electrode has four contact points , and the LFP data were converted off-line to give three bipolar contact pairs ( 01 , 12 and 23 ) per electrode .", "Nonetheless , in acknowledgement of the fact that not all electrode contacts could be expected to lie in the STN per se , we term the area sampled by the electrode contact the STN region ( STNr ) .", "Continuous wavelet transform , with Morlet wavelet and cycle number of 7 , was then applied to LFP recordings from each bipolar contact pairs for time-frequency decomposition .", "The average power changes relative to the pre-movement baseline over the three trials of maximal effort gripping was calculated for each bipolar contact in the contralateral STNr .", "Three features from each bipolar LFP signal were extracted: the power change in the theta/alpha ( 4–12 Hz ) band , beta ( 13–30 Hz ) and gamma ( 55–90 Hz ) frequency bands .", "For each electrode , the bipolar signal with the largest movement related reduction in the beta power was selected for analysis .", "Further , the average power in each frequency band and at each time point was compared against the distribution of the average power for that frequency over a one second period of time before the cue .", "Significant movement-related modulations were defined as those trials in which there were at least 50% of time points during the second after movement onset with power smaller than the 5% boundary ( to capture event related desynchronization ) or larger than the 95% boundary ( to capture event related synchronisation ) of the power distribution before movement for that frequency band .", "This procedure identified 9 STNs ( from six different patients ) with significant movement-related modulations in both the beta band and the gamma band; three more STNs showed significant movement-related modulations in either the beta or gamma band .", "However , there was no significant movement-related modulation in either beta or gamma band in the remaining 9 STNs .", "Details of the patient and the average power changes during the second after cue onset for maximal effort gripping in the beta and gamma bands for each STN are presented in Table 1 .", "For force decoding , time-frequency decomposition using continuous wavelet transform was applied to the STN LFPs from the bipolar contact previously selected .", "For each individual trial of gripping , the power change for each frequency at each time point was calculated by normalizing the power at that time point against the average power during the 1 s before cue presentation , so 0 indicates the power being the same as the baseline activity before cue , positive values indicate power increase ( referred to as ERS ) and negative value indicate power decrease ( referred to as ERD ) .", "Then average power changes in theta/alpha ( α: 4–12 Hz ) band , the beta ( β: 13–30 Hz ) and gamma ( γ: 55–90 Hz ) frequency bands at each time point were calculated .", "The latter frequency range of 55–90 Hz was selected on the basis of our previous study showing that STN LFP activities within this range increase during the onset of a grip ( Anzak et al . , 2012 ) and correlate with the stable force achieved during a grip ( Tan et al . , 2013 ) .", "Our previous study ( Tan et al . , 2013 ) showed that activity changes in the beta and gamma bands make major contributions to the encoding of efforts in gripping .", "In the present study , we tested different hypotheses about the dynamic relationship between LFP features and force .", "Based on the results from the previous study showing that the difference between gamma ( γ ) and beta ( β ) modulations correlates with effort at the force holding phase , the first model to be tested used the signal of γ−β as the as a control input: ( Model 1 ) Force= ( γ−β ) * KpTp∙s+1e−Td∙s Where β and γ are beta and gamma band activity change in the STNr LFP , respectively .", "KpTp∙s+1e−Td∙s is a standard representation of a first order linear dynamic system with a time delay in the Laplace domain , where Kp is the steady state proportional gain , Tp is the time constant which is a measure of how fast the force output responds to brain signal changes , and Td is the time delay between the brain control signal ( LFP ) and the measured force which describes the latency between the timing of brain signal changes and force output onset , s= j ⋅ ω which is a complex variable .", "The equivalent of Model 1 in the time domain is: Force", "( t ) +Tp∙∂Force", "( t ) ∂t= Kp∙ ( γ ( t−Td ) − β ( t−Td ) ) , which suggests that beta and gamma modulations in STN LFP encode the instantaneous amplitude and the differentiation of force over time .", "The second model to be tested assumed that the activities from beta and gamma bands have a different proportional gain , but have the same dynamic relationship in terms of time constant ( Tp ) and time delay ( Td ) in encoding force: ( Model 2 ) Force= ( Kp1∙γ+Kp2∙β ) * 1Tp∙s+1e−Td∙s The third model tested assumed that the dynamic relationship between force and activities at different frequency bands are different , with different values for the proportional gain ( Kp ) , time constant ( Tp ) and time delay ( Td ) .", "Thus the generated force is the sum of the distinct processes with different inputs and different transfer function parameters ( Model 3 ) : ( Model 3 ) Force=γ* Kp1Tp1∙s+1e−Td1∙s+ β* Kp2Tp2∙s+1e−Td2∙s These models were compared against models which include extra information from the STNr LFP in the form of the relative power change in the theta/alpha frequency band ( α ) ( Model 4 and Model 5 ) : ( Model 4 ) Force= ( Kp1∙γ+Kp2∙β+ Kp3∙α ) * 1Tp∙s+1e−Td∙s ( Model 5 ) Force=γ* Kp1Tp1∙s+1e−Td1∙s+ β* Kp2Tp2∙s+1e−Td2∙s+ α* Kp3Tp3∙s+1e−Td3∙s Models with only activities from single frequency bands were also evaluated to see if the combination of activities from different frequency bands was necessary for the decoding for force: ( Model 6 ) Force=α* KpTp∙s+1e−Td∙s ( Model 7 ) Force=β* KpTp∙s+1e−Td∙s ( Model 8 ) Force=γ* KpTp∙s+1e−Td∙s The predicted force was clamped so it did not fall below zero .", "The parameters in different models ( Kp , Tp , Td ) were estimated for each STN separately .", "Cross-validation was used to evaluate the generalisability and decoding performance of the proposed models with the STNr LFP features as inputs for predicting gripping force .", "For each STN/contralateral hand , the parameters of different models were first identified using least-squares optimisation applied on a set of training data to fit the corresponding STNr LFP features and measured force of the training data .", "The decoding accuracy of the models was evaluated by applying the model with identified parameters on another set of testing data .", "When evaluating the performance of the model applied to across-trial averages , the average of STNr LFP features and forces over multiple trials of low effort levels was first used as the training data to identify the model parameters , and the model was then tested on average data from high effort trials; or vice versa , i . e . using the across-trial average of high effort trials as training data and the averages from low effort trials as testing data .", "Bayesian information criterion ( BIC ) was used for model selection .", "The BIC value for each model is calculated as: BIC=n∙ln ( σe2 ) +k∙ln ( n ) ; where σe2 is the error variance: σe2=1n⋅∑t=1n ( F", "( t ) −F^", "( t ) ) 2 with F", "( t ) and F^", "( t ) the actually measured and predicted force of each time point respectively; n is the total number of time points; k is the number of free parameters in the model .", "When evaluating the performance of the model on individual trials , a five-fold cross validation was used .", "All data from each recording session were partitioned into five equal folds .", "During each iteration , one fold was retained for testing , and the other four folds were used as training data to identify model parameters .", "Five iterations would allow for each observation being used for validation exactly once .", "The five results were then combined to produce a single complete estimation for one session .", "Different evaluation parameters including within trial correlation ( WithinTrialR ) , correlation between the measured and predicted stable force were quantified based on the testing data .", "In addition , for cross-subject validation , the 1st order dynamic model with STN LFP features as inputs was used to predict the gripping force of individual trials in another independent patient group .", "Ten patients were recorded in the study when the patients were asked to grip as fast and as hard as they can in each trial in response to an external cue .", "The details of patient information and experimental paradigms were reported in a previous publication ( Anzak et al . , 2012 ) .", "Twenty trials were collected for each hand when the patients were on their normal dopaminergic medication .", "Within each STN in this independent patient group , a 4-fold cross validation was used .", "For each iteration , 15 trials were used to fit the model to estimate model parameters and the model was used to predict force on the remaining five trials .", "The same procedure was repeated four times so each trial was used for prediction exactly once .", "The correlation coefficients between the predicted force and measured force for each individual trial were quantified .", "All analyses were performed in Matlab ( version 2012b ) .", "Median and the range of values are reported if the sample number is smaller than 10 , or the distribution is not normal .", "Otherwise , means ± standard error of means ( SEM ) are presented throughout the text .", "Correlation coefficients were Fisher-z transformed before any statistical test , but the raw values were presented in the text and the figures ." ] ]

[ "The basal ganglia are known to be involved in the planning , execution and control of gripping force and movement vigour .", "Here we aim to define the nature of the basal ganglia control signal for force and to decode gripping force based on local field potential ( LFP ) activities recorded from the subthalamic nucleus ( STN ) in patients with deep brain stimulation ( DBS ) electrodes .", "We found that STN LFP activities in the gamma ( 55–90 Hz ) and beta ( 13–30m Hz ) bands were most informative about gripping force , and that a first order dynamic linear model with these STN LFP features as inputs can be used to decode the temporal profile of gripping force .", "Our results enhance the understanding of how the basal ganglia control gripping force , and also suggest that deep brain LFPs could potentially be used to decode movement parameters related to force and movement vigour for the development of advanced human-machine interfaces ." ]

[ "The basal ganglia are a group of structures deep within the brain .", "Alongside its many other roles , it is thought to be able to control the vigour of movements , including how quickly we move and how much force we use to grip objects .", "Some of the best evidence for this comes from patients with Parkinson’s disease , who show abnormal activity of the basal ganglia .", "These patients move more slowly than healthy individuals and often struggle to grip objects with desired force , making it difficult to perform everyday tasks .", "Inserting electrodes into the brain and using them to electrically stimulate the basal ganglia is one of the most effective treatments for severe Parkinson’s disease .", "To examine the relationship between activity in the basal ganglia and grip strength , Tan et al . studied activity in the basal ganglia of patients as the individuals attempted to grip an object .", "On each trial specific types of coordinated activity within the basal ganglia predicted when movements would start and how much force the person would use .", "By constructing a mathematical model of the data , Tan et al . showed that the coordinated activity of cells within the basal ganglia indirectly controls motor vigour .", "The model also suggested how this process might go wrong in Parkinson’s disease .", "Activity in the basal ganglia predicts grip strength with such accuracy that it might even be possible to exploit this relationship to help individuals with paralysis .", "If imagining a movement triggers the same basal ganglia activity as performing it , patients could in principle use this activity to control robotic devices rather than limbs .", "To test this idea , future work should examine whether the features of basal ganglia activity can drive a Brain Machine Interface for robotic control in real-time .", "If so , the next question is whether signals from the basal ganglia are sufficient to control robotic devices , or if signals from other parts of the brain are needed too ." ]

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

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

[ [ "Signalling ligands are often synthesised as transmembrane domain ( TMD ) containing precursors .", "Upon their delivery to the plasma membrane , protease activity is required to shed the bioactive extracellular domain to allow signal release and subsequent binding to receptors on signal-receiving cells .", "TACE ( also known as ADAM17 ) is a primary shedding enzyme and as such regulates multiple signalling pathways through its ability to cleave and release numerous membrane-tethered signalling ligands and receptors ( Peschon et al . , 1998 ) .", "Of particular interest , it controls the shedding of TNFα , the principal inflammatory cytokine ( Black et al . , 1997; Moss et al . , 1997 ) , amphiregulin , TGFα and most other ligands of the epidermal growth factor ( EGF ) family ( Sunnarborg et al . , 2002; Sahin et al . , 2004 ) .", "Disturbances in these cytokine and growth factor signalling pathways are hallmarks of inflammation and cancer , respectively , as well as other diseases .", "This illustrates the potentially dangerous consequence of unregulated TACE activity and explains the tight post-translational regulation to which TACE is subject .", "TACE is first synthesised in the endoplasmic reticulum ( ER ) as an immature form containing an inhibitory pro-domain that prevents its proteolytic activity .", "We and others identified the iRhom proteins , catalytically inactive members of the rhomboid-like superfamily , as essential regulators of TACE maturation ( Adrain et al . , 2012; McIlwain et al . , 2012; Siggs et al . , 2012; Issuree et al . , 2013 ) .", "We reported that iRhoms control transport of TACE from the ER to the Golgi apparatus , where removal of its pro-domain by pro-protein convertases such as furin occurs ( Endres et al . , 2003 ) .", "In this way , iRhoms regulate the conversion of TACE from an inactive immature form to a mature proteolytically competent shedding enzyme ( Adrain and Freeman , 2012; Freeman , 2014; Lemberg and Adrain , 2016 ) .", "Without iRhoms there is no TACE maturation and therefore no TACE activity ( Christova et al . , 2013; Li et al . , 2015 ) .", "Of the two mammalian iRhoms , iRhom1 is broadly expressed , whereas macrophages express only iRhom2 .", "Since macrophages are the major TNFα-releasing cell type ( Parameswaran and Patial , 2010 ) , this makes iRhom2 an important regulator of inflammation .", "Accordingly iRhom2 knock-out mice have profound inflammatory defects , are sensitive to bacterial infection and are resistant to LPS-induced toxic shock and the development of inflammatory arthritis ( Adrain et al . , 2012; McIlwain et al . , 2012; Issuree et al . , 2013 ) .", "More recently iRhom2 was shown to modulate innate immunity to DNA viruses by regulating the ER-to-Golgi transport and the stability of the immune adaptor STING ( Luo et al . , 2016 ) .", "Although there are also hints that iRhom2 might regulate TACE protein stability ( Maney et al . , 2015 ) , this has not been explored in detail .", "iRhoms comprise a seven transmembrane rhomboid-like domain , with a short luminal C-terminus , a well conserved loop between TMDs 1 and 2 ( termed the iRhom homology domain , IRHD ) , and a long cytoplasmic N-terminus ( Lemberg and Freeman , 2007 , 2016 ) .", "There is accumulating physiological and cell biological evidence that mutation or deletion of the cytoplasmic N-terminus of iRhoms leads to alterations in TACE function ( Johnson et al . , 2003; Blaydon et al . , 2012; Saarinen et al . , 2012; Brooke et al . , 2014; Hosur et al . , 2014; Siggs et al . , 2014; Liu et al . , 2015; Maney et al . , 2015 ) .", "Moreover , iRhoms act downstream of G-protein coupled receptor ( GPCR ) signalling in the transactivation of EGFR signalling ( Christova et al . , 2013; Li et al . , 2015 ) .", "Taken together , these observations indicate that the cytosolic N-terminal domain of iRhoms regulates TACE , but the underlying mechanisms are unclear .", "Importantly , TACE-induced ligand shedding is rapid: it is activated within ten minutes by physiological and pharmacological stimulation ( Le Gall et al . , 2010 ) , and this is compatible with the timescale on which iRhoms have been reported to influence TACE substrate selection ( Maretzky et al . , 2013 ) .", "However , this speed of TACE activation is not easily reconciled with its relatively slow ER-to-Golgi trafficking and maturation ( 3–6 hr , [Schlöndorff et al . , 2000] ) , the process by which iRhoms are known to control TACE .", "Here we resolve this apparent paradox by demonstrating that iRhom2-mediated TACE maturation represents just the first step in the relationship between them .", "Once mature TACE has been trafficked to the cell surface , it requires a further iRhom2-dependent activation step , mediated by phosphorylation of the iRhom2 cytoplasmic domain .", "We reveal two unanticipated molecular roles for iRhom2 beyond its described function in TACE maturation .", "First , iRhom2 interacts with mature TACE at the plasma membrane , simultaneously stabilising TACE from lysosomal degradation and limiting its proteolytic activity .", "Second , iRhom2 acts as a signalling hub whereby phosphorylation of its N-terminus and subsequent 14-3-3 protein binding controls stimulated TACE proteolytic activity and ligand shedding .", "We show that this regulation of mature TACE activity occurs not only in immortalised cell models but also regulates inflammatory TNFα release from primary macrophages .", "Overall , we conclude that iRhom2 binds to TACE and controls its function at multiple stages , indicating that iRhom2 can be treated as a multifunctional regulatory subunit of TACE ." ], [ "iRhom2 is an essential regulator of TACE maturation and therefore TACE-dependent inflammatory and growth factor signalling ( Adrain et al . , 2012; McIlwain et al . , 2012; Siggs et al . , 2012; Christova et al . , 2013; Issuree et al . , 2013; Li et al . , 2015 ) .", "Accumulating data about the significance of the iRhom2 N-terminus led us to hypothesise that iRhom2 control over TACE was subject to further regulation .", "We discovered that treatment with phorbol-12-myristate-13-acetate ( PMA ) , a common activator of TACE-dependent signalling , leads to serine phosphorylation of mouse iRhom2 within 15 min ( Figure 1a ) .", "Furthermore , even without stimulation , mass spectrometric analysis revealed multiple phosphorylated serines within the human iRhom2 cytoplasmic N-terminus ( Figure 1b; Figure 1—figure supplement 1 a-b .", "Combined , this indicates that the iRhom2 N-terminus is subject to a basal level of phosphorylation , which is enhanced by a stimulator of TACE enzyme activity . 10 . 7554/eLife . 23968 . 003Figure 1 . TACE activity , but not TACE maturation is regulated by iRhom2 phosphorylation .", "( a ) HA immunoprecipitates ( IP ) and lysates ( lys ) from HEK293 cells stably expressing GFP or mouse iRhom2WT-3xHA were immunoblotted for phosphorylated serine , HA and beta-actin after treatment with or without 200 nM PMA for 15 min .", "( b ) Phosphorylation sites identified ( in red ) in human iRhom2 by iRhom2-3xHA immunoprecipitation and mass spectrometry .", "Ion scores derive from separate peptides from at least two independent datasets .", "For comparison , the corresponding conserved residue in mouse iRhom2 is stated", "( c ) A model of mouse iRhom2 ( in red ) , with the conserved N-terminal serine/threonine phosphorylation sites ( yellow stars ) identified in Figure 1b and from public source databases .", "All of these sites have been mutated to alanine in iRhom2pDEAD .", "The iRhom homology domain ( IRHD ) is indicated between the first two transmembrane domains .", "( d ) Lysates from iRhom1/2 double knockout ( DKO ) mouse embryonic fibroblasts ( MEFs ) expressing GFP or different versions of HA-tagged iRhom2 enriched for glycoproteins with Concanavalin A ( ConA ) were immunoblotted for TACE , HA , and transferrin receptor ( TfR ) as a labelling control .", "Lysates were probed with beta-actin antibody .", "Black arrowhead: immature TACE .", "Grey arrowhead: mature TACE .", "( e ) Immunofluorescence of DKO MEFs transduced with different versions of iRhom2-3xHA , labelled with HA ( green ) , GM130 label for the cis-Golgi ( red ) and DAPI for DNA ( blue ) .", "GM130-labelled regions ( within white boxes ) have been magnified .", "Scale bar = 10 µm .", "( f ) Lysates and neutravidin-enriched preparations from MEFs transduced with GFP , or HA-tagged iRhom2WT and iRhom2pDEAD labelled with ( biotin pull down ) or without biotin ( no biotin control ) , probed with HA , pan-cadherin ( cell surface positive control; arrowhead indicates specific band ) and p97 ( cell surface negative control ) antibodies .", "( g–j )", "DKO MEFs transduced with GFP or different versions of iRhom2-3xHA transfected with alkaline-phosphatase tagged amphiregulin ( Areg ) , transforming growth factor ( TGFα ) , tumour necrosis factor ( TNFα ) , epidermal growth factor ( EGF ) or betacellulin ( BTC ) , stimulated for indicated times with 200 nM PMA .", "Values represent the level of released alkaline phosphatase/total alkaline phosphatase multiplied by 100 .", "Error bars representing standard deviations and values for two-tailed student t-tests are depicted . DOI: http://dx . doi . org/10 . 7554/eLife . 23968 . 00310 . 7554/eLife . 23968 . 004Figure 1—figure supplement 1 . Human iRhom2 phosphorylation can be detected by mass spectrometry .", "( a ) Protein sequence coverage of human iRhom2 in mass spectrometry analysis .", "The peptides identified in mass spectrometry are indicated in red .", "The identified phosphorylation sites are marked in grey .", "( b ) Spectra of the identified human iRhom2 phosphopeptides .", "Mascot software was used to identify peptides of the human proteome from mass spectrometry data . DOI: http://dx . doi . org/10 . 7554/eLife . 23968 . 00410 . 7554/eLife . 23968 . 005Figure 1—figure supplement 2 . Characterisation of iRhom2KDEL .", "( a ) Lysates ( lys ) and neutravidin-enriched preparations ( biotin PD ) from MEFs transduced with GFP , or HA-tagged iRhom2WT , iRhom2pDEAD and iRhom2KDEL labelled with biotin and probed with HA antibody .", "( b ) Cell surface FACS staining .", "Left panel: cell surface labelling with anti-HA antibody of DKO MEFs transduced with iRhom2WT ( red ) and iRhom2KDEL ( blue ) .", "Cells stained only with the secondary antibody ( grey ) served as control .", "One experiment of 5 replicates is shown .", "Right panel: quantification of cell surface staining of iRhom2WT ( red ) and iRhom2KDEL ( blue ) .", "The percentage of positive stained cells ( relative to control ) from 5 replicates has been analysed with a Student’s t-test ( **p value < 0 . 01 ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 23968 . 005 We focused on the conserved phosphorylation sites in the N-terminus of iRhom2 , combined with phosphosites reported in other screens ( http://www . phosphosite . org/ ) .", "An extensive mutagenesis of mouse iRhom2 was performed , mutating all potential phosphorylation sites in the N-terminus to alanine ( referred to as iRhom2pDEAD , Figure 1c ) .", "We found that blocking iRhom2 N-terminus phosphorylation had no effect on iRhom regulation of the ER-to-Golgi transport and subsequent maturation of TACE .", "Expression of iRhom2pDEAD in iRhom1/2 double knock-out ( DKO ) MEFs rescued TACE maturation to the same extent as iRhom2WT ( Figure 1d , grey arrowhead ) .", "Therefore iRhom2 phosphorylation is not essential for the maturation of TACE , suggesting that any phosphorylation-dependent function is restricted to an unknown post-TACE maturation role of iRhom2 .", "Notably , a form of iRhom2 C-terminally tagged with a KDEL motif ( Munro and Pelham , 1987 ) , which enhances ER retrieval from the cis-Golgi , did not support TACE maturation ( Figure 1d , Figure 1—figure supplement 2 a-c , implying that iRhom2 itself must be trafficked beyond the ER to promote TACE maturation .", "In support of the conclusion that iRhom2 phosphorylation does not control ER-to-Golgi trafficking of TACE , iRhom2WT and iRhom2pDEAD , but not iRhom2KDEL , were both detected in the Golgi apparatus , co-localising with the cis-Golgi marker GM130 ( Figure 1e ) .", "Finally , consistent with a previous report ( Maney et al . , 2015 ) , we found that iRhom2 can reach the cell surface: using cell surface biotinylation we detected both iRhom2WT and iRhom2pDEAD at the plasma membrane ( Figure 1f ) .", "Overall , this demonstrates that iRhom2 is not limited to the early secretory pathway , but also reaches the plasma membrane , and that the ER-to-Golgi traffic of iRhom2 and TACE is independent of iRhom2 N-terminal phosphorylation .", "The independence of TACE maturation from iRhom2 phosphorylation – combined with the plasma membrane localisation of iRhom2 – hinted that phosphorylation might regulate an as yet unknown post-Golgi iRhom2 function .", "We therefore tested whether iRhom2 phosphorylation was required for TACE shedding activity .", "We found that iRhom2pDEAD was greatly impaired in its ability to promote TACE-dependent shedding of EGF-like ligands ( Figure 1g–h ) and TNFα ( Figure 1i ) in response to PMA activation .", "The requirement for iRhom2 phosphorylation was specific to TACE substrates since the shedding of EGF or betacellulin , which depends on the related protease ADAM10 ( Sahin et al . , 2004 ) , was unaffected by absence of iRhom2 phosphorylation ( Figure 1j ) .", "We conclude that phosphorylation of the iRhom2 N-terminus does not regulate its role of controlling ER-to-Golgi trafficking and maturation of TACE , but instead specifically controls the proteolytic activity of mature TACE at the cell surface .", "These data demonstrate that phosphorylation of the cytoplasmic N-terminus of iRhom2 specifically regulates the ability of mature TACE to release growth factors and cytokines from the cell surface .", "To further investigate this newly identified function we performed individual and combined alanine-scanning of the putative phosphorylation sites in iRhom2 ( Figure 2a , Figure 2—figure supplement 1a–c , and data not shown for non-contributing residues ) .", "This revealed three distinct sites that strongly contribute to PMA-induced TACE-dependent shedding: Site1 comprises S58 and S60; Site2 comprises S83 , S85 , and S87; and Site3 comprises S357 , S359 , S360 and T361 ( Figure 2a - highlighted in red text , and Figure 1c ) .", "Assessment of the three phosphorylation site mutants individually ( iRhom2Site1 , iRhom2Site2 and iRhom2Site3 ) and in combination ( iRhom2Site1-3 ) reveals that they additively regulate TACE-dependent shedding of both TGFα and TNFα ( Figure 2b and", "c ) .", "PMA is not a physiological trigger for TACE activity , so to confirm the physiological significance of our observations , we showed that GPCR stimulation with histamine triggers TACE-dependent release of amphiregulin and TGFα in an iRhom2 phosphorylation-dependent manner ( Figure 2d ) .", "Finally , we confirmed that stimulated phosphorylation of iRhom2 is lost upon simultaneous alanine mutation of all three of the identified sites required for TACE activity ( Figure 2e ) . 10 . 7554/eLife . 23968 . 006Figure 2 . Mature TACE activity is dependent upon iRhom2 phosphorylation at three distinct sites , which co-ordinate binding to 14-3-3 family proteins .", "( a–c )", "DKO MEFs stably transduced with GFP or different versions of iRhom2-3xHA transfected with alkaline-phosphatase tagged TGFα or TNFα stimulated for 30 min with 200 nM PMA .", "The contributing residues of Sites 1–3 in iRhom2 are highlighted in red text .", "( a–c ) .", "( d ) As stated for ( a–c ) , except DKO MEFs were co-transfected with Areg , TGF or EGF and the histamine HA1 receptor , then stimulated for 60 min with 300 µM histamine a–d .", "Values represent the level of released alkaline phosphatase/total alkaline phosphatase multiplied by 100 .", "Error bars representing standard deviations and values for two-tailed student t-tests are depicted .", "ns = p>0 . 05 , *p<0 . 05 , **p<0 . 01 , ***p<0 . 001 .", "( e ) HA immunoprecipitates from HEK293 cells stably expressing GFP or HA-tagged mouse iRhom2WT or iRhom2Site1-3 were immunoblotted for phosphorylated serine and HA after treatment with or without 200 nM PMA for 15 min .", "Lysate was probed with HA antibody .", "( f ) Lysates from HEK293T cells overexpressing HA-tagged human iRhom2WT , iRhom2ΔN ( lacking aa 1–382 ) and unc93B1 were subjected to anti-HA immunoprecipitation and analysed by semi-quantitative label-free mass spectrometry .", "The ratio of the spectral index of iRhom2WT is compared to a control pull-down with the polytopic membrane protein unc93b1 to compensate for unspecific binding to hydrophobic TMDs .", "The ratio of the spectral index of iRhom2WT and iRhom2ΔN shows the decreased binding of 14-3-3 proteins to iRhom2ΔN .", "( g ) HA immunoprecipitations and lysates from DKO MEFs transduced with GFP , HA-tagged mouse iRhom2WT or stated phosphorylation site-to-alanine mutants , treated with or without 200 nM PMA for 30 min , immunoblotted for HA , TACE , pan-14-3-3 and beta-actin . DOI: http://dx . doi . org/10 . 7554/eLife . 23968 . 00610 . 7554/eLife . 23968 . 007Figure 2—figure supplement 1 . Analysis of the contribution to TGF shedding of each iRhom2 residue within each phosphorylation site .", "( a–c )", "DKO MEFs transduced with GFP or different versions of HA-tagged iRhom2 transfected with alkaline-phosphatase tagged TGFα stimulated for 30 min with 200 nM PMA .", "Site 1 is depicted in", "( a ) , Site 2 in", "( b ) , and Site 3 in c .", "( d-g )", "HA immunoprecipitations and lysates from DKO MEFs transduced with GFP , mouse iRhom2WT-3xHA , stated phosphorylation site mutants or iRhom2KDEL , treated with or without 200 nM PMA for 15 min , immunoblotted for HA , indicated 14-3-3 isoforms ( epsilon , eta in d , g , alpha/beta , gamma in e , tau , zeta in", "f ) and beta-actin . DOI: http://dx . doi . org/10 . 7554/eLife . 23968 . 007 We hypothesised that phosphorylation might affect iRhom2 interactions with cytoplasmic partners .", "In a proteomic screen for proteins that interact with iRhom2 , we identified multiple 14-3-3 family proteins binding to the cytoplasmic domain ( Figure 2f ) .", "14-3-3 proteins bind phosphorylated residues and have roles in signalling ( Fu et al . , 2000 ) , so we further investigated their interaction with iRhom2 .", "Using both iRhom2pDEAD and iRhom2Site1-3 , we found that 14-3-3 proteins strongly bind to the N-terminus of iRhom2 upon PMA stimulation , in a phosphorylation-dependent manner ( Figure 2g ) .", "Of the seven 14-3-3 isoforms , we could confirm by western blot that all bound phosphorylated iRhom2 apart from 14-3-3α/β , although binding to 14-3-3ζ was weak ( Figure 2—figure supplement 1d–f; 14-3-3σ antibody failed , data not shown ) .", "Last , we found that the ER-Golgi retention mutant , iRhom2KDEL , also recruited 14-3-3 proteins upon stimulation with PMA ( Figure 2—figure supplement 1g ) , indicating that iRhom2 is phosphorylated throughout the secretory pathway .", "Overall , these data show that 14-3-3 proteins bind to the functionally important iRhom2 phosphorylation sites .", "Our mass spectrometry screen reproducibly identified ribosomal protein S6 kinase alpha-3 ( RSK3 ) and extracellular signal-regulated kinases 1/2 ( ERK1/2 , also known as MAPK3/1 respectively ) as potential binding partners of iRhom2 ( Figure 3a ) .", "Using PMA stimulated TACE-dependent shedding of amphiregulin as a readout , we examined the role of these kinases .", "We screened the activity of RSK3 with the well described inhibitor , BI-D1870 ( Sapkota et al . , 2007 ) ; ERK1/2 activity was screened with a potent inhibitor of upstream activator kinases MEK1/2 , U0126 ( Favata et al . , 1998 ) .", "As PMA stimulation is a known activator of protein kinase C ( Ryves et al . , 1991 ) , the PKC inhibitors Go6976 and Go6983 were used as positive controls ( Martiny-Baron et al . , 1993; Gschwendt et al . , 1996 ) .", "This experiment demonstrated that PMA stimulated amphiregulin release is insensitive to RSK inhibition , but reduced upon MEK1/2 inhibition ( Figure 3b ) , in line with a previous study ( Xu et al . , 2012 ) .", "We also tested more physiological stimulation by the GPCR agonist histamine .", "In this case , too , stimulated release of amphiregulin by TACE was inhibited by U0126 ( Figure 3c ) .", "Importantly , we showed that both PMA and histamine treatments indeed do cause upregulated ERK1/2 activity , monitored through enhanced ERK1/2 phosphorylation ( Figure 3d–f ) .", "These results are consistent with iRhom2 phosphorylation being downstream of ERK1/2 , and this was confirmed by the observation that stimulated ERK1/2 phosphorylation is unaffected in DKO MEFs expressing the iRhom2Site1-3 transgene ( Figure 3e–f ) .", "Since TACE itself is phosphorylated downstream of ERK1/2 signalling ( Soond et al . , 2005 ) , it is possible that the observed inhibition of TACE-dependent shedding was independent of iRhom2 phosphorylation .", "We therefore assessed the effects of U0126 directly on serine phosphorylation of iRhom2 and the subsequent recruitment of 14-3-3 proteins .", "First , we show that phosphorylation of iRhom2 is dependent on MEK1/2 activity ( Figure 3d ) .", "Second , PMA and histamine induced recruitment of 14-3-3 proteins to iRhom2 is abrogated by MEK1/2 inhibition with U0126 ( Figure 3d–f ) .", "We conclude from these data that iRhom2 is phosphorylated downstream of activated ERK signalling , and that the activity of these kinases is required for 14-3-3 recruitment to the N-terminus of iRhom2 and TACE-dependent shedding . 10 . 7554/eLife . 23968 . 008Figure 3 . iRhom2 phosphorylation and 14-3-3 binding is dependent on active ERK signalling .", "( a ) Lysates from cells overexpressing HA tagged human iRhom2WT and iRhom2∆N ( lacking aa 1–382 ) and unc93B1 were subjected to HA immunoprecipitation and analysed by semi-quantitative label-free mass spectrometry .", "The ratio of the spectral index of iRhom2WT is compared to the control pull-down with unc93b1 to compensate for unspecific binding to hydrophobic TMDs .", "The ratio of the spectral index of iRhom2WT and iRhom2∆N shows the decreased binding of the identified kinases upon deletion of the N-terminus .", "( b ) DKO MEFs stably transduced with HA tagged murine iRhom2WT transfected with alkaline-phosphatase tagged amphiregulin or EGF , pretreated for 1 hr with the indicated kinase inhibitors ( 20 µM ) followed by stimulation for 1 hr with 20 nM PMA .", "( c ) DKO MEFs stably transduced with HA tagged murine iRhom2WT transfected with alkaline-phosphatase tagged amphiregulin and the histamine HA1 receptor were pretreated with U0126 ( 20 µM ) for 1 hr followed by stimulation for 1 hr with 300 µM histamine .", "In b–c , ***p<0 . 001 .", "( d-e )", "HA immunoprecipitates ( IP ) and lysates ( lys ) from DKO MEFs transduced with GFP and HA-tagged iRhom2WT ( and iRhom2Site1-3 , in", "e ) pre-treated with U0126 ( 20 µM ) for 1 hr and stimulated with 20 nM PMA for 15 min , immunoblotted for 14-3-3 , HA , ERK1/2 and phosphorylated ERK1/2 .", "Arrowheads indicate the iRhom2 phosphoserine positive bands .", "( f ) HA immunoprecipitates ( IP ) and lysates ( lys ) from DKO MEFs transduced with GFP and HA-tagged iRhom2WT and iRhom2Site1-3 , transfected with 4 µg histamine HA1 receptor ( per 10 cm plate ) , pre-treated with U0126 ( 20 µM ) for 1 hr and stimulated with 300 µM histamine for 15 min , immunoblotted for 14-3-3 , HA , ERK1/2 and phosphorylated ERK1/2 . DOI: http://dx . doi . org/10 . 7554/eLife . 23968 . 008 We found that multiple 14-3-3 proteins interact with the iRhom2 N-terminus upon its phosphorylation and that iRhom2 N-terminal phosphorylation strongly promotes TACE activity .", "The two proteins are known to interact ( Adrain et al . , 2012; Figure 4—figure supplement 1a ) , but the nature of the iRhom2-TACE complex is still largely uncharacterised .", "Strikingly , iRhom2 lacking its cytoplasmic domain ( iRhom2∆NT ) did not bind mature TACE , although its binding to immature TACE was unaffected ( Figure 4a ) .", "In contrast , deletion of the iRhom homology domain ( iRhom2∆IRHD , Figure 1c ) strongly impaired immature TACE binding in DKO MEFs ( Figure 4b ) .", "Overall , we could detect no significant difference in the traffic of iRhom2∆NT or iRhom2∆IRHD , relative to their expression level , using cell surface biotinylation ( Figure 4—figure supplement 1b ) .", "These data reveal that the N-terminus of iRhom2 is specifically required for its interaction with mature TACE , but is not required for iRhom2 binding to the immature form of TACE , which instead depends on the iRhom2 IRHD . 10 . 7554/eLife . 23968 . 009Figure 4 . The amino-terminus of iRhom2 is required for binding to , and stabilisation of , mature TACE .", "( a–b )", "HA immunoprecipitates ( IP ) and lysates ( lys ) from WT MEFs ( in", "a ) and DKO MEFs ( in", "b ) transduced with GFP and HA-tagged iRhom2WT , iRhom2ΔIRHD or iRhom2ΔNT , probed for HA , TACE and beta-actin .", "Lysates from WT MEFs , enriched for glycoproteins with Concanavalin A ( ConA ) , show equal levels of mature TACE and transferrin receptor ( TfR ) ( in", "a ) .", "( c ) Lysates from DKO MEFs expressing GFP or different versions of HA-tagged iRhom2 constructs were enriched for glycoproteins with ConA and were immunoblotted for TACE , HA , ADAM10 and TfR as a labelling control .", "Lysates were probed with HA and beta-actin antibody .", "Where indicated , cells had been treated with the lysosomal degradation inhibitor bafilomycin A1 ( Baf A , 100 nM ) or the proteosomal degradation inhibitor MG132 ( 10 µM ) for 4 hr .", "Lane numbers have been added for clarity .", "All panels: black arrowhead: immature TACE; grey arrowhead: mature TACE . DOI: http://dx . doi . org/10 . 7554/eLife . 23968 . 00910 . 7554/eLife . 23968 . 010Figure 4—figure supplement 1 . iRhom2 interaction with TACE , and further characterisation of the deletion mutants of iRhom2 .", "( a ) HA immunoprecipitates ( IP ) and lysates ( lys ) from DKO MEFs transduced with GFP or HA-tagged iRhom2WT , immunoblotted for HA , TACE and beta-actin .", "Empty beads ( without conjugated HA antibody ) are used to show the specificity of the co-immunoprecipitation .", "( b ) Lysates ( lys ) and neutravidin-enriched preparations ( biotin PD ) from MEFs transduced with GFP , or HA-tagged iRhom2WT , iRhom2∆NT and iRhom2∆IRHD labelled with biotin and probed with HA and transferrin receptor antibodies .", "( c ) Lysates from DKO MEFs expressing HA-tagged iRhom2WT and iRhom2∆NT enriched for glycoproteins with ConA were immunoblotted for TACE , HA , and transferrin receptor ( TfR ) as a labelling control .", "Where indicated , cells had been treated with the lysosomal degradation inhibitor bafilomycin A1 ( Baf A , 100 nM ) or the proteosomal degradation inhibitor MG132 ( 10 µM ) for 4 hr .", "Lysates were probed for beta-actin , and immunoblotted for ubiquitin to demonstrate the efficacy of MG132 treatment . DOI: http://dx . doi . org/10 . 7554/eLife . 23968 . 010 Despite its ability to bind immature TACE , iRhom2∆NT was unable to support TACE maturation ( Figure 4c , compare lanes 4 and 7 ) .", "This is consistent with previous reports ( Siggs et al . , 2014; Maney et al . , 2015 ) but establishes an apparent paradox – that iRhom2∆NT is able to bind immature TACE but cannot promote its trafficking and maturation .", "The fact that we did not observe mature TACE could also be explained by degradation of the mature form .", "To test this , we blocked the two major protein degradation pathways: lysosomal and proteasomal degradation were inhibited with Bafilomycin A1 and MG132 , respectively .", "This showed that iRhom2∆NT can in fact support TACE maturation , but in the absence of the iRhom2 cytoplasmic domain , mature TACE is unstable and degraded in lysosomes ( Figure 4c , compare lanes 7 and 8; Figure 4—figure supplement 1c ) .", "This result uncovers an unanticipated molecular function for iRhom2 , regulating the post-Golgi stability of mature TACE .", "It also provides an explanation for the requirement for the iRhom2 N-terminus in TACE-dependent stimulated shedding of TGFα ( Figure 2a and [Maretzky et al . , 2013] ) .", "By comparison , iRhom2∆IRHD , which is strongly impaired in its ability to bind immature TACE , did not support TACE maturation: the absence of mature TACE was not rescued by either of the degradation inhibitors ( Figure 4c , compare lanes 4 and 10–12 ) .", "This implies that , unlike the N-terminus , the iRhom2 IRHD is essential for the ER-to-Golgi traffic and maturation of TACE , further distinguishing the newly discovered post-Golgi function of iRhom2 from its previously reported ER-to-Golgi trafficking role .", "Collectively , these results show that the iRhom2 cytoplasmic N-terminus is not needed for ER-to-Golgi trafficking and maturation of TACE , but instead mediates its binding to mature TACE at the plasma membrane .", "This binding stabilises TACE by preventing its lysosomal degradation .", "The N-terminus of iRhom2 stabilises mature TACE at the plasma membrane .", "How does this affect proteolytic activation of TACE ?", "We hypothesised that this might be the step that depends on the N-terminal phosphorylation of iRhom2 and subsequent 14-3-3 protein binding that we had discovered .", "To test this , we first questioned whether 14-3-3 recruitment to iRhom2 is sufficient to promote TACE activity .", "The phosphorylation Sites 1 and 2 in iRhom2 were replaced with the R18 peptide sequence that constitutively binds 14-3-3 proteins ( Wang et al . , 1999 ) ( iRhom2R18 , Figure 5a ) .", "iRhom2R18 not only showed constitutive recruitment of 14-3-3 proteins ( Figure 5b ) but also constitutively elevated TACE activity ( Figure 5c ) , indicating that 14-3-3 recruitment to the iRhom2 cytoplasmic domain is indeed sufficient to activate the shedding activity of TACE .", "Elevated levels of mature TACE did not cause this increase in shedding , as iRhom2R18 supported TACE maturation to the same extent as iRhom2WT ( Figure 5d ) .", "Furthermore , the insertion of two R18 peptides in iRhom2 did not affect its plasma membrane levels , assessed by surface biotinylation ( Figure 5—figure supplement 1 ) .", "Significantly , we found that this constitutive 14-3-3 binding form binds mature TACE less well than iRhom2WT ( Figure 5e ) .", "Reduction of binding was not caused simply by the loss of the Site 1 and 2 phosphorylation sites in iRhom2R18 , as iRhom2Site1-2 could still bind to mature TACE normally ( Figure 5e ) .", "This suggests that 14-3-3 recruitment to iRhom2 modifies the complex to weaken mature TACE binding . 10 . 7554/eLife . 23968 . 011Figure 5 . Phosphorylation and 14-3-3 protein recruitment uncouples the interaction between iRhom2 and mature TACE to drive its activity at the cell surface .", "( a ) A model of mouse iRhom2 ( in red ) , with the two R18 sequence insertions in the N-terminus to facilitate the recruitment of 14-3-3 proteins ( in green ) .", "Underneath , the sequence of the R18 insertion is indicated .", "( b ) HA immunoprecipitates ( IP ) and lysates ( lys ) from DKO MEFs transduced with GFP , or HA-tagged iRhom2WT , iRhom2Site1-3 or iRhom2R18 , treated with or without 200 nM PMA for 30 min , immunoblotted for HA , 14-3-3 and beta-actin .", "( c ) DKO MEFs transduced with GFP or different versions of HA-tagged iRhom2 transfected with alkaline-phosphatase tagged Areg or TGF .", "Values represent the level of released alkaline phosphatase overnight/total alkaline phosphatase multiplied by 100 .", "Error bars representing standard deviations and values for two-tailed student t-tests , compared to iRhom2Site1-3 are depicted .", "*p<0 . 05 .", "( d ) Lysates from DKO MEFs expressing GFP or different versions of HA-tagged iRhom2 enriched for glycoproteins with Concanavalin A ( Con A ) were immunoblotted for HA , TACE and transferrin receptor ( TfR ) as a labelling control .", "Grey arrowhead: immature TACE .", "White arrowhead: mature TACE .", "( e ) HA immunoprecipitates ( IP ) and lysates ( lys ) from DKO MEFs transduced with GFP , or HA-tagged iRhom2WT , iRhom2Site1-2 , iRhom2Site1-3 or iRhom2R18 , treated with or without 200 nM PMA for 30 min , immunoblotted for HA , TACE , 14-3-3 and beta-actin .", "Grey arrowhead: immature TACE .", "White arrowhead: mature TACE .", "( f ) HA immunoprecipitates ( left ) and neutravidin-enriched cell surface biotinylated protein preparations ( right ) with corresponding lysates ( lys ) from DKO MEFs transduced with GFP , or HA-tagged iRhom2WT , iRhom2Site1-3 , treated with or without 200 nM PMA for 5 min , immunoblotted for HA , TACE and beta-actin .", "As a molecular marker for immature and mature TACE , in the last lane , a lysate from DKO MEFs expressing HA-tagged iRhom2WT enriched for glycoproteins with Concanavalin A ( ConA ) was loaded .", "Lane numbers have been added for clarity .", "On the right-hand side , a quantification of the interaction between iRhom2 and mature TACE from three independent experiments has been plotted .", "This was achieved by dividing densitometry values of mature TACE by levels of iRhom2WT or iRhom2Site1-3 , and normalised to the values before addition of PMA ( a . u ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 23968 . 01110 . 7554/eLife . 23968 . 012Figure 5—figure supplement 1 . Cell surface presentation of iRhom2R18 . Lysates ( lys ) and neutravidin-enriched preparations ( biotin PD ) from MEFs transduced with GFP , or HA-tagged iRhom2WT , iRhom2Site1-3 and iRhom2R18 labelled with biotin and probed with HA , transferrin receptor and beta-actin antibodies . DOI: http://dx . doi . org/10 . 7554/eLife . 23968 . 012 In the light of the emerging model that 14-3-3 recruitment alters the iRhom2-TACE complex and triggers enzymatic activation of mature TACE , we noted an apparent inconsistency: activation of cells with PMA did not appear to affect binding between iRhom2 and mature TACE ( Figure 2g ) .", "Furthermore , iRhom2WT , iRhom2pDEAD and iRhom2Site1-3 appeared to bind mature TACE equally ( Figure 2g ) .", "We hypothesised that any alteration in binding might be hard to detect if it were limited to the physiologically active TACE and iRhom complex at the plasma membrane , which represents only a small fraction of total protein .", "To test this , we investigated the interaction between iRhom2 and TACE specifically at the cell surface .", "Intact cells on ice were incubated with an antibody that recognises the extracellular epitope of iRhom2 so that we could specifically isolate plasma membrane iRhom2 before and after ( 5 min ) PMA stimulation .", "This showed that iRhom2 and mature TACE do indeed form a complex at the plasma membrane , and that this interaction is reduced upon PMA stimulation ( Figure 5f , compare the reduction in TACE co-immunoprecipitation in lanes 3 and 4 , and in lanes 5 and 6; relative to total surface levels of mature TACE in lanes 9–12 ) .", "Furthermore , the complex is more stable with the non-phosphorylatable mutant , iRhom2Site1-3 ( Figure 5f , see graph for quantification ) .", "These data also make the converse point: a more stable iRhom2-TACE complex leads to less TACE activity .", "Overall , these results lead us to propose a new signalling mechanism that governs TACE activity .", "iRhom2 binds to and stabilises mature TACE at the plasma membrane but this complex inhibits TACE activity .", "Phosphorylation of the iRhom2 cytoplasmic domain drives the recruitment of 14-3-3 proteins , weakening the interaction between iRhom2 and TACE , an essential requirement for TACE proteolytic activity as a shedding enzyme .", "TACE and its cytokine and growth factor substrates are profoundly implicated in pathophysiology .", "One of its best-understood and most biologically important roles is as the primary controller of inflammation through TNFα release ( Black et al . , 1997; Moss et al . , 1997 ) .", "iRhom2 is the sole iRhom in macrophages , and iRhom2 KO mice are viable but compromised in macrophage secretion of TNFα , with consequent inflammatory defects ( Adrain et al . , 2012; McIlwain et al . , 2012; Siggs et al . , 2012 ) .", "We therefore investigated whether the phosphorylation-dependent regulatory mechanism we have discovered participates in regulating inflammation in vivo .", "We reconstituted primary bone marrow-derived macrophages from iRhom2 KO mice with iRhom2WT and iRhom2Site1-3 transgenes .", "TACE maturation was rescued by both constructs , indicating that , as in MEFs , phosphorylation of the cytoplasmic domain of iRhom2 is not needed for TACE trafficking and maturation in vivo ( Figure 6a ) .", "Furthermore , in response to PMA activation or lipopolysaccharide ( LPS ) treatment ( which mimics bacterial infection ) , we observed both iRhom2 serine phosphorylation , and 14-3-3 binding in primary macrophages ( Figure 6b and Figure 6c ) .", "Finally , we found that LPS-triggered shedding of TNFα from iRhom2 KO macrophages was rescued by iRhom2WT , but not by its phospho-deficient form , iRhom2Site1-3 ( Figure 6d ) .", "We conclude that iRhom2 phosphorylation and 14-3-3 binding mediate regulation of TACE activity in vivo , and therefore represent a pathophysiologically relevant mechanism that controls macrophage-mediated inflammation . 10 . 7554/eLife . 23968 . 013Figure 6 . Acute regulation of TACE by iRhom2 phosphorylation controls TNFα release in primary macrophages .", "( a ) Lysates from WT or iRhom2 KO primary macrophages transduced with GFP , or HA-tagged iRhom2WT , iRhom2Site1-3 , enriched for glycoproteins with Concanavalin A ( ConA ) and probed for TACE , HA , transferrin receptor ( TfR ) and ADAM10 .", "NB: The separation of immature and mature TACE is not always apparent in macrophage preparations ( Adrain et al . , 2012 ) .", "( b–c )", "HA immunoprecipitations ( IP ) and lysates ( lys ) from iRhom2 KO primary macrophages transduced with GFP , or HA-tagged mouse iRhom2WTor iRhom2Site1-3 , treated with or without 200 nM PMA", "( c ) or 100 ng/ml LPS", "( d ) for 15 min , immunoblotted for HA , phosphorylated serine , pan-14-3-3 , TACE and beta-actin .", "( d ) Levels of secreted TNFα by ELISA from primary macrophages described in a treated with 100 ng/ml LPS for indicated times .", "Error bars representing standard deviations and values for two-tailed student t-tests are depicted . DOI: http://dx . doi . org/10 . 7554/eLife . 23968 . 013" ], [ "We have discovered that iRhom2 regulates the metalloprotease TACE by multiple , distinct mechanisms throughout the secretory pathway .", "Because of its ability to trigger both cytokine and growth factor signalling , TACE activity is potentially dangerous , so it is unsurprising that it is itself subject to multiple layers of control .", "What is more unexpected is that iRhom2 , a catalytically inactive relative of rhomboid intramembrane proteases , should be so intimately involved in TACE regulation throughout its lifecycle .", "We and others have already reported that iRhom2 is responsible for the first step of generating mature TACE , controlling its ER to Golgi trafficking ( Adrain et al . , 2012; McIlwain et al . , 2012; Siggs et al . , 2012; Issuree et al . , 2013 ) ; we now show that this was only the first of several stages at which iRhom2 controls TACE .", "Once TACE has been exported from the ER under the influence of iRhom2 ( Figure 7a ) , and its inhibitory pro-domain is removed in the trans-Golgi network , interaction with iRhom2 is essential to stabilise it at the plasma membrane , preventing its lysosomal degradation ( Figure 7b ) .", "Stimulation of TACE proteolytic activity is then also mediated by iRhom2: the cytoplasmic N-terminus of iRhom2 is phosphorylated in response to , for example , GPCR signalling , leading to 14-3-3 binding .", "Our data support a model in which phosphorylation and 14-3-3 binding elicit a change in the interaction between iRhom2 and mature TACE that licenses TACE activity to shed substrates from the cell surface ( Figure 7c ) . 10 . 7554/eLife . 23968 . 014Figure 7 . iRhom2 regulates TACE at multiple steps in its lifecycle to control growth factor and cytokine signalling .", "( a ) iRhom2 and immature TACE must move together from the ER through the Golgi apparatus ( Adrain et al . , 2012 ) also shown by iRhom2KDEL ( Figure 1d ) , the site of furin cleavage of the TACE prodomain ( in yellow ) , and therefore production of mature TACE .", "This event requires a contribution from the iRhom2 IRHD .", "Without iRhom2 , there is no mature TACE .", "( b ) iRhom2 and mature TACE are in complex with one another at the plasma membrane .", "The iRhom2 N-terminus stabilises mature TACE levels at the cell surface; in its absence mature TACE is degraded in lysosomes .", "( c ) Upon delivery and maintenance of mature TACE at the plasma membrane , GPCR stimulation or macrophage activation with LPS leads to iRhom2 phosphorylation and recruitment of 14-3-3 proteins .", "The recruitment of 14-3-3 proteins is sufficient to alter the inhibitory interaction between iRhom2 and mature TACE , allowing TACE to cleave its cytokine and growth factor substrates . DOI: http://dx . doi . org/10 . 7554/eLife . 23968 . 014 The cytoplasmic domain of iRhom2 maintains the plasma membrane stability of mature TACE .", "Endocytic clearance of mature TACE from the cell surface has been previously demonstrated after stimulation with PMA ( Doedens and Black , 2000; Lorenzen et al . , 2016 ) , although more physiological stimuli do not alter the plasma membrane abundance of the protease ( Lorenzen et al . , 2016 ) , suggesting that this is not a primary control point of TACE-dependent shedding .", "Instead , as with many cell surface proteins , there appears to be a constitutive endocytic recycling of mature TACE , mediated by PACS-2 ( Dombernowsky et al . , 2015 ) .", "The loss of PACS-2 triggers activation-independent mature TACE lysosomal degradation , and it will be interesting to investigate further whether there is a mechanistic link between iRhom2 and PACS-2 in the stabilisation of mature TACE .", "Interestingly , this resonates with a recent report that iRhom2 maintains the stability of another client protein , the innate immune response adaptor protein , STING ( Luo et al . , 2016 ) , although in this case , STING is degraded by the proteasome .", "Signals that lead to the phosphorylation of the cytoplasmic N-terminal domain of iRhom2 cause 14-3-3 binding to iRhom2 .", "The consequence of this signal-dependent 14-3-3 binding is a detectable alteration of the interaction between mature TACE and iRhom2 at the plasma membrane , and activation of TACE-dependent shedding of substrates ( Figure 7c ) .", "Importantly , we have confirmed that this control of stimulated shedding occurs not only in embryonic fibroblasts , but is also an essential regulator of TNFα production in primary macrophages .", "Furthermore , we find that iRhom2 phosphorylation and 14-3-3 binding is dependent on ERK1/2 signalling , which is itself essential for TACE-dependent TNFα release in macrophages ( Dumitru et al . , 2000; Eliopoulos et al . , 2003 ) .", "Consistent with the physiological significance of iRhom2 phosphorylation as a signalling mechanism , Chanthaphavong et al . reported its phosphorylation in primary hepatocytes in response to nitric oxide signalling ( Chanthaphavong et al . , 2012 ) .", "It is significant that stimulated TACE activity does not depend on its own cytoplasmic domain ( Reddy et al . , 2000; Horiuchi et al . , 2007; Le Gall et al . , 2010 ) .", "Instead , the TACE TMD is required for its rapid activation ( Le Gall et al . , 2010 ) , but the molecular mechanism underlying this observation has been mechanistically obscure .", "iRhoms have evolved from rhomboid intramembrane proteases , which are polytopic membrane proteins that recognise and cleave transmembrane domains , and it is therefore probable that TMD interaction remains a core function of iRhoms ( Freeman , 2016 ) .", "It is possible that the iRhom2-TACE interaction resembles a rhomboid-substrate complex , but that instead of then cleaving the TACE TMD , iRhom2 binding and phosphorylation alters the state of TACE , licensing its activation .", "Another possible mechanism that reflects the presumed TMD binding role of iRhom2 would be that the phosphorylation of the iRhom2 N-terminus may regulate its binding to TMD-containing TACE substrates , such as TNFα; in other words , that iRhom2 provides a substrate presentation or selection mechanism for TACE .", "It will be important to understand how this newly identified phosphorylation-dependent regulatory mechanism relates to other previously identified modes of acute TACE regulation .", "Le Gall et al . ( 2010 ) reported that stimulation rapidly alters the accessibility of the TACE active site to a tight binding inhibitor , suggesting conformational changes in TACE itself .", "It has also been observed that iRhom2 controls substrate selectivity of mature TACE ( Maretzky et al . , 2013 ) .", "Recently , Sommer et al . provided another model for acute TACE activation ( Sommer et al . , 2016 ) .", "A variety of external signals cause the translocation of the lipid phosphatidylserine ( PS ) from the inner to outer leaflet of the plasma membrane .", "It has been shown that positive amino-acids in the extracellular membrane-proximal domain of TACE interact with the anionic head groups of exposed of PS , and it was proposed that this supports the positioning of TACE to cleave its substrates ( Sommer et al . , 2016 ) .", "Another model for TACE activation is its conversion from dimers to monomers , which is associated with decreased TIMP3 association ( Xu et al . , 2012 ) .", "Again , this model has significant overlap with our findings , as it is dependent on p38 MAPK and ERK signalling .", "The challenge is now to unify these earlier data with our discovery that phosphorylation and 14-3-3 binding of the iRhom2 cytoplasmic domain is necessary and sufficient to trigger TACE-dependent shedding of its substrates .", "A possible unifying model is that iRhom2 phosphorylation and subsequent conformational change in the iRhom2/TACE complex is necessary to allow mature TACE to engage with the above events of PS exposure , monomerisation , conformational change of the active site , and substrate selection .", "It must be stressed that this sequence of events is entirely speculative , but it is consistent with all published data , and does highlight the extraordinarily complex and tight regulation of TACE .", "Further study of the structure of iRhom2 and TACE should yield further insight into the enzymatic activation process .", "Overall , we propose that iRhoms have evolved as long-term molecular partners of TACE: first escorting and controlling TACE secretion and maturation; then stabilising it at the cell surface; and subsequently acting as a signalling hub at the plasma membrane that licenses stimulated TACE activity upon receipt of appropriate signals .", "These results greatly strengthen the relationship between iRhom2 and TACE , leading us to propose that iRhom2 should perhaps be considered as a multifunctional cofactor or regulatory subunit of TACE .", "The incidence of inflammatory diseases is increasing , and there is a need for new anti-inflammatory therapies ( Myasoedova et al . , 2010a , 2010b ) .", "TNFα blocking antibodies are used to treat rheumatoid arthritis , inflammatory bowel disease and psoriasis , and this therapy is one of the biggest-selling pharmaceuticals in the world .", "It is , however , unsuccessful for approximately a third of patients , requires injection , and is expensive ( Palladino et al . , 2003; Scott and Kingsley , 2006; Esposito and Cuzzocrea , 2009; Monaco et al . , 2015 ) .", "By expanding substantially the scope of the relationship between iRhom2 and TACE , the work we report here strengthens the case for the therapeutic potential of targeting iRhom2 .", "Its previously reported role in regulating TNFα signalling by the ER-to-Golgi trafficking and maturation of TACE makes iRhom2 conceptually a valuable target , but that function will be hard to target pharmaceutically as it depends on a TMD-based interaction in the ER ( our unpublished observation ) .", "In contrast , the regulatory mechanism between iRhom2 and mature TACE at the plasma membrane described here is cytoplasmic and thus perhaps more targetable .", "Moreover , the molecular components such as 14-3-3 proteins and the kinases that directly phosphorylate iRhom2 are more conventional , and therefore accessible , pharmaceutical targets .", "Finally , beyond targeting inflammatory cytokine signalling , pharmaceutical manipulation of the iRhoms-TACE interface could also influence growth factor signalling through the EGF receptor family , which is heavily implicated in a variety of diseases , most notably cancer ( Kenny and Bissell , 2007; Ciardiello and Tortora , 2008 ) ." ], [ "LPS , PMA , Bafilomycin A1 , MG132 were all purchased through either Sigma-Aldrich or Roche .", "U0126 was purchased from Abcam .", "BI-D1870 was purchased through Cambridge Scientific .", "Go6973 and Go6983 were purchased from Calbiochem .", "All drug concentrations are indicated in figure legends and in respective methods sections .", "The following antibodies were used: mouse anti-beta-actin ( Santa Cruz , catalogue number sc-47778; WB 1:2000 ) , rabbit anti-TACE/ADAM17 ( Abcam , catalogue number ab39161; WB 1:2000 ) , rabbit anti-HA ( Santa-Cruz , catalogue number sc-805; IF 1:1000 ) , mouse anti-HA ( ENZO , catalogue number ENZ-ABS120-0200; PM IP 1:1000 ) , mouse anti-transferrin receptor ( Invitrogen , catalogue number 13–6800; 1:1000 ) , rabbit anti-phosphoserine ( Invitrogen , catalogue number 618100; WB 1:1000 ) , rat HA-HRP , clone 3F10 ( Roche , catalogue number 12013819001; WB 1:2000 ) , mouse anti-p97 ( Pierce/Thermo , catalogue number MA1-21412; WB 1:1000 ) , rabbit anti-pan14-3-3 ( Cell Signalling , catalogue number 8312; WB 1:1000 ) , mouse anti-GM130 ( BD Transduction labs , catalogue number 610823; IF 1:1000 ) , rabbit anti-pan-cadherin ( Cell Signalling , catalogue number 4068S; WB 1:1000 ) , rabbit anti-ADAM10 ( Abcam , catalogue number ab1997; WB 1:1000 ) , rabbit anti-ERK1/2 ( Cell Signalling , catalogue number 9102 , WB 1:1000 ) , rabbit anti-pERK1/2 ( Cell Signalling , catalogue number 4377 , WB 1:1000 ) .", "Corresponding species-specific HRP or fluorescently coupled secondary antibodies were used from Santa Cruz and Cell Signaling ( WB ) or Invitrogen ( IF ) .", "pM6P . blast-GFP was a kind gift from Felix Randow ( LMB , Cambridge ) .", "pM6P . blast iRhom2-3xHA and iRhom2cub-3xHA has been described previously ( Siggs et al . , 2014 ) .", "pM6P . blast iRhom2-3xHA was used as a template for generation of the described individual and combined alanine point mutant constructs e . g . iRhom2Site1 , iRhom2Site2 , iRhom2Site3 , iRhom2Site1-2 , iRhom2Site1-3 and iRhom2pDEAD .", "Primers were designed using the Agilent Technologies website .", "QuickChange mutagenesis of putative phosphorylation sites was performed according to manufacturer’s instructions ( Agilent Technologies ) .", "PCR products were treated with DpnI at 37°C for 2–3 hr and transformed into XL10 Gold ultracompetent cells ( Agilent Technologies ) .", "Single colonies were picked and verified by DNA sequencing .", "For iRhom2R18 , we synthesised a gBlocks gene fragment ( Integrated DNA Technologies ) with the insertion of nucleotides coding for amino acids PHCVPRDLSWLDLEANMCLP in the place of regions coding for S58-V59-S60 and S83-L84-S85-Q86-S87 .", "Using this fragment , and an overlapping PCR fragment encoding the remaining sequence of mouse iRhom2 , the full iRhom2R18 product was generated by PCR .", "For iRhom2KDEL , a reverse primer coding for the KDEL epitope at the 3’ end of the 3xHA epitope was used for PCR amplification , using pM6P . blast iRhom2-3xHA as a template .", "For mouse iRhom2ΔNT , a product that codes for amino acids 367–829 of iRhom2 with sequence coding for 3xHA was PCR amplified using pM6P . blast iRhom2-3xHA as a template .", "For iRhom2ΔIRHD , using sequential overlapping PCRs , an iRhom2 product lacking nucleotides encoding for amino acids 404–629 was generated .", "These PCR products were then inserted into digested pM6P . blast constructs by In-Fusion ( Clontech ) cloning and bacterial transformation , according to manufacturer’s instructions .", "Human unc93B1 and human iRhom2WT or iRhom2ΔNT ( missing nucleotides coding for amino-acids 1–382 ) plasmids used for large-scale IP and mass spectrometry were amplified from human unc93B1 ( BC025669 . 1; GI:19343917 ) and iRhom2 cDNA ( NM_024599 . 5; GI:306035188 ) by PCR and cloned into the NotI cleavage site of lentiviral vector pLEX . puro using Gibson assembly , according to manufacturer’s instructions ( NEB ) .", "All plasmids were verified by DNA sequencing ( Source Bioscience , Oxford ) .", "Mouse embryonic fibroblasts ( MEFs ) were isolated from Rhbdf1−/−/Rhbdf2−/− ( referred to by protein name as iRhom1/iRhom2 KO ) E13 . 5 embryos and wild-type C57BL/6J ( RRID: IMSR_JAX:000664 ) controls , and immortalised by lentiviral transduction with SV40 large T antigen .", "All MEFs were generated in house and identity confirmed by in-house genotyping .", "HEK293T cell lines ( RRID:CVCL_0063 ) stably expressing unc93B1 and iRhom2 versions were generated by lentiviral transduction as described in Adrain et al . ( 2012 ) .", "HEK293 cells expressing mouse iRhom2-3xHA and mouse iRhom2site1-3 were generated by the pLEX-based lentiviral method , exactly as described in Siggs et al . ( 2014 ) .", "For mass spectrometry based experiments , HEK293T cells were transduced with pLEX . puro vector containing human unc93B1 , human iRhom2WT and human iRhom2ΔNT .", "Cells were selected by adding 2 . 5 μg/ml puromycin ( Life Technologies ) .", "All cells used were maintained in regular high-glucose DMEM , supplemented with 10% FCS , 100 μg/ml penicillin , and 100 μg/ml streptomycin .", "All cells used in this study were subject to regular mycoplasma testing .", "This study was performed in strict accordance with University of Oxford and UK Government rules and guidelines .", "The procedures and justification for the research was approved under UK PPL 80/2584 .", "Bone marrow was flushed from WT C57BL/6J ( RRID: IMSR_JAX:000664 ) and iRhom2 KO femurs into serum free DMEM/F12 ( 1:1 ) ( Gibco ) , passed through 70 μm nylon cell strainers and pelleted at 250 g for 5 min .", "Cells were resuspended in macrophage growth medium ( DMEM/F12 ( 1:1 ) , containing 10% FCS , 15% of a conditioned culture supernatant from confluent L929 cells , 100 μg/ml penicillin , and 100 μg/ml streptomycin .", "Cells were grown on non-tissue culture petri dishes , and were maintained and differentiated in macrophage growth medium over 7 days .", "This resulted in a ~90–95% pure macrophage population as determined by flow cytometry with F4/80 antibody .", "Prior to experiments , macrophages were detached by PBS/EDTA washes followed by trypsin , and plated at 1 × 105 in 12 well tissue culture dishes ( for ELISA ) and at 5 × 106 in 10 cm tissue culture dishes ( for ConA enrichment/HA immunoprecipitation ) .", "To generate retrovirus , a pCL-based retrovirus packaging system was used .", "HEK293 cells grown to 40–50% confluence were transfected with Lipofectamine in 35 mm plates with 1 μg of the pM6P . blast expression plasmids ( e . g . pM6P . blast-GFP , pM6P . blast iRhom2-3xHA etc ) and 1 μg of the packaging plasmid pCL-Eco .", "The following day , medium was changed and transfected cells were allowed to secrete virus for 24–48 hr in 2 ml of medium .", "Culture supernatants were then centrifuged at 20 , 000 x g , and clarified by filtration with Sartorius Minisart syringe filters ( 0 . 45 µm pore size ) .", "For infection of target iRhom1;iRhom2 double knock-out ( DKO ) cells or macrophages , cells were replated the day before , and viral supernatants were diluted 2-fold in fresh medium for transduction .", "Transduction was carried out in the presence of 50 μg/ml polybrene and a medium change was made 24 hr later .", "For selection of pM6P plasmids , DKO cells were treated with 5 μg/ml blasticidin 48 hr later .", "For macrophages , experiments were performed when levels of GFP in pM6P . blast-GFP transduced macrophages had reached detectable levels using a tissue culture fluorescence microscope ( FLoid Cell Imaging Station; Life Technologies ) ( on average , 3–4 days post-transduction ) .", "DKO MEFs ( 3 × 105 ) transduced with indicated constructs were plated on 13 mm glass coverslips in six well dishes .", "Cells were washed 3x in room temperature PBS and fixed with 4% paraformaldehyde in PBS at room temperature for 20 min .", "Fixative was quenched with 50 mM NH4Cl for 5 min .", "Cells were permeabilised in 0 . 2% TX-100 in PBS for 30 min and epitopes blocked with 1% fish-skin gelatin ( Sigma ) in PBS for 1 hr .", "Coverslips were then incubated overnight with rabbit anti-HA and mouse anti-GM130 in 1% fish-skin gelatin/PBS .", "After 3x PBS washes , coverslips were incubated with fluorescently-coupled secondary antibodies ( Invitrogen ) for 45min .", "Cells were subsequently washed 3x with PBS and 1x with H2O , prior to mounting on glass slides with mounting medium containing DAPI ( ProLong Gold; ThermoFisher Scientific ) .", "Images were acquired with a laser scanning confocal microscope ( Fluoview FV1000; Olympus ) with a 60 × 1 . 4 NA oil objective , and processed using Fiji ( Image J ) .", "To test amphiregulin ( Areg ) , transforming growth factor ( TGFα ) , tumour necrosis factor ( TNFα ) , betacellulin ( BTC ) or epidermal growth factor ( EGF ) shedding , MEFs were plated at a density of 5 × 104 in a 24 well plate followed by transfection 16 hr later with alkaline phosphatase ( AP ) conjugated Areg , TGFα , EGF ( kind gifts from Prof . Carl Blobel ) or TNFα ( a kind gift from Dr Stefan Düsterhöft ) .", "For transfection , 200 ng DNA and 0 . 8 µl of Fugene-6 ( Promega ) were used , following standard protocols .", "For GPCR stimulations , MEFs were co-transfected with a construct expressing histamine HA1 receptor ( kindly provided by Dr . Stefan Düsterhöft ) ( 400 ng per 24 well ) .", "Two days later a stimulation assay was performed as described previously ( Christova et al . , 2013 ) .", "In short , cells were washed twice in PBS and incubated in 300 µl Opti-MEM with or without indicated concentrations of PMA ( 200 nM ) , or histamine ( 300 µM ) for stated times ( Invitrogen ) .", "For kinase screening experiments , cells were pretreated with indicated kinase inhibitors for 1 hr in 300 µl Opti-MEM .", "100 µl Opti-MEM containing 80 nM PMA or 1 . 2 mM histamine ( 4x ) was then added for 1 hr , in the continued presence of kinase inhibitors .", "AP activity was detected in stimulated and un-stimulated supernatant or in cell lysates ( using Triton-X100 buffer ) by adding equal volumes of PNPP buffer ( Pierce ) and incubating at 37°C followed by measurement of absorbance at 405 nm .", "The percentage of the total material shed from each well ( i . e . signal from supernatant divided by total signal from lysate and supernatant ) was then used to calculate the percentage-stimulated release .", "In the kinase screens , the percentage induction above treatment with 100 µl control Opti-MEM is plotted .", "The kinase inhibitors Go6973 , Go6983 , U0126 and BI-D1870 were used at a final concentration of 20 µM .", "Error bars represent standard deviations , and all two-tailed student t-tests were performed comparing indicated mutants with iRhom2WT .", "In the case of kinase screen experiments , two-tailed student t-tests were performed comparing stimulated AP release in the presence of kinase inhibitors with DMSO treatments .", "3–4 days post infection , macrophages were stimulated with 200 nm PMA ( Invitrogen ) or 100 ng/ml LPS ( Sigma ) in OPTIMEM supplemented with 1 mg/ml BSA for indicated times .", "ELISAs were performed with a Quantikine TNF ELISA kit according to the manufacturer’s instructions ( R&D Systems , catalogue number MTA00B ) .", "Error bars represent standard deviations , and all two-tailed student t-tests represent a comparison with iRhom2WT data points .", "For conventional immunoprecipitations ( IPs ) , HEK293 cells or DKO MEFs stably transduced with different versions of iRhom2-3xHA were grown to ~90% confluence in 10 cm plates , on the day of IP .", "For detection of phosphorylation , or plasma membrane IPs , 24 hr before stimulation , cells were cultured overnight in OPTIMEM .", "Cells were then stimulated with or without 200 nM PMA in OPTIMEM for indicated times .", "For plasma membrane immunoprecipitations , after treatment for 5 min , DKO MEFs were placed on ice and washed 3x with ice-cold PBS .", "Intact cells were then incubated on ice with mouse anti-HA antibody diluted in PBS for 1 hr .", "Cells were then washed 4x in ice-cold PBS .", "After all treatments , cells were washed 3x with ice-cold PBS and then lysed in 1 ml TX-100 lysis buffer ( 1% Triton X-100 , 150 mM NaCl , 50 mM Tris-HCl , pH 7 . 4 ) supplemented with protease inhibitor cocktail ( Roche ) , 10 mM 1 , 10-phenanthroline and PhosSTOP ( Roche ) .", "Cell lysates were cleared by centrifugation at 20 , 000 x g for 10 min at 4°C .", "Protein concentrations were measured by a BCA assay kit ( Pierce ) .", "The lysates were then immunoprecipitated for 2–3 hr with 15 μl pre-washed HA antibody-coupled beads ( conventional IPs ) , or 25 μl ProteinA-coupled agarose beads ( Pierce/Thermo ) at 4°C on a rotor .", "After 4–5 washes with lysis buffer , the immunocomplexes were incubated at 65°C for 15 min in 1x LDS sample buffer .", "Typically , 25% of the immunoprecipitates and 1% of lysates were resolved on SDS-PAGE gels for subsequent western blotting .", "As stated in Siggs et al . ( 2014 ) .", "For ConA enrichment experiments , cells grown in 10 cm dishes were grown to ~80% confluence were washed twice in ice-cold PBS and then lysed for 10 min in TX-100 lysis buffer ( 1% Triton X-100 , 150 mM NaCl , 50 mM Tris-HCl , pH 7 . 4 ) containing complete protease inhibitor cocktail ( Roche ) and 10 mM 1 , 10-phenanthroline , to prevent autocatalysis of TACE .", "Cells/lysates were then scraped and centrifuged at 20 , 000 x g .", "Protein concentration was then measured using by a BCA assay ( Life Technogies ) .", "Lysates were then mixed with washed 50 μl ConA beads ( washed in lysis buffer ) and incubated for 2–3 hr at 4°C on a rotor .", "Beads were washed three times in lysis buffer and glycoproteins were eluted with 1 x sample buffer supplemented with 15% sucrose , by incubation at 65°C for 15 min in a thermomixer .", "20% of the ConA preparations and 1% of lysates were resolved on SDS-PAGE gels for subsequent Western blotting .", "After any indicated treatments , cells were placed on ice and washed 3x with PBS .", "Surface proteins were labelled on ice with 1 mg/ml Sulfo-NHS-LC-Biotin diluted in PBS ( Thermo Scientific , catalogue number 21335 ) according to manufacturer’s instructions .", "Cells then underwent 1x PBS wash , 1 × 10 min incubation in 100 mM glycine/PBS to quench and remove excess biotin , and 3x PBS washes .", "Cells were lysed in TX-100 lysis buffer ( 1% Triton X-100 , 150 mM NaCl , 50 mM Tris-HCl , pH 7 . 4 ) containing complete protease inhibitor cocktail ( Roche ) and 10 mM 1 , 10-phenanthroline .", "After pelleting at 20 , 000g , clarified supernatants were incubated with 30 μl high-capacity neutravidin agarose beads for 2–3 hr to capture labelled proteins ( Thermo Scientific , catalogue number 29204 ) .", "Beads were then washed 3x with ice-cold lysis buffer and eluted with 1x LDS sample buffer at 65°C for 15 min in a thermomixer .", "DKO MEFs transduced with HA-tagged iRhom2WT or iRhom2KDEL were detached using 2 mM EDTA and then washed with ice-cold FACS buffer ( 0 . 25% BSA and 0 . 1% sodium azide in PBS ) .", "The cells were stained on ice with anti-HA antibody ( Santa Cruz rabbit polyclonal ( sc-805 ) ; 0 . 5 µg per 5 × 105 cells ) diluted in FACS buffer for 45 min .", "After two washing steps with ice-cold FACS buffer , the cells were incubated with anti-rabbit Alexa Fluor 488 antibody ( Thermo Fisher Scientific donkey polyclonal ( A21206 ) , 1:1000 ) on ice for 30 min .", "Cells stained only with the secondary antibody served as control .", "Cells were washed twice with ice-cold FACS buffer and then analysed with BD FACSCalibur ( BD Biosciences ) and FlowJo software .", "Samples were typically electrophoresed at 150V on 4–12% Bis-Tris gels ( Invitrogen ) until the dye front had migrated off the gel ( approx . 10–15 kDa ) .", "Gels were transferred onto PVDF membranes and blocked in PBS or TBS containing Tween 20 ( 0 . 05% ) and 5% milk or 1% BSA , before detection with the indicated primary antibodies and species-specific HRP-coupled secondary antibodies .", "Band visualisation was achieved with Enhanced Chemiluminescence ( Amersham Biosciences ) using X-ray film .", "HEK293T cells were kept on ice and washed with PBS .", "Proteins were cross-linked in vivo as described in ( Adrain et al . , 2012 ) .", "Cells were lysed in DDM RIPA lysis buffer ( 1% n-Dodecyl β-D-Maltopyranoside ( DDM ) , 150 mM NaCl , Tris-HCl pH 7 . 5 , 50 mM Tris-HCl pH7 . 5 , 0 . 1% SDS , 0 . 5% sodium deoxycholate ) supplemented with EDTA-free protease inhibitor mix ( Roche ) and 10 mM 1 , 10-phenanthroline ( Sigma ) for 20 min on ice .", "A centrifugation step ( 10 , 000 g , 10 min , 4°C ) was performed to precipitate nuclei and insoluble cell fragments .", "Cell lysates were pre-cleared by incubation with mouse IgG ( Sigma ) for 1 hr at 4°C .", "Magnetic anti-HA beads ( Pierce ) were then added to the supernatant and incubated for 1 hr at 4°C .", "Beads were washed with DDM RIPA lysis buffer containing 300 mM NaCl , and once with PBS before bound peptides were eluted three times by incubation with HA peptide ( 1 mg/ml ) for 15 min at 37°C .", "The eluates were pooled and concentrated with a vivaspin concentrator 500 ( 10 , 000 kDa MWCO , Sartorius ) .", "The spin filter was washed five times with PBS by centrifugation at 12 , 000 g , RT .", "Proteins samples were prepared for mass spectrometry using filter-aided sample preparation , as described ( Wiśniewski et al . , 2011 ) .", "Peptides were eluted from the filter with 0 . 1% formic acid , 0 . 1% formic acid in 50% acetonitrile ( ACN ) and finally with 0 . 1% formic acid in 80% ACN .", "The eluate was concentrated and peptides were loaded on PepClean C18 spin columns ( Pierce ) to remove salt .", "Peptides were eluted with 0 . 1% formic acid in 50% ACN and with 0 . 1% formic acid in 80% ACN and concentrated .", "Peptides were resolved in 0 . 1% triflouroacidic acid in 5% ACN and injected into the Ultimate 3000 RSLCnano HPLC ( Dionex ) – tandem mass spectrometry Q Exactive Orbitrap machine .", "Data was analysed using the semi-quantitative label-free quantitation method SINQ ( Trudgian et al . , 2011 ) of the central proteomics facilities pipeline ( CPFP ) Oxford ( Trudgian et al . , 2010; Trudgian and Mirzaei , 2012 ) .", "Phosphopeptides were identified and analysed with the MASCOT software ." ] ]

[ "Proteolytic cleavage and release from the cell surface of membrane-tethered ligands is an important mechanism of regulating intercellular signalling .", "TACE is a major shedding protease , responsible for the liberation of the inflammatory cytokine TNFα and ligands of the epidermal growth factor receptor .", "iRhoms , catalytically inactive members of the rhomboid-like superfamily , have been shown to control the ER-to-Golgi transport and maturation of TACE .", "Here , we reveal that iRhom2 remains associated with TACE throughout the secretory pathway , and is stabilised at the cell surface by this interaction .", "At the plasma membrane , ERK1/2-mediated phosphorylation and 14-3-3 protein binding of the cytoplasmic amino-terminus of iRhom2 alter its interaction with mature TACE , thereby licensing its proteolytic activity .", "We show that this molecular mechanism is responsible for triggering inflammatory responses in primary mouse macrophages .", "Overall , iRhom2 binds to TACE throughout its lifecycle , implying that iRhom2 is a primary regulator of stimulated cytokine and growth factor signalling ." ]

[ "Injury or infection can cause tissues in the body to become inflamed .", "The immune system triggers this inflammation to help repair the injury or fight the infection .", "A signal molecule known as TNF – which is produced by immune cells called macrophages – triggers inflammation .", "This protein is normally attached to the surface of the macrophage , and it only activates inflammation once it has been cut free .", "An enzyme called TACE cuts and releases TNF from the surface of macrophages .", "This enzyme is made inside the cell and is then transported to the surface .", "On the way , TACE matures from an inactive form to a fully functional enzyme .", "Previous work revealed that a protein called iRhom2 controls TACE maturation , but it has been unclear whether iRhom2 affects TACE in any additional ways .", "Grieve et al . studied the relationship between iRhom2 and TACE in more detail .", "The experiments show two new roles for iRhom2: in protecting TACE from being destroyed at the cell surface , and prompting TACE to release TNF to trigger inflammation .", "Injury or infection causes small molecules called phosphate groups to be attached to iRhom2 in macrophages , which causes TACE to release TNF .", "The findings of Grieve et al . provide the first evidence that iRhom2 influences the activity of TACE throughout the enzyme’s lifetime .", "Excessive inflammation , often triggered by the uncontrolled release of TNF , can lead to rheumatoid arthritis , cancer and many other diseases .", "Therefore , iRhom2 could be a promising new target for anti-inflammatory drugs that may help to treat these conditions ." ]

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

[ "ecology", "evolutionary biology" ]

[ [ "Symbiosis between animals and bacteria can enable both organisms to adapt to harsh environments or expand into new habitats , which impacts the ecology and evolution of both bacterial and host lineages ( McFall-Ngai et al . , 2013; Moran , 2007; Moya et al . , 2008 ) .", "Symbiosis with luminescent bacteria has evolved independently multiple times in diverse squid and fish species ( Davis et al . , 2016; Dunlap and Urbanczyk , 2013 ) and has been correlated with host diversification ( Davis et al . , 2016; Ellis and Oakley , 2016 ) .", "Bioluminescence is considered an adaptive phenotype across multiple taxa and a ubiquitous function in the largest habitat on the planet , the bathypelagic biome ( ocean’s midwaters below 1000 m ) ( Martini and Haddock , 2017 ) .", "Four genera of bacteria in the family Vibrionaceae engage in luminescent symbiosis ( Dunlap and Urbanczyk , 2013; Hendry et al . , 2018; Schaechter , 2009 ) , including the model species Aliivibrio fischeri , but comparatively little is known about the bioluminescent symbionts of deep-sea anglerfishes .", "Ceratioid anglerfishes ( suborder Ceratioidei ) consist of 167 species from 11 families ( Froese and Pauly , 2018 ) and are the most speciose fish suborder in the bathypelagic zone ( Pietsch , 2009 ) .", "Most female ceratioid anglerfishes host extracellular luminous symbiotic bacteria in a lure-like projection ( esca ) above the animal’s head ( Munk , 1999 ) .", "The genera Cryptopsaras and Ceratias harbor bacterial symbionts in additional pouch-like symbiont-filled protuberances anterior to the dorsal fin , known as caruncles .", "Bioluminescent symbiosis is thought to be essential to the survival of adult anglerfishes , although the exact function has not been observed .", "The lure has been proposed to attract prey , confound predators , or signal mates ( Pietsch , 2009 ) .", "Recent research by Hendry et al . ( 2018 ) investigated symbiont genomes from two commonly collected anglerfish species , Cryptopsaras couesii and Melanocetus johnsonii , host lineages that diverged approximately 100 million years ago ( Miya et al . , 2010; Pietsch , 2009 ) .", "Each host harbored a distinct species of bacterial symbiont: C . couesii hosts ‘Candidatus Enterovibrio luxaltus’ and M . johnsonii hosts ‘Candidatus Enterovibrio escacola , ’ referred to here as E . luxaltus and E . escacola for ease ( Hendry et al . , 2018 ) .", "Most luminescent bacterial symbionts are facultatively symbiotic , have genome sizes typical of nonsymbiotic , free-living relatives , and are acquired by hosts from environmental populations ( Bongrand et al . , 2016; Bright and Bulgheresi , 2010; Dunlap et al . , 2012; Dunlap and Urbanczyk , 2013; Ruby et al . , 2005; Urbanczyk et al . , 2011 ) .", "In contrast , anglerfish symbiont genomes are reduced ~50% relative to closely related free-living bacteria , a pattern more commonly seen in intracellular , obligate symbiosis ( Fisher et al . , 2017; Kuwahara et al . , 2007; Manzano-Marín and Latorre , 2016; Shigenobu et al . , 2000 ) .", "Anglerfish symbionts , which have not been successfully cultured ( Haygood et al . , 1984 ) , appear to be obligately dependent on their hosts for growth , as the metabolic capacity to use carbon sources other than glucose are absent from the genome and glucose is an extremely limited resource in the deep sea ( Hansell , 2013; Hendry et al . , 2018 ) .", "Genomic degeneration in obligate symbionts is thought to occur as a result of relaxed purifying selection on genes that are unnecessary within the host habitat ( Bright and Bulgheresi , 2010; Kenyon and Sabree , 2014; Fisher et al . , 2017; Sachs et al . , 2011 ) .", "This process may be mediated in part by relaxed regulation of transposable elements ( TEs ) ( McCutcheon and Moran , 2011 ) , which was observed for both E . escacola and E . luxaltus genomes ( Hendry et al . , 2018 ) .", "Transposon expansions and pseudogenization were evident in both symbionts , with TE pseudogenes making up about 30% of each bacterial genome ( Hendry et al . , 2018 ) .", "Phylogenetic investigation of transposon families found independent expansions within each symbiont species , suggesting that genome reduction may have occurred independently within each lineage ( Hendry et al . , 2018 ) .", "Although the aforementioned evolutionary patterns tend to result from physical restriction to hosts and vertical transmission between host generations ( Bright and Bulgheresi , 2010; McCutcheon and Moran , 2011; Moran , 1996 ) , several characteristics of anglerfish and their symbionts suggest that these bacteria may be environmentally acquired .", "The anglerfish symbiont genomes retain genes predicted to be under selection outside the host , such as genes involved in cell wall synthesis and complete motility and chemotaxis pathways .", "Symbionts are also capable of producing polyhydroxybutyrate ( PHB ) , a carbon storage molecule that is hypothesized to aid in environmental persistence until colonizing suitable hosts ( Haygood , 1993; Hendry et al . , 2018; Hendry et al . , 2016 ) .", "Furthermore , anglerfish life history traits could preclude the possibility of vertical transmission .", "Anglerfishes reproduce through the production of an ‘egg raft’ or ‘veil’ that delivers eggs to the surface .", "Juvenile anglerfishes do not have lures; as anglerfishes near sexual maturity they descend to bathypelagic depths and develop their lure ( Pietsch , 2009 ) .", "This developmental process and the anglerfish’s poor swimming abilities , coupled with differences in surface and deep-sea currents ( Etter and Bower , 2015; Pazos and Lumpkin R , 2007; Pietsch , 2009 ) , likely result in generations separated by several kilometers of ocean , making it unlikely that juveniles acquire symbionts from their parents in the deep sea .", "It also appears unlikely that anglerfishes acquire symbionts from their egg raft , as juveniles have not been found with symbiotic bacteria in their developing lures ( Freed et al . , 2019; Munk , 1999 ) .", "An alternative hypothesis to vertical transmission is that anglerfishes acquire bacterial symbionts from persistent environmental populations despite the limited metabolic capacity of symbionts .", "Fishes known to harbor luminous bacteria regularly release symbionts into the environment ( Haygood , 1993 ) .", "In ceratioid anglerfishes , the extracellular symbiotic bacteria are likely released from the host via a small opening in the lure ( Haygood et al . , 1984; Munk , 1999 ) .", "Environmental samples of bacteria taken concurrently with anglerfish collections in the Gulf of Mexico found 16S rDNA sequences resembling symbionts ( Freed et al . , 2019 ) , which suggests that anglerfish symbionts could be environmentally acquired .", "Symbiont transmission between host generations via environmental populations , referred to here as environmental acquisition , is not uncommon in the deep sea; for instance , it occurs in the symbiosis of tubeworms ( Feldman et al . , 1997; Nussbaumer et al . , 2006 ) and mussels ( Won et al . , 2008; Won et al . , 2003 ) with their chemosynthetic bacteria .", "However , these bacteria have genome sizes typical of free-living relatives and lack signatures of reduction ( Kleiner et al . , 2012; Li et al . , 2018; Ponnudurai et al . , 2017 ) .", "Marine symbionts with reduced genomes have been found , such as the symbionts of deep-sea clams in the genus Calyptogena ( Kuwahara et al . , 2007; Newton et al . , 2007 ) or the luminous symbionts of anomalopid flashlight fishes ( Hendry et al . , 2016; Hendry et al . , 2014 ) .", "However , these symbionts are characterized as having vertical ( Goffredi et al . , 2003; Hurtado et al . , 2003 ) and possibly pseudovertical transmission ( Hendry and Dunlap , 2014 ) .", "Because strictly vertically transmitted symbionts will codiverge with their hosts ( Bright and Bulgheresi , 2010; Clark et al . , 2000; Jousselin et al . , 2009; Zhang et al . , 2018 ) , we assessed the likelihood of hypothesized transmission modes of anglerfish symbionts by testing for symbiont-host codivergence .", "This analysis included multiple specimens of the previously studied host species C . couesii and M . johnsonii , as well as less common genera of anglerfishes , including Ceratias , Chaenophryne , Linophryne , and Oneirodes .", "The geographic distribution of these genera is poorly known , but based on collection data the rarest species in our study ( Linophryne maderensis ) , has only four documented museum samples ( Pietsch , 2009 ) .", "These host species originate from four of the eleven families of ceratioid anglerfishes and span much of the phylogenetic diversity of the suborder Ceratioidei .", "We hypothesized that a high degree of congruence between host and symbiont phylogenies will indicate codivergence due to vertical transmission .", "Additionally , codivergence could result in diverse symbiont species associated with diverse host lineages .", "Alternatively , if symbiont and host phylogenies lack congruence and different host species share symbionts , this indicates likely acquisition of symbionts from environmental populations ." ], [ "Contigs closely matching genome sequences of the previously reported luminous symbionts E . escacola or E . luxaltus were found in all samples .", "These symbiont species were never recovered together and no additional luminescence genes from other taxa were found in any assemblies , confirming prior findings that individual anglerfish host a single species of symbiont ( Hendry et al . , 2018 ) .", "Additionally , hosts for which both esca and caruncle samples were available ( Cryptopsaras ) hosted the same symbiont species in both light organs ( Table 1 ) .", "Phylogenetic analysis of conserved housekeeping genes confirmed that all new symbiont samples in this study are closely related to previously documented species ( Hendry et al . , 2018 ) ( Figure 1 ) .", "Short or nonexistent branch lengths within each symbiont species clade suggest that there are few genetic differences between samples , which was supported by ANI values ( Table 1 ) .", "Within a species there was greater than 99% ANI to the previously identified symbiont species ( Hendry et al . , 2018 ) and the between symbiont species ANI was less than 74% .", "All genomes had an average coverage depth of 15x or greater .", "Comparison of the host and symbiont phylogenies showed very little congruence , suggesting that neither symbiont species has co-diverged with their host ( Figure 2 ) .", "A symbiont phylogeny was constructed using 205 single-copy protein-coding genes shared by anglerfish symbionts and closely-related free-living bacteria .", "The construction of a protein-coding phylogeny was employed to get higher-resolution of the relationship between symbionts relative to the house keeping phylogeny .", "Both analyses showed similar relationships between symbionts ( Figure 2 ) .", "This symbiont topology was compared to a host phylogeny constructed using mitochondrial genes , which matches previous analysis of anglerfish evolutionary relationships ( Miya et al . , 2010 ) ( Figure 2 ) .", "Comparison of host and symbiont phylogenies found E . luxaltus was only associated with the fish species C . couesii , and that all fish in this clade hosted the same species of symbiont , indicating that E . luxaltus and C . couesii may have a specific interaction .", "In contrast , E . escacola was the symbiont associated with every other anglerfish sample evaluated .", "These other fish samples cover much of the diversity in the suborder , including four of the 11 ceratioid families distributed across the phylogeny .", "These diverse anglerfishes all hosted very genetically similar symbiont lineages that are polyphyletic with respect to host identity .", "None of the symbiont phylogenies , including those constructed with single-copy protein-coding genes and conserved housekeeping genes ( Figure 3 ) , resulted in significant congruence of E . escacola and host phylogeny after statistical testing for symbiont-host codivergence using Procrustean superposition of the symbiont phylogeny .", "This is not surprising as E . escacola symbionts from the same genus of host did not form monophyletic groups in any analysis .", "In the analysis of conserved single-copy protein-coding genes , only two of the Atlantic samples of E . luxaltus ( symbiont CC32E and CC62E ) were significantly congruent with C . couesii hosts .", "Analysis using the housekeeping gene phylogeny resulted in significant congruence with only some host-symbiont pairs , specifically symbionts CC26E , CCS1E , and CCS2E , suggesting that any significant congruence in this clade is not robust .", "Symbiont and host phylogenies had limited congruence across the anglerfish suborder , and we found no reliable signals of codivergence which might indicate vertical transmission .", "In order to further evaluate the possibility of anglerfish symbionts persisting environmentally , we attempted to amplify DNA from environmental samples with symbiont-specific primers .", "We used a PCR assay for a highly conserved and species specific 198-basepair portion of the cheA locus from each symbiont on seawater bacterial samples .", "This locus was successfully amplified and sequenced from a subset of the samples , with sequences identified as E . luxaltus and E . escacola found in distinct environmental samples .", "Four samples ( 8% of those evaluated ) were confirmed to contain the E . luxaltus cheA gene ( Supplementary file 6 ) .", "These nucleotide sequences were 99–100% similar to the cheA locus in all E . luxaltus genomes available ( Hendry et al . , 2018; this study ) .", "The amplicon sequences do not appear to be from other known bacteria , such as closely related Enterovibrio .", "The most similar match to the environmental sequences in GenBank databases ( non-redundant , Refseq genome , and whole genome shotgun ) with >60% coverage shared only 80% nucleotide identity .", "Four different samples ( 8% of those evaluated ) were identified as E . escacola .", "Sequences of E . escacola cheA from the environment did not have significant matches in GenBank databases , but were 99% similar to the E . escacola locus from available genomes ( Hendry et al . , 2018; this study ) .", "The amplified cheA region is only 78% similar between E . escacola and E . luxaltus and phylogenetic analysis confirmed that the environmental sequences clustered with E . escacola and E . luxaltus sequences rather than cheA orthologs from the highest non-symbiont BLAST matches in GenBank ( Figure 4 ) .", "This phylogenetic clustering and high nucleotide identity suggest that the cheA locus is highly conserved within each species and distinctive from closely related bacteria , so we conclude that successful amplifications from seawater indicate that the symbionts were present in the environment .", "There was very little genetic diversity within both E . escacola and E . luxaltus at the loci analyzed above , which could possibly obscure codivergence between symbionts and hosts .", "To investigate this possibility , as well as any geographic patterns in symbiont distribution , phylogenies were constructed using more data in the form of genome-wide single nucleotide polymorphisms ( SNPs ) ( Figure 5A B ) .", "Fewer SNPs present across samples were found in E . luxaltus genomes ( 2252 ) compared to E . escacola genomes ( 15272 ) .", "The E . luxaltus SNP phylogeny does further differentiate between samples with high support ( Figure 5A ) .", "Specifically , a host esca ( E ) and caruncle ( C ) from the North Atlantic were divergent from those collected in the Gulf of Mexico .", "However , with the limited number of available samples , we are not able to fully investigate if this is due to geographic patterns or the substantial time between sampling ( ~20 years ) ( Supplementary file 1 ) .", "However , samples collected from the same location from different months ( with a maximum difference in collection time of 12 months ) did not form distinct clades .", "The SNP phylogeny constructed for E . escacola also showed more divergence than phylogenies from conserved genes , but samples did not form distinct clades by location or collection date ( Figure 5B ) .", "Consistent with other analyses , E . escacola samples isolated from the same host genera were polyphyletic and there was often greater variation between symbionts isolated from the same anglerfish genus than between symbionts from different host genera .", "Some Ceratias symbiont samples did form a long branch that was distinct from all other E . escacola lineages , with the exception of a single Ceratias sample ( Csp75C ) .", "To confirm a lack of codivergence with improved phylogenetic resolution , we performed the Procrustean analysis using the SNP phylogenies , but neither symbiont was significantly codiverging with their hosts ( p>0 . 05 ) in this analysis ." ], [ "Within the broad phylogenetic spectrum of ceratioid anglerfishes sampled in this study we identified only two symbiont species , E . luxaltus and E . escacola .", "These symbiont species were previously described as the symbionts of two commonly collected anglerfish species , Cryptopsaras couesii and Melanocetus johnsonii .", "The fact that sampling from four additional anglerfish genera from two different ocean basins did not uncover more symbiont diversity suggests that there are a low number of luminescent symbiont species that can associate with deep-sea anglerfishes .", "However , it should be noted that this study was not all-inclusive , particularly since new anglerfish species are still being described ( Pietsch and Sutton , 2015 ) , and further sampling could reveal more symbiont diversity .", "In addition to the lack of species-level diversity , the low intra-specific diversity in each symbiont species was notable .", "Similar trends have been found in other obligate symbionts , such as the aphid endosymbiont Buchnera and in the obligate luminous symbionts of flashlight fishes ( Hendry et al . , 2014 ) , but not in facultative luminous symbionts ( Abbot and Moran , 2002; Funk et al . , 2001; Hendry et al . , 2014 ) .", "Host population bottlenecks may lead to low genetic diversity within Buchnera ( Abbot and Moran , 2002; Funk et al . , 2001 ) , but this seems unlikely to account for low diversity in obligate symbionts of fishes .", "It is unclear why anglerfish symbionts , even from distinct hosts and geographic locations , are so genetically similar .", "It is possible that although the hosts are separated by ocean basins , low mutation rates and long doubling times have resulted in a fairly stable and widespread symbiont population .", "Anglerfish symbionts lack many DNA repair pathways ( Hendry et al . , 2018 ) , which has been implicated in increased mutation rates in obligate symbionts ( Lind and Andersson , 2008 ) , but the connection between the loss of these pathways and genomic evolution is not always clear ( Tamas et al . , 2002 ) .", "Our finding of low genetic diversity in anglerfish symbionts supports the idea that a loss of DNA repair mechanisms does not necessarily lead to high mutation rates in bacteria .", "We speculate that anglerfish symbionts may instead have long doubling times , as this adaptation is common for bacteria surviving in the low-nutrient , high-hydrostatic pressure of the deep sea ( Lauro and Bartlett , 2008; Wirsen and Molyneaux , 1999 ) .", "Lowered metabolic rates are also common for cooperative symbionts ( An et al . , 2014 ) .", "Collectively these factors may have led to the bacterial genomes being relatively static when free-living and resulted in the limited diversity observed in this study .", "Although some obligate bacteria show low genetic diversity within a host species , obligate symbionts from different host species are often distinct due to codivergence with their hosts ( Clark et al . , 2000; Jousselin et al . , 2009 ) .", "This pattern has been found in numerous symbionts that are known to be vertically transmitted and often results in phylogenetic congruence between distinct host and symbiont taxa ( Fisher et al . , 2017; Sachs et al . , 2011 ) , as has been documented in the bacterial symbionts of insects ( Dale and Moran , 2006; Moran et al . , 2008 ) and deep-sea clams ( Goffredi et al . , 2003; Hurtado et al . , 2003 ) .", "Within vertically transmitted symbionts , symbiont replacements or horizontal transfers can often be observed in specific lineages where congruence breaks down ( Bright and Bulgheresi , 2010 ) , as has been observed in Wolbachia-harboring insects ( Kikuchi and Fukatsu , 2003; Lefoulon et al . , 2016 ) , bacterial symbionts of marine worms ( Blazejak et al . , 2006 ) , and Prochloron associated with sea-squirts ( Münchhoff et al . , 2007 ) .", "Neither of these patterns is seen in our data .", "Anglerfish symbionts and their hosts lack consistently congruent phylogenies and it does not seem likely that congruence is being obscured by symbiont replacements or transfers , since very diverse host species all share low diversity symbionts .", "These results support the hypothesis that anglerfish symbionts are not codiverging with their host species .", "This conclusion is robust for E . escacola and associated hosts , but we may not have enough samples , and genetic diversity within those samples , to rule out the possibility that E . luxaltus and C . couesii could be codiverging due to vertical transmission .", "An alternative hypothesis is that C . couesii and E . luxaltus have a specific interaction , and that either the host or the bacterium excludes the other symbiont species ( Bongrand and Ruby , 2019; Koch et al . , 2014 ) .", "A lack of robust and statistically significant codivergence between hosts and symbionts contradicts the hypothesis that either symbiont species is vertically transmitted .", "This is consistent with previous studies of the luminous symbionts of squid and fish hosts , as they show no congruence between host and symbiont species ( Dunlap et al . , 2007 ) .", "The most likely conclusion based on these data is that anglerfishes acquire their bacteria from an environmental symbiont pool that interacts with diverse anglerfish species .", "However , anglerfish symbiont genomes resemble vertically transmitted symbionts in multiple ways , including having extreme gene loss , expansion of transposable elements , and limited metabolic capacity ( Hendry et al . , 2018 ) .", "Similar genomic patterns are seen in ‘Candidatus Photodesmus’ species , the luminous symbiont of anomalopid flashlight fishes ( Hendry et al . , 2016; Hendry et al . , 2014 ) .", "Both anglerfish and anomalopid symbionts have evaded culturing efforts and are divergent from known species of luminous bacteria in the Vibrionaceae ( Haygood et al . , 1992; Haygood and Distel , 1993; Hendry et al . , 2018; Hendry and Dunlap , 2011 ) .", "However , anglerfishes do not appear to school , nor do they exhibit diurnal cave dwelling , that is hypothesized to assist in pseudovertical transmission of Photodesmus species to flashlight fishes ( Hendry et al . , 2016; Hendry et al . , 2014 ) .", "Flashlight fishes and their symbionts lack sufficient sampling to test for codivergence , but symbiont sequencing from four fish species found high symbiont-host specificity ( Hendry et al . , 2014 ) , a distinct pattern from the results presented here for E . escacola and six host species .", "We are not aware of other bacterial symbionts that have undergone extensive , degenerative genome reduction while maintaining environmental populations and associations with diverse and widespread hosts , as is seen with deep-sea anglerfish symbionts ( For an overview of transmission modes and evolutionary patterns in symbionts , see Table 2 ) .", "Based on catch rates and limited observation , anglerfishes are thought to be relatively solitary , and different life stages are separated by hundreds to thousands of meters of ocean ( Pietsch , 2009 ) .", "Anglerfishes are unlikely to encounter environmental symbionts regularly , as symbionts are unlikely to establish widespread populations due to their limited metabolic capabilities ( Hendry et al . , 2018 ) .", "Other environmentally transmitted luminescent symbionts have much higher host densities to enrich populations in the local environment ( Nealson and Hastings , 1991 ) .", "Anglerfishes may have evolved mechanisms to similarly increase the concentration of symbionts in their local environment .", "A small pore in the lure is likely seeding the environment with symbiotic bacteria ( Munk , 1999 ) , but the caruncles on Ceratias and Cryptopsaras species are also a likely source of symbiotic bacteria .", "The caruncle is not externally luminescent and its function for the fish is not established ( Pietsch , 2009 ) .", "Although it is not connected to the esca , the caruncle does connect to the surrounding water through a small distal pore ( Pietsch , 2009 ) .", "The conclusion that anglerfishes must acquire their symbionts from potentially sparse environmental populations leads us to propose that the caruncle evolved as a mechanism to increase the concentration of symbiotic bacteria in the environment , thereby increasing the likelihood of symbionts being transmitted to new fish generations .", "Low host densities could also drive bacterial evolution in this system .", "Although M . johnsonii is relatively abundant among anglerfishes , as is C . couesii , all other anglerfish genera investigated in this study are less prevalent than the most common species ( Pietsch , 2009 ) .", "With extremely low host densities , E . escacola may remain a viable symbiont for diverse anglerfish species due to selection against host specificity .", "The lack of apparent host specificity in this symbiont may increase the likelihood that these bacteria in the environment will encounter a permissive host before losing viability .", "In the case of E . luxaltus , this symbiont may encounter sufficient abundances of C . couesii individuals to allow for host specificity , either as a result of selection or by chance .", "For example , E . luxaltus and E . escacola differ in the genes present in the structural symbiosis polysaccharide ( syp ) pathway , the regulation of which influences host specificity in the luminous symbiont A . fischeri ( Mandel et al . , 2009 ) .", "Both sypF and sypG are exclusive to E . luxaltus , that is neither gene is present in annotations and cannot be found in a BLAST search of any E . escacola genome .", "In A . fischeri , SypF is predicted to be a sensor kinase that regulates biofilm formation ( Darnell et al . , 2008; Thompson et al . , 2018 ) and SypG is a response regulator that directly activates the syp locus ( Hussa et al . , 2008; Thompson et al . , 2018; Yip et al . , 2005 ) .", "Although both symbionts maintain genes in the syp pathway , the loss of these regulatory genes in E . escacola could facilitate their colonization of a greater diversity of hosts .", "The finding that anglerfish symbionts are likely environmentally transmitted further supports the hypothesis that the limited functional capacity of anglerfish luminous symbionts is sufficient to persist in the deep sea before contacting a new host .", "Motile symbionts capable of chemotaxis may be able to out-compete other non-motile deep-sea bacteria for access to the high nutrient environment of the host esca ( DeLong et al . , 2006 ) .", "Additionally , polyhydroxybutyrate ( PHB ) may be a sufficient carbon source to sustain the symbiont before it arrives at a new host ( Hendry et al . , 2018 ) .", "PHB has been estimated to sustain rhizobia for years , and may assist these microbes to survive thousands of years in a dormant state ( Johnson et al . , 2007; Muller and Denison , 2018 ) ; we hypothesize that PHB should function similarly for flashlight fish and anglerfish symbionts .", "Environmental samples of free-living bacteria collected by the DEEPEND Consortium taken concurrently with anglerfish collection found multiple samples containing 16S rDNA matching anglerfish symbionts , at various depths , and from multiple sampling efforts ( Freed et al . , 2019 ) .", "Our characterization of symbionts using a portion of the chemotaxis protein cheA found multiple environmental samples containing either E . luxaltus or E . escacola .", "This result confirms previous reports that the symbiont persists in the water column ( Freed et al . , 2019 ) , and further supports our conclusion that symbionts are acquired from environmental populations .", "The low genetic diversity within the anglerfish symbionts made it difficult to determine if symbiont distribution was impacted by geographic origin or host identity .", "Using SNPs we were able to discern differences between E . luxaltus samples collected from different times at different locations , however , this result was limited to a single C . couesii individual from the North Atlantic , with esca and caruncle sampled ( symbiont CCS1E and CCS2C ) .", "A subset of the E . escacola hosted by Ceratias formed a distinct clade and included two of the Northern Atlantic samples and a sample from the Gulf of Mexico .", "This confounding result suggests that further sampling of Ceratias may provide more insight into how location and time impact the diversity of E . escacola .", "Alternatively , it is possible that because the two collection sites are connected by deep-sea currents ( Loop Current/Gulf Stream system ) , that symbionts were acquired by fishes in a similar location although they were collected hundreds of kilometers apart .", "The deep sea is the earth’s largest and most understudied ecosystem , where studying symbiosis is both challenging and costly .", "In this study we use genomic analysis on rare samples of one of the deep sea’s most prominent symbioses to answer an outstanding question , how are deep-sea anglerfish symbionts transmitted between generations ?", "Our findings demonstrate the value of studying relatively rare organisms in this ecosystem , as we can uncover new findings that may contrast with model systems .", "Bioluminescent symbiosis in anglerfishes breaks with several expectations from well-studied symbioses; symbionts that leave the host and establish environmental populations typically do not undergo genome degeneration .", "Yet , here we show that a luminous bacterial symbiont with an extremely reduced genome is able to traverse the low-nutrient , high-pressure environment of the deep sea to establish a symbiosis with a dispersed and relatively rare host .", "As samples of these fishes and symbionts become available , we may be able to address additional outstanding questions , such as the lack of diversity in anglerfish symbionts and their biogeographic population structure ." ], [ "Anglerfish samples were collected in the Gulf of Mexico by the DEEPEND Consortium and from east of the Cape Verde Islands by Spencer Nyholm and Peter Herring on the RRS Discovery expedition D243 ( sample information in Supplementary file 1 ) .", "Morphological identification was done on ship by Tracey Sutton ( DEEPEND ) or Spencer Nyholm and Peter Herring .", "Molecular genetic confirmation of morphological identification is discussed below .", "Samples were named according to: initial morphological identification , order collected , and anglerfish light organ sampled--either esca ( E ) or caruncles ( C ) .", "Lures were collected immediately after identification using a sterile scalpel and stored in ethanol or RNAlater ( Qiagen , Hilden , Germany ) at −80°C until processing .", "DNA extraction from samples collected in the Gulf of Mexico was performed at the Marine Microbiology and Genetics Laboratory at Nova Southeastern University’s Halmos College of Natural Sciences and Oceanography using the PowerLyzer PowerSoil kit ( MoBio ) as is described in Hendry et al . ( 2018 ) Samples collected in the Northern Atlantic were extracted using the DNeasy Blood and Tissue Kit ( Qiagen ) .", "Paired-end 250 base pair Illumina sequence libraries were prepared using the Nextera kit ( Illumina , San Diego , CA ) and sequenced using HiSeq2500 at the Cornell University Institute of Biotechnology Resource Center Genomics Facility .", "Contigs were assembled using DISCOVAR de novo and binned and assessed for quality using multiple approaches which are detailed in the Supplementary Information .", "Binned symbiont genomes and sequences mapped to the reference genomes for E . luxaltus and E . escacola ( GCA_002381345 . 1 and GCA_002300443 . 1 ) were submitted to NCBI ( Supplementary file 1 ) .", "High concentrations of an evident monoculture of symbionts within anglerfish escae enable assembly and study of symbiont genomes from samples that are technically metagenomic , as they include symbiont and host DNA as well as DNA from contaminant bacteria likely on the surface of the light organ ( Hendry et al . , 2018 ) .", "After assembly using DISCOVAR de novo , bacterial genomes were binned using metabat2 , which bins similar contigs according to tetranucleotide frequency and sequencing depth ( Kang et al . , 2015 ) .", "Sequences that failed to bin using metabat2 were binned using the Pathosystems Resource Integration Center ( PATRIC 3 . 5 . 23 ) ( Wattam et al . , 2014 ) .", "Three C . couesii-associated samples were not successfully binned using metabat2 or PATRIC; these sample assemblies were processed as is outlined , with the exception of finding average nucleotide identity or annotating gene content .", "Binned contigs were evaluated through a local BLAST search for genes within the luciferase operon ( luxA , luxB , and luxC ) and contigs in the resulting bin were input into the NCBI BLAST database to confirm symbiont identification .", "The average genome coverage depth was calculated using BBmap ( Bushnell , 2014 ) .", "Genome completeness was evaluated using checkM ( Parks et al . , 2015 ) , which previously estimated for E . luxaltus and E . escacola as only nearing 90% completion due to genome reduction ( Hendry et al . , 2018 ) .", "The quality of the genome assemblies unique to this study are similar to previously documented anglerfish symbionts .", "Contig bins which had approximately 90% genomic completion , lux luminescence genes , and high BLAST similarity to previously sequenced anglerfish symbiont genomes were consider complete symbiont genome sequences and were submitted to Rapid Annotation using Subsystem Technology ( RAST ) for annotation .", "All other bins generated by metabat2 and PATRIC did not contain luciferase genes nor did they have sequences that closely resembled symbiont housekeeping genes .", "Anglerfish morphological identification and evolutionary relationships among samples were evaluated using mitochondrial genes .", "Similar methods and comparison species are discussed in Miya et al . ( 2010 ) .", "Anglerfish mitochondrial sequences were identified using a local BLAST search of the unbinned contigs and deposited in GenBank ( accession numbers MK118159-MK118174 ) .", "Reference mitochondrial sequences were selected based on initial morphological identifications and supplemented with sequences of nearest neighbors present in GenBank and Miya et al . , 2010 ( Supplementary file 2 ) .", "Mitochondrial sequences were aligned using MAFFT and a phylogenetic tree was assembled using IQ-TREE ( Katoh et al . , 2002; Nguyen et al . , 2015 ) .", "Within IQ-TREE modelfinder selects a phylogenetic model using a model-selection method that concurrently searches model and tree space to increase the accuracy of phylogenetic estimates ( Kalyaanamoorthy et al . , 2017 ) .", "A consensus tree was constructed using the general time reversible model with empirical base frequencies , allowing for invariable sites , and four rate categories ( GTR+F+I+G4 ) and 1000 bootstrap replicates .", "Based on phylogenetic analysis , samples were assigned to a species if they fell within the same clade as multiple representatives of the same species or by morphological species identification if sequences from representative species were not available for comparison .", "Samples were identified to a genus if there was an indeterminate species designation .", "The genetic identification of a single sample contradicted morphological identification , was reevaluated morphologically , and found to confirm the mitochondrial identification .", "Similarity among symbiont genomes isolated from individual anglerfish samples was evaluated using average nucleotide identity ( ANI ) , housekeeping genes , and conserved single-copy protein-coding genes .", "ANI , a measure of nucleotide-level genomic similarity , was found using orthoANIu ( Yoon et al . , 2017 ) ; comparisons greater than 95% ANI considered the same species ( Konstantinidis and Tiedje , 2005 ) .", "Bacterial species trees were created using conserved housekeeping genes ( 16S rRNA gene , atpA , gapA , gyrB , pyrH , rpoA , topA ) from both symbiont contigs and closely related bacterial genomes downloaded from NCBI ( Supplementary file 3 ) .", "Genes were aligned using MAFFT and a tree was constructed from the concatenated alignments in IQ-TREE as described above ( GTR+F+I+G4 with 1000 bootstrap replicates ) .", "Single-copy protein-coding genes shared by bacterial symbionts and whole genome sequences of closely related free-living bacteria ( Supplementary file 4 ) were found by inputting RAST protein annotations into PhyloPhLan .", "DNA sequence of shared proteins were then extracted from RAST annotations and used to construct a phylogenomic tree by aligning individual genes in MAFFT .", "The 205 shared genes were concatenated , and a tree was constructed from 331103 positions using the GTR+F+I+G4 model selected by modelfinder and using 1000 bootstrap replicates in IQ-TREE .", "Host-symbiont codivergence was evaluated using Procrustean Approach to Cophylogeny ( PACo ) as implemented in R ( Balbuena et al . , 2013; R Development Core Team , 2012 ) .", "PACo is a global fit method that does not require fully resolved phylogenies to evaluate if the symbiont has evolved as a result of codivergence with the host species .", "In PACo , Procrustes superposition manipulates the symbiont genetic distance matrix to fit the host matrix , to evaluate the congruence of the symbiont to the host tree .", "Anglerfish phylogenies input into PACo were constructed as described above for mitochondrial sequences .", "Various bacterial phylogenies were analyzed in PACo , including the conserved housekeeping gene phylogeny , genome-wide SNP phylogenies ( described below ) , and the conserved single-copy protein-coding gene ( identified by PhyloPhLan ) phylogeny ( Segata et al . , 2013 ) .", "Symbiont and bacterial ultrametic trees were input into PACo as distance matrices , and 104 iterations were performed for significance testing .", "The contribution of each bacterial symbiont to the overall global codivergence was evaluated using jackknife estimation of the relative squared residuals; codivergence was indicated in those samples that have a significantly smaller fraction of the sum of squares .", "The 16S rDNA sequences matching anglerfish symbionts were previously found in environmental samples taken concurrently with anglerfish collections ( Freed et al . , 2019 ) , suggesting that symbionts persist outside the host .", "To confirm that anglerfish symbionts can persist in the environment , symbiont species-specific primers were developed from whole genomes to amplify multicopy loci of a conserved chemotaxis protein cheA , which should be relatively more abundant than single copy loci in low density samples .", "We performed PCR assays on DNA extracted from environmental bacteria in 52 samples taken concurrently with anglerfish collections during DEEPEND consortium cruises ( D01-D04 ) in the Gulf of Mexico .", "The filtering and extraction protocol used , as well as the 16S rDNA composition of a subset of these samples is described in Easson et al . ( Easson and Lopez , 2019 ) .", "Primers for cheA specific to each symbiont were designed by importing symbiont and closely related sequences found using the BLAST genome searches into DECIPHER ( Wright et al . , 2012 ) ( Supplementary file 5 ) .", "Sequences were amplified using nested PCR primers and the New England Biolabs standard taq polymerase kit ( NE Biolabs , Ipswich , MA , USA ) using the recommended protocol for amplifications under 500 base pairs .", "Reactions were prepared in a UV sterilized biosafety cabinet with surface sterilized implements .", "Negative controls prepared with sterile water were included in each round of PCR .", "No negative controls resulted in visible amplification .", "Amplifications were gel extracted using the Qiaquick gel extraction kit ( Qiagen , Venlo , Netherlands ) and Sanger Sequenced ( Genewiz , New Jersey , USA ) .", "Sequence identity was evaluated using blastx and blastn searching and a phylogenetic tree was constructed using MAFFT and IQ-TREE ( GTR+F+I+G4 and 1000 bootstrap replicates ) from environmental amplifications and cheA sequences annotated from genomes available in RAST ( Supplementary file 6 ) .", "Evolution within each symbiont species was evaluated using genome-wide SNPs .", "Bacteria were grouped into different species based on the result of ANI and the conserved housekeeping gene phylogeny .", "SNPs were identified using snippy v . 4 . 0-dev , which implements bwa mem and freebayes to compare reads from haploid genomes to a reference genome ( Garrison and Marth , 2012; Li , 2013; R Development Core Team , 2012; Seemann , 2015 ) .", "The reference genome was selected from the previously characterized anglerfish symbionts described in Hendry et al . ( 2018 ) .", "SNPs were identified in sequence reads and snippy-core was used to generate a core alignment of SNPs common to all samples .", "A phylogenetic tree was constructed using this core alignment in IQ-TREE for each symbiont species with 1000 bootstrap replicates , with the models selected for by modelfinder .", "The E . luxaltus SNPs phylogeny was constructed using the Kimura 3-parameter and ascertainment bias correction model ( K3P+ASC ) and the E . escacola SNPs phylogeny was constructed using the symmetric model with unequal rates and an ascertainment bias correction model ( SYM+ASC ) ." ] ]

[ "Deep-sea anglerfishes are relatively abundant and diverse , but their luminescent bacterial symbionts remain enigmatic .", "The genomes of two symbiont species have qualities common to vertically transmitted , host-dependent bacteria .", "However , a number of traits suggest that these symbionts may be environmentally acquired .", "To determine how anglerfish symbionts are transmitted , we analyzed bacteria-host codivergence across six diverse anglerfish genera .", "Most of the anglerfish species surveyed shared a common species of symbiont .", "Only one other symbiont species was found , which had a specific relationship with one anglerfish species , Cryptopsaras couesii .", "Host and symbiont phylogenies lacked congruence , and there was no statistical support for codivergence broadly .", "We also recovered symbiont-specific gene sequences from water collected near hosts , suggesting environmental persistence of symbionts .", "Based on these results we conclude that diverse anglerfishes share symbionts that are acquired from the environment , and that these bacteria have undergone extreme genome reduction although they are not vertically transmitted ." ]

[ "The deep sea is home to many different species of anglerfish , a group of animals in which females often display a dangling lure on the top of their heads .", "This organ shelters bacteria that make light , a partnership ( known as symbiosis ) that benefits both parties .", "The bacteria get a safe environment in which to grow , while the animal may use the light to confuse predators as well as attract prey and mates .", "The genetic information of these bacteria has changed since they became associated with their host .", "Their genomes have become smaller and more specialized , limiting their ability to survive outside of the fish .", "This phenomenon is also observed in other symbiotic bacteria , but mostly in microorganisms that are directly transmitted from parent to offspring , never having to live on their own .", "Yet , some evidence suggests that the bacteria in the lure of anglerfish may be spending time in the water until they find a new host , crossing thousands of meters of ocean in the process .", "To explore this paradox , Baker et al . looked into the type of bacteria carried by different groups of anglerfish .", "If each type of fish has its own kind of bacteria , this would suggest that the microorganisms are passed from one generation to the next , and are evolving with their hosts .", "On the other hand , if the same sort of bacteria can be found in different anglerfish species , this would imply that the bacteria pass from host to host and evolve independently from the fish .", "Genetic data analysis showed that amongst six groups of anglerfishes , one species of bacteria is shared across five groups while another is specific to one type of fish .", "The analyses also revealed that anglerfish and their bacteria are most likely not evolving together .", "This means that the bacteria must make the difficult journey from host to host by persisting in the deep sea , which was confirmed by finding the genetic information of these bacteria in the water near the fish .", "Anglerfish and the bacteria that light up their lure are hard to study , as they live so deep in the ocean .", "In fact , many symbiotic relationships are equally difficult to investigate .", "Examining genetic information can help to give an insight into how hosts and bacteria interact across the tree of life ." ]

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

[ "physics of living systems" ]

[ [ "Over the last decade , hopeful analogies between genetic and electronic circuits have posed the challenge of predicting the output gene expression of a DNA regulatory sequence in much the same way that the output current of an electronic circuit can be predicted from its wiring diagram ( Endy , 2005 ) .", "This challenge has been met with a plethora of theoretical works , including thermodynamic models , which use equilibrium statistical mechanics to calculate the probability of finding transcription factors bound to DNA and to relate this probability to the output rate of mRNA production ( Ackers et al . , 1982; Buchler et al . , 2003; Vilar and Leibler , 2003; Bolouri and Davidson , 2003; Bintu et al . , 2005a; Bintu et al . , 2005b; Sherman and Cohen , 2012 ) .", "Thermodynamic models of bacterial transcription launched a dialogue between theory and experiments that has largely confirmed their predictive power for several operons ( Ackers et al . , 1982; Bakk et al . , 2004; Zeng et al . , 2010; He et al . , 2010; Garcia and Phillips , 2011; Brewster et al . , 2012; Cui et al . , 2013; Brewster et al . , 2014; Sepúlveda et al . , 2016; Razo-Mejia et al . , 2018 ) with a few potential exceptions ( Garcia et al . , 2012; Hammar et al . , 2014 ) .", "Following these successes , thermodynamic models have been widely applied to eukaryotes to describe transcriptional regulation in yeast ( Segal et al . , 2006; Gertz et al . , 2009; Sharon et al . , 2012; Zeigler and Cohen , 2014 ) , human cells ( Giorgetti et al . , 2010 ) , and the fruit fly Drosophila melanogaster ( Jaeger et al . , 2004a; Zinzen et al . , 2006; Segal et al . , 2008; Fakhouri et al . , 2010; Parker et al . , 2011; Kanodia et al . , 2012; White et al . , 2012; Samee et al . , 2015; Sayal et al . , 2016 ) .", "However , two key differences between bacteria and eukaryotes cast doubt on the applicability of thermodynamic models to predict transcriptional regulation in the latter .", "First , in eukaryotes , DNA is tightly packed in nucleosomes and must become accessible in order for transcription factor binding and transcription to occur ( Polach and Widom , 1995; Levine , 2010; Schulze and Wallrath , 2007; Lam et al . , 2008; Raveh-Sadka et al . , 2009; Li et al . , 2011; Fussner et al . , 2011; Bai et al . , 2011; Li et al . , 2014b; Hansen and O'Shea , 2015 ) .", "Second , recent reports have speculated that , unlike in bacteria , the equilibrium framework may be insufficient to account for the energy-expending steps involved in eukaryotic transcriptional regulation , such as histone modifications and nucleosome remodeling , calling for non-equilibrium models of transcriptional regulation ( Kim and O'Shea , 2008; Estrada et al . , 2016; Li et al . , 2018; Park et al . , 2019 ) .", "Recently , various theoretical models have incorporated chromatin accessibility and energy expenditure in theoretical descriptions of eukaryotic transcriptional regulation .", "First , models by Mirny , 2010 , Narula and Igoshin , 2010 , and Marzen et al . , 2013 accounted for chromatin occluding transcription-factor binding by extending thermodynamic models to incorporate the Monod-Wyman-Changeux ( MWC ) model of allostery ( Figure 1A; Monod et al . , 1965 ) .", "This thermodynamic MWC model assumes that chromatin rapidly transitions between accessible and inaccessible states via thermal fluctuations , and that the binding of transcription factors to accessible DNA shifts this equilibrium toward the accessible state .", "Like all thermodynamic models , this model relies on the ‘occupancy hypothesis’ ( Hammar et al . , 2014; Garcia et al . , 2012; Phillips et al . , 2019 ) : the probability pb⁢o⁢u⁢n⁢d of finding RNA polymerase ( RNAP ) bound to the promoter , a quantity that can be easily computed , is linearly related to the rate of mRNA production dmRNAdt , a quantity that can be experimentally measured , such that ( 1 ) dmRNAdt=Rpbound .", "Here , R is the rate of mRNA production when the system is in an RNAP-bound state ( see Appendix section 1 . 1 for a more detailed overview ) .", "Additionally , in all thermodynamic models , the transitions between states are assumed to be much faster than both the rate of transcriptional initiation and changes in transcription factor concentrations .", "This separation of time scales , combined with a lack of energy dissipation in the process of regulation , makes it possible to consider the states to be in equilibrium such that the probability of each state can be computed using its Boltzmann weight ( Garcia et al . , 2007 ) .", "Despite the predictive power of thermodynamic models , eukaryotic transcription may not adhere to the requirements imposed by the thermodynamic framework .", "Indeed , Narula and Igoshin , 2010 , Hammar et al . , 2014 , Estrada et al . , 2016 , Scholes et al . , 2017 , and Li et al . , 2018 have proposed theoretical treatments of transcriptional regulation that maintain the occupancy hypothesis , but make no assumptions about separation of time scales or energy expenditure in the process of regulation .", "When combined with the MWC mechanism of DNA allostery , these models result in a non-equilibrium MWC model ( Figure 1B ) .", "Here , no constraints are imposed on the relative values of the transition rates between states and energy can be dissipated over time .", "To our knowledge , neither the thermodynamic MWC model nor the non-equilibrium MWC model have been tested experimentally in eukaryotic transcriptional regulation .", "Here , we performed a systematic dissection of the predictive power of these MWC models of DNA allostery in the embryonic development of the fruit fly Drosophila melanogaster in the context of the step-like activation of the hunchback gene by the Bicoid activator and the pioneer-like transcription factor Zelda ( Driever et al . , 1989; Nien et al . , 2011; Xu et al . , 2014 ) .", "Specifically , we compared the predictions from these MWC models against dynamical measurements of input Bicoid and Zelda concentrations and output hunchback transcriptional activity .", "Using this approach , we discovered that no thermodynamic or non-equilibrium MWC model featuring the regulation of hunchback by Bicoid and Zelda could describe the transcriptional dynamics of this gene .", "Following recent reports of the regulation of hunchback and snail ( Desponds et al . , 2016; Dufourt et al . , 2018 ) and inspired by discussions of non-equilibrium schemes of transcriptional regulation ( Coulon et al . , 2013; Wong and Gunawardena , 2020 ) , we proposed a model in which Bicoid and Zelda , rather than passively biasing thermal fluctuations of chromatin toward the accessible state , actively assist the overcoming of an energetic barrier to make chromatin accessible through the recruitment of energy-consuming histone modifiers or chromatin remodelers .", "This model ( Figure 1C ) recapitulated all of our experimental observations .", "This interplay between theory and experiment establishes a clear path to identify the molecular steps that make DNA accessible , to systematically test our model of transcription-factor-driven chromatin accessibility , and to make progress toward a predictive understanding of transcriptional regulation in development ." ], [ "During the first 2 hr of embryonic development , the hunchback P2 minimal enhancer ( Margolis et al . , 1995; Driever et al . , 1989; Perry et al . , 2012; Park et al . , 2019 ) is believed to be devoid of significant input signals other than activation by Bicoid and regulation of chromatin accessibility by both Bicoid and Zelda ( Perry et al . , 2012; Xu et al . , 2014; Hannon et al . , 2017 ) .", "As a result , the early regulation of hunchback provides an ideal scaffold for a stringent test of simple theoretical models of eukaryotic transcriptional regulation .", "Our implementation of the thermodynamic MWC model ( Figure 1A ) in the context of hunchback states that in the inaccessible state , neither Bicoid nor Zelda can bind DNA .", "In the accessible state , DNA is unwrapped and the binding sites become accessible to these transcription factors .", "Due to the energetic cost of opening the chromatin ( Δ⁢εchrom ) , the accessible state is less likely to occur than the inaccessible one .", "However , the binding of Bicoid or Zelda can shift the equilibrium toward the accessible state ( Adams and Workman , 1995; Miller and Widom , 2003; Mirny , 2010; Narula and Igoshin , 2010; Marzen et al . , 2013 ) .", "In our model , we assume that all binding sites for a given molecular species have the same binding affinity .", "Relaxing this assumption does not affect any of our conclusions ( as we will see below in Sections 'The thermodynamic MWC model fails to predict activation of hunchback in the absence of Zelda' and 'No thermodynamic model can recapitulate the activation of hunchback by Bicoid alone' ) .", "Bicoid upregulates transcription by recruiting RNAP through a protein-protein interaction characterized by the parameter ωb⁢p .", "We allow cooperative protein-protein interactions between Bicoid molecules , described by ωb .", "However , since to our knowledge there is no evidence of direct interaction between Zelda and any other proteins , we assume no interaction between Zelda and Bicoid , or between Zelda and RNAP .", "In Figure 2A , we illustrate the simplified case of two Bicoid binding sites and one Zelda binding site , plus the corresponding statistical weights of each state given by their Boltzmann factors .", "Note that the actual model utilized throughout this work accounts for at least 6 Bicoid-binding sites and 10 Zelda-binding sites that have been identified within the hunchback P2 enhancer ( Section 'Predicting Zelda binding sites'; Driever and Nüsslein-Volhard , 1988; Driever and Nüsslein-Volhard , 1989; Park et al . , 2019 ) .", "This general model is described in detail in Appendix section 1 . 2 .", "The probability of finding RNAP bound to the promoter is calculated by dividing the sum of all statistical weights featuring RNAP by the sum of the weights corresponding to all possible system states .", "This leads to ( 2 ) pbound= ( 1+z ) nzp ( 1+∑i=1nb ( nbi ) biωbi−1ωbpi ) eΔεchrom/kBT⏟inaccessible state+ ( 1+z ) nz⏟Zelda binding ( 1+p+∑j=0 , 1∑i=1nb ( nbi ) biωbi−1pjωbpij ) ⏟Bicoid and RNAP binding , where b=[B⁢i⁢c⁢o⁢i⁢d]/Kb , z=[Z⁢e⁢l⁢d⁢a]/Kz , and p=[R⁢N⁢A⁢P]/Kp , with [B⁢i⁢c⁢o⁢i⁢d] , [Z⁢e⁢l⁢d⁢a] , and [R⁢N⁢A⁢P] being the concentrations of Bicoid , Zelda , and RNAP , respectively , and Kb , Kz , and Kp their dissociation constants ( see Appendix sections 1 . 1 and 1 . 2 for a detailed derivation ) .", "Given a set of model parameters , plugging pb⁢o⁢u⁢n⁢d into Equation 1 predicts the rate of RNAP loading as a function of Bicoid and Zelda concentrations as shown in Figure 2B .", "Note that in this work , we treat the rate of transcriptional initiation and the rate of RNAP loading interchangeably .", "In order to experimentally test the theoretical model in Figure 2 , it is necessary to measure both the inputs – the concentrations of Bicoid and Zelda – as well as the output rate of RNAP loading .", "Typically , when testing models of transcriptional regulation in bacteria and eukaryotes , input transcription-factor concentrations are assumed to not be modulated in time: regulation is in steady state ( Ackers et al . , 1982; Bakk et al . , 2004; Segal et al . , 2008; Garcia and Phillips , 2011; Sherman and Cohen , 2012; Cui et al . , 2013; Little et al . , 2013; Raveh-Sadka et al . , 2009; Sharon et al . , 2012; Zeigler and Cohen , 2014; Xu et al . , 2015; Sepúlveda et al . , 2016; Estrada et al . , 2016; Razo-Mejia et al . , 2018; Zoller et al . , 2018; Park et al . , 2019 ) .", "However , embryonic development is a highly dynamic process in which the concentrations of transcription factors are constantly changing due to their nuclear import and export dynamics , and due to protein production , diffusion , and degradation ( Edgar and Schubiger , 1986; Edgar et al . , 1987; Jaeger et al . , 2004b; Gregor et al . , 2007b ) .", "As a result , it is necessary to go beyond steady-state assumptions and to predict and measure how the instantaneous , time-varying concentrations of Bicoid and Zelda at each point in space dictate hunchback output transcriptional dynamics .", "In order to quantify the concentration dynamics of Bicoid , we utilized an established Bicoid-eGFP line ( Sections 'Fly Strains' , 'Sample preparation and data collection' and 'Image analysis'; Figure 3A and Appendix 1—figure 3A; Video 1; Gregor et al . , 2007b; Liu et al . , 2013 ) .", "As expected , this line displayed the exponential Bicoid gradient across the length of the embryo ( Appendix section 2 . 1; Appendix 1—figure 3B ) .", "We measured mean Bicoid nuclear concentration dynamics along the anterior-posterior axis of the embryo , as exemplified for two positions in Figure 3A .", "As previously reported ( Gregor et al . , 2007b ) , after anaphase and nuclear envelope formation , the Bicoid nuclear concentration quickly increases as a result of nuclear import .", "These measurements were used as inputs into the theoretical model in Figure 2 .", "Zelda concentration dynamics were measured in a Zelda-sfGFP line ( Sections 'Fly Strains' , 'Sample preparation and data collection' , and 'Image analysis'; Figure 3B; Video 2; Hamm et al . , 2017 ) .", "Consistent with previous results ( Staudt et al . , 2006; Liang et al . , 2008; Dufourt et al . , 2018 ) , the Zelda concentration was spatially uniform along the embryo ( Appendix 1—figure 3 ) .", "Contrasting Figure 3A and B reveals that the overall concentration dynamics of both Bicoid and Zelda are qualitatively comparable .", "As a result of Zelda’s spatial uniformity , we used mean Zelda nuclear concentration dynamics averaged across all nuclei within the field of view to test our model ( Appendix section 2 . 1; Figure 3B ) .", "Given the high reproducibility of the concentration dynamics of Bicoid and Zelda ( Appendix 1—figure 3 ) , we combined measurements from multiple embryos by synchronizing their anaphase in order to create an ‘averaged embryo’ ( Appendix section 2 . 1 ) , an approach that has been repeatedly used to describe protein and transcriptional dynamics in the early fly embryo ( Garcia et al . , 2013; Bothma et al . , 2014; Bothma et al . , 2015; Berrocal et al . , 2018; Lammers et al . , 2020 ) .", "Our model assumes that hunchback output depends on the instantaneous concentration of input transcription factors .", "As a result , at each position along the anterior-posterior axis of the embryo , the combined Bicoid and Zelda concentration dynamics define a trajectory over time along the predicted input-output function surface ( Figure 3C ) .", "The resulting trajectory predicts the rate of RNAP loading as a function of time .", "However , instead of focusing on calculating RNAP loading rate , we used it to compute the number of RNAP molecules actively transcribing hunchback at each point in space and time , a more experimentally accessible quantity ( Section 'The thermodynamic MWC model fails to predict activation of hunchback in the absence of Zelda' ) .", "This quantity can be obtained by accounting for the RNAP elongation rate and the cleavage of nascent RNA upon termination ( Appendix section 2 . 2; Appendix 1—figure 4; Bothma et al . , 2014; Lammers et al . , 2020 ) yielding the predictions shown in Figure 3D .", "Instead of examining the full time-dependent nature of our data , we analyzed two main dynamical features stemming from our prediction of the number of RNAP molecules actively transcribing hunchback: the initial rate of RNAP loading and the transcriptional onset time , to⁢n , defined by the slope of the initial rise in the predicted number of RNAP molecules , and the time after anaphase at which transcription starts as determined by the x-intercept of the linear fit to the initial rise , respectively ( Figure 3D ) .", "Examples of the predictions generated by our theoretical model are shown in Figure 3E and F , where we calculate the initial rate of RNAP loading and to⁢n for different values of the Bicoid dissociation constant Kb .", "This framework for quantitatively investigating dynamic input-output functions in living embryos is a necessary step toward testing the predictions of theoretical models of transcriptional regulation in development .", "In order to test the predictions of the thermodynamic MWC model ( Figure 3E and F ) , we used the MS2 system ( Bertrand et al . , 1998; Garcia et al . , 2013; Lucas et al . , 2013 ) .", "Here , 24 repeats of the MS2 loop are inserted in the 5′ untranslated region of the hunchback P2 reporter ( Garcia et al . , 2013 ) , resulting in the fluorescent labeling of sites of nascent transcript formation ( Figure 4A; Video 3 ) .", "This fluorescence is proportional to the number of RNAP molecules actively transcribing the gene ( Garcia et al . , 2013 ) .", "The experimental mean fluorescence as a function of time measured in a narrow window ( 2 . 5% of the total embryo length , averaged across nuclei in the window ) along the length of the embryo ( Figure 4B ) is in qualitative agreement with the theoretical prediction ( Figure 3D ) .", "To compare theory and experiment , we next obtained the initial RNAP loading rates ( Figure 4C , blue points ) and to⁢n ( Figure 4D , blue points ) from the experimental data ( Appendix section 2 . 3; Appendix 1—figure 5B ) .", "The step-like shape of the RNAP loading rate ( Figure 4C , blue points ) agrees with previous measurements performed on this same reporter construct ( Garcia et al . , 2013 ) .", "The plateaus at the extreme anterior and posterior positions were used to constrain the maximum and minimum theoretically allowed values in the model ( Appendix section 1 . 3 ) .", "With these constraints in place , we attempted to simultaneously fit the thermodynamic MWC model to both the initial rate of RNAP loading and to⁢n .", "For a given set of model parameters , the measurements of Bicoid and Zelda concentration dynamics predicted a corresponding initial rate of RNAP loading and to⁢n ( Figure 3E and F ) .", "The model parameters were then iterated using standard curve-fitting techniques ( Section 'Data analysis' ) until the best fit to the experimental data was achieved ( Figure 4C and D , blue lines ) .", "Although the model accounted for the initial rate of RNAP loading ( Figure 4C , blue line ) , it produced transcriptional onset times that were much lower than those that we experimentally observed ( Appendix 1—figure 6B , purple line ) .", "We hypothesized that this disagreement was due to our model not accounting for mitotic repression , when the transcriptional machinery appears to be silent immediately after cell division ( Shermoen and O'Farrell , 1991; Gottesfeld and Forbes , 1997; Parsons and Spencer , 1997; Garcia et al . , 2013 ) .", "Thus , we modified the thermodynamic MWC model to include a mitotic repression window term , implemented as a time window at the start of the nuclear cycle during which no transcription could occur; the rate of mRNA production is thus given by ( 3 ) dmRNAdt={0if t < tMitRepRpboundif t ≥ tMitRep , where R and pb⁢o⁢u⁢n⁢d are as defined in Equations 1 and 2 , respectively , and tM⁢i⁢t⁢R⁢e⁢p is the mitotic repression time window over which no transcription can take place after anaphase ( Appendix sections 1 . 2 and 3 ) .", "After incorporating mitotic repression , the thermodynamic MWC model successfully fit both the rates of RNAP loading and to⁢n ( Figure 4C and D , blue lines , Appendix 1—figure 6A and B , blue lines ) .", "Given this success , we next challenged the model to perform the simpler task of explaining Bicoid-mediated regulation in the absence of Zelda .", "This scenario corresponds to setting the concentration of Zelda to zero in the models in Appendix section 1 . 2 and Figure 2 .", "In order to test this seemingly simpler model , we repeated our measurements in embryos devoid of Zelda protein ( Video 4 ) .", "These z⁢e⁢l⁢d⁢a- embryos were created by inducing clones of non-functional zelda mutant ( 𝑧𝑒𝑙𝑑𝑎294 ) germ cells in female adults ( Sections 'Fly Strains' , 'Zelda germline clones'; Liang et al . , 2008 ) .", "All embryos from these mothers lack maternally deposited Zelda; female embryos still have a functional copy of zelda from their father , but this copy is not transcribed until after the maternal-to-zygotic transition , during nuclear cycle 14 ( Liang et al . , 2008 ) .", "We confirmed that the absence of Zelda did not have a substantial effect on the spatiotemporal pattern of Bicoid ( Appendix section 4; Xu et al . , 2014 ) .", "While close to 100% of nuclei in wild-type embryos exhibited transcription along the length of the embryo ( Figure 4E , blue; Video 5 ) , measurements in the zelda− background revealed that some nuclei never displayed any transcription during the entire nuclear cycle ( Video 6 ) .", "Specifically , transcription occurred only in the anterior part of the embryo , with transcription disappearing completely in positions posterior to about 40% of the embryo length ( Figure 4E , red ) .", "We confirmed that no visible transcription spots were present in zelda− embryo posteriors by imaging in the posteriors of three zelda− embryos .", "These embryos are not included in our total embryo counts .", "From those positions in the mutant embryos that did exhibit transcription in at least 30% of observed nuclei , we extracted the initial rate of RNAP loading and to⁢n as a function of position .", "Interestingly , these RNAP loading rates were comparable to the corresponding rates in wild-type embryos ( Figure 4C , red points ) .", "However , unlike in the wild-type case ( Figure 4D , blue points ) , to⁢n was not constant in the z⁢e⁢l⁢d⁢a- background .", "Instead , to⁢n became increasingly delayed in more posterior positions until transcription ceased posterior to 40% of the embryo length ( Figure 4D , red points ) .", "Together , these observations indicated that removing Zelda primarily results in a delay of transcription with only negligible effects on the underlying rates of RNAP loading , consistent with previous fixed-embryo experiments ( Nien et al . , 2011; Foo et al . , 2014 ) and with recent live-imaging measurements in which Zelda binding was reduced at specific enhancers ( Dufourt et al . , 2018; Yamada et al . , 2019 ) .", "We speculate that the loss of transcriptionally active nuclei posterior to 40% of the embryo length is a direct result of this delay in to⁢n: by the time that onset would occur in those nuclei , the processes leading to the next mitosis have already started and repressed transcriptional activity .", "Next , we attempted to simultaneously fit the model to the initial rates of RNAP loading and to⁢n in the zelda− mutant background .", "Although the model recapitulated the observed initial RNAP loading rates ( Figure 4C , red line ) , we noticed a discrepancy between the observed and fitted transcriptional onset times of up to ∼5 min ( Figure 4D , red ) .", "While the mutant data exhibited a substantial delay in more posterior nuclei , the model did not produce significant delays ( Figure 4D , red line ) .", "Further , our model could not account for the lack of transcriptional activity posterior to 40% of the embryo length in the zelda− mutant ( Figure 4E , red ) .", "These discrepancies suggest that the thermodynamic MWC model cannot fully describe the transcriptional regulation of the hunchback promoter by Bicoid and Zelda .", "However , the attempted fits in Figure 4C and D correspond to a particular set of model parameters and therefore do not completely rule out the possibility that there exists some parameter set of the thermodynamic MWC model capable of recapitulating the zelda− data .", "In order to determine whether this model is at all capable of accounting for the zelda− transcriptional behavior , we systematically explored how its parameters dictate its predictions .", "To characterize and visualize the limits of our model , we examined two relevant quantitative features of our data .", "First , we defined the offset in the transcriptional onset time as the value of the onset time at the position 20% along the embryo length , the most anterior position studied here ( Figure 5A ) , namely ( 4 ) offset=to⁢n ( x=20% ) where x is the position along the embryo .", "Second , we measured the average transcriptional onset delay along the anterior-posterior axis ( Figure 5A ) .", "This quantity is defined as the area under the curve of to⁢n versus embryo position , from 20% to 37 . 5% along the embryo ( the positions where the zelda− embryos display transcription in at least 30% of nuclei ) , divided by the corresponding distance along the embryo ( 5 ) ⟨onset delay⟩=137 . 5%−20%∫20%37 . 5% ( ton ( x ) −ton ( x=20% ) ) dx , where the offset in the onset time was used to define the zero of this integral ( Appendix section 5 . 1 ) .", "While the offset in to⁢n is similar for both wild-type and zelda− backgrounds ( approximately 4 min ) , the average to⁢n delay corresponding to the wild-type data is close to 0 min , and is different from the value of about 0 . 7 min obtained from measurements in the zelda− background within experimental error ( Figure 5C , ellipses ) .", "Based on Estrada et al . , 2016 and as detailed in Appendix section 5 . 1 , we used an algorithm to efficiently sample the parameter space of the thermodynamic MWC model ( dissociation constants Kb and Kz , protein-protein interaction terms ωb and ωb⁢p , energy to make the DNA accessible Δ⁢εchrom , and length of the mitotic repression window tM⁢i⁢t⁢R⁢e⁢p ) , and to calculate the corresponding to⁢n offset and average to⁢n delay for each parameter set .", "Figure 5B features three specific realizations of this parameter search; for each combination of parameters considered , the predicted to⁢n is calculated and the corresponding to⁢n offset and average to⁢n delay computed .", "Although the wild-type data overlap with the thermodynamic MWC model region , the range of the to⁢n offset and average to⁢n delay predicted by the model ( Figure 5C , green ) did not overlap with that of the zelda− data .", "We concluded that our thermodynamic MWC model is not sufficient to explain the regulation of hunchback by Bicoid and Zelda .", "Since the failure of the thermodynamic MWC model to predict the zelda− data does not necessarily rule out the existence of another thermodynamic model that can account for our experimental measurements , we considered other possible thermodynamic models .", "Conveniently , an arbitrary thermodynamic model featuring nb Bicoid binding sites can be generalized using the mathematical expression ( 6 ) d⁢mRNAd⁢t= ( ∑i=0nbP1 , i⁢R⁢[B⁢i⁢c⁢o⁢i⁢d]i ) pi⁢n⁢a⁢c⁢c+∑r=01∑i=0nbPr , i⁢[B⁢i⁢c⁢o⁢i⁢d]i , where pi⁢n⁢a⁢c⁢c and Pr , i are arbitrary weights describing the states in our generalized thermodynamic model , R is a rate constant that relates promoter occupancy to transcription rate , and the r and i summations refer to the numbers of RNAP and Bicoid molecules bound to the enhancer , respectively ( Appendix section 6 . 1; Bintu et al . , 2005a; Estrada et al . , 2016; Scholes et al . , 2017 ) .", "Note , that this generalized thermodynamic model also included the possibility of Bicoid binding to the inaccessible chromatin state ( Appendix section 6 . 3 ) .", "Although this generalized thermodynamic model contains many more parameters than the thermodynamic MWC model previously considered , we could still systematically explore reasonable values of these parameters and the resulting to⁢n offsets and average to⁢n delays ( Appendix section 6 . 2 ) .", "For added generality , and to account for recent reports suggesting the presence of more than six Bicoid-binding sites in the hunchback minimal enhancer ( Park et al . , 2019 ) , we expanded this model to include up to 12 Bicoid-binding sites .", "The generalized thermodynamic model also failed to explain the zelda− data ( Appendix section 6 . 2; Figure 5C , yellow ) .", "Note that the region of parameter space occupied by the generalized thermodynamic model does not entirely include that of the thermodynamic MWC model due to differences in the constraints of parameter values used in the parameter exploration , as described in Appendix sections 1 . 3 and 6 . 2 .", "Nevertheless , our results strongly suggest that no thermodynamic model of Bicoid-activated hunchback transcription can predict transcriptional onset in the absence of Zelda , casting doubt on the general applicability of these models to transcriptional regulation in development .", "Qualitatively , the reason for the failure of thermodynamic models to predict hunchback transcriptional is revealed by comparing Bicoid and Zelda concentration dynamics to those of the MS2 output signal ( Appendix 1—figure 10 ) .", "The thermodynamic models investigated in this work have assumed that the system responds instantaneously to any changes in input transcription factor concentration .", "As a result , since Bicoid and Zelda are imported into the nucleus by around 3 min into the nuclear cycle ( Figure 3A and B ) , these models always predict that transcription will ensue at approximately that time .", "Thus , thermodynamic models cannot accommodate delays in the to⁢n such as those revealed by the zelda− data ( see Appendix section 6 . 4 for a more detailed explanation ) .", "Rather than further complicating our thermodynamic models with additional molecular players to attempt to describe the data , we instead decided to examine the broader purview of non-equilibrium models to attempt to reach an agreement between theory and experiment .", "Thermodynamic models based on equilibrium statistical mechanics can be seen as limiting cases of more general kinetic models that lie out of equilibrium ( Appendix section 6 . 5; Figure 1B ) .", "Following recent reports ( Estrada et al . , 2016; Li et al . , 2018; Park et al . , 2019 ) that the theoretical description of transcriptional regulation in eukaryotes may call for models rooted in non-equilibrium processes – where the assumptions of separation of time scales and no energy expenditure may break down – we extended our earlier models to produce a non-equilibrium MWC model ( Appendix sections 6 . 5 and 7 . 1; Kim and O'Shea , 2008; Narula and Igoshin , 2010 ) .", "This model , shown for the case of two Bicoid binding sites in Figure 6A , accounts for the dynamics of the MWC mechanism by positing transition rates between the inaccessible and accessible chromatin states , but makes no assumptions about the relative magnitudes of these rates , or about the rates of Bicoid and RNAP binding and unbinding .", "Since this model can operate out of steady state , we calculate the probabilities of each state as a function of time by solving the system of coupled ordinary differential equations ( ODEs ) associated with the system shown in Figure 6A .", "Consistent with prior measurements ( Blythe and Wieschaus , 2016 ) , we assume that chromatin is inaccessible at the start of the nuclear cycle .", "Over time , the system evolves such that the probability of it occupying each state becomes nonzero , making it possible to calculate the fraction of time RNAP is bound to the promoter and , through the occupancy hypothesis , the rate of RNAP loading .", "Mitotic repression is still incorporated using the term tM⁢i⁢t⁢R⁢e⁢p .", "For times t<tM⁢i⁢t⁢R⁢e⁢p , the system can evolve in time but the ensuing transcription rate is fixed at zero .", "We systematically varied the magnitudes of the transition rates and solved the system of ODEs in order to calculate the corresponding to⁢n offset and average to⁢n delay .", "Due to the combinatorial increase of free parameters as more Bicoid-binding sites are included in the model , we could only explore the parameter space for models containing up to five Bicoid-binding sites ( Appendix section 7 . 2; Figure 6B and Appendix 1—figure 9 ) .", "Regardless , none of the non-equilibrium MWC models with up to five Bicoid-binding sites came close to reaching the mutant to⁢n offset and average to⁢n delay ( Figure 6B ) .", "Additionally , an alternative version of this non-equilibrium MWC model where the system could not evolve in time until after the mitotic repression window had elapsed yielded similar conclusions ( see Appendix section 7 . 3 for details ) .", "We conjecture that the observed behavior extends to the biologically relevant case of six or more binding sites .", "Thus , we conclude that the more comprehensive non-equilibrium MWC model still cannot account for the experimental data , motivating an additional reexamination of our assumptions .", "Since even non-equilibrium MWC models incorporating energy expenditure and non-steady behavior could not explain the zelda− data , we further revised the assumptions of our model in an effort to quantitatively predict the regulation of to⁢n along the embryo .", "In accordance with the MWC model of allostery , all of our theoretical treatments so far have posited that the DNA is an allosteric molecule that transitions between open and closed states as a result of thermal fluctuations ( Narula and Igoshin , 2010; Mirny , 2010; Marzen et al . , 2013; Phillips et al . , 2013 ) .", "In the MWC models considered here , the presence of Zelda and Bicoid does not affect the microscopic rates of DNA opening and closing; rather , their binding to open DNA shifts the equilibrium of the DNA conformation toward the accessible state .", "However , recent biochemical work has suggested that Zelda and Bicoid play a more direct role in making chromatin accessible .", "Specifically , Zelda has been implicated in the acetylation of chromatin , a histone modification that renders nucleosomes unstable and increases DNA accessibility ( Li et al . , 2014a; Li and Eisen , 2018 ) .", "Further , Bicoid has been shown to interact with the co-activator dCBP , which possesses histone acetyltransferase activity ( Fu et al . , 2004 ) .", "Additionally , recent studies by Desponds et al . , 2016 in hunchback and by Dufourt et al . , 2018 in snail have proposed the existence of multiple transcriptionally silent steps that the promoter needs to transition through before transcriptional onset .", "These steps could correspond to , for example , the recruitment of histone modifiers , nucleosome remodelers , and the transcriptional machinery ( Li et al . , 2014a; Park et al . , 2019 ) , or to the step-wise unraveling of discrete histone-DNA contacts ( Culkin et al . , 2017 ) .", "Further , Dufourt et al . , 2018 proposed that Zelda plays a role in modulating the number of these steps and their transition rates .", "We therefore proposed a model of transcription-factor-driven chromatin accessibility in which , in order for the DNA to become accessible and transcription to ensue , the system slowly and irreversibly transitions through m transcriptionally silent states ( Appendix section 8 . 1; Figure 7A ) .", "We assume that the transitions between these states are all governed by the same rate constant π .", "Finally , in a stark deviation from the MWC framework , we posit that these transitions can be catalyzed by the presence of Bicoid and Zelda such that ( 7 ) π=cb⁢[B⁢i⁢c⁢o⁢i⁢d]+cz⁢[Z⁢e⁢l⁢d⁢a] .", "Here , π describes the rate ( in units of inverse time ) of each irreversible step , expressed as a sum of rates that depend separately on the concentrations of Bicoid and Zelda , and cb and cz are rate constants that scale the relative contribution of each transcription factor to the overall rate ( see Appendix section 8 . 2 for a more detailed discussion of this choice ) .", "We emphasize that this is only one potential model , and there may exist several other non-equilibrium models capable of describing our data .", "In this model of transcription-factor-driven chromatin accessibility , once the DNA becomes irreversibly accessible after transitioning through the m non-productive states , we assume that , for the rest of the nuclear cycle , the system equilibrates rapidly such that the probability of it occupying any of its possible states is still described by equilibrium statistical mechanics .", "Like in our previous models , transcription only occurs in the RNAP-bound states , obeying the occupancy hypothesis .", "Further , our model assumes that if the transcriptional onset time of a given nucleus exceeds that of the next mitosis , this nucleus will not engage in transcription .", "Thus , this transcription-factor-driven model is an extension of the non-equilibrium MWC model with two crucial differences:", "( i ) we allow for multiple inaccessible states preceding the transcriptionally active state , and", "( ii ) the transitions between these states are actively driven by Bicoid or Zelda .", "Unlike the thermodynamic and non-equilibrium MWC models , this model of transcription-factor-driven chromatin accessibility quantitatively recapitulated the observation that posterior nuclei in zelda− embryos do not engage in transcription as well as the initial rate of RNAP loading , and to⁢n for both the wild-type and zelda− mutant data ( Figure 7B and C ) .", "Additionally , we found that a minimum of m=3 steps was required to sufficiently explain the data ( Appendix section 8 . 3; Appendix 1—figure 14 ) .", "Interestingly , unlike all previously considered models , the model of transcription-factor-driven chromatin accessibility did not require mitotic repression to explain to⁢n ( Appendix sections 3 and 8 . 1 ) .", "Instead , the timing of transcriptional output arose directly from the model’s initial irreversible transitions ( Appendix 1—figure 14 ) , obviating the need for an arbitrary suppression window in the beginning of the nuclear cycle .", "The only substantive disagreement between our theoretical model and the experimental data was that the model predicted that no nuclei should transcribe posterior to 60% of the embryo length , whereas no transcription posterior to 40% was experimentally observed in the embryo ( Figure 7B and C ) .", "Finally , note that this model encompasses a much larger region of parameter space than the thermodynamic and non-equilibrium MWC models and , as expected from the agreement between model and experiment described above , contained both the wild-type and zelda− data points within its domain ( Figure 7D ) ." ], [ "For four decades , thermodynamic models rooted in equilibrium statistical mechanics have constituted the null theoretical model for calculating how the number , placement and affinity of transcription factor binding sites on regulatory DNA dictates gene expression ( Bintu et al . , 2005a; Bintu et al . , 2005b ) .", "Further , the MWC mechanism of allostery has been proposed as an extra layer that allows thermodynamic and more general non-equilibrium models to account for the regulation of chromatin accessibility ( Mirny , 2010; Narula and Igoshin , 2010; Marzen et al . , 2013 ) .", "In this investigation , we tested thermodynamic and non-equilibrium MWC models of chromatin accessibility and transcriptional regulation in the context of hunchback activation in the early embryo of the fruit fly D . melanogaster ( Driever et al . , 1989; Nien et al . , 2011; Xu et al . , 2014 ) .", "While chromatin state ( accessibility , post-translational modifications ) is highly likely to influence transcriptional dynamics of associated promoters , specifically measuring the influence of chromatin state on transcriptional dynamics is challenging because of the sequential relationship between changes in chromatin state and transcriptional regulation .", "However , the hunchback P2 minimal enhancer provides a unique opportunity to dissect the relative contribution of chromatin regulation on transcriptional dynamics because , in the early embryo , chromatin accessibility at hunchback is granted by both Bicoid and Zelda ( Hannon et al . , 2017 ) .", "The degree of hunchback transcriptional activity , however , is regulated directly by Bicoid ( Driever and Nüsslein-Volhard , 1989; Driever et al . , 1989; Struhl et al . , 1989 ) .", "Therefore , while genetic elimination of Zelda function interferes with acquisition of full chromatin accessibility , the hunchback locus retains a measurable degree of accessibility and transcriptional activity stemming from Bicoid function , allowing for a quantitative determination of the contribution of Zelda-dependent chromatin accessibility on the transcriptional dynamics of the locus .", "With these attributes in mind , we constructed a thermodynamic MWC model which , given a set of parameters , predicted an output rate of hunchback transcription as a function of the input Bicoid and Zelda concentrations ( Figure 2B ) .", "In order to test this model , it was necessary to acknowledge that development is not in steady-state , and that both Bicoid and Zelda concentrations change dramatically in space and time ( Figure 3A and B ) .", "As a result , we went beyond widespread steady-state descriptions of development and introduced a novel approach that incorporated transient dynamics of input transcription-factor concentrations in order to predict the instantaneous output transcriptional dynamics of hunchback ( Figure 3C ) .", "Given input dynamics quantified with fluorescent protein fusions to Bicoid and Zelda , we both predicted output transcriptional activity and measured it with an MS2 reporter ( Figures 3D and 4B ) .", "This approach revealed that the thermodynamic MWC model sufficiently predicts the timing of the onset of transcription and the subsequent initial rate of RNAP loading as a function of Bicoid and Zelda concentration .", "However , when confronted with the much simpler case of Bicoid-only regulation in a zelda mutant , the thermodynamic MWC model failed to account for the observations that only a fraction of nuclei along the embryo engaged in transcription , and that the transcriptional onset time of those nuclei that do transcribe was significantly delayed with respect to the wild-type setting ( Figure 4D and E ) .", "Our systematic exploration of all thermodynamic models ( over a reasonable parameter range ) showed that that no thermodynamic model featuring regulation by Bicoid alone could quantitatively recapitulate the measurements performed in the zelda mutant background ( Figure 5C , yellow ) .", "This disagreement could be resolved by invoking an unknown transcription factor that regulates the hunchback reporter in addition to Bicoid .", "However , at the early stages of development analyzed here , such a factor would need to be both maternally provided and patterned in a spatial gradient to produce the observed position-dependent transcriptional onset times .", "To our knowledge , none of the known maternal genes regulate the expression of this hunchback reporter in such a fashion ( Chen et al . , 2012; Perry et al . , 2012; Xu et al . , 2014 ) .", "We conclude that the MWC thermodynamic model cannot accurately predict hunchback transcriptional dynamics .", "To explore non-equilibrium models , we retained the MWC mechanism of chromatin accessibility , but did not demand that the accessible and inaccessible states be in thermal equilibrium .", "Further , we allowed for the process of Bicoid and RNAP binding , as well as their interactions , to consume energy .", "For up to five Bicoid-binding sites , no set of model parameters could quantitatively account for the transcriptional onset features in the zelda mutant background ( Figure 6B ) .", "While we were unable to investigate models with more than five Bicoid-binding sites due to computational complexity ( Estrada et al . , 2016 ) , the substantial distance in parameter space between the mutant data and the investigated models ( Figure 6B ) suggested that a successful model with more than five Bicoid-binding sites would probably operate near the limits of its explanatory power , similar to the conclusions from studies that explored hunchback regulation under the steady-state assumption ( Park et al . , 2019 ) .", "Thus , despite the simplicity and success of the MWC model in predicting the effects of protein allostery in a wide range of biological contexts ( Keymer et al . , 2006; Swem et al . , 2008; Martins and Swain , 2011; Marzen et al . , 2013; Rapp and Yifrach , 2017; Razo-Mejia et al . , 2018; Chure et al . , 2019; Rapp and Yifrach , 2019 ) , the observed transcriptional onset times could not be described by any previously proposed thermodynamic MWC mechanism of chromatin accessibility , or even by a more generic non-equilibrium MWC model in which energy is continuously dissipated ( Tu , 2008; Kim and O'Shea , 2008; Narula and Igoshin , 2010; Estrada et al . , 2016; Wang et al . , 2017 ) .", "Since Zelda is associated with histone acetylation , which is correlated with increased chromatin accessibility ( Li et al . , 2014a; Li and Eisen , 2018 ) , and Bicoid interacts with the co-activator dCBP , which has histone acetyltransferase activity ( Fu et al . , 2004; Fu and Ma , 2005; Park et al . , 2019 ) , we suspect that both Bicoid and Zelda actively drive DNA accessibility .", "A molecular pathway shared by Bicoid and Zelda to render chromatin accessible is consistent with our results , and with recent genome-wide experiments showing that Bicoid can rescue the function of Zelda-dependent enhancers at high enough concentrations ( Hannon et al . , 2017 ) .", "Thus , the binding of Bicoid and Zelda , rather than just biasing the equilibrium toward the open chromatin state as in the MWC mechanism , may trigger a set of molecular events that locks DNA into an accessible state .", "In addition , the promoters of hunchback ( Desponds et al . , 2016 ) and snail ( Dufourt et al . , 2018 ) may transition through a set of intermediate , non-productive states before transcription begins .", "We therefore explored a model in which Bicoid and Zelda catalyze the transition of chromatin into the accessible state via a series of slow , effectively irreversible steps .", "These steps may be interpreted as energy barriers that are overcome through the action of Bicoid and Zelda , consistent with the coupling of these transcription factors to histone modifiers , nucleosome remodelers ( Fu et al . , 2004; Li et al . , 2014a; Li and Eisen , 2018; Park et al . , 2019 ) , and with the step-wise breaking of discrete histone-DNA contacts to unwrap nucleosomal DNA ( Culkin et al . , 2017 ) .", "In this model , once accessible , the chromatin remains in that state and the subsequent activation of hunchback by Bicoid is described by a thermodynamic model .", "Crucially , this transcription-factor-driven chromatin accessibility model successfully replicated all of our experimental observations .", "A minimum of three transcriptionally silent states were necessary to explain our data ( Figure 7D and Appendix 1—figure 14C ) .", "Interestingly , recent work dissecting the transcriptional onset time distribution of snail also suggested the existence of three such intermediate steps in the context of that gene ( Dufourt et al . , 2018 ) .", "Given that , as in hunchback , the removal and addition of Zelda modulates the timing of transcriptional onset of sog and snail ( Dufourt et al . , 2018; Yamada et al . , 2019 ) , we speculate that transcription-factor-driven chromatin accessibility may also be at play in these pathways .", "Thus , taken in consideration with similar works examining the dynamics of transcription onset ( Desponds et al . , 2016; Dufourt et al . , 2018; Fritzsch et al . , 2018; Li et al . , 2018 ) , our results strongly suggest that chromatin state does not fluctuate thermodynamically , but rather progresses through a series of stepwise , transcription-factor-driven transitions into a final RNAP-accessible configuration ( Coulon et al . , 2013 ) .", "Intriguingly , accounting for these intermediate states also obviated the need for the ad hoc imposition of a mitotic repression window ( Appendix sections 3 and 8 . 1 ) , which was required in the thermodynamic MWC model ( Appendix 1—figure 6 ) .", "Our results thus suggest a mechanistic interpretation of the phenomenon of mitotic repression after anaphase , where the promoter must traverse through intermediary transcriptionally silent states before transcriptional onset can occur .", "These clues into the molecular mechanisms of action of Bicoid , Zelda , and their associated modifications to the chromatin landscape pertain to a time scale of a few minutes , a temporal scale that is inaccessible with widespread genome-wide and fixed-tissue approaches .", "Here , we revealed the regulatory action of Bicoid and Zelda by utilizing the dynamic information provided by live imaging to analyze the transient nature of the transcriptional onset time , highlighting the need for descriptions of development that go beyond steady state and acknowledge the highly dynamic changes in transcription-factor concentrations that drive developmental programs .", "While we showed that one model incorporating transcription-factor-driven chromatin accessibility could recapitulate hunchback transcriptional regulation by Bicoid and Zelda , and is consistent with molecular evidence on the modes of action of these transcription factors , other models may have comparable explanatory power .", "In the future , a systematic exploration of different classes of models and their unique predictions will identify measurements that determine which specific model is the most appropriate description of transcriptional regulation in development and how it is implemented at the molecular level .", "While all the analyses in this work relied on mean levels of input concentrations and output transcription levels , detailed studies of single-cell features of transcriptional dynamics such as the distribution of transcriptional onset times ( Narula and Igoshin , 2010; Dufourt et al . , 2018; Fritzsch et al . , 2018 ) could shed light on these chromatin-regulating mechanisms .", "Simultaneous measurement of local transcription-factor concentrations at sites of transcription and of transcriptional initiation with high spatiotemporal resolution , such as afforded by lattice light-sheet microscopy ( Mir et al . , 2018 ) , could provide further information about chromatin accessibility dynamics .", "Finally , different theoretical models may make distinct predictions about the effect of modulating the number , placement , and affinity of Bicoid and Zelda sites ( and even of nucleosomes ) in the hunchback enhancer .", "These models could be tested with future experiments that implement these modulations in reporter constructs .", "In sum , here we engaged in a theory-experiment dialogue to respond to the theoretical challenges of proposing a passive MWC mechanism for chromatin accessibility in eukaryotes ( Mirny , 2010; Narula and Igoshin , 2010; Marzen et al . , 2013 ) ; we also questioned the suitability of thermodynamic models in the context of development ( Estrada et al . , 2016 ) .", "At least regarding the activation of hunchback , and likely similar developmental genes such as snail and sog ( Dufourt et al . , 2018; Yamada et al . , 2019 ) , we speculate that Bicoid and Zelda actively drive chromatin accessibility , possibly through histone acetylation .", "Once chromatin becomes accessible , thermodynamic models can predict hunchback transcription without the need to invoke energy expenditure and non-equilibrium models .", "Regardless of whether we have identified the only possible model of chromatin accessibility and regulation , we have demonstrated that this dialogue between theoretical models and the experimental testing of their predictions at high spatiotemporal resolution is a powerful tool for biological discovery .", "The new insights afforded by this dialogue will undoubtedly refine theoretical descriptions of transcriptional regulation as a further step toward a predictive understanding of cellular decision-making in development ." ], [ "Zelda-binding sites in the hunchback promoter were identified as heptamers scoring three or higher using a Zelda alignment matrix ( Harrison et al . , 2011 ) and the Advanced PASTER entry form online ( http://stormo . wustl . edu/consensus/cgi-bin/Server/Interface/patser . cgi ) ( Hertz et al . , 1990; Hertz and Stormo , 1999 ) .", "PATSER was run with setting ‘Seq . Alphabet and Normalization’ as ‘a:t 3 g:c 2’ to provide the approximate background frequencies as annotated in the Berkeley Drosophila Genome Project ( BDGP ) /Celera Release 1 .", "Reverse complementary sequences were also scored .", "Bicoid nuclear concentration was imaged in embryos from line yw;his2av-mrfp1;bicoidE1 , egfp-bicoid ( Gregor et al . , 2007b ) .", "Similarly , Zelda nuclear concentration was determined by imaging embryos from line sfgfp-zelda;+;his-irfp .", "The sfgfp-zelda transgene was obtained from Hamm et al . , 2017 and the his-iRFP transgene is courtesy of Kenneth Irvine and Yuanwang Pan .", "Transcription from the hunchback promoter was measured by imaging embryos resulting from crossing female virgins yw;HistoneRFP;MCP-NoNLS ( 2 ) with male yw;P2P-MS2-LacZ/cyo;+ ( Garcia et al . , 2013 ) .", "In order to image transcription in embryos lacking maternally deposited Zelda protein , we crossed mother flies whose germline was w , his2av-mrfp1 , zelda ( 294 ) , FRT19A;+;MCP-egfp ( 4F ) /+ obtained through germline clones ( see below ) with fathers carrying the yw;P2P-MS2-LacZ/cyo;+ reporter .", "The 𝑧𝑒𝑙𝑑𝑎294 transgene is courtesy of Christine Rushlow ( Liang et al . , 2008 ) .", "The MCP-egfp ( 4F ) transgene expresses approximately double the amount of MCP than the MCP-egfp ( 2 ) ( Garcia et al . , 2013 ) , ensuring similar levels of MCP in the embryo in all experiments .", "Imaging Bicoid nuclear concentration in embryos lacking maternally deposited Zelda protein was accomplished by replacing the MCP-egfp ( 4F ) transgene described in the previous paragraph with the bicoidE1 , egfp-bicoid transgene used for imaging nuclear Bicoid in a wild-type background .", "We crossed mother flies whose germline was w , his2av-mrfp1 , zelda ( 294 ) , FRT19A;+;bicoidE1 , egfp-bicoid/+ obtained through germline clones ( see below ) with yw fathers .", "In order to generate mother flies containing a germline homozygous null for zelda , we first crossed virgin females of w , his2av-mrfp1 , zelda ( 294 ) , FRT19A/FM7 , y , B;+;MCP-egfp ( 4F ) /TM3 , ser ( or w , his2av-mrfp1 , zelda ( 294 ) , FRT19A;+;bicoidE1 , egfp-bicoid/+ to image nuclear Bicoid ) with males of ovoD , hs-FLP , FRT19A;+;+ ( Liang et al . , 2008 ) .", "The resulting heterozygotic offspring were heat-shocked in order to create maternal germline clones as described in Liang et al . , 2008 .", "The resulting female virgins were crossed with male yw;P2P-MS2-LacZ/cyo;+ ( Garcia et al . , 2013 ) to image transcription or male yw to image nuclear Bicoid concentration .", "Male offspring are null for zygotic zelda .", "Female offspring are heterozygotic for functional zelda , but zygotic zelda is not transcribed until nuclear cycle 14 ( Liang et al . , 2008 ) , which occurs after the analysis in this work .", "All embryos lacking maternally deposited Zelda showed aberrant morphology in nuclear size and shape ( data not shown ) , as previously reported ( Liang et al . , 2008; Staudt et al . , 2006 ) .", "Sample preparation followed procedures described in Bothma et al . , 2014 , Garcia and Gregor , 2018 , and Lammers et al . , 2020 .", "Embryos were collected and mounted in halocarbon oil 27 between a semipermeable membrane ( Lumox film , Starstedt , Germany ) and a coverslip .", "Data collection was performed using a Leica SP8 scanning confocal microscope ( Leica Microsystems , Biberach , Germany ) .", "Imaging settings for the MS2 experiments were the same as in Lammers et al . , 2020 , except the Hybrid Detector ( HyD ) for the His-RFP signal used a spectral window of 556–715 nm .", "The settings for the Bicoid-GFP measurements were the same , except for the following .", "The power setting for the 488 nm line was 10 μW .", "The confocal stack was only 10 slices in this case , rather than 21 , resulting in a spacing of 1 . 11 μm between planes .", "The images were acquired at a time resolution of 30 s , using an image resolution of 512 × 128 pixels .", "The settings for the Zelda-sfGFP measurements were the same as the Bicoid-GFP measurements , except different laser lines were used for the different fluorophores .", "The sf-GFP excitation line was set at 485 nm , using a power setting of 10 μW .", "The His-iRFP excitation line was set at 670 nm .", "The HyD for the His-iRFP signal was set at a 680–800 nm spectral window .", "All specimens were imaged over the duration of nuclear cycle 13 .", "Images were analyzed using custom-written software following the protocol in Garcia et al . , 2013 .", "Briefly , this procedure involved segmenting individual nuclei using the histone signal as a nulear mask , segmenting each transcription spot based on its fluorescence , and calculating the intensity of each MCP-GFP transcriptional spot inside a nucleus as a function of time .", "Additionally , the nuclear protein fluorescences of the Bicoid-GFP and Zelda-sfGFP fly lines were calculated as follows .", "Using the histone-labeled nuclear mask for each individual nucleus , the fluorescence signal within the mask was extracted in xyz , as well as through time .", "For each timepoint , the xy signal was averaged to give an average nuclear fluorescence as a function of z and time .", "This signal was then maximum projected in z , resulting in an average nuclear concentration as a function of time , per single nucleus .", "These single nucleus concentrations were then averaged over anterior-posterior position to create the protein concentrations reported in the main text .", "All fits in the main text were performed by minimizing the least-squares error between the data and the model predictions .", "Unless stated otherwise , error bars reflect standard error of the mean over multiple embryo measurements .", "See Appendix section 2 . 1 for more details on how this was carried out for model predictions ." ] ]

[ "Thermodynamic models of gene regulation can predict transcriptional regulation in bacteria , but in eukaryotes , chromatin accessibility and energy expenditure may call for a different framework .", "Here , we systematically tested the predictive power of models of DNA accessibility based on the Monod-Wyman-Changeux ( MWC ) model of allostery , which posits that chromatin fluctuates between accessible and inaccessible states .", "We dissected the regulatory dynamics of hunchback by the activator Bicoid and the pioneer-like transcription factor Zelda in living Drosophila embryos and showed that no thermodynamic or non-equilibrium MWC model can recapitulate hunchback transcription .", "Therefore , we explored a model where DNA accessibility is not the result of thermal fluctuations but is catalyzed by Bicoid and Zelda , possibly through histone acetylation , and found that this model can predict hunchback dynamics .", "Thus , our theory-experiment dialogue uncovered potential molecular mechanisms of transcriptional regulatory dynamics , a key step toward reaching a predictive understanding of developmental decision-making ." ]

[ "Cells in the brain , liver and skin , as well as many other organs , all contain the same DNA , yet behave in very different ways .", "This is because before a gene can produce its corresponding protein , it must first be transcribed into messenger RNA .", "As an organism grows , the transcription of certain genes is switched on or off by regulatory molecules called transcription factors , which guide cells towards a specific ‘fate’ .", "These molecules bind to specific locations within the regulatory regions of DNA , and for decades biologist have tried to use the arrangement of these sites to predict which proteins a cell will make .", "Theoretical models known as thermodynamic models have been able to successfully predict transcription in bacteria .", "However , this has proved more challenging to do in eukaryotes , such as yeast , fruit flies and humans .", "One of the key differences is that DNA in eukaryotes is typically tightly wound into bundles called nucleosomes , which must be disentangled in order for transcription factors to access the DNA .", "Previous thermodynamic models have suggested that DNA in eukaryotes randomly switches between being in a wound and unwound state .", "The models assume that once unwound , regulatory proteins stabilize the DNA in this form , making it easier for other transcription factors to bind to the DNA .", "Now , Eck , Liu et al . have tested some of these models by studying the transcription of a gene involved in the development of fruit flies .", "The experiments showed that no thermodynamic model could accurately mimic how this gene is regulated in the embryos of fruit flies .", "This led Eck , Liu et al . to identify a model that is better at predicting the activation pattern of this developmental gene .", "In this model , instead of just ‘locking’ DNA into an unwound shape , transcription factors can also actively speed up the unwinding of DNA .", "This improved understanding builds towards the goal of predicting gene regulation , where DNA sequences can be used to tell where and when cell decisions will be made .", "In the future , this could allow the development of new types of therapies that can regulate transcription in different diseases ." ]

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

[ "neuroscience" ]

[ [ "CB1R is one of the most abundant G-protein-coupled receptors ( GPCRs ) in the CNS and endocannabinoid signalling through CB1R is a neuromodulatory system that influences a wide range of brain functions including pain , appetite , mood , and memory ( Soltesz et al . , 2015; Lu and Mackie , 2016 ) .", "Furthermore , CB1R function and dysfunction are implicated in multiple neurodegenerative disorders ( Basavarajappa et al . , 2017 ) .", "Thus , modulation of endocannabinoid pathways is of intense interest as a potential therapeutic target ( Reddy , 2017 ) .", "CB1R is present in both excitatory and inhibitory neurons , and also in astroglia , where it plays important roles in synaptic plasticity and memory ( Han et al . , 2012; Robin et al . , 2018; Busquets-Garcia et al . , 2018 ) .", "In hippocampal neurons , ~80% of CB1R is present in intracellular vesicular clusters in the soma and dendrites ( Leterrier et al . , 2006 ) .", "Strikingly , however , CB1R is not stably surface expressed on somatodendritic plasma membrane .", "Rather , it has a highly polarised axonal surface expression ( Irving et al . , 2000; Coutts et al . , 2001 ) where it acts to attenuate neurotransmitter release ( Katona , 2009 ) and modulate synaptic plasticity ( Lu and Mackie , 2016 ) .", "How this near exclusive axonal surface expression of CB1R is established remains the subject of debate .", "One suggestion is that high rates of endocytosis due to constitutive activity selectively remove CB1Rs from the somatodendritic plasma membrane , resulting in an accumulation at the axonal surface ( Leterrier et al . , 2006 ) .", "These internalised somatodendritic CB1Rs may then be either sorted for degradation or recycled to axons via a transcytotic sorting pathway ( Simon et al . , 2013 ) .", "Alternatively , newly synthesized CB1Rs may be constitutively targeted to lysosomes , but under appropriate circumstances the CB1Rs destined for degradation are retrieved and rerouted to axons ( Rozenfeld and Devi , 2008; Rozenfeld , 2011 ) .", "Surprisingly , a direct role for the 73-residue intracellular C-terminal domain of CB1R ( ctCB1R ) in axonal/somatodendritic trafficking or polarised surface expression has not been identified .", "It has , however , been reported that motifs within ctCB1R are required for receptor desensitization and internalization ( Hsieh et al . , 1999; Jin et al . , 1999 ) ( reviewed by Mackie , 2008 ) .", "Interestingly , there are two putative amphipathic helical domains in ctCB1R ( H8 and H9 [Ahn et al . , 2009] ) .", "H8 has been proposed to play a role in ER assembly and/or exit during biosynthesis ( Ahn et al . , 2010; Stadel et al . , 2011 ) .", "The role of the 21-residue H9 motif is unknown , although analogous regions have been reported to act as a Gαq-binding site in both squid rhodopsin ( Murakami and Kouyama , 2008 ) and bradykinin receptors ( Piserchio et al . , 2005 ) .", "Here we systematically investigated how axonal surface polarity of CB1R arises by tracking newly-synthesised CB1Rs through the secretory pathway to their surface destination .", "We demonstrate that a population of CB1R is preferentially targeted to the axon through the biosynthetic pathway .", "CB1Rs that reach the dendritic membrane are rapidly removed by endocytosis whereas CB1Rs surface expressed on the axonal membrane have a longer residence time .", "We further show that the putative helical domain H9 in ctCB1R plays a key role in CB1R surface expression and endocytosis in hippocampal neurons .", "Taken together our data suggest that CB1R polarity is determined , at least in part , by a novel determinant in the C-terminus of CB1R that contributes to targeted delivery to the axonal compartment and the rapid removal of CB1Rs that reach the somatodendritic membrane ." ], [ "To investigate how CB1R surface polarity is established we used the retention using selective hooks ( RUSH ) system ( Boncompain et al . , 2012 ) and antibody feeding techniques to examine its secretory pathway trafficking and surface expression ( Figure 1 ) .", "We used CB1R tagged at the N-terminus with streptavidin binding peptide ( SBP ) and EGFP ( SBP-EGFP-CB1R ) .", "When co-expressed with a Streptavidin-KDEL ‘hook’ that localises to the lumen of the Endoplasmic Reticulum ( ER ) , SBP-EGFP-CB1R is anchored at the ER membrane .", "The retained SBP-EGFP-CB1R can then be synchronously released by addition of biotin and its trafficking through the secretory pathway and surface expression in both axons and dendrites can be monitored ( Evans et al . , 2017 ) .", "The intracellular ctCB1R is implicated in desensitization and internalization ( reviewed in Mackie , 2008; Stadel et al . , 2011 ) and structural motifs and potential interaction partners have been identified ( Figure 3A; Stadel et al . , 2011 ) .", "However , the role of this region in determining axonal polarity has not been investigated and the function of the H9 structural motif is unknown .", "We therefore wondered whether ctCB1R , and H9 in particular , contribute to CB1R surface polarisation .", "To test the role of the C-terminal domain we initially used CD4 , a single-pass membrane protein that has no intrinsic localisation signals and is normally surface expressed in a non-polarised manner ( Garrido , 2001; Fache et al . , 2004 ) .", "We expressed chimeras of CD4 alone or CD4 fused to either ctCB1RWT or a ctCB1R lacking the H9 domain ( ctCB1RΔH9; Figure 3B ) in hippocampal neurons and examined surface expression by immunostaining ( Figure 3C ) .", "Analysis of the axon to dendrite ratio of surface expression ( the surface polarity index ) revealed that CD4-ctCB1RWT was markedly more axonally polarised than CD4 alone , indicating that ctCB1R may play a role in polarisation despite its lack of defined canonical localisation signals .", "Moreover , although still significantly axonally polarised , the degree of polarisation was significantly lower for CD4-ctCB1RΔH9 , suggesting that H9 may also contribute to this process ( Figure 3D ) .", "We next analysed constitutive endocytosis of CD4-ctCB1RWT and CD4-ctCB1RΔH9 ( Figure 4A , B ) .", "H9 does not determine surface polarity by driving differential constitutive endocytosis from either the dendritic or axonal membrane since there was no difference between the internalisation of CD4-ctCB1RWT or CD4-ctCB1RΔH9 .", "Interestingly , both CD4-ctCB1RWT and CD4-ctCB1RΔH9 were significantly more internalised than CD4 alone in dendrites , but not in axons ( Figure 4A , B ) .", "These results suggest that a domain other than H9 promotes constitutive , but reportedly not activity-dependent ( McDonald et al . , 2007a ) , internalisation in dendrites .", "Importantly , however , because this increase in internalisation in identical between CD4-ctCB1RWT and ctCB1RΔH9 , this endocytic mechanism does not account for the failure of CD4-ctCB1RΔH9 to polarise to the level of CD4-ctCB1RWT .", "To investigate the role of H9 in membrane stability , we next compared surface expression ( Figure 6A ) and endocytosis ( Figure 6B ) of EGFP-CB1RWT and EGFP-CB1RΔH9 in axons and dendrites at steady-state .", "EGFP-CB1RΔH9 displayed lower levels of surface expression ( Figure 6C ) , as well as increased endocytosis ( Figure 6D ) in both axons and dendrites compared to EGFP-CB1RWT , suggesting H9 plays a role in stabilising CB1R at the surface of both axons and dendrites .", "Moreover , similar to our findings using RUSH , there was no difference in surface polarity between EGFP-CB1RWT and EGFP-CB1RΔH9 ( Figure 6E ) .", "These results suggest that , while H9 plays a role in CB1R surface expression and endocytosis , its potential to mediate surface polarity is masked in the full-length receptor .", "Because CB1R surface expression and polarisation has been linked to its activity ( Leterrier et al . , 2006; Ladarre et al . , 2014 ) , we investigated if deleting H9 affects CB1R downstream signalling pathways .", "Measuring the signalling efficiency of EGFP-CB1RΔH9 in neurons would require the complete removal of endogenous CB1R so we expressed EGFP-CB1RWT or EGFP-CB1RΔH9 in HEK293T cells , which do not express endogenous CB1R ( Atwood et al . , 2011 ) and are routinely used to measure activation of the ERK pathway .", "Cells were treated with vehicle ( EtOH ) or stimulated with the selective CB1R agonist ACEA ( arachidonyl-2'-chloroethylamide ) ( Hillard et al . , 1999 ) and blotted for ERK1/2 phosphorylation as a measure of signalling downstream of CB1R ( Daigle et al . , 2008 ) .", "There was no significant difference in ERK1/2 phosphorylation in cells expressing EGFP-CB1RWT or EGFP-CB1RΔH9 under basal conditions in the absence of ACEA .", "However , upon ACEA stimulation , the level of ERK1/2 activation was significantly reduced in EGFP-CB1RΔH9-transfected cells compared to EGFP-CB1RWT-transfected cells expressing equivalent amounts of receptor ( Figure 7A–C ) .", "Because the ΔH9 mutant is more internalised than the wild-type in neurons , we examined whether the deficiency in ERK1/2 phosphorylation was due to a similarly reduced surface expression in HEK283T cells .", "However , EGFP-CB1RWT and EGFP-CB1RΔH9 were surface expressed at equivalent levels in HEK293T cells , as determined by surface biotinylation experiments ( Figure 7D–E ) , suggesting the ΔH9 mutant is deficient in its ability to activate downstream signalling pathways .", "We next monitored ACEA-induced internalisation of EGFP-CB1RWT and EGFP-CB1RΔH9 in axons of hippocampal neurons ( Figure 7F ) .", "ACEA-induced internalisation of EGFP-CB1RΔH9 was significantly greater than that observed for EGFP-CB1RWT ( Figure 7F , G ) .", "Taken together , these data indicate that CB1RΔH9 is less stable at the axonal surface under basal conditions and that it is more susceptible to agonist-induced internalisation .", "Our data thus far indicate that ctCB1R , and the H9 domain in particular , can mediate surface polarity of a CD4 chimera ( Figure 3 ) , and promote polarised surface delivery of CB1R ( Figure 5 ) .", "In contrast , deletion of H9 has no effect on CB1R surface polarity at steady-state ( Figure 6 ) .", "However , deletion of H9 does have a striking effect on the surface stability of CB1R with CB1RΔH9 being less surface expressed in both axons and dendrites and displaying increased endocytosis ( Figures 4 and 6 ) .", "Furthermore , CB1R ΔH9 is more responsive to agonist-induced internalisation ( Figure 7 ) .", "We therefore wondered whether the difference between the CD4 chimeras and the full-length receptor and between surface + endocytosed and surface polarity may be attributable to the agonist binding capability of the full-length receptor .", "To test this we used the CB1R-specific inverse agonist AM281 to prevent the receptor entering an active conformation and which has previously been shown to increase somatodendritic surface expression similar to treatment with an endocytosis inhibitor ( Leterrier et al . , 2006 ) .", "We reasoned that AM281 might reveal a difference in surface polarity between EGFP-CB1RWT and EGFP-CB1RΔH9 , like that observed with the CD4 chimeras and in surface + endocytosed polarity .", "In hippocampal neurons treated with the DMSO control both EGFP-CB1RWT and EGFP-CB1RΔH9 displayed similar levels of surface polarity ( Figure 8B ) .", "In the presence of AM281 , however , EGFP-CB1RΔH9 had significantly reduced surface polarity compared EGFP-CB1RWT ( Figure 8B ) due to a significantly increased amount of dendritic surface expression ( Figure 8C ) .", "These results indicate that in the absence of constitutive activity of the receptor , H9 plays a role in mediating CB1R surface polarity .", "Furthermore , these data suggest that the increased internalisation observed in dendrites with H9 deletion may be mediated by the presence of endogenous agonist and reaffirm the importance of the neuronal milieu on CB1R trafficking ." ], [ "The mechanism behind polarised membrane trafficking in neurons is a fundamental question and our data suggest a sorting mechanism at the level of the secretory pathway that preferentially targets CB1R to the axon .", "Since dendritic and axonal cargo are synthesized in the somatodendritic compartment , selective sorting to the correct domain is crucial .", "While several sorting signals and adaptors have been described for dendritic cargo , the mechanisms behind selective sorting to axons are less well known ( Lasiecka and Winckler , 2011; Bentley and Banker , 2016 ) .", "For example , a recent study in C . elegans has suggested that sorting of cargos to axons or dendrites depends on binding to different types of clathrin-associated adaptor proteins ( AP ) ; axonal cargo bind to AP-3 whereas dendritic cargo bind to AP-1 ( Li et al . , 2016 ) .", "Interestingly , AP-3 binding has been associated with CB1R trafficking to the lysosome in the dendritic compartment ( Rozenfeld and Devi , 2008 ) .", "One possibility is that H9 may modulate CB1R binding to AP-3 , allowing for preferential delivery to axons and sorting to dendritic lysosomes , causing an decrease in dendritic membrane CB1R .", "More studies are needed to examine the possibility of H9 influencing AP-3 and CB1R interaction .", "We used time-resolved RUSH experiments to investigate the initiation of CB1R polarity .", "We measured the transit through the secretory pathway and incorporation into , and passage through , the highly organised axon initial segment ( AIS ) that acts as a ‘gate-keeper’ for proteins entering the axonal compartment .", "Our data show that CB1R polarisation was initiated in the first 90 min since they were directly targeted to , and surface expressed within , proximal axonal regions .", "Immunocytochemistry in brain sections using immunogold electron microscopy ( Katona et al . , 1999; Nyíri et al . , 2005 ) or STORM super-resolution imaging ( Dudok et al . , 2015 ) detect CB1R predominantly at the presynaptic terminal , consistent with a disto-proximal gradient of expression at the axonal plasma membrane ( Simon et al . , 2013 ) .", "Therefore , given the highly branched morphology of typical CB1R expressing neurons , correct axonal polarization likely requires specific distal targeting mechanisms in addition to the processes we describe using time-resolved RUSH experiments .", "CB1Rs are highly mobile and diffuse rapidly in the plasma membrane ( Mikasova et al . , 2008; Oddi et al . , 2012 ) , so the accumulation of surface CB1R we observe may be followed by lateral diffusion and ‘capture’ of surface CB1R at presynaptic sites , analogous to the diffusion and retention models proposed for AMPARs and GABAARs at the postsynaptic membrane ( Hastings and Man , 2018; Kneussel and Hausrat , 2016 ) .", "Another , non-exclusive possibility is that CB1R-containing vesicles originating from the secretory system and/or endosomal system traffic to distal sites inside the axon ( Lasiecka and Winckler , 2011 ) .", "Indeed , it has been reported that CB1R in somatodendritic endosomes can be rerouted and trafficked to distal axonal surfaces ( Simon et al . , 2013 ) .", "Our observation that intracellular CB1R is present at least 100 µm along the axon before it appears at the surface supports the concept of a rapid and direct trafficking of CB1R-containing secretory vesicles to more distal areas of the axon , although more detailed tracking of these secretory vesicles to presynaptic boutons would be required to confirm this .", "Overall , we interpret our data to suggest that arrival at , and progression through , the AIS constitutes the initial phase of CB1R axonal polarisation .", "Once within the axonal compartment trafficking to more distal locations and to presynaptic sites is then mediated by additional mechanisms that probably include both intracellular transport and lateral diffusion and trapping .", "Our data suggest that H9 stabilises CB1R at the membrane , regardless of compartment .", "While the H8 domain is highly conserved in GPCRs , structural domains analogous to H9 have only been reported in squid rhodopsin ( Murakami and Kouyama , 2008 ) and the bradykinin receptor ( Piserchio et al . , 2005 ) .", "NMR and circular dichroism studies suggest that H9 , like H8 , is an amphipathic α-helix , associating with the lipid bilayer via a cluster of hydrophobic residues on the non-polar face of the helix ( Ahn et al . , 2009 ) .", "Furthermore , CB1R has been shown to be palmitoylated just downstream of H8 at C416 , which affects its membrane association and G-protein coupling ( Oddi et al . , 2012; Oddi et al . , 2018 ) .", "H9 also contains a cysteine residue , raising the possibility that post-translational modifications such as palmitoylation , prenylation , or farnesylation at this site could modulate membrane association .", "Since our data suggest that H9 stabilises CB1R at the membrane , it is possible that the membrane association of H9 could mask internalisation signals or interacting motifs .", "Consistent with this possibility , ctCB1R interacting proteins regulate CB1R endocytosis .", "For example , SGIP1 , a protein linked to clathrin-mediated endocytosis , binds at an as yet undefined site on ctCB1R downstream of H8 , preventing internalisation of activated CB1R ( Hájková et al . , 2016 ) .", "Similarly , cannabinoid receptor interacting protein 1a ( CRIP1a ) , which interacts directly with a motif in the last 9 C-terminal amino acids ( Niehaus et al . , 2007 ) , reduces constitutive CB1R internalisation ( Mascia et al . , 2017 ) by competing with β-Arrestin binding ( Blume et al . , 2017 ) .", "Therefore , it is possible that H9 mediates the interactions between CB1R and SGIP1 and/or selectively promotes β-Arrestin rather than CRIP1a binding .", "Further studies examining the interaction between CB1RWT , CB1RΔH9 , CRIP1a , β-Arrestin1/2 , and SGIP1 are needed to examine the mechanism by which H9 stabilises surface CB1R .", "Given the increased interest in CB1R as a clinical target , understanding the fundamental cell biology and trafficking behaviour of CB1R is an increasingly active and important area of research .", "Taken together , our results reveal that the C-terminal domain , and H9 in particular , play important roles in trafficking of CB1R .", "These findings provide important insight into the mechanisms of CB1R polarity and highlight H9 as an important regulator of CB1R endocytosis and surface expression ." ], [ "A previously characterised rat CB1R construct in which the first 25 N-terminal amino acids were omitted to avoid possible cleavage of the SBP-EGFP tag ( McDonald et al . , 2007b; Nordström and Andersson , 2006 ) was used as a template for sub-cloning into pcDNA3 . 1 .", "This construct displays normal plasma membrane trafficking ( McDonald et al . , 2007b; Hebert-Chatelain et al . , 2016 ) and removes the region reported to constitute a mitochondrial targeting motif ( Hebert-Chatelain et al . , 2016 ) .", "Helix 9 ( residues 440–460 ) was removed by site-directed mutagenesis .", "These WT and ΔH9 constructs were subsequently used as a template to clone into the RUSH vector system ( interleukin-2 signal peptide followed by SBP and EGFP N-terminal tags ) as previously described ( Evans et al . , 2017; Boncompain and Perez , 2013 ) .", "Non-ER-retained SBP-EGFP-tagged versions were obtained by re-cloning these inserts from the RUSH vector into pcDNA3 . 1 ( pcDNA-SPIl-2-SBP-EGFP-CB1R ) .", "The SBP tag was deleted for surface biotinylation experiments by site-directed mutagenesis ( pcDNA-SPIl-2-EGFP-CB1R ) .", "Chimeric CD4-ctCB1R WT and ΔH9 were generated by overlap extension PCR followed by cloning into a plasmid expressing CD4 lacking its own C-terminus ( Garrido , 2001 ) .", "Chicken anti-GFP was from Abcam ( ab13970 ) ; mouse anti-Ankyrin-G was from NeuroMab ( clone N106/36 ) ; rabbit anti-MAP2 was from Synaptic Systems ( 188 003 ) ; mouse anti-CD4 was from BioLegend ( clone OKT4 ) ; rat anti-GFP was from ChromoTek ( 3H9 ) ; anti-phosphoERK ( M7802 ) , and anti-non-phosphoERK ( M3807 ) were from Sigma; mouse anti-GAPDH ( 6C5 ab8245 ) was from Abcam .", "All fluorescent secondaries were from Jackson Immunoresearch Laboratories and HRP conjugated secondaries were from Sigma .", "ACEA and AM281 were from Tocris bio-techne .", "Dissociated hippocampal cultures were prepared from E17-E18 Wistar rats as previously described ( Martin and Henley , 2004 ) .", "Glass coverslips were coated in poly-D-lysine or poly-L-lysine ( 1 mg/mL , Sigma ) in borate buffer ( 10 mM borax , 50 mM boric acid ) overnight and washed in water .", "Dissociated hippocampal cells were plated at different densities in plating medium ( Neurobasal , Gibco supplemented with 10% horse serum , Sigma; 2 mM GlutaMAX , Gibco; and either GS21 , GlobalStem , or B27 , Thermo Fisher ) which was changed to feeding medium ( Neurobasal supplemented with 1 . 2 mM GlutaMAX and GS21 or B27 ) after 24 hr .", "For RUSH experiments , cells were plated and fed in media containing GS21 instead of B27 because it does not contain biotin .", "Cells were incubated at 37°C and 5% CO2 for up to 2 weeks .", "Animal care and procedures were carried out in accordance with UK Home Office and University of Bristol guidelines .", "Transfection of neuronal cultures was carried out at DIV 12 using Lipofectamine2000 ( Invitrogen ) according to the manufacturer’s instructions with minor modifications .", "Cells were left for 20–48 hr before fixation .", "HEK293T cells ( ECACC ) were passaged and maintained in complete DMEM ( DMEM + 10% FBS + 2 mM L-Glutamine ) .", "HEK293T cells were regularly treated with ciprofloxacin ( 10 µg/mL ) to prevent mycoplasma contamination .", "HEK293T cells were transfected with SBP-EGFP-CB1RWT , SBP-EGFP-CB1RΔH9 , or empty pcDNA3 . 1 and left for 24 hr .", "The cells were serum-starved overnight and then treated with 1 μM ACEA or 0 . 01% EtOH for 5 min before being lysed in lysis buffer ( 50 mM Tris-HCl; 150 mM NaCl; 1% CHAPS , ThermoFisher Scientific; protease inhibitors , Roche ) with phosphatase inhibitors ( Pierce , ThermoFisher Scientific ) .", "SDS-PAGE and Western blotting procedures were carried out according to standard protocols .", "HEK293T cells were transfected with EGFP-CB1RWT or EGFP-CB1RΔH9 for 48 hr , then cooled to 4°C on ice and washed 3 times in ice-cold PBS .", "0 . 3 mg/mL of EZ-Link Sulfo-NHS-SS-Biotin in PBS was added for 10 min , then the cells were washed 3 times in PBS .", "50 mM NH4Cl in PBS was added for 2 min to quench any remaining unreacted biotin , and the cells were washed another 3 times in PBS before being lysed in lysis buffer .", "Biotinylated surface proteins were isolated using streptavidin coated agarose beads according to standard protocols .", "To measure surface staining , cultured neurons were cooled at room temperature for 5–10 min , then incubated with the appropriate antibody ( chicken anti-GFP or mouse anti-CD4 ) in conditioned media for 10–20 min at RT .", "The neurons were washed multiple times in PBS before fixation .", "For agonist and inverse agonist experiments , the neurons were treated with 5 μM ACEA ( in EtOH ) or vehicle control ( 0 . 1% EtOH ) for 3 hr or 10 μM AM281 ( in DMSO ) or vehicle control ( 0 . 2% DMSO ) for 3 hr in conditioned media at 37°C and 5% CO2 , and then subsequently surface stained .", "To measure endocytosed receptors , neurons were fed with the appropriate antibody ( chicken anti-GFP or mouse anti-CD4 ) for 2 hr in conditioned media at 37°C and 5% CO2 .", "Neurons were washed several times in PBS and then surface antibody was stripped by two quick washes with ice-cold pH 2 . 5 PBS ( anti-GFP ) or a 4 min incubation with 0 . 5M NaCl and 0 . 2M acetic acid ( anti-CD4 ) followed by several washes in PBS before fixation .", "Neurons were transfected with RUSH constructs at DIV 12 for no longer than 24 hr to prevent ER stress resulting from accumulation of unreleased receptors .", "Neurons were incubated in conditioned media containing D-biotin ( 40 μM , Sigma ) and chicken anti-GFP ( 1:1 , 000 ) for different lengths of time at 37°C and 5% CO2 .", "The 0 min timepoint was only incubated with chicken anti-GFP without biotin for 60 min .", "For the O/N timepoint , neurons were incubated in 40 μM D-biotin immediately following transfection and then left overnight at 37°C and 5% CO2 before being incubated with biotin and chicken anti-GFP for 60 min to label surface CB1R .", "Every independent experiment included a 60 min timepoint to which values were normalised and a 0 min control .", "Following biotin treatment , neurons were washed several times in PBS and cooled to 4°C to prevent further internalisation .", "They were then live labelled with 647-labelled anti-chicken in conditioned media for 15 min at 4°C before being fixed and permeabilised and stained with Cy3-labelled anti-chicken .", "In the text , ‘surface’ thus refers to 647 fluorescence acquisition , whereas ‘surface + endocytosed’ refers to Cy3 fluorescence acquisition .", "Cultured neurons were fixed in 4% formaldehyde in PBS for 12 min , then washed 3x in PBS , 1x in 100 mM Glycine in PBS , and 3x in PBS .", "The neurons were then blocked and permeabilised in PBS + 3% BSA + 0 . 1% Triton X-100 before being incubated in fluorescent secondary ( 1:400 ) in PBS + 3% BSA .", "Subsequently , the neurons were re-incubated in primary antibody ( anti-GFP or anti-CD4 ) to measure total levels of expression and stained with either anti-MAP2 ( dendritic marker ) or anti-Ankyrin-G ( axonal initial segment marker ) in PBS + 3% BSA .", "The neurons were then washed several times in PBS and mounted onto glass slides using Fluoromount-G ( ThermoFisher Scientific ) .", "Images were acquired using either a Leica SPE single channel confocal laser scanning microscope or a Leica SP8 AOBS confocal laser scanning microscope ( Wolfson Bioimaging Facility , University of Bristol ) .", "All settings were kept the same within experiments .", "Neurons used for data acquisition were selected only on their total staining .", "All quantification was performed using FIJI ( ImageJ ) software .", "Based on previous experiments , at least five cells were analysed per experiment , and at least three independent experiments ( i . e . on different neuronal cultures on different days ) were performed .", "Images were max projected , and regions of interest ( ROIs ) of approximately similar lengths were drawn around axons and 3–4 proximal and secondary dendrites based on the total channel only .", "Axons were defined either as processes whose initial segment was positive for Ankyrin-G or as processes negative for MAP2 .", "The mean fluorescence was measured for each channel and the dendritic values were averaged .", "‘Surface’ or ‘endocytosed’ mean fluorescence values were normalised to the ‘total’ mean fluorescence value for each ROI to account for varying levels of expression of transfected constructs .", "These values were then normalised to the axon value of the control ( WT or CD4 ) .", "For drug treatments , surface values were normalised to their respective vehicle treated controls and sampled at the same time-point ( i . e . WT + drug was normalised to WT + vehicle and ΔH9 + drug was normalised to ΔH9 + vehicle ) to account for possible differences in steady-state surface expression between the constructs and/or constitutive internalisation .", "Because of the change in total mean fluorescence in axons throughout the different conditions , the above image analysis was slightly modified for RUSH experiments .", "In these experiments , neurites were traced using NeuronJ so that only the mean fluorescence of exactly the first 50 μm of the axons and 30–40 μm of 2–4 primary dendrites for each channel was measured .", "All ‘surface’ and ‘surface + endocytosed’ values ( of both axons and dendrites ) were normalised to the average total dendritic value for each neuron .", "Axon total mean fluorescence was also normalised to the average total dendritic value within each cell .", "All values were then normalised to the WT 60 min axon value within each experiment .", "In a slightly smaller subset of RUSHed neurons , axons were traced for ~100 µm using NeuronJ .", "Line plots were generated and the mean fluorescence was averaged in 10 µm segments .", "The averages for each 10 µm segment from each cell were normalised first to the dendritic total value , then to the axonal 60 min value of the first 50 µm .", "Polarity indices ( A/D ratio ) were calculated by dividing the axonal mean fluorescence value by the average dendritic mean fluorescence value .", "The scalebar for all images represents 20 μm .", "All statistics were performed using GraphPad Prism .", "The ROUT method was used to identify outliers for all parameters measured before normalising to control .", "Neurons were removed from analysis if any one parameter was found to be an outlier .", "As is the convention in the field ( Leterrier et al . , 2006; Coutts et al . , 2001; Simon et al . , 2013; Evans et al . , 2017; McDonald et al . , 2007b; Leterrier et al . , 2017 ) , ‘N’ denotes the number of separate neuronal cultures prepared from litters of pups from separate dams and ‘n’ denotes the total number of neurons across the separate cultures assessed .", "To determine statistical significance between two groups , a D’Agostino and Pearson normality test was performed .", "Unpaired t-tests were performed on data that passed the normality test whereas the Mann-Whitney test was used if it did not .", "One- or Two-way ANOVAs with Tukey’s or Sidak’s post hoc test were used to determine statistical significance between more than two groups depending on the comparisons required .", "*p ≤ 0 . 05 , **p ≤ 0 . 01 , ***p ≤ 0 . 001 , ****p ≤ 0 . 0001 .", "All data are presented as mean ± SEM ." ] ]

[ "Cannabinoid type one receptor ( CB1R ) is only stably surface expressed in axons , where it downregulates neurotransmitter release .", "How this tightly regulated axonal surface polarity is established and maintained is unclear .", "To address this question , we used time-resolved imaging to determine the trafficking of CB1R from biosynthesis to mature polarised localisation in cultured rat hippocampal neurons .", "We show that the secretory pathway delivery of CB1R is axonally biased and that surface expressed CB1R is more stable in axons than in dendrites .", "This dual mechanism is mediated by the CB1R C-terminus and involves the Helix 9 ( H9 ) domain .", "Removal of the H9 domain increases secretory pathway delivery to dendrites and decreases surface stability .", "Furthermore , CB1RΔH9 is more sensitive to agonist-induced internalisation and less efficient at downstream signalling than CB1RWT .", "Together , these results shed new light on how polarity of CB1R is mediated and indicate that the C-terminal H9 domain plays key roles in this process ." ]

[ "The brain contains around 100 billion neurons that are in constant communication with one another .", "Each consists of a cell body , plus two components specialized for exchanging information .", "These are the axon , which delivers information , and the dendrites , which receive it .", "This exchange takes place at contact points between neurons called synapses .", "To send a message , a neuron releases chemicals called neurotransmitters from its axon terminals into the synapse .", "The neurotransmitters cross the synapse and bind to receptor proteins on the dendrites of another neuron .", "In doing so , they pass on the message .", "Cannabinoid type 1 receptors ( CB1Rs ) help control the flow of information at synapses .", "They do this by binding neurotransmitters called endocannabinoids , which are unusual among neurotransmitters .", "Rather than sending messages from axons to dendrites , endocannabinoids send them in the opposite direction .", "Thus , it is dendrites that release endocannabinoids , which then bind to CB1Rs in axon terminals .", "This backwards , or 'retrograde' , signalling dampens the release of other neurotransmitters .", "This slows down brain activity , and gives rise to the 'mellow' sensation that recreational cannabis users often describe .", "Like most other proteins , CB1Rs are built inside the cell body .", "So , how do these receptors end up in the axon terminals where they are needed ?", "Are they initially sent to both axons and dendrites , with the CB1Rs that travel to dendrites being rerouted back to axons ?", "Or do the receptors travel directly to the axon itself ?", "Fletcher-Jones et al . tracked newly made CB1Rs in rat neurons growing in a dish .", "The results revealed that the receptors go directly to the axon , before moving on to the axon terminals .", "A specific region of the CB1R protein is crucial for sending the receptors to the axon , and for ensuring that they do not get diverted to the dendrite surface .", "This region stabilizes CB1Rs at the axon surface , and helps to make the receptors available to bind endocannabinoids .", "CB1Rs also respond to medical marijuana , a topic that continues to generate interest as well as controversy .", "Activating CB1Rs could help treat a wide range of diseases , such as chronic pain , epilepsy and multiple sclerosis .", "Future studies should build on our understanding of CB1Rs to explore and optimize new therapeutic approaches ." ]

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

[ "plant biology", "genetics and genomics" ]

[ [ "Most parasites have a restricted host range and are often unable to exploit even closely related hosts ( Thompson , 2005; Poulin and Keeney , 2014 ) .", "Compared to necrotrophs that reproduce on dead plant material , obligate biotrophic parasites can only reproduce on living tissue , and thus are intimately associated with their hosts ( Thines , 2014 ) .", "This might be expected to result in host specialization .", "The adaptive evolution of , for example , new effectors that enable more efficient exploitation of one host species , increases the risk of detection in other host species by triggering their immune system ( Martin and Kamoun , 2012 ) .", "Due to this trade-off , the saying ‘Jack of all trades , master of none’ is especially true for obligate biotrophic parasites , because natural selection that maximises parasite fitness on one particular host species might lead to specialisation and reduced host range ( e . g . , Dong et al . , 2014 ) .", "Yet there are generalist biotrophic parasites that appear to have overcome this evolutionary dilemma and show virulence on diverse hosts .", "Some ‘generalist’ parasite species have solved the dilemma by evolving multiple specialised races , each of which infect different hosts .", "For example , the eukaryotic oomycete order Albuginales consists entirely of obligate biotrophic pathogens that cause disease on a broad range of plant hosts ( Biga , 1955; Choi and Priest , 1995; Walker and Priest , 2007 ) .", "Its largest genus , Albugo , comprises ∼50 ( usually ) specialist pathogens ( Choi et al . , 2009; Thines et al . , 2009; Ploch et al . , 2010; Choi et al . , 2011 ) , yet Albugo candida ( Pers . ) Roussel can infect over 200 species of plants in 63 genera from the families of Brassicaceae , Cleomaceae and Capparaceae ( Saharan and Verma , 1992; Choi et al . , 2009 ) .", "A . candida infections are the causal agent of ‘white blister rust’ disease resulting in 9–60% losses on economically important oilseed and vegetable Brassica crops .", "( Harper and Pittma , 1974; Barbetti and Carter , 1986; Saharan and Verma , 1992; Meena et al . , 2002 ) .", "A . candida consists of different physiological races that each usually show high host specificity ( Hiura , 1930; Pound and Williams , 1963; Petrie , 1988 ) .", "∼24 races of A . candida have been defined , based primarily on their host range ( Saharan and Verma , 1992 ) .", "A . candida is a diploid organism that reproduces both asexually and sexually ( Holub et al . 1995 ) , but the relative importance of both reproductive modes is not well established .", "During asexual reproduction , diploid zoospores are formed in a special propagule ( the zoosporangium ) on a thallus of hyphae beneath the leaf epidermis .", "White blisters comprising large numbers of dehydrated sporangia eventually rupture the epidermis to release inoculum for dispersal .", "When reproducing sexually , fertilization between two isolates results in non-motile diploid thick-walled oospores that can resist extreme temperatures and desiccation .", "Although the relative importance of different mechanisms of reproduction in the Albugo life cycle remains poorly understood , clonal reproduction enables rapid population expansion , especially in genetically uniform crop monocultures .", "A . candida is considered a single species despite the fact it comprises several specialised physiological races that colonize different host plants ( cf . Drès and Mallet , 2002 ) .", "According to evolutionary and population genetic theory , adaptations and trade-offs associated with host-specialisation combined with strong population structuring can result in adaptive radiation and speciation ( Abbott et al . , 2013; Stukenbrock , 2013 ) .", "Conceivably , A . candida is an adaptive radiation ‘in progress’ , and the broad host range is realised by ongoing specialisation of independent races that are on the road to speciation ( Drès and Mallet , 2002 ) .", "This raises the important question; does parasite specialization inevitably lead to speciation ?", "Albugo spp .", "infection strongly suppresses host innate immunity and Albugo spp .", "are unique compared to other microbial plant pathogens in enhancing host susceptibility to secondary infection by otherwise avirulent pathogens , including downy mildews ( Cooper et al . , 2008 ) .", "It has been suggested that enhanced susceptibility imposed by Albugo might accelerate adaptation of other pathogen species to an Albugo-susceptible host ( Thines , 2014 ) .", "However , no evolutionary rationale has been put forward to explain why it might be adaptive for Albugo sp .", "to render its hosts so susceptible to other pathogens that could compete for access to the same resources ( Cooper et al . , 2008 ) .", "Arguably , suppression of host innate immunity could facilitate cohabitation of distinct physiological races and thus may enable genetic exchange between them .", "Introgression , here defined as the introduction by recombination of syntenic nucleotide variation from a parental donor race into the genome of a recipient race ( Hedrick , 2013 ) , could slow down genetic divergence , and hence , retard speciation .", "On the other hand , introgression between races that are well-adapted to exploit different host plants could be maladaptive and strongly selected against because hybrids will inherit effector alleles derived from both parental races .", "Given that immune recognition of even a single effector is sufficient to trigger the immune response and stop an infection , hybrids that possess an expanded repertoire of effector alleles are likely to have a strong fitness disadvantage on most potential host plants .", "Much of the ecology and evolution of A . candida remains unknown , but with its many specialized races and a broad host range , this ‘generalist’ plant pathogen is a fascinating study organism .", "The questions we addressed in the present study are: ( 1 ) Are the distinct physiological A . candida races genetically isolated and ‘on the road to speciation’ ?", "( 2 ) Does suppression of host innate immunity enable cohabitation and growth of races with non-overlapping host ranges ?", "To answer these questions , we generated genome sequence assemblies of five isolates that were collected from four host species ( Brassica oleracea , Brassica juncea , Capsella bursa-pastoris , and Arabidopsis thaliana ) .", "We sequenced one isolate of one race , and two isolates of each of two additional races .", "We show that these races are not genetically isolated despite having non-overlapping host ranges .", "Recombination analysis shows there is widespread genetic exchange between A . candida races , and that hybridisation leading to introgression has occurred numerous times , which include exchanges in the recent past .", "To explain this observation , we examined whether pre-infection of Arabidopsis and Brassica with virulent A . candida races results in enhanced host susceptibility , and found that pre-infection with a virulent strain enables proliferation of an A . candida isolate that would otherwise not colonize that host .", "For the two races with two isolates , we show that population expansion is by clonal reproduction .", "We discuss the impact of genetic exchange on A . candida evolution , and consider the implications for pathogen evolution and reproduction in an agro-ecological environment ." ], [ "AcNc2 was recovered from infected leaves of A . thaliana Eri-1 field-grown plants in Norwich ( UK ) in 2007 .", "AcEm2 was isolated from wild C . bursa-pastoris in Kent ( UK ) in 1993 ( Borhan et al . , 2008 ) .", "Isolate AcBoT was harvested from infected inflorescences of B . oleracea cultivar ‘Bordeaux F1’ in Lincolnshire in May 2009 , and another isolate AcBoL was harvested from infected leaves of B . oleracea in Lincolnshire ( UK ) in January 2009 .", "Races were single spore purified ( Kemen et al . , 2011 ) .", "The Ac2V isolate virulent on B . juncea was provided by M Borhan ( Agriculture and Agri-Food , Canada [Links et al . , 2011] ) and single-spore purified .", "We tested the virulence of single race infections of AcNc2 , Ac2V and AcBoT on different host species and cultivars .", "AcNc2 was propagated on A . thaliana Ws-2 .", "Two B . juncea cultivars ( ‘Cutlass’ and ‘Czerniac’ ) and 18 cultivars of B . oleracea were resistant to AcNc2 .", "We screened 356 A . thaliana accessions for their susceptibility to AcNc2 and 38 . 5% were susceptible ( Table 1 , Supplementary files 1 , 2 ) .", "Race Ac2V was originally isolated in Canada on B . juncea ( Rimmer et al . , 2000 ) .", "We confirmed virulence of Ac2V on B . juncea by spray inoculation of B . juncea ‘Cutlass’ and ‘Czerniac’ .", "On 3-week-old A . thaliana plants , all 107 tested accessions were resistant .", "Two B . oleracea cultivars were also resistant to Ac2V ( Table 1 , Supplementary files 1 , 2 ) .", "Race AcBoT was virulent on all 15 tested cultivars of B . oleracea .", "In contrast , 34 accessions of A . thaliana were fully resistant to AcBoT , as were Brassica rapa and B . juncea cultivars ( Table 1 , Supplementary file 1 ) .", "These tests confirm that A . candida races show pronounced host specificity to distinct host species .", "While we cannot prove that there are no host species in nature that support growth of more than one of the three races we define here , our experiments and all literature strongly suggest that Ac2V only grows on B . juncea , and on some accessions of B . rapa , a diploid ancestor of tetraploid B . juncea ( Kole et al . , 2002 ) , AcBoT and AcBoL only grow on B . oleracea , and AcNc2 and AcEm2 can only grow on a subset of Arabidopsis and Capsella genotypes .", "Genetic exchange between races is unlikely to occur unless they colonize the same host .", "In our study , only the immune-compromised A . thaliana Ws-2-eds1 mutant was susceptible to all races . 10 . 7554/eLife . 04550 . 003Table 1 . Virulence of the A . candida races on different plant host accessionsDOI: http://dx . doi . org/10 . 7554/eLife . 04550 . 003A .", "candida raceArabidopsis thalianaBrassica rapaBrassica junceaeBrassica oleraceae+−+−+−+−AcNc21372190101018AcBoT1*340102150Ac2V1* , †1071‡4‡6‡0‡02+ Host-pathogen compatible interactions ( number of susceptible accessions ) .", "− Host-pathogen incompatible interactions ( number of resistant accessions ) .", "*A .", "thaliana Ws-eds1 ( enhanced disease susceptibility ) mutants were susceptible to all tested A . candida races .", "†In the laboratory conditions , the cotyledons of the A . thaliana accession Ws-3 were found to be susceptible to the Ac2V ( Cooper et al . , 2008 ) .", "‡Data from ( Rimmer et al . , 2000 ) incorporated; in this study , one cultivar B . rapa ( CrGC1-18 , rapid-cycling accession ) was infected by Ac2V race and four other tested cultivars ( ‘Torch’ , ‘Colt’ , ‘Horizon’ , ‘Reward’ ) were incompatible with Ac2V race .", "All analysed cultivars of B . juncea ( CrGC4-1S , ‘Burgonde’ , ‘Domo’ , ‘Cutlass’ ) were susceptible to Ac2V .", "The AcNc2 A . candida assembly was used as the reference in this study , and comprises 34 Mb in 5212 contigs of ∼160-fold coverage ( Table 2 ) .", "We assembled ∼73% of an estimated 45 Mb genome of A . candida AcNc2 ( Voglmayr and Greilhuber , 1998; Links et al . , 2011 ) .", "In a previous study , a similar proportion ( 76% ) of the A . candida Ac2V race genome was assembled ( Links et al . , 2011 ) .", "The unassembled part of the genome ( ∼11 Mb ) is likely to include repeats and duplicated sequences .", "Repeat sequences constitute ∼17 . 4% of the AcNc2 assembly .", "Approximately 8% of annotated repeats represent collapsed regions with coverage several times higher than average , so the real repeat content may be higher . 10 . 7554/eLife . 04550 . 004Table 2 . Summary of the A . candida AcNc2 , AcEm2 , AcBoT , AcBoL and Ac2V genome assembliesDOI: http://dx . doi . org/10 . 7554/eLife . 04550 . 004AcNc2AcEm2AcBoTAcBoLAc2VNumber of contigs521211 , 58111 , 92911 , 14312 , 210N50 length ( bp ) 41 , 07829 , 32614 , 67314 , 95324 , 005N50 number231284584581353Mean contig length ( bp ) 66102668278129982770Assembly size ( bp ) 34 , 454 , 16933 , 409 , 14633 , 184 , 52633 , 409 , 85633 , 823 , 601GC content ( % ) 43 . 19%43 . 09%43 . 15%43 . 15%43 . 11%CEGMA gene space coverage ( % ) *93 . 55%92 . 74%93 . 55%93 , 13%92 . 34%Average genome coverage160150140140200Repeat content ( % ) 17 . 4%NANANANAPredicted genes10 , 907NANANANA*Completeness of the gene space in the different genome assemblies was estimated using CEGMA pipeline; the presence of over 90% of core eukaryotic genes in the assembly serves as an indication of a overall complete gene space .", "Ab initio gene predictions were conducted with several gene prediction programs , resulting in 10 , 907 predicted gene models .", "About 90% ( 9830 ) of the predicted proteins have homologous sequences in the proteome of A . candida Ac2V race ( 15 , 824 genes ) ( Links et al . , 2011 ) .", "In about 1000 cases , when we predict a single copy gene in the AcNc2 race , Links and co-authors ( 2011 ) have predicted multi-gene families in the Ac2V race , explaining the discrepancy in the predicted gene number for two assemblies .", "Only 37% of AcNc2 proteins showed significant sequence similarity to known proteins ( BLASTP E-value ≤ 10−5 ) .", "Using the Tribe-MCL algorithm , 3522 genes ( 32% of the predicted AcNc2 gene repertoire ) were clustered into 1020 gene families .", "The largest gene tribes are protein kinases ( 187 members ) , kinesin- ( 59 genes ) and myosin- ( 44 genes ) like proteins and 35 genes homologous to the secreted ‘CHXC’ proteins of Albugo laibachii ( Kemen et al . , 2011 ) .", "915 genes in the AcNc2 assembly are predicted to encode putative proteins with amino-terminal secretory signal peptides , but no trans-membrane domain .", "Only 34% of the predicted secretome was functionally annotated ( BLASTP , E-value ≤ 10−5 ) , including 115 proteins ( proteases , hydrolases , elicitin-like proteins , elicitors , protease inhibitors ) that could be involved in plant cell wall degradation and protection against host defense enzymes .", "In addition to the 35 CHXC proteins ( Tyler et al . , 2006 ) , further candidate virulence factors were identified including 19 homologs of the Phytophthora Crinkler effectors ( Haas et al . , 2009 ) , and another 23 secreted proteins with ‘RXLR’ and 35 proteins with similar ‘RXLQ’ motifs .", "Both motifs are located in the N-terminal part of protein after the predicted signal peptide , thus resembling the RXLR effectors of Phytophthora infestans and Hyaloperonospora arabidopsidis ( Haas et al . , 2009; Baxter et al . , 2010 ) , but not having any other significant sequence similarity to these proteins .", "After carrying out several assemblies based on different k-mer lengths , the quality of each assembly was assessed with various parameters and one best assembly was chosen for each isolate ( Table 2 ) .", "The high similarity of the five A . candida isolates enabled us to conclude we had sequenced three ‘races’ , within which AcNc2 and AcEm2 were isolates of the same race and AcBoT and AcBoL were also isolates of the same race ( Figure 1 ) .", "Therefore , genome comparisons were first conducted on one representative from each race ( AcNc2 , Ac2V and AcBoT ) , from each of which ∼33–34 Mb of genome was assembled ( Table 2 ) . 10 . 7554/eLife . 04550 . 005Figure 1 . Phylogeny shows that the five sequenced Albugo candida isolates fall into three divergent races . BEAST tree constructed based on using contig 1 ( 398 , 508 bp ) shows the divergence among the three A . candida races ( AcEm2 and AcNc2; AcBoT and AcBoL; Ac2V ) .", "Blue bars represent the 95% Higher Posterior Density ( HPD ) .", "Here , we used a strict molecular clock fixed at 1 . 0 in order to show the relationship in the scale of substitutions per site . DOI: http://dx . doi . org/10 . 7554/eLife . 04550 . 005 To assess the overall genome-wide similarity between races , we performed alignments of reads against the AcNc2 reference assembly .", "For the majority of the AcNc2 genome , we observed a significant positive correlation between read depth in the reference assembly and mapping depth of the Ac2V and AcBoT reads ( r = 0 . 65 , p < 2 . 2e-16; Figure 2A ) .", "Some AcNc2 regions ( 3–4% of the assembly ) showed low or zero coverage by Ac2V and/or AcBoT reads ( Figure 2—figure supplement 1 ) , suggesting the presence of highly divergent or unique regions amongst the races .", "These are gene sparse regions ( 150 and 234 genes predicted in the AcNc2 , respectively ) , without apparent enrichment for genes encoding for secreted proteins ( χ2 = 0 . 11 , d . f . = 1 , p > 0 . 7 ) .", "Amplification of randomly selected AcNc2 genes from these regions revealed that four of the selected five genes are indeed absent/or highly diverged in the AcBoT and Ac2V races , and present in the AcNc2 genome . 10 . 7554/eLife . 04550 . 006Figure 2 . Comparison of A . candida races using alignments of Illumina reads against the AcNc2 assembly .", "( A ) Positive correlation between the depths of coverage of the reference assembly ( AcNc2 ) by the Ac2V and AcBoT reads .", "For the reference contigs less than 20 kb , the mean coverage was calculated across the whole contig length and log-transformed .", "For the contigs over 20 kb , the mean coverage was calculated for the sliding window of 20 kb and log-transformed .", "Y-axis shows the log-transformed depth of the reference coverage by the Ac2V reads; X-axis shows the log-transformed depth of the reference coverage by the AcBoT reads .", "( B ) Nucleotide identity amongst the homologous genomic regions of Ac2V , AcBoT and AcNc2 .", "The mean identity was calculated for the sliding window of 20 kb . DOI: http://dx . doi . org/10 . 7554/eLife . 04550 . 00610 . 7554/eLife . 04550 . 007Figure 2—figure supplement 1 . Coverage of the reference assembly ( AcNc2 ) by Ac2V and AcBoT . Proportion of mean coverage is a measure relative to the depth of coverage of the reference reads mapped backed to the reference assembly .", "The proportion of AcNc2 assembly not covered by AcBoT and/or Ac2V reads ( ≤10% of the mean coverage ) was 3 . 17% ( 971 , 030 bp ) in Ac2V and 4 . 21% ( 1 , 285 , 877 bp ) in AcBoT . DOI: http://dx . doi . org/10 . 7554/eLife . 04550 . 007 The overall mean level of nucleotide identity in the homologous genomic regions amongst races is ∼99% ( Figure 2B ) .", "We verified 25 polymorphic genomic regions by Sanger sequencing ( Supplementary file 3 ) .", "We used the longest of all contigs from AcNc2 , ‘contig 1’ ( 398 , 508 bp ) , to compare the levels of divergence between races vs the number of heterozygous positions within each race .", "An extremely low proportion of sites ( 0 . 03% and 0 . 01% ) on ‘contig 1’ are heterozygous within AcNc2 , AcEm2 and Ac2V races , respectively ( Figure 3 ) .", "Races AcBoT and AcBoL are more heterozygous than Ac2V and AcNc2 , with 0 . 65% of nucleotide positions in ‘contig 1’ being heterozygous in AcBoT ( Figure 3 ) .", "Importantly , >97% of all heterozygous positions are shared in AcBoL and AcBoT ( see below ) .", "In between-race comparisons , ∼1 . 0% of nucleotide positions on ‘contig 1’ have diverged between AcBoT , Ac2V and AcNc2 . 10 . 7554/eLife . 04550 . 008Figure 3 . Nucleotide polymorphism within and between A . candida isolates . Mean ( ±5–95%CI ) polymorphism expressed as the percentage observed heterozygote sites ( solid symbols ) and percentage nucleotide divergence ( open symbols ) at contig 1 .", "Confidence intervals were calculated using a bootstrap of contig 1 after removal of indels .", "Isolates infecting the same host plant ( i . e . , AcBoT-AcBoL and AcEm2- AcNc2 ) show little nucleotide divergence , which indicates that they are genotypically almost identical ( i . e . , diverged by less than 0 . 05% ) .", "Nevertheless , the Brassica oleracea infecting race ( AcBoT and AcBoL ) possess a relatively high heterozygosity compared to the isolates of the Arabidopsis thaliana infecting race .", "Moreover , most of this heterozygous polymorphism is shared ( low nucleotide divergence ) and presence of the majority of heterozygous sites is consistent with clonal reproduction . DOI: http://dx . doi . org/10 . 7554/eLife . 04550 . 008 Polymorphisms are not homogeneously distributed among A . candida races .", "Some regions of the genome are identical for up to 10 , 000 base pairs , whereas the local nucleotide identity is as low as 89% in other regions of up to 5 kb ( Figure 2B ) .", "We examined 133 contigs ( 12 , 373 , 253 bp ) , covering 38% of the reference assembly .", "Stretches of nucleotide similarity amongst races are distributed in a block-like structure; there are regions where AcNc2 is highly similar ( or identical ) to AcBoT and significantly ( nucleotide divergence π > 1% ) diverged from Ac2V , and vice versa ( Figure 4 ) .", "By using multiple different algorithms that can detect recombination in DNA sequence data incorporated in the software RDP3 ( Martin et al . , 2010 ) , we examined whether this pattern can be explained by genetic introgression amongst races .", "In addition , we used the software HybRIDS ( http://www . norwichresearchpark . com/HybRIDS ) to perform a probabilistic recombination analysis , calculate the coalescence time of each recombinant block , and visualize the mosaic-like genome structure . 10 . 7554/eLife . 04550 . 009Figure 4 . Variation in sequence similarity between races .", "( A ) Alignment of nucleotides in between positions 158 , 779 and 167 , 382 within ‘contig 1’ of three A . candida races ( AcNc2 , AcBoT and Ac2V ) illustrating two recombination blocks coloured blue and green .", "Both blocks show high sequence similarity between races .", "Also shown is the sequence divergence in between blocks .", "Alignment gaps and monomorphic sites have been removed .", "( B ) The sequence similarity at ‘contig 1’ amongst three A . candida races was visualised using the colours of a RBG colour triangular in the software HybRIDS ( http://www . norwichresearchpark . com/HybRIDS ) .", "Areas where two contigs have the same colour ( yellow , purple or turquoise ) are indicative of two races sharing the same polymorphisms .", "The linear plot of the proportion of SNPs shared between the three pairwise comparisons between the races .", "Shown on the X-axis is the actual base position .", "The graphs were made in the R package HybRIDS ( http://www . norwichresearchpark . com/HybRIDS ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 04550 . 009 Recombination analysis of 133 contigs highlighted a total of 675 recombined blocks on 127 contigs that were significant ( after Bonferroni correction ) in three or more tests using RDP3 ( Supplementary file 4 ) .", "The combined length of all identified blocks is nearly 3 Mb or ∼25% of the analysed regions .", "These blocks indicate regions of genome in one race that derive from another race ( or the ancestor of another race ) .", "Figure 4 illustrates the effect of genetic introgression on the pattern of nucleotide similarity between the three races in the largest contig of ∼400 kb .", "The sequence ( dis ) similarity between the three races shows a mosaic-like genome structure with large regions where races AcNc2 and AcBoT show near sequence identity ( yellow blocks in Figure 4B ) , whilst other areas show a high level of sequence similarity between AcNc2 and Ac2V , and AcBoT and Ac2V ( indicated by the purple and turquoise regions , respectively ) .", "Note that the presence of such well-defined blocks of high sequence similarity in an otherwise diverged genome is characteristic for rare introgression between organisms that show a high ( yet incomplete ) level of reproductive isolation .", "Noteworthy too is the fact that a high level of recombination ( relative to the mutation rate ) would homogenise the sequence divergence between the races , and hence , that this would not result in the observed mosaic-like structure .", "Despite the fact that introgression between races is rare , it must have occurred multiple times between the ancestors of the three races given that the coalescence times varies markedly between the different blocks ( Figure 5 ) .", "Assuming a base mutation rate of µ = 10−8 per cell cycle , with 100 cell cycles per year ( i . e . , a combined mutation rate of 10−6 per year ) , analysis in the software HybRIDS show that the most recent introgression event has occurred circa 220 years ago , whilst the oldest event occurred almost 200 , 000 years ago .", "The mean age calculated across all introgression events equals 6237 ( ±12 , 594 ) years ( Figure 5 ) .", "( With a combined mutation rate of µ = 10−7 per base per year , the range in the age of introgression would span from 2200 to 2 , 000 , 000 years ) .", "Irrespective of the mutation rate , the principal finding is that genetic introgression amongst A . candida races is an ongoing evolutionary process occurring across a wide range of evolutionary times , and that it gives rise to mosaic genomes with the introgression blocks interspersed in the recipient genomic background . 10 . 7554/eLife . 04550 . 010Figure 5 . Age of recombination blocks .", "( A ) Age of the 675 recombination blocks ( mutation rate of μ = 10−6 ) estimated using binomial mass function; ( B ) Boxplot of the median ( plus first nation blocks and third quartile ) log-age of recombination events in contigs .", "Only contigs with eight or more events are shown .", "There is no significant difference in age of events between contigs ( GLM: F22 , 233 = 1 . 06 , p = 0 . 387 ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 04550 . 010 A total of 1655 predicted genes are located in the recombined regions , and amongst these , 125 are predicted to encode secreted proteins .", "In the introgression regions , we identified 14 genes encoding secreted proteases and hydrolases that in some pathogens act as virulence factors ( Monod et al . , 2002; Soanes et al . , 2007; Lebrun et al . , 2009 ) .", "Thus , recombination between races , resulting in introgression can act as a mechanism for exchange of virulence gene alleles .", "However , neither gene density nor dN/dS are enriched or depleted in regions affected by introgression , compared to regions not affected ( Paired t-test: T = −0 . 05 , p = 0 . 958; Mann–Whitney test: W = 3 . 15 × 166 , p = 0 . 152 , respectively ) .", "It is however entirely likely that by studying only a small subset of all the known races , we have underestimated the actual level of introgression across all races .", "To better understand the evolution and diversification of A . candida genomes , we analysed the pattern of recombination and nucleotide divergence with the inclusion of two additional A . candida isolates .", "AcBoL is an additional isolate from the AcBoT race and AcEm2 is an additional isolate of the AcNc2 race ( see Figure 1 ) .", "We found evidence for 581 recombination events , of which 335 included AcBoT or AcBoL as recombinants .", "These two isolates shared a recombinant block in 97 . 3% of recombination events ( AcBoT and AcBoL = 326; AcBoT = 6; AcBoL = 3 ) .", "AcNc2 and AcEm2 shared a recombinant block in 99 . 6% of events ( AcNc2 and AcEm2 = 246; AcNc2 = 0; AcEm2 = 1 ) .", "This demonstrates that the AcBoT/AcBoL and AcNc2/AcEm2 races have remained largely unchanged since their initial emergence .", "The nucleotide diversity within genomes ( i . e . , the observed heterozygosity ) and the nucleotide divergence between genomes ( i . e . , genetic differentiation ) can be used to further understand A . candida population biology .", "Isolates AcBoT and AcBoL were both collected in Lincolnshire in 2009 , and the observed heterozygosity in these isolates was at least 13 times higher than that of any of the other races ( percentage heterozygous positions in contig 1: AcBoT = 0 . 653%; AcBoL = 0 . 644%; AcNc2 = 0 . 047%; AcEm2 = 0 . 044%; Ac2V = 0 . 028% ) ( see Figure 3 ) .", "Remarkably , AcBoT and AcBoL are heterozygous for almost all of the same sites; 97 . 2% of the sites that are heterozygous in AcBoT are also heterozygous in AcBoL ( and 98 . 5% vice versa ) .", "This is only consistent with clonal reproduction because after just one generation of sexual reproduction ( or selfing with recombination ) , Mendelian segregation would eradicate this high level of genotypic similarity .", "Notably , such evidence for clonal propagation of a race would be difficult to obtain for haploid fungal pathogens .", "With little evidence for recombination and gene flow , most nucleotide divergence between AcBoT and AcBoL must have accumulated through mutation .", "The nucleotide divergence between these isolates is just 0 . 030% ( 121 polymorphisms in 398 , 508 bp in contig 1 ) ( Figure 3 ) .", "Assuming a mutation rate of 1 × 10−8 per cell division , and 100 cell divisions per lineage per year , we estimate that these isolates could have diverged 305 ( 262–353 ) years ago .", "The other pair of isolates , AcNc2 and AcEm2 , were collected in Norfolk in 2007 and in Kent in 1993 ( 160 km apart ) , respectively .", "Similar to the former two isolates , AcNc2 and AcEm2 are also nearly identical ( nucleotide divergence π = 0 . 046% , that is , they are >99 . 95% identical ) ( Figure 3 ) .", "If we again assume that their nucleotide divergence arose by mutation alone , their estimated divergence time is 890 ( 814–970 ) years .", "However , unlike AcBoT and AcBoL , AcNc2 and AcEm2 are much less heterozygous ( see Figure 3 ) , which suggests that another genetic mechanism , for example , loss of heterozygosity ( Lamour et al . , 2012 ) , might be operating which has eradicated the gene diversity in the clonally propagating races over time .", "A . candida infection compromises host resistance against otherwise avirulent pathogen species ( Cooper et al . , 2008 ) .", "Conceivably , A . candida could suppress host defenses to otherwise avirulent races of A . candida , enabling co-infection and sexual exchange .", "To test this we performed sequential inoculation experiments , identifying races using the genome sequences to create race-specific DNA markers .", "Race-specific PCR of pre-inoculated plants ( Supplementary files 5 , 6 ) shows preinfection by AcNc2 suppresses resistance in A . thaliana accession Ws-2 leaves towards the B . juncea-infecting race , Ac2V ( Figure 6A ) .", "Also , preinfection by AcBoT suppresses B . oleracea resistance towards Ac2V ( Figure 6B ) .", "Furthermore , defense suppression was so effective that Ac2V was able to complete its life cycle on both A . thaliana Ws-2 and B . oleracea as observed by successful subsequent infection on B . juncea from the sequentially inoculated plants .", "In a reciprocal experiment , preinfection of B . juncea with Ac2V enabled AcNc2 growth on B . juncea ( Figure 6C ) .", "Therefore , AcNc2 not only can suppress Ac2V recognition on A . thaliana , but Ac2V is also capable of suppressing B . juncea resistance towards AcNc2 .", "TIR-NB-LRR resistance genes likely confer Ac2V resistance in Arabidopsis ( McHale et al . , 2006 ) , as Ac2V grows on an eds1-1 mutant of A . thaliana Ws-2 ( Supplementary file 6; [Parker et al . , 1996] ) .", "It has long been noted that Albugo sp .", "have a remarkable capacity to suppress immunity in their hosts ( Cooper et al . , 2008 ) .", "We hypothesise that suppression of host innate immunity enables co-infection of hosts by races with otherwise non-overlapping host ranges , thus providing a remarkable mechanism to enable sexual genetic exchange between specialised A . candida races . 10 . 7554/eLife . 04550 . 011Figure 6 . PCR with race-specific markers on DNA prepared from plant tissue generated from co-infection assays .", "( A ) Co-infection assay of AcNc2 followed by Ac2V onto Ws-eds1 and Ws-2 .", "( B ) Co-infection of AcBoT followed by Ac2V onto Ws-eds1 and B . oleracea ( B . o ) .", "( C ) Co-infection of Ac2V followed by AcNc2 onto Ws-eds1 and B . juncea ( B . j ) .", "Bands highlighted in orange indicate amplification of secondary inoculum on usually non-host plants upon primary inoculation with virulent A . candida .", "These experiments were repeated multiple times with similar results ( see Supplementary file 6 ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 04550 . 011" ], [ "A . candida comprises distinct races that specialize on different plant species ( Liu et al . , 1996; Rimmer et al . , 2000 ) and its physiological races can infect over 200 species of plants .", "Here , we describe the genomes of five isolates from three races of A . candida that colonize distinct host plant species and appear to have non-overlapping host ranges .", "Genome analyses show that the races have a mosaic-like genome structure that is consistent with genetic introgression between races that have significantly diverged ( mean nucleotide divergence ∼1% ) .", "Despite this divergence , ∼25% of the nucleotide sequence within the analysed genome ( 3 . 2 Mb out of 12 . 4 Mb ) is derived from other A . candida races .", "675 introgressed blocks were identified in the 127 analysed contigs and each block was confirmed by at least three independent recombination algorithms .", "Based on the high nucleotide similarity of the recombined regions , it appears that some genetic exchange must have occurred recently and that introgression has happened multiple times between the ancestors of the three races .", "An alternative hypothesis for the observed mosaic-like genome structure is that the polymorphisms were already present in the ancestor of A . candida , and that those may have sorted stochastically among descendant host races .", "This is known as incomplete lineage sorting ( Pamilo and Nei , 1988; Hobolth et al . , 2007 ) , and such ancestrally shared polymorphism is notoriously difficult to distinguish from polymorphisms shared through secondary contact and introgression .", "However , by estimating the coalescence time of the introgressed regions we showed that hybridisation between the races is an ongoing evolutionary process , with some introgression events occurring as recent as 220 years ago .", "We therefore reject the hypothesis of incomplete lineage sorting and propose that genetic exchanges between the A . candida host races have occurred through rare but nevertheless significant introgression events .", "If host-ranges are determined by multiple loci , one might expect that recombination will quickly lead to ‘super genotypes’ with very wide host ranges .", "However , this is unlikely because of the dual consequences of effector alleles; they not only can contribute to virulence , but if recognized by Resistance ( R ) gene alleles in a particular host , they can result in the complete loss of virulence on that host ( Dodds and Rathjen , 2010 ) .", "Effectors selected to facilitate infection and evade recognition by R genes in one host might be recognised by the R genes in another host and be maladaptive .", "Thus , genotypes that are highly virulent on multiple hosts are unlikely to arise by recombination between races that specialize on distinct specific hosts , although occasionally , acquiring an effector from one race by introgression might offer an adaptive advantage assuming that the effector is not recognised .", "Strikingly , each race shared a mosaic pattern of large genomic regions ( >10 , 000 bp ) that were virtually identical between two races ( and diverged from the third ) .", "Given that mutations accumulate over time , this implies that the exchange must have occurred relatively recently .", "Yet , A . candida is an obligate biotrophic parasite that is highly host-specific .", "The host-specificity of A . candida races was confirmed in our experiments , and this raises the question of how distinct A . candida races can achieve the physical contact required for them to have sex and recombine .", "We addressed this question using experimental infections of host plants with multiple races .", "Crucially , we showed that infection with a virulent race of A . candida suppresses host immunity sufficiently to enable subsequent co-colonization by an otherwise non-virulent race ( see also Cooper et al . , 2008 ) .", "Although this opens up competition between races for the same ( limited ) resource , it also brings a significant evolutionary/ecological advantage by blurring the borders of host range , thereby creating secondary contact zones that enable sexual reproduction and genetic exchange between races .", "Given the high-host specificity of obligate biotrophic parasites , this ability of A . candida to enable occasional genetic exchange between specialised races could create novel repertoires of effector alleles that would enable colonization of new hosts ( ‘host jumps’ ) .", "For example , Arabidopsis resistance to race Ac2V can at least in part be explained by the WRR4 gene , a classical NB-LRR R gene , which presumably recognizes an Ac2V effector ( Borhan et al . , 2008 ) .", "Hypothetically , if this Ac2V effector would segregate away in hybrid offspring , or by loss of heterozygosity , these offspring could become virulent on WRR4-carrying Arabidopsis hosts .", "Once a new hybrid race has been established , it can reproduce asexually and clonally on susceptible hosts without continued genetic exchange with other races .", "We base this inference on the exceptionally high genotypic similarity between independent isolates that infect the same host plant ( i . e . , AcBoT-AcBoL and AcEm2-AcNc2 ) .", "For example , AcBoT and AcBoL share >97% of their heterozygous sites .", "This observation is significant because it rules out sexual reproduction ( or selfing with recombination ) , given that Mendelian segregation would eradicate this high level of genotypic similarity within a single generation .", "Hence , we conclude that reproduction of AcBoT and AcBoL is asexual and clonal , and we speculate that these races may have derived from a common ancestor that was selected coincident with the onset of widespread B . oleracea cultivation in Europe .", "The A . thaliana-infecting isolates ( AcNc2 and AcEm2 ) were sampled at a broad spatiotemporal scale ( 14 years and 100 miles apart ) , and yet they showed a very high nucleotide similarity ( >99 . 95% identical ) .", "This shows that clonal reproduction coincided with their rapid population expansion .", "Remarkably though , compared to the former two isolates , AcEm2 and AcNc2 had a relatively low level of observed heterozygosity , and the number of heterozygous sites was >13 times less than that of AcBoT and AcBoL .", "Since these races do not self-fertilize , this suggests that gene conversion or another mechanism might be operating to reduce the nucleotide diversity within their genomes over time .", "Such loss of heterozygosity ( LOH ) has also been reported for Phytophthora capsici ( Lamour et al . , 2012 ) , and in yeast , LOH events can encompass entire chromosomes , which is thought to be explained by the break-induced replication ( BIR ) mechanism ( Diogo et al . , 2009 ) .", "‘Hybrid speciation’ or ‘recombinational speciation’ is often associated with a mosaic genome ( Baack and Rieseberg , 2007; Stukenbrock et al . , 2012 ) and because introgressed genes have been ‘pre-tested’ by selection , they are more likely to be adaptive than random changes to the genetic code by mutations ( Hedrick , 2013 ) .", "For example , Helianthus anomalus is a wild sunflower species derived via hybridization between two parental species , and its genome is characterised by parental species blocks .", "In this case , however , hybridisation and speciation occurred over a short evolutionary time-span ( 10–60 generations ) ( Ungerer et al . , 1998 ) , which differs from hybridisation in A . candida , where it appears to be a persistent evolutionary process .", "Darwin's finches are a classic example of a young adaptive radiation , and recent research by Lamichhaney et al . ( 2015 ) showed that species from different islands show extensive genomic exchange through recent hybridisation .", "Introgressive hybridization was found to occur throughout the radiation , fuelling the genetic variation in beak shape , and thereby facilitating adaptive evolution .", "Perhaps more pertinently , the relationships of members of the parasite's principal host , plants of the Brassica genus , are themselves considered to be the result of a number of hybridisation events , as defined by the Triangle of U ( Nagahara , 1935 ) .", "Hybridisation events have also been implicated in speciation and adaptive radiation in vertebrates ( Nichols et al . , 2015 ) .", "Furthermore , rare introgression events among Heliconius butterflies are believed to have facilitated the exchange of mimicry genes across multiple time points post speciation ( Martin et al . , 2013 ) .", "Evidence for more frequent introgression has been observed in the malaria vector species complex ( Anopheles gambiae ) ( Fontaine et al . , 2015 ) .", "More examples of such introgression can be anticipated to emerge given the continued improvement and reduction in cost of DNA sequencing methods , and the development of novel software that enable analysis of whole genome recombination ( Ward and van Oosterhout , submitted ) .", "Perhaps more than any other system , studies on yeasts have offered valuable insights into how introgression and hybridisation can affect genome architectures of eukaryotic microorganisms ( reviewed by Dujon , 2010 ) .", "Genetic exchanges amongst three strains of Saccharomyces cerevisiae have been quantified using genomic data which show that the rate of outcrossing is remarkably low , with only 314 outcrossing events during circa 16 million cell divisions ( Ruderfer et al . , 2006 ) .", "With such a low rate of genetic exchange , the strains accumulate sequence variation by mutation and thus genetically diverge .", "Given the low level of genetic exchange amongst the A . candida host races , this may also explain their genetic divergence .", "In yeast , natural hybrids have been reported in many species ( Dujon , 2010 ) , with hybridisation leading to the nonreciprocal genetic exchange accompanied by the loss of genes and genomic regions .", "This results in chimeric sequences from which novel lineages could emerge ( Greig et al . , 2002; Usher and Bond , 2009 ) .", "For example , Lachancea kluyveri possesses mega-base long chromosomal fragments of distinct composition ( Payen et al . , 2009 ) .", "Furthermore , the genomes of wine strains of S . cerevisiae contain introgressed regions from Saccharomyces paradoxus , S . kudriavzevii kudriavzevii , S . uvarum uvarum , and Zygosaccharomyces bailii ( Dujon , 2010 ) .", "Given that the introgressed sequences in the genome of S . cerevisiae are nearly identical to those in the donor genomes , hybridisation must have occurred recently , which is similar to what we observe in A . candida .", "Although introgression appears to be a general phenomenon in yeast genomes , its importance for evolution has yet to be determined ( Dujon , 2010 ) .", "The mosaic-like genome structure of A . candida suggests that hybridisation and genetic introgression may play an equally important role in the biology of this oomycete .", "Introgression can introduce novel adaptive trait combinations ( Seehausen , 2004; Hedrick , 2013 ) as well as enable the loss by segregation of host-specific ‘avirulent’ effector alleles that can trigger the immune response in potential hosts .", "In rare novel recombinant races , such maladapted effectors that trigger the response of a specific host may be segregated away , allowing the recombinant to avoid immune recognition and colonise this new host .", "Once this happens , the new hybrid can rapidly expand its geographic range and population size through clonal reproduction .", "Hybrids between Phytophthora spp .", "races can show expanded host range compared to their parental lineages ( Ersek et al . , 1995 ) while the importance of virulence gene transfer that subsequently leads to the expansion of pathogens' host range has also been reported for bacterial and fungal pathogens ( Doolittle , 1999; Mehrabi et al . , 2011 ) .", "The ability of pathogens to recombine and generate novel recombinant genotypes and subsequently proliferate clonally may be particularly favoured in the agro-ecological environment .", "Recent fusion between genetically distinct plant pathogens has been shown in Mycosphaerella graminicola ( Stukenbrock et al . , 2012 ) , where a hybrid speciation event has generated a generalist pathogen of grass species , and in Blumeria graminis ( Hacquard et al . , 2013 ) , where a mosaic genome structure has been generated by sex between divergent isolates .", "Adaptation of pathogens to agro-ecosystems can be correlated with a reduction in diversity of recently emerged lineages and at the same time , high levels of genome plasticity ( Stukenbrock and Bataillon , 2012 ) .", "This genome plasticity has also been observed in B . graminis ( Hacquard et al . , 2013 ) .", "Race specialization observed in the potato late blight pathogen P . infestans , the rice blast pathogen Magnaporthe oryzae , and the wheat yellow rust pathogen Puccinia striiformis ( Stukenbrock and Bataillon , 2012 ) , may represent the result of a broader mechanism of pathogen adaptation to a crop monoculture .", "A . candida , which is known to live on both wild weeds ( e . g . , A . thaliana ) and important crop species ( e . g . , B . juncea ) , thus provides remarkable insights into the impact of recombination in generating new virulent races and subsequent clonal propagation of a novel race .", "These findings are particularly relevant to modern agricultural methods and the emergence of new epidemic pathogen strains on crop monocultures ." ], [ "A . candida races were isolated and propagated by first washing zoosporangia from infected leaves and then infecting A . thaliana Ws-eds1 ( enhanced disease susceptibility [Parker et al . , 1996] ) plants .", "After 2 weeks , one pustule was punched out and spores were treated on ice for 30 min to release zoospores .", "Unhatched zoosporangia were removed by filtration and zoospores were diluted to ∼10 zoospore per ml and sprayed on A . thaliana Ws–eds1 plants ( ∼100 μl/plant ) .", "This procedure was repeated four times until spores were bulked up on A . thaliana Ws-eds1 plants .", "Zoosporangia were harvested using a homemade cyclone spore collector ( Mehta and Zadoks , 1971 ) .", "Subsequently , A . candida races AcEm2/AcNc2 , AcBoT/AcBoL and Ac2v were propagated and maintained on A . thaliana Ws-2 , B . oleracea and B . juncea , respectively .", "Host specificity was tested for the AcNc2 , Ac2V and AcBoT races on a number of A . thaliana and Brassica spp .", "accessions ( Supplementary files 1 , 2 ) .", "A . candida inoculations were performed using the following method: zoospores were suspended in water ( 105 spores/ml ) and incubated on ice for 30 min; the spore suspension was then sprayed on plants using a spray gun ( ∼700 µl/plant ) , and plants were incubated in a cold room in the dark over night .", "Infected plants were kept under 10-hr light and 14-hr dark cycles with 20°C day and 16°C night temperature .", "Plants were scored susceptible if a pathogen was capable of accomplishing its life cycle and sporulation was macroscopically visible within 3 weeks after plant inoculations .", "For sequential infection analyses , we developed A . candida race specific PCR primers from genome sequences ( Supplementary file 5 ) .", "Regions in all vs all alignments were identified that lacked read coverage by other isolates .", "Primers were designed within these regions to amplify products of between 300 and 800 bases and were tested on pure genomic DNA extracts from each isolate .", "Primary inoculum was sprayed onto control and test plants .", "In the case of AcNc2 defence suppression assays , both A . thaliana Ws-2 and Ws-eds1 were inoculated; for AcBoT assays , B . oleracea and Ws-eds1 were inoculated; for Ac2v assays , B . juncea and Ws-eds1 were inoculated .", "Following inoculations , plants were incubated in the dark in a cold room over night before transferring to a growth cabinet set to the conditions described above .", "In addition to pathogen treatment , the same numbers of plants were also treated with water in order to serve as a negative infection control .", "At 7 days post-inoculation a secondary infection with the avirulent A . candida race was performed on 50% of the plants while the remaining 50% were water treated ( Supplementary file 6 ) .", "Plants were returned to the growth cabinet and cultivated for a further 8 days .", "Inoculated plant tissue was harvested , washed in sterile water to remove surface adhering spores , and flash frozen in liquid nitrogen .", "DNA was prepared using a DNeasy Plant Mini Kit ( Qiagen , Valencia , CA ) as described in manufacturers instructions .", "PCR was performed using race-specific primers and products visualised on a 1% agarose gel .", "Plants from which tissue was harvested were maintained for a further 7 days before re-inoculation onto the original host of the otherwise non-virulent pathogen .", "This was done in order to confirm the completion of the secondarily inoculated pathogen race's lifecycle on immunosuppressed non-host plants .", "DNA was extracted from zoosporangia according to the method described in Mckinney et al . ( 1995 ) , and Illumina libraries for sequencing were constructed according to Farrer et al . ( 2009 ) .", "Paired-end libraries of 800 bp and 400 bp insert lengths ( for the race Ac2V only one library of ∼400 bp ) were sequenced using Illumina Genome Analyzer II platform at the Sainsbury Laboratory Sequencing Centre ( GA2 ) .", "The base calling was done on the Illumina GAP v1 . 3 pipeline .", "A . thaliana Ws-0 plants were infected with A . candida AcNc2 and infected plants were harvested at 0 , 2 , 4 , 6 , 8 and 10 days after infection .", "RNA was isolated using TRI Reagent RNA Isolation Reagent ( Sigma , UK ) , and subsequently enriched for mRNA with Dynabeads ( Invitrogen ) .", "cDNA was prepared using the SMART cDNA Library Construction Kit ( Clontech , Sunnyvale , CA ) according to manufacturer's instructions .", "These libraries were normalized using Evrogen Duplex-specific nuclease ( DSN ) .", "Normalized cDNA libraries were fragmented using Covaris sonicator and libraries prepared according to Illumina genomic library preparation kit .", "Libraries were sequenced on the Illumina GA2 platform .", "The sequence data have been deposited at the EMBL Nucleotide Sequence Database , with the accession numbers for A . candida AcNc2: SRR1811450 , SRR1811464 , AcEm2: SRR1806791 , AcBoT: SRR1811472 , SRR1811473 , AcBoL: SRR1811474 , Ac2V: SRR1811471 .", "The genomic assemblies were produced using program Velvet 1 . 0 . 19 ( Zerbino and Birney , 2008 ) .", "Using BLAST ( Altschul et al . , 1990 ) , resulting contigs were searched ( BLASTN , E-value ≤ 10−5 ) against genomic sequences of A . thaliana TAIR 9 . 0 ( The Arabidopsis Genome Initiative , 2000 ) , fungi Neurospora crassa ( Galagan et al . , 2003 ) , a collection of bacterial genomes ( such as , Xanthomonas sp . and Pseudomonas sp . : microbialgenomics . energy . gov ) , and against mitochondrion of Pythium ultimum ( Levesque et al . , 2010 ) to remove potential contamination and mitochondrial DNA .", "The assembly of AcNc2 was processed by merging the overlapping contigs from two velvet assemblies ( based on the k-mers 55 and 61 ) with the Minimus2 genome merge pipeline ( Sommer et al . , 2007 ) and in-house perl scripts .", "Read alignment and mapping was performed using programs BWA ( 0 . 7 . 3 ) and SAMtools ( 0 . 1 . 17 ) ( Li and Durbin , 2009; Li et al . , 2009 ) and BedTools ( Quinlan and Hall , 2010 ) .", "Duplicates were removed from mapped reads and SNP calling and filtering was done with BCFtools view and varFilter ( -D100 ) .", "Where required , conversion to fasta format from vcf was done using the modified version of vcf2fq which has been modified to include indels ( http://sourceforge . net/p/vcftools/feature-requests/19/ ) and then sequences were aligned using mafft online server ( http://mafft . cbrc . jp/alignment/server/ ) .", "Illumina sequenced cDNA from the AcNc2 infected A . thaliana Ws-0 leaves was assembled using Velvet/Oases ( Schulz et al . , 2012 ) with different k-mer lengths ( 43 , 45 , 47 , 51 , 55 , 57 , 61 , 63 ) .", "We used various characteristics ( total number of contigs , assembly size , longest contig length and mean contig length , and the proportion of core eukaryotic genes ( KOGs ) predicted by CEGMA ) to assess assembly quality .", "Two best assemblies based on the k-mers 55 and 57 were merged using VMATCH ( http://www . vmatch . de/ ) .", "The cDNA orientation was predicted using Illumina generated cDNA 5′ tags .", "Using Bowtie aligner ( Langmead et al . , 2009 ) , cDNA 5′ tags were aligned against the assembled cDNA and , based on tag counts , orientated in the 5′–3′ direction .", "Ab initio gene predictions were performed for A . candida AcNc2 using the Augustus gene prediction package ( Stanke et al . , 2006 ) , Geneid ( Blanco et al . , 2002 ) and GeneMark ( Lomsadze et al . , 2005 ) .", "Alternative splice variants were predicted with Augustus .", "To improve gene predictions , the ‘hints’ files were created using cDNA evidence and gene homology information .", "The generated library of AcNc2 transcripts was aligned to the AcNc2 assembly using BLAT ( Kent , 2002 ) , setting minimal identity to 92; the ‘hints’ file was produced with script blat2hints . pl provided with the Augustus package .", "The parameters previously obtained for the gene prediction in the A . laibachii genome project were utilized when running the Augustus and Geneid .", "GeneMark predictions were made with the default settings .", "Consensus gene models were generated with Evigan ( Liu et al . , 2008 ) .", "Subsequently , the catalog of non-overlapping gene models was created from the Evigan , Augustus , Genemark and Geneid predictions .", "The gene space coverage was assessed with CEGMA ( Parra et al . , 2007 ) .", "Functional annotations of AcNc2 proteins were performed via comparison of the predicted protein sequences with the protein databases; UniProtKB ( Suzek et al . , 2007 ) and NCBI non-redundant RefSeq ( Pruitt et al . , 2009 ) databases were scanned using BLASTP algorithm ( E-value ≤ 10−5 ) ; and Pfam database ( Punta et al . , 2012 ) was searched with the program hmmscan from HMMER3 ( Eddy , 2011 ) with the default settings .", "GO terms were assigned with BLAST2GO pipeline ( Conesa et al . , 2005 ) .", "Gene families were predicted using Tribe-MCL algorithm that implements Markov cluster ( MCL ) approach for the clustering of proteins into families based on the pre-computed sequence similarity information ( Enright et al . , 2002 ) .", "Signal peptides and cleavage sites were predicted by the hidden Markov Model and the neutral network algorithm implemented in SignlP 4 . 0 program ( Petersen et al . , 2011 ) .", "Transmembrane helices were predicted with the hidden Markov Model in TMHMM 2 . 0 ( Moller et al . , 2001 ) .", "Candidate cytoplasmic effectors carrying ‘RXLR’ motif were identified through the string search of the predicted secreted proteins using ‘R[A-Z]L[RQ]’ regular expression in the first 100 residues downstream of the signal peptide cleavage site .", "Crinkler's homologs were detected using BLASTP searches ( E-value ≤ 10−5 ) against NCBI non-redundant RefSeq database ( Pruitt et al . , 2009 ) .", "Homologs of A . laibachii ‘CHXC’ proteins were identified through Tribe-MCL clustering , also using BLASTP searches ( E-value ≤ 10−5 ) of the A . laibachii predicted proteome , and string search for the ‘CH[A-Z]C’ motif in the first 100 residues after predicted signal peptide .", "The library of repeats in AcNc2 assembly was constructed with RepeatScout ( Price et al . , 2005 ) and was joined with the previously made library for A . laibachii ( Kemen et al . , 2011 ) .", "This updated library in combination with RepeatMasker ( http://www . repeatmasker . org/ ) was used for the identification of repeats and their frequencies .", "Transposon elements were annotated using TBLASTX searches ( E-value ≤ 10−5 ) against the database of transposon elements , RepBase ( Jurka et al . , 2005 ) .", "Phylogenetic trees were built ( MrBayes program [Huelsenbeck and Ronquist , 2001] ) for the nucleotide sequence alignments of the orthologous genes present in four A . candida races and A . laibachii .", "Trees were built for 100 single copy genes ( 50 core eukaryotic genes and 50 singletons with unknown function ) Phylogenetic Bayesian inference and Markov chain Monte Carlo ( MCMC ) methods were used to estimate the posterior distribution of model parameters .", "We used lset = 6 , gamma model , mcmc of 10 million , samfreq = 7000 and burnin = 375 .", "Population genetic parameter ‘theta’ ( Θ = 4Neμ ) was estimated using mlRho program ( Haubold et al . , 2010 ) .", "Four different topologies were inferred with equal support which warranted further investigation into the role of introgression .", "Bayesian Evolutionary Analysis by Sampling Trees ( BEAST ) software package version 1 . 7 ( Drummond and Rambaut , 2007 ) was used to produce the race phylogeny ( based on contig 1 ) .", "BEAST implements Markov chain Monte Carlo ( MCMC ) algorithms for Bayesian for divergence time dating ( Drummond and Rambaut , 2007 ) .", "Bayesian phylogenetic trees were constructed with a HKY+G nucleotide substitution model under a strict molecular clock ( with units in mutations per site ) and a Yule tree prior .", "We ran ten independent MCMC analyses each of 10 million steps and a 10% burn-in .", "MCMC chain mixing was assessed using Tracer 1 . 5 which showed ESS >3000 for each statistic .", "Recombination events were statistically identified on contigs ≥10 , 000 bp using the software RDP3 using five independent detection algorithms: RDP ( Heath et al . , 2006 ) , GENECONV ( Padidam et al . , 1999 ) , Maxchi ( Smith , 1992 ) , Chimaera ( Posada and Crandall , 2001 ) , and 3Seq ( Boni et al . , 2007 ) .", "Tests were conducted using a critical value α = 0 . 05 and p-values were Bonferroni corrected for multiple comparisons of sequences .", "Sequences were linear using unphase base calling and the random assignment of one of the nucleotides at each polymorphic site .", "Given that recombination algorithms use cis mutations to define regions of a sequence that share the same phylogenetic history , the statistical power to detect recombination is reduced when using unphased data ( Darren Martin pers . comm . ) .", "This is because the signal of unphase base calling erodes any underlying signal of recombination .", "This procedure is conservative and underestimates the true number of recombination events because it reduces sequence similarity between the recombinant and parental sequence .", "Phylogenetic evidence of recombination was required to confirm a recombination event .", "Window sizes for each detection method were set to defaults .", "Only events for which the software identified the parental sequences ( i . e . , no ‘unknowns’ ) without ambiguous start and end position of the recombination block are reported and used in the analyses .", "Furthermore , events were only considered to be genuine if they were supported by at least three of the five detection algorithms .", "Hence , the estimates of the number of recombination events are conservative .", "The effects of recombination on the sequence similarity between three genomes was visualised using a newly developed code in the R package HybRIDS ( Hybrid Recombination , Identification and Dating , Software , http://www . elsa . ac . uk/ ) .", "HybRIDS uses a colour triangle to visualise the sequence similarity between aligned sequences .", "It calculates the colour of each 100 bp window based on the proportion of SNPs shared between the pairwise sequences .", "All monomorphic sites were excluded in this calculation .", "HybRIDS uses the additive colour system in which the primary colours used are red , green , and blue .", "These colours are plotted on the corners of the RGB colour triangle , which is shown in the legend as a reference .", "In cases where all SNPs are shared between just two of the three races , the hybrid colour is an exact 50% mix of two primary colours .", "The hybrid colours are yellow , purple and turquoise , and these colours suggest recent gene exchange between the two races .", "At such recombined regions , the third race receives its primary colour ( because by definition , it must be unique at the 100 bp window and completely dissimilar from the other two races ) .", "Older recombined regions are predicted to have accumulated SNPs unique to each race , causing the block of two hybridizing races to diverge .", "In the graph , the colour of such areas contains more than 50% of the primary colour of that race .", "The colours in the centre of the triangle are pale and reflect areas where the three races share approximately similar numbers of polymorphisms .", "The SNPs in these pale regions , and in regions where the colours are close to the primary colours , are more likely caused by mutations than by genetic exchange .", "Note that primary colours can also be assigned to regions that may have recombined , but which originate from a race that was not sampled , and hence , which polymorphisms were not included in this analysis .", "We used the sequence divergence of the recombination block between the recombinant and the minor parent ( i . e . , the sequence donating the recombinant region ) to estimate the divergence time since a recombination event .", "We used two dating methods , a binomial mass function and an analysis with the Bayesian Evolutionary Analysis by Sampling Trees ( BEAST ) software package version 1 . 7 ( Drummond and Rambaut , 2007 ) .", "A binomial mass function was used to estimate the mean divergence time of a block of given size with an observed number of SNPs .", "In order to correct for mutation saturation , homoplasy , back mutations and transition/transversion ratios , we converted the observed number of SNPs into the number of mutations using a JC correction ( Jukes and Cantor , 1969 ) .", "The probability of finding a number of SNPs less or equal to the observed number in a block of known size was calculated .", "The mean time is found when the binomial mass function returns a probability value p = 0 . 5 .", "This approach finds the most probable age of the recombination event , and it assumes that since the recombination event , the block evolved neutrally over t years , and that each base has the chance to mutate with a probability µ per year ( µ = 10−6 and 10−7 ) .", "The algorithm uses a strict molecular clock , and because the mutation rate in oomycetes is unknown , we assumed µ = 10−6 as well as µ = 10−7 per base per year .", "The lower value of the mutation rate of µ = 10−7 was used as a more conservative estimate .", "Given that we do not know the mutation rate of oomycetes , the estimated dates are merely an approximation and shown to illustrate that the exchanged blocks are dated back to a wide range of evolutionary times .", "The simple dating method based on the binomial mass function was compared to more computationally intensive analysis with BEAST by dating 20 recombination events from the ‘contig 1’ of AcNc2 and performing a linear regression analysis to confirm application of the faster binomial algorithm to all 675 recombination events .", "The principle aim of these analyses was to identify whether or not recombinant regions span a range of dates ( in line with the expectation under an introgression model ) .", "BEAST bayesian phylogenetic trees were constructed with a HKY+G nucleotide substitution model under a strict molecular clock ( µ = 10−6 ) and a Yule tree prior .", "We ran 10 independent MCMC analyses each of 10 million steps and a 10% burn-in for each of the 20 recombination events .", "MCMC chain mixing was assessed using Tracer 1 . 5 which showed ESS >3000 for each statistic .", "Note however that the divergence estimates made by the binomial mass function and the analysis with BEAST are conservative ( i . e . , the true time of the recombination event are probably more recent ) given that we had only three sequences in the analysis .", "Consequently , the ‘true’ parental sequence has probably not been sampled , which means that the observed divergence is larger than that of the actual parental ( donor ) sequence .", "The substitution rates ( non-synonymous substitution rate per non- synonymous site ( dN ) and synonymous substitution rate per synonymous site ( dS ) ) and the ratio of the dN/dS for the orthologous protein-coding sequences between three isolates were estimated with the M0 model in PAML ( Yang , 2007 ) .", "The dN/dS ratio is traditionally used as an indicator of the strength and type of selective constrains acting upon a gene .", "Values of dN/dS ≈ 1 indicate neutral evolution .", "Values of dN/dS significantly less than unity indicate purifying selection , whereas dN/dS significantly larger than unity suggest positive selection .", "The Tajima's D statistics ( Tajima , 1989 ) were not calculated because we do not possess allele frequency data ." ] ]

[ "How generalist parasites with wide host ranges can evolve is a central question in parasite evolution .", "Albugo candida is an obligate biotrophic parasite that consists of many physiological races that each specialize on distinct Brassicaceae host species .", "By analyzing genome sequence assemblies of five isolates , we show they represent three races that are genetically diverged by ∼1% .", "Despite this divergence , their genomes are mosaic-like , with ∼25% being introgressed from other races .", "Sequential infection experiments show that infection by adapted races enables subsequent infection of hosts by normally non-infecting races .", "This facilitates introgression and the exchange of effector repertoires , and may enable the evolution of novel races that can undergo clonal population expansion on new hosts .", "We discuss recent studies on hybridization in other eukaryotes such as yeast , Heliconius butterflies , Darwin's finches , sunflowers and cichlid fishes , and the implications of introgression for pathogen evolution in an agro-ecological environment ." ]

[ "Many microorganisms live as parasites inside another living organism , and gain nutrients at their host's expense .", "Plants and animals have immune systems that serve to protect against this kind of exploitation , but successful parasites have evolved ways to avoid detection by their hosts' immune systems , and/or to suppress hosts' defence mechanisms .", "Parasites often avoid detection by releasing molecules that interfere with specific aspects of a host's immune system .", "The same molecules , however , can be recognised by the immune systems of other species and trigger defence responses that eradicate the parasites; this explains why most parasites can colonise only a limited number of host species .", "It is less clear how parasites evolve to become ‘generalists’ that can infect many host species .", "However , some generalist parasites have several distinct subgroups—each of which is specialised to infect a limited number of host species .", "Albugo candida is a generalist parasite that infects over 200 plant species , including mustard greens , oilseed and vegetable crops .", "Even though its looks and its lifestyle resemble those of a fungus , A . candida is actually an oomycete: a group of organisms that are more closely related to golden-brown algae than they are to fungi .", "About 24 subgroups ( or ‘races’ ) of this generalist parasite have been identified to date , but it remains unclear how these subgroups have evolved .", "McMullan , Gardiner et al . tested different isolates of A . candida—four from southeast England and one from western Canada—which had been collected from different plant species and confirmed that each could only infect a narrow range of plant hosts .", "Next , the genome sequences of these five A . candida strains were assembled and compared .", "This analysis revealed that the five strains represented three distinct subgroups ( or races ) of A . candida .", "Moreover , some parts of one subgroup's genome were most similar to those found in a second subgroup; and other parts were more like sections of the third subgroup's genome .", "McMullan , Gardiner et al . point out that such ‘mosaic-like genomes’ indicate crossbreeding between the different subgroups .", "But as A . candida must infect a plant in order to reproduce , and different subgroups infect different host plants , how can different subgroups meet in order to mate and reproduce ?", "In answer to this question , McMullan , Gardiner et al . showed that a plant that is infected with one subgroup of A . candida becomes susceptible to co-infection with other subgroups , including those that couldn't normally infect this plant species on their own .", "These findings reveal that generalist parasites can therefore evolve new subgroups via a mechanism that is similar to the way that crossbreeding ( or hybridization ) between existing species can lead to the evolution of new species ." ]

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

[ "cancer biology" ]

[ [ "One of the hallmarks of cancer is increased genomic instability ( Hanahan and Weinberg , 2011 ) .", "In addition to DNA replication errors and /or mutations induced by exposure to DNA damaging agents , over-expression of oncogenes have been shown to induce genomic instability ( Bartkova et al . , 2005; Gorgoulis et al . , 2005 ) .", "One such oncogene is Myc .", "As one of the most widely studied oncogenes in cancer biology , it is mutated or over-expressed in multiple types of cancer ( Pelengaris et al . , 2002 ) .", "Myc is involved in driving cellular proliferation and promoting stem cell self-renewal under normal circumstances .", "When myc is overexpressed in a cell , it can cause increased genomic instability and promote carcinogenesis ( Karlsson et al . , 2003; Ray et al . , 2006 ) .", "Despite numerous studies , the mechanisms involved in myc-induced genomic instability and transformation remain controversial .", "There are conflicting reports on the mechanism of myc-induced genomic instability and transformation .", "Several studies suggest that myc-induced genomic instability and carcinogenesis is a result of an overabundance of reactive oxygen species ( ROS ) ( Vafa et al . , 2002; Felsher and Bishop , 1999 ) .", "However , it has also been reported that myc overexpression can cause DNA damage and transformation in the absence of ROS ( Ray et al . , 2006 ) .", "Over-expression of myc has been shown to induce apoptosis ( Evan et al . , 1992; Harrington et al . , 1994 ) .", "Until recently , apoptosis has been widely recognized as an anti-carcinogenic process based on the assumption that it is utilized by the host to eliminate damaged cells , including those suffering DNA damage ( Hanahan and Weinberg , 2011; Reed , 1999 ) .", "However , there has been increasing evidence that apoptosis may in fact been involved in promoting carcinogenesis ( Tang et al . , 2012; Ichim et al . , 2015; Liu et al . , 2015 ) .", "Mammalian cells exposed to external stress can survive activation of the apoptotic cascade and incur increased genetic instability and oncogenic transformation .", "Studies show that mammalian cells that survive apoptosis experience increased mitochondria membrane permeability ( MOMP ) ( Ichim et al . , 2015 ) and sublethal caspase 3 activation ( Liu et al . , 2015 ) , which lead to activation of downstream endonucleases such as CAD and endoG , which in turn cause increased genetic instability and oncogenic transformation .", "In the current study , we carried out experiments to examine the potential roles of the cellular apoptotic machinery in Myc-induced mutagenesis and carcinogenesis .", "We show sublethal activation of caspase 3 and endonuclease G plays an essential role in Myc-induced genetic instability and oncogenic transformation in human cells ." ], [ "Previously , we and others have shown that mammalian cells exposed to external stress such radiation and chemicals could survive the activation of apoptotic caspases .", "Among the cells that survive caspase activation , elevated DNA damage , such as DNA double strand breaks were observed .", "In this study , we set out to examine the hypothesis that sublethal activation of apoptotic caspases are involved in Myc-induced genetic instability .", "Our hypothesis is based on the well-established evidence that Myc over-expression in mammalian cells promotes caspase activation and cell death ( Karlsson et al . , 2003; Ray et al . , 2006 ) .", "To investigate the effects of Myc expression we used a recombinant lentivirus to transduce the c-Myc gene under the control of a constitutively active CMV promoter into MCF10A cells , an immortalized but not transformed human mammary epithelial cell line ( Soule et al . , 1990; Tait et al . , 1990 ) .", "We reasoned that if some of the Myc-expressing cells can survive caspase activation , they may possess higher levels genomic instability , similar to those cells that were exposed to ionizing radiation ( Liu et al . , 2015 ) .", "We quantified Myc’s ability to activate Casp3 by immunofluorescence staining of cleaved Casp3 ( Figure 1A , B ) .", "Roughly 6% of Myc-expressing MCF10A cells were observed with having relatively normal nuclei and cleaved caspase 3 , as compared to control MCF10A cells where only ~1% of the cells were observed with cleaved caspase 3 .", "We also examined the relationship between Casp3 activation and cellular survival by use of a reporter system described in a prior publication ( Liu et al . , 2015 ) .", "Our data indicate that irrespective of Casp3 activation status in the presence or absence of Myc expression , 40% or more of the individually sorted MCF10A cells can form colonies in 96-well plates ( Figure 1—figure supplement 1 ) .", "Further flow cytometry analysis showed that despite increased Casp3EGFP activities in Myc-expressing MCF10A cells , the levels of Annexin V staining , a well-recognized marker of apoptosis , did not increase significantly ( Figure 1—figure supplement 2 ) .", "Those data provide clear evidence that a significant fraction of MCF10A cells can survive spontaneous or Myc-induced Casp3 activation . 10 . 7554/eLife . 26371 . 003Figure 1 . Requirement for Casp3 in Myc-induced genomic instability .", "( A ) Representative immunofluorescence staining of MCF10A cells with cleaved caspase 3 ( green ) and DAPI ( blue ) .", "Scale bar represents 10 μm .", "( B ) The proportion of nonapoptotic MCF10A cells presenting with normal nuclear morphology and cleaved caspase 3 signal .", "( C ) Western blot analysis of Caspase-3 status in MCF10A cells with or without CASP3 gene knockout .", "( D ) Representative immunofluorescence γH2AX foci ( green ) and DAPI staining in MCF10A cells with wild type ( left panel ) and CASP3KO .", "Scale bar represents 20 μm .", "( E ) Fraction of cells which stained positive for a γH2AX foci in control and Casp3-deficient MCF10A with or without exogenous expression of Myc , n = 3 .", "( F ) A representative image of chromosome staining in MCF10A cells .", "A dicentric chromosome is indicated by an arrow .", "( G ) Fraction of cells containing at least one chromosome aberration in control and Casp3-deficient MCF10A with or without exogenous expression of Myc .", "In B , E , and G , error bars represent standard error of the mean ( SEM ) , * indicates a p value < 0 . 01 , ** indicates a p value > 0 . 1 , Student’s t-test , n = 3 .", "For B , E , each data point was derived from the average of three triplicate groups of 150 cells each .", "In G , each data point was derived from the average of three triplicate groups of 50 cells each . DOI: http://dx . doi . org/10 . 7554/eLife . 26371 . 003 10 . 7554/eLife . 26371 . 004Figure 1—source data 1 . Data for Figure 1B . DOI: http://dx . doi . org/10 . 7554/eLife . 26371 . 004 10 . 7554/eLife . 26371 . 005Figure 1—source data 2 . Data for Figure 1E . DOI: http://dx . doi . org/10 . 7554/eLife . 26371 . 005 10 . 7554/eLife . 26371 . 006Figure 1—source data 3 . Data for Figure 1G . DOI: http://dx . doi . org/10 . 7554/eLife . 26371 . 006 10 . 7554/eLife . 26371 . 007Figure 1—source data 4 . Data for Figure 1—figure supplement 3A . DOI: http://dx . doi . org/10 . 7554/eLife . 26371 . 007 10 . 7554/eLife . 26371 . 008Figure 1—source data 5 . Data for Figure 1—figure supplement 3B . DOI: http://dx . doi . org/10 . 7554/eLife . 26371 . 008 10 . 7554/eLife . 26371 . 009Figure 1—source data 6 . Data for Figure 1—figure supplement 5 . DOI: http://dx . doi . org/10 . 7554/eLife . 26371 . 009 10 . 7554/eLife . 26371 . 010Figure 1—source data 7 . Data for Figure 1—figure supplement 6B . DOI: http://dx . doi . org/10 . 7554/eLife . 26371 . 010 10 . 7554/eLife . 26371 . 011Figure 1—source data 8 . Data for Figure 1—figure supplement 6C . DOI: http://dx . doi . org/10 . 7554/eLife . 26371 . 011 10 . 7554/eLife . 26371 . 012Figure 1—source data 9 . Data for Figure 1—figure supplement 7B . DOI: http://dx . doi . org/10 . 7554/eLife . 26371 . 012 10 . 7554/eLife . 26371 . 013Figure 1—source data 10 . Data for Figure 1—figure supplement 7C . DOI: http://dx . doi . org/10 . 7554/eLife . 26371 . 013 10 . 7554/eLife . 26371 . 014Figure 1—figure supplement 1 . Clonogenic abilities of control and Myc-expressing MCF10A cells with high and low Casp3 reporter ( Casp3GFP ) activities after being individually sorted into 96-well plates by use of FAC based on their reporter activities . DOI: http://dx . doi . org/10 . 7554/eLife . 26371 . 01410 . 7554/eLife . 26371 . 015Figure 1—figure supplement 2 . Flow cytometry analysis of annexin v-PE staining in Casp3-GFP transduced MCF10A cells with or without Myc expression . There was not a significant increase in annexin V-high cells in Casp3-GFP high cells . DOI: http://dx . doi . org/10 . 7554/eLife . 26371 . 01510 . 7554/eLife . 26371 . 016Figure 1—figure supplement 3 . Additional data for the influence of Casp3 on Myc-induced genetic instability in MCF10A cells .", "( A ) Average number of γH2AX foci per cell in Myc-transduced MCF10A cells with or without CASP3 gene ablation .", "( B ) Average number of chromosomal aberrations per cell in Myc-transduced MCF10A cells with or without CAPS3 gene ablation .", "Error bars represent standard error of the mean ( SEM ) .", "*p>0 . 05; **p<0 . 05; Students t-test , n = 3 .", "In A , each data point was derived from the average of three triplicate groups of 150 cells each .", "In B , each data point was derived from the average of three triplicate groups of 50 cells each . DOI: http://dx . doi . org/10 . 7554/eLife . 26371 . 01610 . 7554/eLife . 26371 . 017Figure 1—figure supplement 4 . Immunofluorescence co-staining of vector control and Myc-transduced MCF10A cells . Notice the heterogeneous nature of Myc expression . DOI: http://dx . doi . org/10 . 7554/eLife . 26371 . 01710 . 7554/eLife . 26371 . 018Figure 1—figure supplement 5 . DNA double-strand repair kinetics in MCF10A cells with various genetic backgrounds . Cells were synchronized and irradiated with 3Gys of X-rays .", "At different time points after radiation exposure , they were fixed and stained with fluorescence-labeled γH2AX antibody .", "The fraction of cells with γH2AX foci were then enumerated from fluorescence images .", "MCF10A Myc vs Casp3KO Myc , **p<0 . 001 , n = 5 , Student’s t-test . DOI: http://dx . doi . org/10 . 7554/eLife . 26371 . 01810 . 7554/eLife . 26371 . 019Figure 1—figure supplement 6 . Additional data confirming the role of Casp3 in mediating Myc-induced genomic instability .", "( A ) Western blot confirmation of Myc over-expression and re-expression Casp3 in Casp3KO cells .", "( B ) The effect of Casp3 re-expression on Myc-induced γH2AX foci in MCF10A-CASP3KO cells .", "( C ) The effect of dominant-negative Casp3 ( C163A mutation ) on Myc-induced γH2AX foci in MCF10A-CASP3KO cells .", "Error bars represent standard error of the mean ( SEM ) .", "*p>0 . 05; **p<0 . 05 .", "In B and C , each data point was derived from the average of three triplicate groups of 150 cells each . DOI: http://dx . doi . org/10 . 7554/eLife . 26371 . 01910 . 7554/eLife . 26371 . 020Figure 1—figure supplement 7 . Role of Casp3 in Myc-induced genetic instability in BJ-1 human fibroblasts .", "( A ) Western blot showing CASP3 gene knockout in immortalized human BJ1 fibroblasts .", "( B ) Quantitative estimate of Myc induced γH2AX foci in control and CASP3 knockout BJ1 fibroblasts .", "( C ) Quantitative estimate of Myc-induced chromosomal aberrations in control and CAPS3 knockout BJ1 human fibroblast cells .", "In B , C , error bars represent SEM .", "*p>0 . 05 , **p<0 . 001 , Student’s t-test , n = 3 .", "In B , each data point was derived from the average of three triplicate groups of 150 cells each .", "In C , each data point was derived from the average of three triplicate groups of 50 cells each . DOI: http://dx . doi . org/10 . 7554/eLife . 26371 . 020 To examine if Casp3 plays a causative role in Myc-induced genomic instability and carcinogenesis , we generated MCF10A and BJ-1hTERT cells with CASP3 gene knockout by use of the CRISPR/Cas9 technology ( Figure 1C , Tables 1–3 ) .", "Control and Casp3-deficient MCF10A cells with and without exogenous Myc expression were then analyzed for both chromosome aberrations and γH2AX foci , two well-established markers of genomic instability .", "In control MCF10A cells , Myc overexpression caused significant increases in both the fraction of cells with and the average numbers per cell of γH2AX foci and chromosomal aberrations ( Figure 1D–G , Figure 1—figure supplement 3 ) .", "In contrast , Myc overexpression in Casp3-deficient cells induced no increases in the numbers of either chromosome aberrations or γH2AX foci when compared to control MCF10A or CASP3 knockout ( CASP3KO ) cells without Myc overexpression ( Figure 1D–G , Figure 1—figure supplement 3 ) .", "To examine the relationship between c-Myc expression and γH2AX foci induction , we carried out immunofluorescence staining of c-Myc and γH2AX ( Figure 1—figure supplement 4 ) .", "Our results indicate that c-Myc expression was not homogeneous , perhaps a reflection of the silencing of the CMV promoter that controlled c-Myc expression .", "Furthermore , γH2AX foci presence did not always correlate with high c-Myc expression , perhaps indicating a stochastic nature of γH2AX foci induction .", "However , Myc expression did have a strong influence on both the baseline and induced levels of γH2AX foci and their repair kinetics in MCF10A cells .", "A systematic analysis on the repair of radiation-induced γH2AX foci showed that Myc expression caused not only higher background levels of γH2AX foci but also higher residual foci levels after a significant number of the induced foci were repaired .", "On the other hand , CASP3 knockout eliminated most of the basal and residual levels of γH2AX foci in MCF10A cells ( Figure 1—figure supplement 5 ) . 10 . 7554/eLife . 26371 . 021Table 1 . Primary antibodies used in this study . DOI: http://dx . doi . org/10 . 7554/eLife . 26371 . 021Target proteinAntibody sourceClone informationγH2AX ( Ser139 ) Upstate BiotechnologyJBW301 , Mouse mAbCaspase-3 ( full length ) Cell Signaling Technology8G10 , Rabbit mAbCaspase-3 ( cleaved , Asp175 ) Cell Signaling Technology5A1E , Rabbit mAbEndoGChemiconRabbit polyclonalHA epitopeNovus BiologicalsGoat polyclonalβ-ActinNovus BiologicalsMouse mAbc-MycCell Signaling TechnologyRabbit mAbMito MarkerThermo Fisher ScientificN/A10 . 7554/eLife . 26371 . 022Table 2 . Single guided RNA ( sgRNA ) sequences used in this study . DOI: http://dx . doi . org/10 . 7554/eLife . 26371 . 022GeneAccessionsgRNA oligo ( 5`−3` ) *Targeted exonCASPASE3NC_000004CACCGcatacatggaagcgaatcaa AAACttgattcgcttccatgtatgCExon4CASPASE3NC_000004CACCGggaagcgaatcaatggactc AAACgagtccattgattcgcttccCExon4EndoGNC_000009CACCGgggctgggtgcggtcgtcga AAACtcgacgaccgcacccagcccCExon1EndoGNC_000009CACCGcgacttccgcgaggacgact AAACagtcgtcctcgcggaagtcgCExon1*Capital letters: enzyme overhangs; non-capital letters: sgRNA target guide sequence . 10 . 7554/eLife . 26371 . 023Table 3 . Mutations at target sequences in various CRISPR knockout MCF10A and BJ-1 hTERT cells . DOI: http://dx . doi . org/10 . 7554/eLife . 26371 . 023 5`……3`MutationCasp3 KOMCF10AClone1: AAAGATCATACATGGAAGCGAATCAATGGA - - - - - - - ATAT Casp3: AAAGATCATACATGGAAGCGAATCAATGGACTCTGGAATAT7 bp deletionCasp3 KOBJ-1hTERTClone28: AAAGATCATACATGGAAGCGAATCAATG - - - deletion-------- Casp3: AAAGATCATACATGGAAGCGAATCAATGGACTCTGGAATAT193 bp deletionEndoG KOMCF10AClone13: -------- deletion-------- EndoG: TGCCACCAACGCCGACTACCGCGGCAGTGGCTTCGACCGCG169 bp deletionNote: Red: sgRNA sequence; Yellow: PAM sequence; Bold: insertion sequence; -: deletion sequence .", "In all cases , knockout clones that showed both clear absence of target protein expression and gene mutations were chosen .", "In addition , in most cases , only those clones with homozygous mutations ( where both copies of the gene showed the same mutation ) were chosen for convenience .", "In order to rule out the possibility that our CASP3KO cells suffered off-target effects during the generation process , we re-expressed Casp3 in CASP3KO MCF10A cells ( Figure 1—figure supplement 6A ) and examined for Myc-induced γH2AX foci .", "Our results indicate that Casp3 re-expression restored Myc-induced DNA damage foci ( Figure 1—figure supplement 6B ) .", "In a parallel experiment , we expressed a dominant-negative CAPS3 gene ( dnCASP3 ) in Casp3KO cells .", "DnCasp3 differs from wild-type Casp3 in only a single amino acid that eliminates its cleavage activities ( Stennicke and Salvesen , 1997 ) .", "In contrast to wild-type Casp3 re-expression , dnCASP3 re-expression did not restore the ability of Myc to induce γH2AX foci ( Figure 1—figure supplement 6C ) .", "In order to make sure that our observations so far are not restricted to MCF10A cells , we generated CASP3 gene knockout cells from hTERT immortalized BJ1 human fibroblast cells ( Figure 1—figure supplement 7A , Table 3 ) to assess the effects of Casp3 on myc-induced genomic instability .", "Similar to MCF10A cells , we observed that Myc overexpression resulted in statistically significant increases in γH2AX foci ( Figure 1—figure supplement 7B ) and chromosomal aberrations ( Figure 1—figure supplement 7C ) in cells overexpressing Myc .", "However , such increases were almost completely eliminated in CASP3KO BJ1 cells ( Figure 1—figure supplement 7B , C ) , similar to CASP3KO MCF10A cells .", "Since Myc-induced genomic instability is intimately associated with its ability to transform mammalian cells , we investigated Myc-induced tumorigenicity in MCF10A cells .", "We initially evaluated Myc-induced tumorigenicity of MCF10A cells by use of the soft agar colony forming assay , a well-establish assay that evaluates the anchorage independence ability of putative tumor cells .", "Our results indicate that Myc overexpression in control MCF10A resulted in a significant increase in the number of observed soft agar colonies ( Figure 2A , B ) .", "However , such increases were completely absent in Casp3 knockout cells ( Figure 2A , B ) .", "The causative role for Casp3 in this process was further demonstrated in Casp3KO cells with Casp3 re-expression , the ability of Myc to induce soft agar colony formation was restored ( Figure 2C ) .", "In control MCF10A cells , expression of an exogenous Casp3 caused no increase in Myc-induced soft agar colony formation ( Figure 2C ) . 10 . 7554/eLife . 26371 . 024Figure 2 . Requirement for Casp3 in Myc-induced transformation .", "( A ) Depicts colonies which grew in soft agar .", "( B ) Average number of colonies in soft agar in control and Casp3-deficient cells with or without Myc expression .", "( C ) Average number of colonies in soft agar in wild type and Casp3 knockout MCF10A cells with Casp3 re-expression in the absence or presence of Myc over-expression .", "( D ) Tumor growth from control , Myc-overexpressing , and Casp3KO cells with Myc over-expressiong in nude mice .", "( E ) Individual tumor sizes in nude mice form wild-type cells with Myc over-expression .", "The error bars in B , D , and E represent standard error of the mean ( SEM ) .", "* Indicates p value < 0 . 001 , ** indicates p value << 1e−5 , *** indicates a p value > 0 . 1 .", "Student’s t-test was used to calculate the p-values in B and C . N = 3 for B and C . N = 5 for D . DOI: http://dx . doi . org/10 . 7554/eLife . 26371 . 024 10 . 7554/eLife . 26371 . 025Figure 2—source data 1 . Data for Figure 2B . DOI: http://dx . doi . org/10 . 7554/eLife . 26371 . 025 10 . 7554/eLife . 26371 . 026Figure 2—source data 2 . Data for Figure 2C . DOI: http://dx . doi . org/10 . 7554/eLife . 26371 . 026 10 . 7554/eLife . 26371 . 027Figure 2—source data 3 . Data for Figure 2D&E . DOI: http://dx . doi . org/10 . 7554/eLife . 26371 . 027 In a further experiment , we examined the ability of Myc-transduced control or CASP3KO cells to form tumors in nude mice .", "Despite injection of 4 × 106 cells each , only the group of nude mice that were injected with Myc-transduced control MCF10A cell were able to form tumors after 8 weeks of observation ( Figure 2D ) , thereby confirming that both Myc over-expression and an intact CAPSP3 gene are required for oncogenic transformation .", "A more detailed examination showed that in Myc over-expressing MCF10A cells , five out of eight injected sites formed tumors , albeit with varying growth kinetics ( Figure 2E ) .", "The in vivo data here are striking in that Casp3 deficiency completely blocked the ability of Myc to transform MCF10A cells .", "In vitro , although Casp3 deficiency significantly reduced Myc-induced soft agar colony formation , it was not completely blocked .", "The discrepancy between the in vitro and in vivo assays perhaps reflected the different properties that the two assays are measuring .", "We next sought to determine the downstream effectors of Casp3 in mediating Myc-induced genomic instability and oncogenic transformation .", "In a previous study , endonuclease G ( endo G ) , an apoptotic endonuclease that normally resides within the mitochondria and migrates to the nucleus after activation of apoptotic cascade ( Parrish et al . , 2001; Li et al . , 2001 ) , is responsible for much of the Casp3-induced genomic instability after stress exposure .", "In order to evaluate if endoG is involved in Myc-induced genomic stability , we determined the cellular location of endoG in control and Myc expressing MCF10A cells by use of immunofluorescence staining .", "Our results show that there was a significant increase in the fraction of cells with endoG nuclear migration in Myc-expressing MCF10A vs control cells ( 11% vs 2% , Figure 3A , B ) .", "However , the increase was completely eliminated in CASP3 knockout cells ( Figure 3B ) . 10 . 7554/eLife . 26371 . 028Figure 3 . Requirement of EndoG in Myc-induced genomic instability and transformation .", "( A ) Immunofluorescence staining of of MCF10A with antibodies to EndoG ( green ) , mitochondria ( Orange ) and DAPI ( blue ) .", "Scale bar represents 20 μm .", "( B ) Fraction of MCF10A cells with activated EndoG ( EndoG signal within the nucleus ) .", "Error bar indicates SEM .", "( C ) Western blot analysis fo EndoG expression in wild type or endoG knockout MCF10A cells .", "( D ) Fraction of cells which stained positive for a γH2AX foci in control and EndoG-deficient MCF10A with or without exogenous expression of Myc .", "( E ) Influence of endoG status on Myc-induced transformation of MCF10A cells , as indicted by soft agar ( SA ) colony formation .", "* Indicates p value < 0 . 001 , **p>0 . 05 .", "Student’s t-test in B , D , E . Error bars represent SEM .", "In B , D , each data point was derived from the average of three triplicate groups of 150 cells each .", "In E , n = 3 . DOI: http://dx . doi . org/10 . 7554/eLife . 26371 . 028 10 . 7554/eLife . 26371 . 029Figure 3—source data 1 . Data for Figure 3B . DOI: http://dx . doi . org/10 . 7554/eLife . 26371 . 029 10 . 7554/eLife . 26371 . 030Figure 3—source data 2 . Data for Figure 3D . DOI: http://dx . doi . org/10 . 7554/eLife . 26371 . 030 10 . 7554/eLife . 26371 . 031Figure 3—source data 3 . Data for Figure 3E . DOI: http://dx . doi . org/10 . 7554/eLife . 26371 . 031 10 . 7554/eLife . 26371 . 032Figure 3—source data 4 . Data for Figure 3—figure supplement 1 . DOI: http://dx . doi . org/10 . 7554/eLife . 26371 . 032 10 . 7554/eLife . 26371 . 033Figure 3—source data 5 . Data for Figure 3—figure supplement 4 . DOI: http://dx . doi . org/10 . 7554/eLife . 26371 . 033 10 . 7554/eLife . 26371 . 034Figure 3—figure supplement 1 . Additional data showing the average number of γH2AX foci per cell in control and EndoG deficient MCF10A cells with or without exogenous expression of Myc . Error bar indicates SEM .", "* indicates p<0 . 01 , Student’s t-test , n = 3 .", "Each data point was derived from the average of three triplicate groups of 150 cells each . DOI: http://dx . doi . org/10 . 7554/eLife . 26371 . 03410 . 7554/eLife . 26371 . 035Figure 3—figure supplement 2 . Flow cytometry analysis of various MCF10A cells for PI and annexin V staining to quantitate the fraction of cell undergoing early ( lower right quadrants ) , late ( top right quadrants ) apoptosis , and necrosis ( top left quadrants ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 26371 . 03510 . 7554/eLife . 26371 . 036Figure 3—figure supplement 3 . Flow cytometry level analysis of reactive oxygen species ( ROS ) levels in various MCF10A cells . Top panels .", "ROS levels in MCF10A vs CASP3KO cells ( left ) and MCF10A vs ENDOGKO cells ( right ) .", "Lower panels , ROS levels in wild type ( left ) , CASP3KO ( middle ) , and ENDOGKO ( right ) MCF10A cells with or without Myc expression . DOI: http://dx . doi . org/10 . 7554/eLife . 26371 . 03610 . 7554/eLife . 26371 . 037Figure 3—figure supplement 4 . Relative levels of mitochondrial DNA that leaked into the cytoplasm in control as wells genetically modified MCF10A cells . Cytoplasmic mtND5 of indicated cells were analyzed by use of quantitative RT-PCR .", "The levels of 18S rDNA were also determined to serve as genomic DNA control .", "Error bars represent standard error of the mean ( SEM ) , n = 3 , p values derived from Student’s t-test . DOI: http://dx . doi . org/10 . 7554/eLife . 26371 . 037 In order to determine if endoG plays a causative role in Myc-induced genomic damage and transformation , we created MCF10A cells with ENDOG gene knockout by use of CRISPR/Cas9 ( Figure 3C ) .", "We then evaluated the abilities of Myc to induce γH2AX foci and oncogenic transformation .", "Our results show that ENDOG deletion was able to eliminate both Myc-induced γH2AX foci ( Figure 3D and Figure 3—figure supplement", "1 ) and oncogenic transformation as evaluated by use of the soft agar colony-forming assay ( Figure 3E ) .", "To further determine the relationships among Myc expression , CASP3 status , ENDOG status , and apoptosis , we carried out flow cytometry analysis of Annexin V and PI ( propidium iondine ) staining , which allowed for the identification of different stages of cellular apoptosis .", "Our results ( Figure 3—figure supplement", "2 ) indicate that CASP3KO caused small reductions in both Annexin V+ and Annexin V+/PI+ MCF10A cells when compared with control cells .", "However , ENDOG knockout caused small increases in fractions of Annexin V+/PI- and Annexin V-/PI+ cells when compared with control cells .", "Myc expression , on the other hand , caused increases in fractions of Annexin V+/PI+ cells I all three cell populations .", "Overall , while the relative changes could be sizable , the absolute changes caused by the knockouts or Myc expression in terms of PI+ or Annexin V+ cells were small .", "Increased ROS has been previously implicated in Myc-induced carcinogenesis .", "Our data so far has suggested strongly that the Casp3 activation and endoG release from the mitochondria played decisive roles in Myc-induced carcinogenesis .", "In order to determine if ROS levels in MCF10A cells correspond with oncogenic transformation , we did DCFDA-based flow cytometry analysis ROS levels in various MCF10A-derived cells ( Figure 3—figure supplement 3 ) .", "Our results indicate that CASP3 or ENDOG knockout caused small increase and decrease in MCF10A cells ( Figure 3—figure supplement 3 , top panels ) , respectively .", "Forced MYC expression increased ROS in wild-type MCF10A cells but not in CASP3KO or ENDOGKO MCF10A cells ( Figure 3—figure supplement 3 , mid-panels ) .", "Further , Myc was able to induce significantly more ROS in wild-type MCF10A cells than CASP3KO cells , but about equal levels of ROS in wild type vs ENDOGKO cells ( Figure 3—figure supplement 3 , lower panels ) .", "Those data suggest that although ROS production appeared to track with carcinogenesis in wild-type and CASP3KO cells , it did not in ENDOGKO cells .", "In order to determine whether Casp3 activation and endoG release is the result of partial damage of many mitochondria vs severe damage to a small number of mitochondria , a quantitative PCR ( qPCR ) analysis of cytoplasmic mitochondrial DNA ( mtDNA ) levels was done ( Figure 3—figure supplement 4 ) .", "Our results indicate that MYC expression in MCF10A cells caused a significant increase in cytoplasmic mtDNA levels , indicating that a significant fraction of mitochondria had compromised membrane integrity .", "On the other hand , CASP3KO significantly reduced cytoplasmic mtDNA levels in MCF10A cells with or without Myc over expression .", "While we are not clear of the exact cause of this , we speculate that Casp3 and other upstream factors that promote mitochondrial leakage form a positive loop to promote mitochondrial leakage with or without Myc expression .", "To further examine if endoG leakage from the mitochondria and migration is sufficient to cause genomic instability and oncogenic transformation , we engineered a modified endoG protein where the native mitochondrial localization signal is switched to a nuclear localization signal ( NLS-EndoG , Figure 4A ) and transduced it into MCF10ACASP3KO cells with or without Myc expression ( Figure 4B ) .", "Immunofluorescence staining confirmed the nuclear localization of the engineered endoG ( Figure 3C ) .", "We then determined the incidence of γH2AX foci in transduced cells .", "Our results indicate the NLS-endoG expression restored the depleted γH2AX foci induction by Myc in CASO3KO cells ( Figure 4D ) .", "In fact , NLS-endoG alone was sufficient to induce γH2X foci in CASP3KO cells to levels induced by Myc ( Figure 4D ) . 10 . 7554/eLife . 26371 . 038Figure 4 . A nucleus-located EndoG restores Myc induced transformation in Casp3-deficient cells .", "( A ) A diagram showing a re-engineered endoG with a nucleus localization signal ( NLS ) at its tagged N-terminal .", "( B ) Western blot demonstrating exogenous myc and NLS-EndoG expression by use of an anti-HA antibody .", "( C ) Immunoflouresence staining of EndoG ( green ) , mito marker ( red ) , and DAPI ( blue ) in NLS-EndoG transduced CASP3KO cells .", "Scale bar indicates 20 µm .", "( D ) Average number of γH2AX foci per cell in MCF10A with or without nEndoG in the absence or presence of Myc or Casp3KO .", "( E ) The influence of nEndoG on soft agar formation in MCF10A-Casp3KO cells with or without Myc gene expression .", "( F ) Xenograft tumor formation in nude mice from MCF10A-Casp3KO cells with NLS-EndoG ( nEndoG ) and/or Myc expression .", "In D , E , F error bars represent standard error of the mean ( SEM ) , * indicates p value < 0 . 001 while ** Indicates p value > 0 . 1 .", "Student’s t-test .", "In D , each data point was derived from the average of three triplicate groups of 150 cells each .", "In E , n = 3 .", "In F , n = 5 . DOI: http://dx . doi . org/10 . 7554/eLife . 26371 . 038 10 . 7554/eLife . 26371 . 039Figure 4—source data 1 . Data for Figure 4D . DOI: http://dx . doi . org/10 . 7554/eLife . 26371 . 039 10 . 7554/eLife . 26371 . 040Figure 4—source data 2 . Data for Figure 4E . DOI: http://dx . doi . org/10 . 7554/eLife . 26371 . 040 10 . 7554/eLife . 26371 . 041Figure 4—source data 3 . Data for Figure 4F . DOI: http://dx . doi . org/10 . 7554/eLife . 26371 . 041 We next examined the influence of NLS-EndoG on oncogenic transformation by use of the soft agar assay .", "Our results show that NLS-EndoG restored the ability of Myc to induce oncogenic transformation in Casp3 knockout MCF10A cells ( Figure 4E ) .", "However , NLS-EndoG expression alone was not sufficient to induce oncogenic transformation despite its ability to induce DNA damage foci to levels similar to Myc expression ( Figure 4E ) .", "Those results suggest while endoG nuclear migration is a necessary condition for Myc-induced oncogenic transformation , it is not sufficient by itself .", "Additional activities of Myc are clearly required in the transformation process .", "A further tumor formation experiment was conducted in nude mice to examine the role of NLS-EndoG in oncogenic transformation .", "NLS-EndoG restored the ability of Myc to induce oncogenic transformation in CASP3KO MCF10A cells ( Figure 4F ) , with five out of eight inoculations formed tumors .", "On the other hand , NLS-EndoG alone was not able to make MCF10A cells with CASP3 gene knockout to become tumorigenic , consistent with results from soft agar colony formation assay ( Figure 4E ) .", "Despite being one of the first oncogenes identified and having numerous studies dedicated to discovering its roles in cancer biology , there are still many gaps in our knowledge about Myc .", "The present study provides significant new insights into the roles of Myc in carcinogenesis .", "In particular , it resolves two apparently paradoxical observations regarding Myc: its ability to stimulate apoptosis and to induce genomic instability and oncogenic transformation .", "In many instances , such as in the case of p53 , apoptosis induction is anti-carcinogenic due to its ability to remove damaged cells from the body .", "However , in the case of Myc , the apoptotic machinery , is exploited by Myc as a vehicle to cause genomic instability and induce oncogenic transformation .", "Most strikingly , our data suggest that Casp3 and Endo G , two well-established apoptosis effectors , are activated and required for Myc-induced oncogenic transformation .", "This finding suggest that Myc-induced activation of apoptosis , instead of being a result of Myc induced cellular stress , is actually part and parcel of Myc’s capacity to induce mammalian transformation .", "One important piece of information that remain missing is how Myc induces mitochondrial leakage and Casp3 activation .", "While we do not have any experimental evidence at present , it is possible that deregulation in mitochondrial biogenesis , which is known to be stimulated by Myc , may be responsible for it , as suggested previously ( Dang et al . , 2005; Zhang et al . , 2007; Ahuja et al . , 2010 ) .", "This possibility should be investigated in future studies since it may holds the key to Myc’s powerful oncogenic abilities .", "On the surface , our discovery appears to be contrary to the established paradigm that apoptosis is a key barrier for carcinogenesis ( Hanahan and Weinberg , 2011 ) .", "However , it is consistent with an increasing body of literature that suggest a pro-oncogenic role for apoptosis and some apoptotic factors ( Tang et al . , 2012; Ichim et al . , 2015; Liu et al . , 2015 ) .", "The key conceptual advance in the studies is the realization of that cells exposed to stress can survive caspase activation ( Tang et al . , 2012; Ichim et al . , 2015; Liu et al . , 2015; Ding et al . , 2016; Ichim and Tait , 2016 ) .", "The observation of such survival in development ( Ding et al . , 2016 ) , chemical exposure ( Tang et al . , 2012; Ichim et al . , 2015 ) , and radiation exposure ( Liu et al . , 2015 ) indicate that it is a wide spread phenomenon .", "Our observation of cells surviving Myc-induced caspase activation is a significant extension of those earlier observations and may provide important insights into the relationship between apoptosis and oncogene-induced transformation beyond that of Myc .", "Taken together , our study provides crucial evidence that Casp3 and endo G , two key factors in the canonical apoptosis pathway , play essential , facilitative roles in Myc-induced oncogenic transformation ." ], [ "Early passage , immortalized , non-transformed human breast epithelial cell line , MCF10A , was a kind gift from Dr . Hatsumi Nagasawa of Colorado State University ( Fort Collins , CO ) .", "MCF10A growth medium was composed of Dulbacco’s modified Eagle’s medium ( DMEM ) /F12 ( Sigma ) supplemented with 5% donor horse serum ( Sigma ) , 20 ng/ml of epidermal growth factor ( EGF; R&D Systems ) , 0 . 5 µg/ml hydrocortisone ( Sigma ) , 100 ng/ml cholera toxin ( Sigma ) , 10 µg/ml insulin ( Invitrogen ) , and 100 units/ml penicillin and 100 µg/ml streptomycin .", "hTERT immortalized , non-transformed human fibroblast cell line , hTERT BJ-1 , was a kind gift from Dr . Takamitsu Kato of Colorado State University .", "hTERT BJ-1 growth medium was composed of DMEM supplemented with 10% fetal bovine serum ( Sigma ) and 100 units/ml penicillin and 100 µg/ml streptomycin .", "The identities of both MCF10A and hTERT-BJ1 were authenticated through STR profiling methods .", "Throughout the course of the experiments , the cells were also checked periodically for the absence of mycoplasma contamination .", "For γH2AX foci assays , the cells were synchronized in G1 using the well-established double-thymidine block protocol ( Bostock et al . , 1971 ) .", "Briefly , cells were plated on glass-bottom 35 mm petri dishes ( MatTek , Ashland , MA ) and cultured with growth medium for overnight .", "They were incubated with 2 mM thymidine for 18 hr , washed 2x with PBS , and incubated for 10–12 hr in growth media .", "They were then incubated for 15–18 hr with 2 mM thymidine .", "After synchronization , cells were fixed with 4% PFA and permeabilized and blocked in PBS containing 0 . 1% Triton X-100 , 5% goat serum , and 1% BSA .", "Cells were incubated with a primary antibody against γH2AX ( Upstate Biotechnology , Lake Placid , NY ) , wash with PBS and incubated with a secondary antibody conjugated with Alexa Fluor 488 ( Invitrogen ) .", "Cells were mounted with mounting medium ( Vector Laboratories ) containing DAPI .", "Fluorescent images of γH2AX were acquired with a Zeiss fluorescence microscope with a 63x oil objective ( Axio Observer Z1 ) .", "For each experimental group we observed 150 cells in triplicate .", "We carried out chromosome aberration analysis in cultured cells following an established protocol ( Savage , 1976 ) .", "We analyzed for various chromosome/chromatid aberrations that include breaks/gaps , dicentrics , centric/acentric rings , and translocations .", "Each data points represent data from 50 cells in triplicate .", "We made various cells deficient in various genes by use of the CRISPR/Cas9 technology .", "Single-guided RNA ( sgRNA ) sequences targeting the genes were generated with the use of a free online CRISPR design tool ( crispr . mit . edu ) .", "The sgRNA sequences used were listed in Table 2 .", "Annealed double stranded sgRNA oligos were ligated into the lentiCRISPR vector ( Cong et al . , 2013 ) ( deposited by Dr . Feng Zhang to Addgene , Cambridge , MA ) at BsmBl site , which co-express cas9 and sgRNA in the same vector .", "The constructed CRISPR lentivirus vectors were then packaged according to a standard protocol .", "To produce lentiviral vectors , lentiviral plasmids with the target genes were transduced into 293 T cells together with second-generation packaging plasmids ( psPAX2 , pMD2 . G ) following previously published procedures: http://tronolab . epfl . ch/lentivectors .", "Cells were cultured on glass-bottom 35 mm petri dishes .", "Cells were fixed with 4% paraformaldehyde ( PFA ) in PBS for 15 min , permeabilized and blocked with PBS containing 5% goat serum , 0 . 1% Triton X-100 , and 1% bovine serum albumin ( BSA ) for 45 min .", "Fixed cells were incubated with primary antibodies for cleaved Caspase-3 , γH2AX , or EndoG overnight at 4C , followed by incubation with appropriate Alexa Fluor 488-conjugated secondary antibodies ( Invitrogen , Carlsbad , CA ) for 1 hr and mounted with mounting medium ( Vector Laboratories , CA ) containing DAPI .", "Fluorescent images were acquired with a Zeiss fluorescence microscope with a 63x oil objective ( Axio Observer Z1 ) .", "The soft-agar assay was carried out following an established procedure ( Cifone and Fidler , 1980 ) .", "About 1 × 104 MCF10A cells in growth medium were plated into six-well plates with 1 . 5 ml 0 . 3% ( m/v ) low-melting agar ( BD , Sparks , MD , which was overlaid onto 1 . 5 ml 0 . 6% ( w/v ) bottom agar layer .", "Soft-agar colonies were maintained at 37°C and fed twice a week by drop-wise addition of growth medium for colony formation .", "After 21 days in culture , the colonies were counted after staining with 0 . 005% crystal violet .", "In order to measure reactive oxygen species , the cells were labeled with DCFDA ( 20 µM ) according to the manufacturer’s instruction that comes with the ROS kit ( Abcam , Cambridge , MA ) .", "The cells were then analyzed by use of flow cytometry .", "To measure cytoplasmic mtDNA in various MCF10A-derived cells , the cytoplasmic fraction of the cellular lysates were isolated according to a published protocol ( Bronner and O’Riordan , 2016 ) .", "To quantify mtDNA , a segment of the mtND5 gene was amplified by use of the primer pair ( Neufeld-Cohen et al . , 2016 ) : forward 5′-ACGAAAATGACCCAGACCTC-3′ , rev 5′-GAGATGACAAATCCTGCAAAGATG-3′ through Q-PCR .", "A pair of primers for 18 s rDNA ( Forward: 5’-TAGAGGGACAAGTGGCGTTC-3’ Reverse: 5’-CGCTGAGCCAGTCAGTGT-3’ ) was used as control .", "Animal experiments conducted in this study were approved by the Duke University Institutional Animal Use and Care Committee ( protocol# A195-14-08 ) .", "To confirm the tumorigenicity of myc overexpressing MCF10A cells , about 4 × 106 cells were injected subcutaneously into the flanks of 6–8 week-old , female athymic nude mice ( Jackson Laboratories ) in 50 µl of 1:1 Matrigel ( Corning ) and PBS .", "After inoculation , the growth of tumors was evaluated once a week for 8 weeks .", "Student’s t-test was used to evaluate the significance of differences between different experimental groups .", "In most cases , a p-value of less than 0 . 05 was deemed as significant while a p-value of more than 0 . 05 was deemed not significant ." ] ]

[ "The mechanism for Myc-induced genetic instability is not well understood .", "Here we show that sublethal activation of Caspase-3 plays an essential , facilitative role in Myc-induced genomic instability and oncogenic transformation .", "Overexpression of Myc resulted in increased numbers of chromosome aberrations and γH2AX foci in non-transformed MCF10A human mammary epithelial cells .", "However , such increases were almost completely eliminated in isogenic cells with CASP3 gene ablation .", "Furthermore , we show that endonuclease G , an apoptotic nuclease downstream of Caspase-3 , is directly responsible for Myc-induced genetic instability .", "Genetic ablation of either CASP3 or ENDOG prevented Myc-induced oncogenic transformation of MCF10A cells .", "Taken together , we believe that Caspase-3 plays a critical , unexpected role in mediating Myc-induced genetic instability and transformation in mammalian cells ." ]

[ "Healthy cells can become cancerous if their DNA is damaged and not repaired properly , leading to changes in the DNA known as mutations .", "The cells tend to accumulate more and more mutations – a phenomenon known as genomic instability – as they transition into cancer cells .", "A protein called Myc is known to promote genomic instability and contributes to many types of cancers .", "However , high levels of Myc proteins in a cell can also activate proteins that trigger a process called apoptosis , which makes the cell commit suicide .", "This role does not appear to fit with the cancer-promoting properties of Myc because apoptosis is generally thought to protect against cancer by helping to remove damaged cells from the body .", "Two of the proteins that Myc activates are known as caspase-3 and endonuclease G . In healthy cells , caspase-3 triggers a series of events that lead to apoptosis , while endonuclease G cuts up DNA in preparation for cell death .", "However , it is not known how these proteins affect the cancer-promoting properties of Myc .", "Here , Cartwright , Liu et al . used a gene editing technique called CRISPR-Cas9 to examine how these apoptosis proteins affect the ability of Myc to promote cancer .", "Increasing the production of Myc in healthy human mammary cells transformed these cells into breast cancer cells that were capable of forming tumors when injected into mice .", "However , in cells that could not make caspase-3 or endonuclease G , increasing the production of Myc did not lead to the cells becoming cancerous .", "Further experiments show that when Myc levels are high , the activation of caspase-3 and endonuclease G is not sufficient to kill the cells .", "As a result , endonuclease G causes damage to the DNA and promotes genomic instability .", "Future studies may focus on understanding exactly how and when the apoptosis proteins can contribute to cancer growth .", "With this knowledge , it may be possible to prevent or treat Myc-driven cancers by changing how these apoptosis proteins behave ." ]

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

[ "epidemiology and global health" ]

[ [ "Engaging in forest activities , such as logging , hunting , or spending the night in the forest , is considered a primary risk factor for malaria infection in the Greater Mekong Sub-region ( GMS ) ( Chaveepojnkamjorn and Pichainarong , 2004; Das et al . , 2004; Lansang et al . , 1997; Erhart et al . , 2005; Trung et al . , 2004 ) with recent outbreaks attributed to deforestation activities ( Lyttleton , 2016 ) .", "This is most likely the result of increased human exposure to the forest dwelling Anopheles dirus and Anopheles minimus , the most efficient and widespread malaria vectors in the GMS ( Obsomer et al . , 2007; Obsomer et al . , 2013 ) .", "However , deforestation may also alter this ‘forest malaria’ ( Sharma and Kondrashin , 1991 ) ecosystem and eventually reduce malaria incidence , as is generally accepted to be the case in Southeast Asia ( Guerra et al . , 2006 ) .", "Several previous studies have assessed the relationship between deforestation and malaria , but the majority focused outside of the GMS , most notably in the Amazonian forest ( Wayant et al . , 2010; Olson et al . , 2010; Hahn et al . , 2014a; Valle and Clark , 2013; Terrazas et al . , 2015 ) where the evidence has been met with conflicting interpretations ( Tucker Lima et al . , 2017 ) .", "As national malaria programs across the GMS target forest-going populations for prevention and treatment efforts ( Guyant et al . , 2015; WHO , 2016 ) , improved understanding of the relationship between deforestation and malaria is critical for programs to assess and meet national 2030 malaria elimination goals ( World Health Organization , 2016; WHO , 2018 ) .", "In the Amazon , the ‘frontier malaria’ hypothesis ( Sawyer , 1988 ) posits that malaria temporarily increases following deforestation efforts to open a human settlement area in the forest .", "Subsequently , after approximately 6–8 years , settlements become more urbanized and isolated from the surrounding forest , and less suitable for malaria vectors , resulting in reduced malaria transmission ( de Castro et al . , 2006 ) .", "Recent work has challenged this hypothesis , however , and found that some older settlements were also likely to have high malaria incidence ( Ilacqua et al . , 2018 ) , highlighting the importance of assessing the relationship between deforestation and malaria at different spatio-temporal scales ( Singer and de Castro , 2006 ) .", "A recent review of the literature on deforestation and malaria in the Amazon ( Tucker Lima et al . , 2017 ) recommended the integration of multiple socio-economic , demographic and ecological mechanisms to disentangle the relationship between deforestation and malaria .", "The complexity of land-use changes driving deforestation such as urbanization , agriculture , irrigation or resource mining can alter the environment in different ways .", "For example , deforestation in the Amazon has been shown to increase mosquitoes’ larval habitat ( Vittor et al . , 2009 ) through the creation of areas with abundant sunlight and pooling water , resulting in increased human biting activity ( Vittor et al . , 2006 ) .", "Alternatively , immigration and rapid population movements , stirring human-vector interactions , are other mechanisms affecting malaria transmission in frontier areas ( Moreno et al . , 2007 ) .", "A modeling study ( Baeza et al . , 2017 ) showed that the temporal pattern of increased incidence followed by a decrease can vary depending on ecological and socio-economic parameters in frontiers areas .", "The importance of addressing complex confounding structures influencing the relationship between deforestation and malaria was also highlighted by Bauhoff and Busch , 2020 .", "Variables such as temperature ( Beck-Johnson et al . , 2013; Mordecai et al . , 2013 ) , precipitation ( Parham and Michael , 2010; Parham et al . , 2012 ) , or seasonality Hay et al . , 1998 are known environmental predictors of malaria , although the spatio-temporal scale of those effects often varies across different areas ( Teklehaimanot et al . , 2004 ) .", "Furthermore , remote areas may experience higher malaria rates because of poor access to public health services , but also have denser forest cover or lower deforestation rates ( Busch and Ferretti-Gallon , 2017 ) .", "Finally , forest-going populations in the GMS are also at higher risk for malaria ( Parker et al . , 2017 ) due to poor adherence to protective measures against mosquitoes such as insecticide-treated bed nets ( ITNs ) or long-lasting insecticidal hammocks ( LLIHs ) ( Peeters Grietens et al . , 2010; WHO , 2016 ) and inadequate access to treatment ( Guyant et al . , 2015 ) .", "Bauhoff and Busch , 2020 identified only 10 empirical studies that assessed the relationship between deforestation and malaria with appropriate adjustments for confounding .", "Of these , seven reported a positive association ( Austin et al . , 2017; Wayant et al . , 2010; Olson et al . , 2010; Terrazas et al . , 2015; Pattanayak et al . , 2010; Garg , 2015; Fornace et al . , 2016 ) , two did not find any associations ( Bauhoff and Busch , 2020; Hahn et al . , 2014a ) , and one disputed study found a negative association ( Valle and Clark , 2013; Hahn et al . , 2014b; Valle , 2014 ) .", "Most recently , a study found deforestation to increase malaria risk and malaria to decrease deforestation activities in the Amazon , using an instrumental variable analysis to disentangle any reverse causality loop ( MacDonald and Mordecai , 2019 ) .", "However , only half of the above-mentioned studies used high-resolution forest data , with most studies using spatially aggregated data and exploring only a limited range of spatial and temporal scales .", "Only three of these studies were conducted in Southeast Asia ( Pattanayak et al . , 2010; Garg , 2015; Fornace et al . , 2016 ) , and none in the GMS .", "Importantly , all three found that malaria increases after deforestation , but all had limitations .", "The two studies in Indonesia Pattanayak et al . , 2010; Garg , 2015 used coarsely aggregated forest data and potentially biased self-reported malaria data .", "The third study , in Malaysia ( Fornace et al . , 2016 ) , focused on a specific and geographically confined malaria parasite , Plasmodium knowlesi , whose primary host is the long-tailed macaque and whose presence in the GMS , where P . falciparum and P . vivax dominate , is limited .", "In this analysis , we examined the relationship between deforestation and malaria incidence by combining high-resolution forest coverage data ( Hansen et al . , 2013 ) and monthly malaria incidence data from 2013 to 2016 from two separate regions in the GMS: northern Lao People’s Democratic Republic ( PDR ) with very low malaria transmission and southern Lao PDR where P . falciparum and P . vivax are seasonal .", "By conducting the analysis at the village level , we were able to explore a wide range of spatial scales ( 1 , 10 , and 30 km around villages ) that might be relevant in characterizing the relationship between deforestation and malaria .", "In addition , we leveraged the longitudinal nature of both the incidence data collected and the forest data produced from annual remote sensing imagery ( Hansen et al . , 2013 ) to explore the most relevant temporal scales .", "Finally , we considered alternative definitions of deforestation , restricted to areas with at least certain levels of forest cover , to investigate the type of deforestation driving the relationship with malaria .", "To date , no prior studies have quantified the relationship between deforestation and malaria incidence in the GMS .", "Understanding this relationship is especially important in the GMS , where forest-going activities are a main source of income generation ( Lao PDR , 2016 ) and malaria clusters in forest-going populations ( World Health Organization , 2016; WHO , 2016 ) .", "To assess the relationship between deforestation and malaria incidence , we modeled the monthly village-level malaria incidence in two regions of Lao PDR using health facility surveillance data and evaluated the most relevant spatio-temporal scale ." ], [ "Figure 1 shows the average tree crown cover within 10 km for the year 2016 and the percent area within 10 km that experienced forest loss between 2011 and 2016 in two regions of southern and northern Lao PDR .", "Overall , the forest cover was denser in the north than in the south and deforestation over this period was higher in the north than in the south .", "Appendix 1—figure 6 and Appendix 1—figure 7 show the distribution of forest and deforestation variables as the temporal scales and spatial scales around study villages were varied .", "For example , the cumulative percent area within 30 km of a village that experienced forest loss between 2011 and 2016 ranged from 0 to 10% in the north , whereas it rarely exceeded 2 . 5% in the south .", "Deforestation rate in 2015 within 10 km of a village was of about 1% in the south and 2 . 5% in the north .", "The average tree crown cover increased with increasing buffer radius around villages ( 1 , 10 , and 30 km ) .", "However , the relationship with the percent area that experienced forest loss was less clear because both the area that experienced forest loss ( numerator ) and the area around villages ( denominator ) increased .", "Appendix 1—figure 15 and Appendix 1—figure 16 show the raw time series of forest cover and percent area with forest loss .", "The 284 villages in the north were overall less populated ( mean 2015 population size: 498 , IQR: [241; 548] ) than the 207 villages in the south ( 2015 mean: 1095 , IQR: [584; 1384] ) , but with some highly populated outliers .", "As expected , altitude differed substantially between villages of the mountainous northern region ( mean: 557 m , IQR: [378; 679] ) and the lowlands of the south ( mean: 120 m , IQR: [98; 130] ) .", "Although both regions exhibited similar seasonal trends in precipitation and temperature , with a rainy season spanning from April to October , villages in the south experienced higher monthly precipitation and temperature than in the north over the study period ( Appendix 1—figure 8 ) .", "For villages with an estimated travel time of 0 hr to the closest health facility ( same 300 m2 pixel ) , the predicted probability of seeking treatment for fever was 0 . 87 ( 95% CI: [0 . 79; 0 . 92] ) in the north and 0 . 78 ( 95% CI: [0 . 63; 0 . 89] ) in the south .", "A 1 hr increase in travel time to the closest health facility was associated with a similar 0 . 79 ( 95% CI: [0 . 55; 1 . 13] ) reduction in the odds of seeking treatment in the north and 0 . 76 ( 95% CI: [0 . 43; 1 . 34] ) in the south , almost reaching statistical significance when pooling data from both regions: 0 . 77 ( 95% CI: [0 . 56; 1 . 04] ) .", "See detailed results in Appendix 1 - S2 ." ], [ "Based on a large dataset of health facility surveillance records in two regions of Lao PDR , we found evidence that deforestation around villages is associated with higher malaria incidence over the short-term but lower incidence over the long-term ( e . g , in the south , within 30 km of villages: IRR = 1 . 16 [1 . 10; 1 . 22] for deforestation in the previous year and IRR = 0 . 93 [0 . 90; 0 . 97] for deforestation in the previous 3 years ) .", "Our evaluation of alternative spatial scales identified strong associations for deforestation within a 30 km radius around villages but not for deforestation in the near ( 10 km ) and immediate ( 1 km ) vicinity .", "Our results incorporated correction for the probability of seeking treatment , modeled as a function of distance to the closest health facility , as well as adjustment for several environmental covariates .", "Results appear driven by deforestation in densely forested areas and the patterns exhibited are clearer for infections with P . falciparum than for P . vivax .", "The wide availability and longitudinal nature of malaria surveillance records collected routinely by the national program enabled exploration of the relationship between deforestation and malaria incidence over multiple spatio-temporal scales and across different levels of forest density .", "The spatio-temporal variability highlighted here provides insights into the causal mechanisms driving local-scale malaria incidence in the GMS .", "This approach not only quantified the deforestation-malaria incidence association in the GMS , but also strengthened the evidence for the key influence of forest-going populations on malaria transmission in the GMS .", "This study’s results echo the frontier malaria hypothesis from the Amazon region , which posits an increase in malaria incidence in the first few years following deforestation and a decrease over the long term .", "However , we found an earlier inflexion point , 1–3 years after deforestation compared to 6–8 years in the Amazon ( de Castro et al . , 2006 ) , most likely because of very different underlying human processes .", "Indeed , the frontier malaria hypothesis considers non-indigenous human settlements sprouting deeper and deeper in the forest whereas forest-going populations in the GMS are primarily members of established forest-fringe communities who regularly tour the forest overnight to hunt and collect wood ( Dysoley et al . , 2008 ) .", "Industrial and agricultural projects or lucrative forest-based activities also attract mobile and migrant populations ( MMPs ) Guyant et al . , 2015 in remote forested areas of the GMS but not on the same scale as the politically and economically driven unique colonization of the Amazon ( de Castro et al . , 2006 ) .", "Our results are also consistent with the three previous multivariable empirical studies ( Garg , 2015; Pattanayak et al . , 2010; Fornace et al . , 2016 ) that assessed the effect of deforestation on malaria in Southeast Asia .", "Our study builds on these findings by using higher resolution forest data and exploring additional spatio-temporal scales .", "Using biennial village census data from Indonesia between 2003 and 2008 and district-aggregated remote sensing forest data , Garg , 2015 reported a 2–10 . 4% increase in the probability of a malaria outbreak in each village of districts that lost 1000 hectares of their forest cover in the same year .", "Using data from a 1996 cross-sectional household survey conducted in a quasi-experimental setting around a protected area in Indonesia , Pattanayak et al . , 2010 found a positive association between disturbed forest ( vs undisturbed ) and malaria in children under 5 , again using no temporal lag .", "Our analysis plan was largely inspired by Fornace et al . , 2016 , which used similar high-resolution forest data ( Hansen et al . , 2013 ) and 2008–2012 incidence data from Sabah , Malaysia .", "They reported a 2 . 22 ( 95% CI: [1 . 53; 2 . 93] ) increase in the P . knowlesi incidence rate for villages where more than 14% ( <8% , being the reference ) of the surrounding area ( within 2 km ) experienced forest loss in the previous 5 years .", "On the other hand , our analysis explored wider spatial scales , bypassed any coarse categorization of forest and deforestation variables , corrected incidence for treatment-seeking probability , and most importantly focused on P . falciparum and P . vivax , the dominant malaria parasites in the GMS .", "Engaging in forest activities , such as logging , hunting or spending the night in the forest , has been reported as a major risk factor by many studies in the region ( Chaveepojnkamjorn and Pichainarong , 2004; Das et al . , 2004; Lansang et al . , 1997; Erhart et al . , 2005; Trung et al . , 2004 ) .", "As countries of the GMS work toward malaria elimination , the literature stresses the key role of forest-going populations ( Guyant et al . , 2015; Nofal et al . , 2019; Bannister-Tyrrell et al . , 2019; Wen et al . , 2016; Smith and Whittaker , 2014 ) , although research programs highlight the challenges of accessing them ( Bennett et al . , 2021 ; Lover et al . , 2019 ) as well as their diversity ( Nofal et al . , 2019; Bannister-Tyrrell et al . , 2019 ) .", "To our knowledge , no previous study has leveraged geo-spatial statistical analyses to characterize the importance of forest-going populations in the GMS .", "Our results suggest that deforestation in dense forests ( Table 3 ) around villages , particularly areas further from the village ( Table 1 ) , is a driver of malaria in Lao PDR .", "We argue that this is indicative of the existence of a key high-risk group linking the deforestation patterns identified to malaria in the villages , namely a forest-going population .", "Deforestation captured by remote sensing in this setting likely reflects locations and times of heightened activity in the forest areas near villages , and therefore greater human-vector contact .", "We suspect longer and deeper trips into the forest result in increased exposure to mosquitoes , putting forest-goers at higher risk .", "We conducted this study in northern and southern Lao PDR , where the malaria species composition differs , and assessed species-specific relationships in the south where P . falciparum and P . vivax are co-endemic .", "Our results highlight the challenges ahead of national programs with P . vivax elimination after successful P . falciparum elimination , as increasingly mentioned in the literature ( Cotter et al . , 2013; Kaehler et al . , 2019 ) .", "This study identified a clear pattern of spatio-temporal associations between P . falciparum and deforestation , but these were not apparent for P . vivax ( Table 2 ) .", "The increase in P . vivax incidence in the first 2 years following deforestation was identified as well but the associations were smaller than for P . falciparum .", "Importantly , deforestation was never associated with lower risks of P . vivax .", "A recent study in the Amazon MacDonald and Mordecai , 2019 reported a similar attenuation of the effects of deforestation on P . vivax compared to P . falciparum , most likely because of P . vivax parasites’ ability to relapse months or even years after infection , which decouples the association between transmission and incidence data .", "These species-specific differences may also explain why the pattern of spatio-temporal associations between malaria and deforestation were markedly clearer in the south than in the north where P . vivax dominates .", "Our results did have some inherent limitations based upon routine health facility surveillance data .", "First , reliability of such records varies across and within countries of the GMS and may depend on malaria incidence level .", "This could lead to unmeasured residual confounding , further exacerbated by the lack of available data on malaria control activities in the region .", "Another challenge with these data is obtaining an accurate denominator for incidence , as not everyone attends a public health center when febrile .", "We addressed this issue by modeling the probability of seeking treatment as a function of travel time to the closest health facility using data from two cross-sectional surveys .", "Last , the village-level geo-referencing of malaria registries ignores the possibility that patients may become infected elsewhere .", "Unfortunately , these surveillance records did not include information about patients’ forest-going trips .", "Research to track and analyse micro-scale movements of forest-goers is needed to understand how they interact with the forest and where are the foci of infection .", "The forest data we used has also been criticized , in particular for not distinguishing tropical forests from agroforestry ( Tropek et al . , 2014; Hansen et al . , 2014 ) or man-made from natural causes of deforestation .", "The lack of temporal resolution for the forest gain variable ( 2000–2017 aggregate ) as well as the assumption that forest loss happens all in 1 year are additional limitations of these data .", "Finally , our relative measure of deforestation , key to consistently compare the effects across different spatial scales , also implies that a 0 . 1% of the area that experienced forest loss within 30 km of a village is a much larger area ( ∼280 hectares ) than within 1 km ( ∼0 . 3 hectare ) and should be interpreted cautiously .", "In conclusion , this study assessed the relationship between deforestation and malaria in Lao PDR .", "Our approach leveraged surveillance records collected by the national malaria program and high-resolution forest data and rigorously explored the spatio-temporal pattern of associations .", "As countries of the GMS work toward malaria elimination , our results highlight the challenges to transition from P . falciparum to P . vivax elimination , confirm and characterize the importance of high-risk populations engaging in forest activities and suggest malaria programs may benefit from monitoring areas of on-going deforestation using remotely sensed data ." ], [ "Lao PDR has seen a 92% reduction in cases between 2000 ( 280 , 000 ) and 2010 ( 23 , 000 ) ( Lao PDR , 2016 ) .", "Much of this progress has been attributed to heightened funding and better testing and treatments ( Okayas , 2018 ) .", "This study was conducted in eight districts ( Figure 6 ) to leverage the ecological and epidemiological diversity of Lao PDR .", "Four districts ( Moonlapamok , Pathoomphone , Sanasomboon , and Sukhuma ) are situated in the southern province of Champasak where both P . falciparum and P . vivax are endemic .", "The four other districts ( Et , Paktha , Nambak , and Khua ) each come from one of four northern provinces ( Bokeo , Huaphanh , Phongsaly , Luang-Prabhang ) where P . vivax is endemic but P . falciparum has reached historical lows ( Lao national malaria database ( dhis2 ) , 2018 ) .", "The four districts in the north were chosen in consultation with district and provincial level malaria staff to represent the epidemiology of malaria in the region .", "They were selected as part of a cross-sectional survey designed to assess the prevalence and risk factors for malaria in northern Lao PDR ( Lover et al . , 2018 ) .", "This region is very mountainous and characterized by a diverse climate , low-population density and limited road access ( UNFPA , 2016 ) .", "Land clearing using fires for agriculture is customary .", "The four districts in the south were selected within a larger cluster randomized controlled trial ( RCT ) study designed to assess the effectiveness of high-risk group targeted active case detection in southern Lao PDR ( Lover et al . , 2019 ) , where more than 95% of the country malaria burden is concentrated ( Lao national malaria database ( dhis2 ) , 2018 ) .", "This region is characterized by a moderately hilly and forested terrain and a workforce primarily engaged in forest-based and agricultural activities ( Bennett et al . , 2021 ) .", "When designing the study , in collaboration with the national control program , we purposefully excluded regions where we knew large programmatic activities where being implemented .", "For every 30 m pixel in Lao PDR , tree crown cover density for the year 2000 and year of forest loss between 2000 and 2017 , were obtained from Hansen et al . , 2013 .", "These layers were produced using decision tree classifiers on Landsat remote sensing imagery ( Hansen et al . , 2013 ) .", "Trees are defined as ‘all vegetation taller than 5 m in height’ ( Hansen et al . , 2013 ) and forest loss as ‘the removal or mortality of all tree cover in a Landsat pixel’ ( Hansen et al . , 2014 ) .", "For example , as depicted in Figure 7 , the Hansen data indicates that the tree crown cover in 2000 in pixel 1 is 54% , meaning that 54% of the 30 m pixel is covered by vegetation taller than 5 m .", "The Hansen data also indicates that forest loss occurred in pixel in 2013 , meaning that all of the tree canopy disappeared in 2013 .", "Village population sizes were needed to estimate monthly malaria incidence .", "2005 and 2015 population estimates for the 491 villages of study districts were obtained from the national census Lao national census , 2020 .", "The annual population growth rate ( 3 . 7% ) was used to impute population values for two villages missing 2005 estimates and for two villages missing 2015 estimates .", "Then , village-level population growth rates were used to estimate villages’ population per year between 2008 and 2016 , assuming linear annual growth rate ( median = 1 . 7% , IQR = [0%; 4 . 5%] ) .", "Altitude , temperature , rainfall , and access to health care were considered as potential village-level confounders of the relationship between malaria and forest cover factors .", "Travel time to closest health facility , computed for the treatment-seeking model , was used as a proxy for health care access and villages’ remoteness .", "Altitude was extracted from SRTM ( Jarvis et al . , 2008 ) 1 km resolution layers .", "Monthly average day and night temperature were extracted from MODIS 1 km resolution product ( MOD11C3 Wan et al . , 2015 ) .", "Finally , monthly total rainfall was extracted from CHIRPS ( Funk et al . , 2014 ) 1 km resolution publicly available data .", "The average and standard deviation of the annual total precipitation and the average monthly temperature from the monthly time series was computed over the 2008–2012 period , which corresponds to the 5 year time period directly before our malaria data ( 2013–2016 ) .", "This ‘long-term’ aggregation of the climatic variables is included in the model to capture the spatial differences in overall climate between the villages of our study area .", "To account for the seasonal effect of these climatic variables , monthly temperatures and precipitation in the previous 1 , 2 , and 3 months were also extracted , as well as the average temperatures and total precipitation over the previous 1 , 2 , and 3 months ( seven ‘short-term’ variations: in current month , in previous 1 , 2 , or 3 months and aggregated over current and previous 1 , 2 , or 3 months ) .", "See ‘Details on covariates’ below .", "Altitude was missing for one village and we used an online elevation finder tool ( FreeMapTools ) for imputation .", "Temperature was missing for 2 . 4% of the village-months over the study period , most likely because of cloud coverage of the MODIS imagery .", "Monthly temperature was never missing more than 2 years in a row at villages of the study’s districts and we imputed the temperature of the same month of the following year ( or prior year when needed ) , adjusting for average district-level monthly temperature differences between the 2 consecutive years .", "Monthly rainfall was not missing at any of the villages ." ] ]

[ "As countries in the Greater Mekong Sub-region ( GMS ) increasingly focus their malaria control and elimination efforts on reducing forest-related transmission , greater understanding of the relationship between deforestation and malaria incidence will be essential for programs to assess and meet their 2030 elimination goals .", "Leveraging village-level health facility surveillance data and forest cover data in a spatio-temporal modeling framework , we found evidence that deforestation is associated with short-term increases , but long-term decreases confirmed malaria case incidence in Lao People’s Democratic Republic ( Lao PDR ) .", "We identified strong associations with deforestation measured within 30 km of villages but not with deforestation in the near ( 10 km ) and immediate ( 1 km ) vicinity .", "Results appear driven by deforestation in densely forested areas and were more pronounced for infections with Plasmodium falciparum ( P . falciparum ) than for Plasmodium vivax ( P . vivax ) .", "These findings highlight the influence of forest activities on malaria transmission in the GMS ." ]

[ "Biting mosquitos spread the malaria parasite to humans .", "Along the Mekong River in Southeast Asia , spending time in the surrounding forest increases a person's risk of malaria .", "This has led to a debate about whether deforestation in this area , which is called the Greater Mekong Sub-region ( GMS ) , will increase or decrease malaria transmission .", "The answer to the debate is not clear because some malaria-transmitting mosquitos thrive in heavily forested areas , in particular in the GMS , while others prefer less forested areas .", "Scientists studying malaria in the Amazon in South America suspect that malaria transmission increases shortly after deforestation but decreases six to eight years later .", "Some studies have tested this ‘frontier malaria’ theory but the results have been conflicting .", "Fewer studies have tested this theory in Southeast Asia .", "But deforestation has been blamed for recent malaria outbreaks in the GMS .", "Using data on malaria testing and forest cover in the GMS , Rerolle et al . show that deforestation around villages increases malaria transmission in the first two years and decreases malaria rates later .", "This trend was driven mostly by a type of malaria called Plasmodium falciparum and was less strong for Plasmodium vivax .", "The location of deforested areas also mattered .", "Deforestation within one to 10 kilometer of villages did not affect malaria rates .", "Deforestation further away in about a 30 kilometer radius did affect malaria transmission .", "Rerolle et al . suggest this may be because villagers have to spend longer times trekking through forests to hunt or harvest wood when the wider area is deforested .", "Currently , National Malaria Control Programs in the GMS focus their efforts on reducing forest-related transmission .", "This study strengthens the evidence supporting this approach .", "The results also suggest that different malaria elimination strategies may be necessary for different types of malaria parasite .", "Using this new information could help malaria control programs better target resources or educate villagers on how to protect themselves .", "The innovative methods used by Rerolle et al . reveal a more complex role of deforestation in malaria transmission and may inspire other scientists to think more carefully about environmental drivers of malaria ." ]

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

[ "physics of living systems" ]

[ [ "Reversible phosphorylation , and post-translational modification ( PTM ) of proteins in general , constitutes a basic way of conferring functionality to proteins in cells .", "This basic unit ( covalent modification ) is built upon in many different ways to result in the complex biochemical pathways encountered in cells .", "A particular elaboration of this mechanism , which is widely encountered , is the reversible multisite modification of proteins by enzymes .", "Here , a number of basic variations are possible depending on whether the enzymes involved in distinct modification steps are different or if a common enzyme effects multiple modifications .", "In the latter case , there are variations depending on whether the enzymatic mechanism is distributive ( enzyme dissociating from substrate after every modification ) or processive ( enzyme remains attached ) .", "Finally , there are variations depending on whether a specific ordering of modifications occurs ( ordered mechanism ) or not ( random mechanism ) .", "In addition to being a widely encountered way in which substrates are reversibly modified to confer functionality ( and consequently of broad interest ) , interest in multisite modification stems from the fact that the basic modification mechanisms are capable of acting as complex molecular information processors ( Conradi and Shiu , 2018 ) .", "Various studies have highlighted the possibilities of these mechanisms exhibiting switching and threshold behaviour ( Markevich et al . , 2004 ) , bistability/multistability ( Thomson and Gunawardena , 2009; Conradi and Mincheva , 2014 , oscillations Suwanmajo and Krishnan , 2015; Rubinstein et al . , 2016; Suwanmajo et al . , 2020 ) , biphasic dose–response curves ( Suwanmajo and Krishnan , 2013 ) , and other complex behaviour ( Suwanmajo and Krishnan , 2018 ) .", "A range of studies have delineated the ingredients required ( from the above possible variations ) to enable or prevent such behaviour ( Conradi et al . , 2017; Eithun and Shiu , 2017; Tung , 2018 ) .", "We emphasize that this rich repertoire of behaviour emerges from the most basic considerations and aspects of enzymatic modification of substrates , and that this behaviour is a feature and a consequence of the modification network ( rather than a single modification ) .", "Information processing capabilities are also at the heart of different strands of work in synthetic biology engineering multisite modification , and reaction networks more broadly ( Valk et al . , 2014; Lyons et al . , 2013; OShaughnessy et al . , 2011; Maguire and Huck , 2019 ) .", "This paper focuses on a distinct aspect of information processing of multisite modification: symmetry and symmetry breaking .", "Symmetry and symmetry breaking are themes encountered across different scales and levels in biology , ranging from the cell population , to the cellular , to the molecular level .", "A fundamental theme in developmental biology is the breaking of symmetry to generate patterns .", "The basic questions here centre around how an apparently homogeneous field of cells can differentiate to exhibit a basic pattern which serves as a precursor for subsequent development .", "Modelling , experiments , and concepts from self-organization have been used to probe this generation of form , which breaks spatial symmetry .", "The underlying mechanisms invoked involve many variations on the classical Turing mechanism or the interplay of mechanics and chemistry ( Green and Sharpe , 2015; Maini et al . , 2012 ) .", "This can be significantly complicated by the presence of many layers of regulation .", "Strong experimental evidence for such mechanisms present at the core of developmental regulation has been demonstrated in multiple model systems ( Onimaru et al . , 2016 ) .", "Symmetry breaking as a basis of generating form at the cellular level , for instance , polarization and polarized or other strongly inhomogeneous patterns of concentration of species , has been explored in a range of contexts .", "Examples include polarity generation in fungi and plant cells ( Khan et al . , 2015 ) , and in neutrophil chemotaxis ( Wang , 2009 ) .", "Symmetry has also been invoked as a key ingredient in the development of the MWC model which has been used to explain allostery in biomolecular information processing ( Changeux , 2012 ) .", "While the theme of symmetry in chemistry is well recognized especially at the molecular structure level ( Hargittai and Hargittai , 1994 ) , there are relatively few studies of symmetry breaking at the molecular reaction level .", "In chemical reaction systems , symmetry is encountered in the context of chirality in racemic mixtures .", "Racemic mixtures comprise equal amounts of the two enantiomeric forms of a chiral molecule with opposite chiralities , and a central question is how a dominant orientation ( chirality ) of the molecular mixture can emerge from this .", "Some studies explain this as an emergent behaviour of the reaction network system governing the two forms of the molecules: even if the network/reaction system is symmetric allowing for equal amounts of the two forms , this symmetry can break , giving rise to a dominant form .", "A recent study ( Hochberg et al . , 2017; Ribó et al . , 2017 ) evaluated and demonstrated the feasibility of such symmetry breaking in a number of potential reaction systems .", "Chiral symmetry breaking has been experimentally observed in crystallization of nanoparticles ( Hananel et al . , 2019 ) , fibril formation from racemic mixtures ( Kushida et al . , 2017 ) , and in the Soai reaction ( Soai et al . , 1995 ) .", "Such symmetry breaking is of particular importance in prebiotic evolution and biology , where biopolymers and biomolecules are characterized by a specific chirality and orientation , even though the original non-life chemical world was chirally symmetric ( Chen and Ma , 2020 ) .", "The establishment of such chirality has been postulated to be important in understanding the origins of life ( Blackmond , 2020 ) .", "This paper focuses on a specific aspect of symmetry breaking at the junction of the biological and the chemical: the breaking of symmetry in basic multisite phosphorylation ( MSP ) systems .", "The motivation for studying symmetry and symmetry breaking in the context of multisite modification stems from different sources: conceptual insights , relevance to systems biology , and potential application in synthetic biology .", "In this regard , we note that", "( i ) many of the basic modification networks accommodate different types of symmetries , as we discuss below .", "( ii ) Certain symmetries , for example , resulting in equal concentrations of different partial phosphoforms of a given level ( Case 2 symmetry , discussed below ) are not only plausible in vivo , but have also been assumed in multiple contexts , sometimes implicitly .", "( iii ) An asymmetric state currently observed may have its genesis traced back to a symmetric state in evolution , which broke symmetry .", "( iv ) Other symmetries ( Case 3 symmetry , discussed below ) have been found to be particularly desirable in enabling oscillatory behaviour: in fact , a thorough parametric analysis of oscillatory behaviour in certain random double-site modification networks reveals clusters in parameter space centred around parameter sets representing networks with this symmetry ( Jolley et al . , 2012 ) .", "Case 3 symmetry involves a combination of two symmetries ( Case 1 and Case 2 ) which we individually study as well .", "( v ) Symmetry breaking can confer new functionality and information processing characteristics enriching the repertoire of MSP .", "( vi ) Our study of symmetric systems allows us to draw important insights about multisite modification even when exact symmetry does not hold good .", "Thus it is also relevant to networks which are approximately symmetric .", "( vii ) In this sense , the symmetric scenarios also serve as valuable ( and sometimes non-obvious ) vantage points from which to investigate important aspects of multisite modification .", "Furthermore , while studying modification networks of larger numbers of modifications , the symmetric networks may represent one of the few tractable vantage points from which to study and elucidate the behaviour of such networks .", "( viii ) These serve as interesting candidates for engineering multisite modification in synthetic biology with desirable features .", "We examine basic models of MSP and evaluate the possibility of spontaneous symmetry breaking in basic and canonical reaction pathways/circuits/networks of MSP .", "We discuss the consequences of the results which emerge for multisite modification networks which may not possess an exact symmetry .", "We then discuss the various consequences of such behaviour for biological systems , and cellular signalling pathways and networks which contain multisite modification .", "The ordering of multisite modification is a fundamental aspect of substrate modification and its regulation , and the deployment of modified substrates in various processes .", "It has been the focus of different studies ( Kocieniewski et al . , 2012; Lyons et al . , 2013; Lössl et al . , 2016; Valk et al . , 2014 ) spanning canonical pathways , important cellular processes , basic principles , and engineering for synthetic biology .", "We show how symmetry breaking could provide a natural mechanism for the creation of ordered multisite modification systems from random multisite modification , which could in turn explain the various degrees of ordering encountered in cells ." ], [ "Our primary focus is on random mechanisms of multisite modification , and we study the case of double-site modification as a tractable , representative case .", "Figure 1A represents random mechanisms of modification ( i . e . modifications can proceed in either order ) and depicts cases where the kinases and phosphatases effecting individual modifications could either be the same or different .", "Taken together , these networks span a range of basic cases of multisite modification , including the possibility of an enzyme performing multiple modifications ( seen in many biological contexts ) and the possibility that this may be associated with one modification direction , but not the other due to the fact that kinases significantly outnumber phosphatases , as seen in genome-wide studies ( Ghaemmaghami et al . , 2003 ) .", "When a common enzyme is involved in effecting multiple modifications , the modification mechanism is assumed to be distributive , unless otherwise stated .", "We note here that such modification circuits are encountered in multiple cellular contexts and can be viewed as building blocks of more complex multisite modification networks .", "Such networks have been the focus of detailed studies in contexts such as circadian oscillations ( Ode and Ueda , 2018 , involving the common kinase common phosphatase network depicted: the substrate represents the Per proteins ) , with additional studies on temperature compensation in this context ( Shinohara et al . , 2017; Hatakeyama and Kaneko , 2012 ) .", "They have also been used to evaluate design principles for both oscillatory and pattern forming behaviour more broadly ( Jolley et al . , 2012; Sugai et al . , 2017 ) .", "For purposes of contrast and elucidating basic effects , we also examine two related modification networks:", "( i ) an ordered double-site modification mechanism ( Figure 1C ) mediated by a common kinase and common phosphatase .", "The specific ordering of modification involves the phosphorylation order being opposite to that of dephosphorylation resulting in one partial phosphoform .", "This has been extensively studied in the literature ( e . g . Thomson and Gunawardena , 2009; Conradi and Shiu , 2018 ) .", "( ii ) A random mechanism where the dephosphorylation is processive ( Figure 1B and Appendix 2—figure 1A ) .", "The model for each of these networks ( depicted in Figure 1A–C , Appendix 2—figure 1A ) is built up from widely used descriptions of individual substrate modification by an enzyme ( involving reversible complex formation and irreversible modification to give the product; see Appendix 2—figure 10 ) .", "Such a description makes no a priori assumptions about the kinetic regimes of modifications .", "Further details are presented in Appendix 1 .", "Throughout the paper , we work with these canonical modification circuits , where the substrate forms are denoted by A , with subscripts denoting the type of modification .", "Depending on the context , these could represent different proteins .", "In order to understand and visualize the types of symmetries we will examine , it is fruitful to examine a ‘square’ reaction network , which has the same network topology as that of the multisite modification networks above .", "Note that in this depiction the nodes of the network represent substrates , while the enzymes are implicitly present in the arrows: both substrates and enzymes together constitute an enzymatic reaction network of this type .", "As depicted in Figure 1D ( left panel ) , there are two types of symmetries which can be encountered .", "( i ) In the first case , the two ‘legs’ of the network are symmetric ( about the axis of symmetry depicted ) , which means that the rates of reaction for corresponding reactions on either side of the axis are the same .", "The associated pair of species ( nodes of this network representing substrates , on either side of the axis of symmetry ) is expected to behave identically ( assuming the same initial conditions ) .", "( ii ) In the second case , the symmetry is associated with two pairs of species simultaneously and can be viewed as a simultaneous occurrence of two of the previous symmetries , along different axes ( see Figure 1D ) .", "Viewed from a general network perspective , in each case the symmetry is a consequence of rates of different pairs of reactions ( intrinsic reaction rate constants as well as total enzyme concentrations ) being identical , thus giving rise to the symmetry .", "Thus enabling such symmetries establishes correspondences/constraints between different pairs of enzymes .", "Note that in this network we have not made any restrictions on which enzymes may be involved in specific steps .", "Establishing the structural requirements for symmetry then allows us to examine when and how the multisite modification networks we study exhibit different symmetries .", "We now focus on the network symmetries in the specific instance of the biochemistry of multisite modification .", "In so doing , we discuss different types of symmetries which multisite modification networks can exhibit .", "While some of these symmetries may appear to be more natural biologically , it is useful to examine all these together to obtain a comprehensive systems understanding .", "Furthermore , some of these have been postulated explicitly or invoked implicitly in multiple different contexts .", "Symmetries in such networks require basic conditions/constraints on the kinetics and enzyme amounts ( refer Figure 1D , right panel ) .", "In particular , equivalence between two reactions ( as represented in the schematic ) requires that the rate constants of their constituent elementary reactions ( binding , unbinding , and catalytic ) remain equal .", "The first two cases of symmetries correspond to a scenario where the two ‘legs’ of the network are symmetric .", "The difference between them is what the symmetric nodes of the network correspond to in the context of multisite modification along with the fundamentally distinct pairings of enzymes in each case ( discussed further below ) .", "In this case , the nodes involved in either leg of the symmetric network are A00 and A11 .", "In such a case , the requirement of a symmetry implies that for these two phosphoforms the action of an enzyme ( kinase ) on one of these substrates ( A00 ) has the same rate as that of another enzyme ( phosphatase ) on the other substrate ( A11 ) ( this is seen by examining the corresponding reaction arrows on the two legs of the network ) .", "Furthermore , an analogous requirement applies to the production of each of these species from the partial phosphoforms .", "With these requirements a symmetry between A00 and A11 is maintained .", "We further note that such a requirement ( of having certain rates of kinase-mediated reactions being equal to that of other phosphate-based reactions ) places a constraint on total enzyme amounts as well .", "Case 1 symmetry is of interest both as a basic independent symmetry and as a constituent of Case 3 symmetry discussed below .", "( i ) The above requirements can be accommodated both in the common kinase common phosphatase case and the separate kinase separate phosphatase case , but not in the separate kinase common phosphatase case ( discussed in Appendix 1 ) .", "( ii ) We also note that a simpler network , which corresponds to ordered double-site modification , also accommodates a symmetry of this type ( while only possessing a single partial phosphoform ) .", "Here too , this is accommodated in the common kinase common phosphatase and separate kinase separate phosphatase cases .", "In this case , the nodes involved in either leg of the symmetric network are A01 and A10 .", "Such a symmetry is realized if the following pairs of reactions have the same rates:", "( i ) phosphorylation of A00 to produce the respective partial phosphoforms ,", "( ii ) dephosphorylation of A11 to produce the respective partial phosphoforms ,", "( iii ) the phosphorylation of the respective partial phosphoforms , and", "( iv ) the dephosphorylation of the two partial phosphoforms .", "Note that equal rates of reaction require the same intrinsic kinetic rate constants ( for binding , unbinding , and catalysis of substrate by enzymes ) as well as total enzyme amounts .", "This is characterized by saying that the rate of modification of all substrates of a given level of modification is the same ( and likewise for demodification ) , and this ensures that progression in substrate modification is equally balanced between the pathways associated with each partial phosphoform ( a feature explicitly/implicitly assumed in multiple instances in the literature ) .", "This symmetry can be accommodated in all cases of separate/common kinases and separate/common phosphatases .", "Case 1 and Case 2 symmetries involve different pairs of symmetric nodes .", "As noted earlier , the symmetries require both intrinsic rate constants and enzyme amounts to be equal for different pairs of enzymes .", "The essential difference between the two cases is the essentially different enzyme pairs associated with this .", "In Case 1 symmetry , the pairing is between enzymes of different types ( a kinase and a phosphatase ) , while in Case 2 it is between enzymes of the same type ( between kinases and between phosphatases ) .", "This is exactly why Case 1 symmetry is not possible in the separate kinase common phosphatase network , while Case 2 symmetry is .", "This involves the combination of the earlier cases .", "Here the action of a kinase enzyme on a substrate occurs at the same rate as that of the phosphatase enzyme on its associated substrate in the diagonally opposite modification leg .", "Thus the action of the kinase on A00 modifying it to A01 is the same as that of the phosphatase action on A11 modifying it to A10 .", "This applies to the modification of all substrates in the network .", "Such symmetries have been implicated in oscillatory networks of multisite modification underlying circadian oscillators ( Jolley et al . , 2012 ) .", "Again , similar to Case 1 symmetry , the separate kinase common phosphatase case cannot accommodate this symmetry .", "The basic questions we address below are", "( i ) Are these symmetries always maintained or can they be broken ?", "( ii ) What network features determine whether or not symmetry breaking is possible ?", "( iii ) What kind of capabilities does symmetry breaking provide ?", "( iv ) When symmetry breaking is possible , can the parameter regimes for symmetry breaking be established ?", "To address the above questions , we employ two approaches in tandem:", "( i ) computational analysis , involving simulations and bifurcation analysis , where we demonstrate the possibility of such behaviour occurring .", "We note here that our bifurcation parameter is the total substrate concentration , though it could apply to other parameters; and", "( ii ) analytical work which rules out the possibility of symmetry breaking in networks irrespective of kinetic parameters , bringing to the fore structural features which prevent the occurrence of the behaviour .", "Analytical work is also used to demonstrate necessary conditions for symmetry breaking ( in terms of kinetic parameters and total enzyme and substrate amounts ) and further that in these cases these conditions are sufficient to guarantee the presence of symmetry breaking .", "Additionally , analytical work also reveals important characteristics of symmetry-broken states .", "We present the results for Case 1 , Case 2 , and Case 3 for the different random modification networks below .", "The ordered double-site modification network can exhibit Case 1 symmetry , as noted above .", "Therefore , in presenting Case 1 symmetry , we start with this simpler network , before proceeding to the random modification networks .", "We first analyse the scenario of Case 1 Symmetry .", "It is instructive to examine an ordered mechanism ( Figure 2A ) in this regard as it is simpler while exhibiting the same behaviour encountered in random mechanisms .", "The system has a symmetric steady state which is characterized by", "( i ) equal concentrations of unmodified ( A ) and fully modified ( Ap⁢p ) phosphoforms and", "( ii ) equal concentrations of free kinase and phosphatase ( note that the total concentrations of these enzymes need to be the same for symmetry to be present ) .", "This steady state simply represents an absence of bias in the direction of modification ( i . e . between the unmodified and fully modified phosphoforms ) .", "However as the total substrate concentration is varied , we find that this steady state loses stability via a supercritical pitchfork bifurcation ( Strogatz , 2001 ) .", "A pair of asymmetric steady states emerge which are stable .", "These correspond to either [Ap⁢p]>[A] or the other way around , and unequal free enzyme concentrations .", "Interestingly on each of these steady-state branches , the value of the intermediate phosphoform Ap remains fixed at the level at the bifurcation point ( Figure 2A , lower panel ) .", "The presence of asymmetric states , as well as the fact that the partial phosphoform concentration is fixed on the branches of asymmetric steady states , is established analytically ( see Appendix 1 , Source code 1 [Section 2 . 1] , and Supplementary file 1 ) .", "The asymmetric steady states represent the establishment of overall directionality in the reaction network output , even in the absence of any a priori bias ( in terms of reaction rates and enzyme concentrations ) .", "Analytical work also reveals the necessary conditions for symmetry breaking to occur in this system: k2>k1 .", "In other words , the catalytic rate constant for the second phosphorylation is greater than that of the first phosphorylation step .", "Note that this does not involve binding or unbinding constants for enzyme/substrate interactions .", "Further analysis indicates that in such a parameter regime a symmetry-broken state is guaranteed to exist for some value of the bifurcation parameter AT⁢o⁢t⁢a⁢l ( see Appendix 1 , Source code 1 [Section 2 . 1] , and Supplementary file 1 ) .", "It is worth emphasizing here that", "( i ) the nonlinearity responsible for the symmetry breaking arises purely from sequestration effects with no explicit feedback present and", "( ii ) an analogous case of single-site modification is incapable of intrinsically exhibiting such behaviour .", "The symmetry breaking observed in this ordered mechanism is seen in the random modification with common kinase and phosphatase ( Figure 2B ) .", "The random modification network can be thought of as two connected ‘legs’ of ordered modification networks , and consequently echoes of the behaviour seen previously are observed here .", "In this instance , beyond the bifurcation point , the concentrations of both partial phosphoforms remain fixed at their values at the bifurcation point .", "This is established analytically ( see Appendix 1 , Source code 1 [Section 3 . 1] , and Supplementary file 1 ) .", "An analytical necessary criterion for the presence of symmetry breaking in this system is presented in Appendix 1 , Source code 1 ( Section 3 . 1 ) , and Supplementary file 1 .", "It takes the form ( k2-k1 ) +α⁢ ( k2/a2 ) ⁢ ( a2-a1 ) >0 .", "Here k1 , k2 are the catalytic rate constants associated with phosphorylation along one leg of modifications ( A00→A01→A11 ) , while a1 , a2 are the catalytic rate constants associated with phosphorylation along the other leg of modifications ( A00→A10→A11 ) .", "Further α is a positive constant determined in terms by rate constants ( including binding and unbinding ) .", "Further work in Source code 1 ( Section 3 . 1 ) and Supplementary file 1 establishes the fact that this is a sufficient condition for the generation of asymmetric states for some value of AT⁢o⁢t⁢a⁢l .", "We can make multiple inferences from this condition in relation to the corresponding condition for ordered mechanisms discussed above .", "( i ) If both k2>k1 and a2>a1 , then the requisite condition is satisfied .", "This means that if each leg ( viewed as an ordered mechanism ) satisfies the conditions for symmetry breaking in ordered mechanisms , this guarantees the possibility of symmetry breaking in the random mechanism .", "( ii ) For the same reason , if neither leg satisfies the condition , then symmetry breaking is precluded in the random modification network .", "( iii ) Interestingly if only one leg satisfies the criterion for symmetry breaking , it is possible for the entire random network to break symmetry ( an example of this is presented in Appendix 2—figure 3 ) .", "In such a case , the random network can be viewed as being comprising two subnetworks , only one of which is the driver of this behaviour .", "Random mechanisms with different kinases and phosphatases can also exhibit the same type of symmetry ( this places constraints on total concentrations of ‘corresponding’ enzymes , in addition to the kinetic constraints already discussed ) .", "As seen in Figure 2C , this system also exhibits a similar symmetry-breaking behaviour as encountered above , and here again the concentration of partial phosphoforms is fixed beyond the bifurcation .", "The case of different kinases and phosphatases represents a much more general case ( not requiring any enzyme to perform more than one modification ) with significantly reduced nonlinearity ( for the same reason ) , which is nonetheless sufficient for symmetry breaking .", "The necessary conditions for symmetry breaking here are established analytically in Source code 1 ( Section 3 . 3 ) and Supplementary file 1 , where it is also shown that these conditions guarantee the existence of a symmetry-broken state .", "This takes the form k2/k1>P2Total/P1Total and a2/a1>P1Total/P2Total ( the equation could also be written in terms of the total concentrations of kinases ) .", "The main difference here is that", "( i ) there are two such conditions to be satisfied and", "( ii ) they also involve total enzyme amounts .", "( iii ) When P2⁢T⁢o⁢t⁢a⁢l=P1⁢T⁢o⁢t⁢a⁢l , this amounts to k2>k1 and a2>a1 , which is a requirement for each of the legs of the modification network .", "( iv ) When P2⁢T⁢o⁢t⁢a⁢l≠P1⁢T⁢o⁢t⁢a⁢l , this amounts to a tighter requirement on one of the legs ( where enzyme P2 is involved in the first dephosphorylation step ) and a weaker requirement on the other ( where P2 is involved in the second dephosphorylation step ) .", "Each leg of the modification corresponds to an ordered modification mechanism with different kinases and phosphatases , which is incapable of symmetry breaking ( as shown in Source code 1 [Section 2 . 2] and Supplementary file 1 ) and multistability in general .", "Thus the observed symmetry breaking is an emergent behaviour of the entire network with both modification legs .", "The implication of Case 1 symmetry breaking is that it is possible to establish a directionality to the modification even if none existed , resulting in an establishment of relative dominance of fully modified phosphoforms vis-a-vis fully unmodified forms or the other way round .", "Case 1 symmetry is also a constituent of Case 3 symmetry , and this has implications in that situation as well .", "Case 2 symmetry implies that there is no bias in the ordering of modifications and this is manifest in the equal steady-state concentrations of the partial phosphoforms A01 and A10 .", "Examining all the cases of random networks together ( Figure 1A–C ) reveals the following insights:", "( i ) the case of common kinase and common phosphatase will not lead to the breaking of this symmetry .", "( ii ) The case of different kinase and common phosphatase will also not lead to the breaking of this symmetry .", "In both cases , this can be shown directly analytically by demonstrating that for any steady states ( irrespective of parameters ) the partial phosphoforms necessarily must have equal concentrations ( see Source code 1 [Sections 3 . 1 and 3 . 2] and Supplementary file 1 ) .", "Incidentally , an identical conclusion can be drawn for the common kinase common phosphatase case , irrespective of the number of modification sites .", "( iii ) On the other hand , the case of different kinase and different phosphatase can indeed exhibit the breaking of this symmetry ( Figure 2D ) via a supercritical pitchfork bifurcation: here the asymmetric steady states are characterized by fixed values of concentrations of unmodified and fully modified phosphoforms .", "This is established analytically ( see Source code 1 [Section 3 . 3] and Supplementary file 1 ) .", "Analytical results provide further insights .", "The necessary requirements for an asymmetric state are k1/k4>PTotal/KTotal and k2/k3<PTotal/KTotal .", "Note here that KT⁢o⁢t⁢a⁢l denotes the total concentration of each of the kinase enzymes while PT⁢o⁢t⁢a⁢l denotes the total concentration of each of the phosphatase enzymes ( the equality , a requirement of symmetry ) .", "k1 , k2 denote the catalytic constants for phosphorylation of A and the partial phosphoforms ( the constants being equal ) , while k3 , k4 denote the catalytic constants of dephosphorylation of A11 and the partial phosphoforms .", "Further analysis shows that this is sufficient for the existence of an asymmetric state .", "From this we can infer that", "( i ) if k1/k4<k2/k3 then this behaviour is precluded .", "( ii ) On the other hand if k1/k4>k2/k3 , then by suitable choices of total enzyme concentrations ( quantities easy to manipulate ) , these conditions can be satisfied .", "( iii ) In such a case , there is only a finite range of ratio of total enzyme concentrations ( between k2/k3 and k1/k4 ) which can result in symmetry breaking .", "( iv ) The presence of multiple kinases and phosphatases proves to have a combination of both sufficient nonlinearity as well as sufficient flexibility ( from the multiplicity of enzymes ) to enable such behaviour .", "As seen previously with multiple kinases/phosphatases , the parameters need to satisfy two inequalities .", "A combination of the naturalness of the symmetry and the widely encountered modification scenario suggests that symmetry breaking here may be encountered quite broadly .", "We aimed to get further insights into the factors driving such behaviour by comparing it with other related networks .", "To do this , we examined associated random modification networks where the dephosphorylation was processive ( Figure 1B , Appendix 2—figure 1A ) .", "Note that this implies that the phosphatase has to be the same for all modifications .", "Here we find that if the kinase is common to different phosphorylation steps , the symmetry does not break; however , surprisingly when the kinases are different , symmetry breaking does indeed happen , as shown in Figure 2E ( again reinforcing the flexibility provided by having different enzymes in enabling such behaviour ) .", "This is supported analytically ( see Source code 1 [Section 4 . 2] and Supplementary file 1 ) .", "A direct comparison with the different kinase common phosphatase random mechanism above reveals a new dimension: having processive dephosphorylation while reducing the nonlinearity in the network actually enables this behaviour which was otherwise precluded .", "Elsewhere we have noted how having processive dephosphorylation could readily enable other behaviour ( oscillations ) which was not observed when the modification was distributive ( Suwanmajo and Krishnan , 2015 ) .", "We also note that different conditions in the cell ( or stimuli ) could effect a transition from distributive to processive mechanisms , as demonstrated ( Aoki et al . , 2013; Aoki et al . , 2011; Kocieniewski et al . , 2012 ) .", "Case 2 symmetry breaking ultimately results in the biasing of one ordered sequence of modifications over another .", "The implications of this as a key step for generating ordering of modifications are discussed subsequently .", "The scenario of Case 3 symmetry is examined in Figure 3 .", "This involves a combination of the earlier cases .", "Figure 3A ( and Appendix 2—figure 2 ) focuses on the common kinase common phosphatase case .", "Here too , the symmetric steady state ( characterized by [A01]=[A10] and [A00]=[A11] ) can lose symmetry to a pair of asymmetric steady states via a supercritical pitchfork bifurcation ( Appendix 2—figure 2 ) .", "The noteworthy point here is that both symmetries necessarily break together in general .", "In addition to previously observed bifurcation patterns , new possibilities arise .", "One is the possibility of a subcritical pitchfork bifurcation ( Figure 3A ) .", "In such a case , the branches of asymmetric states are unstable at the point of inception , but following a saddle node bifurcation , they become stable .", "As a direct consequence of this , when the parameter ( total concentration of substrate ) is varied , a sudden change from a symmetric to asymmetric steady state is observed in simulations , the latter exhibiting a pronounced asymmetry , which is not a small perturbation of the symmetric state .", "The asymmetric steady states are characterized by high levels of A11 and one of the partial phosphoforms , and low levels of A00 and the other phosphoform , or the other way around .", "Furthermore , the grouping in which each partial phosphoform is present is determined by baseline parameters ( and changing baseline parameters can alter the grouping ) .", "Irrespective of the nature of the pitchfork bifurcation ( supercritical or subcritical ) , we find that the sum of partial phosphoform concentrations remains fixed on the asymmetric branches , and this is established analytically ( see Appendix 1 , Source code 1 [Section 3 . 1] , and Supplementary file 1 ) .", "The implications of this distinct type of invariant are discussed in the next section .", "It is interesting to contrast the invariant in this Case 3 symmetry breaking with invariants in symmetry breaking in the constituent symmetries ( in this instance of common kinase common phosphatase , Case 1 symmetry , since Case 2 symmetry does not break ) .", "In Case 1 symmetry breaking , the invariants are the individual partial phosphoform concentrations while here it is the sum .", "This simultaneous breaking of symmetries and pairing of partial phosphoform and fully modified ( or fully unmodified phosphoform ) has a transparent interpretation .", "Symmetry breaking simultaneously imposes directionality to the modification ( i . e . relative dominance of fully modified vs . fully unmodified phosphoform ) as well as a particular route of modification ( via one of the two phosphoforms ) .", "Another behaviour which is observed in a different parameter regime is the emergence of oscillations , via a Hopf bifurcation , and this precedes the subcritical pitchfork bifurcation ( Figure 3A ) .", "The oscillations do not preserve the symmetry of the original system .", "Instead we see correlated changes between the corresponding pairs of substrates at different time intervals .", "As the total substrate concentration increases , the period of oscillations increases , as the periodic trajectory comes close to a steady state in the phase space ( Appendix 2—figure 2 ) .", "Oscillations in such networks can occur without symmetry breaking , and in fact oscillations emerging from such random modification networks have been the focus of earlier studies ( Jolley et al . , 2012 ) , Here we show that in the presence of symmetry ( a condition recognized as a desirable criterion for oscillations ) , oscillations may be present in conjunction with symmetry breaking , which affects the oscillatory range and characteristics of oscillations .", "Analytical work in the case of common kinases and common phosphatases reveals a necessary condition for the realization of an asymmetric state ( which is shown to be sufficient as well ) .", "This takes the form c3⁢ ( 1-k3/k2 ) +c1⁢ ( 1-k1/k4 ) >0 .", "Here c1 and c3 are known positive constants , which depend on kinetic parameters .", "As in the situation of Case 1 symmetry , this hinges on two ratios of catalytic constants , though in contrast to that case it is the phosphorylation and dephosphorylation rate constants associated with the interconversion between A00 and A01 ( k1/k4 ) and dephosphorylation and phosphorylation rate constants for the interconversion between A01 and A11 ( k3/k2 ) .", "As before we can make a range of conclusions:", "( i ) k3/k2>1 and k1/k4>1 will preclude an asymmetric state;", "( ii ) k3/k2<1 and k1/k4<1 will ensure the possibility of an asymmetric state; and", "( iii ) a combination of parts of the above conditions can allow for an asymmetric state depending on the constants c1 , c3 .", "In this regard , we also point out that these results show when symmetry breaking is precluded , and this combined with ( Jolley et al . , 2012 ) yield conditions under which oscillations can occur without interference from symmetry breaking ( and in fact multistability ) .", "Here ( Figure 3B ) , we again find symmetry breaking via supercritical pitchfork bifurcations ( but not subcritical pitchfork bifurcations ) , and the symmetry-broken states ( which necessarily have both symmetries broken: see Source code 1 [Section 3 . 3] and Supplementary file 1 ) are characterized by having a higher level of one pair of substrates ( one partial phosphoform and a completely modified/unmodified phosphoform ) and a lower level for the other pair .", "Here an exact invariant of the form examined previously does not hold: instead we find that ( depending on parameters ) either the sum of partial phosphoform concentrations or the sum of concentrations of A00 and A11 is approximately constant ( see Figure 3 , Appendix 2—figure 4 , Source code 1 [Section 3 . 3] , and Supplementary file 1 ) .", "It is worth noting as a contrast that fixed individual concentrations for pairs of species ( A01 , A10 ) and ( A00 , A11 ) are obtained in this case for symmetry breaking of the constituent Case 1 and Case 2 symmetries , respectively .", "The possibility of oscillations ( stable over a broad range of parameters ) emerging is also seen here ( Figure 3B ) , though we have never found it occurring side-by-side with symmetry breaking ( seen earlier in the common kinase common phosphatase network ) .", "The presence of oscillations expands on and complements ( Jolley et al . , 2012 ) , by revealing oscillations in this network which is desirable as it has additional tuneable dials ( multiple total enzyme amounts ) .", "In Case 3 symmetry breaking in both the common kinase common phosphatase and separate kinase separate phosphatase , we find that in the symmetry-broken state the concentration of one partial phosphoform and either unmodified or fully modified form may be significantly elevated relative to its counterpart .", "This disparity between the two pairs can become pronounced and easily be misinterpreted as suggesting", "( i ) the nonexistence of other active modifications or", "( ii ) a significant disparity in intrinsic kinetic rates of modification in the two legs of the network ." ], [ "The symmetries which arise can be conceptualized and understood by examining symmetries of the underlying ‘square’ reaction network topology ( refer Figure 1D ) .", "That viewpoint , relevant for a broad class of chemical reaction networks , can then be applied to the specific instance of multisite substrate modification networks .", "Our studies involved a systematic analysis of different symmetries which emerge ( noting the reasons above ) : symmetry in the modification direction ( Case 1 symmetry: arising from a symmetry in action of the two enzymes on their corresponding substrates ) , symmetry between different branches of modification ( Case 2 symmetry: arising from the same rates of modification to and from phosphoforms at a given level of modification: see text for distinction from Case 1 ) and combinations of the two ( Case 3 symmetry: arising from a symmetry in the action of the two enzymes in the modification/demodification at a given site , but on opposite legs ) .", "The different types of symmetries are encountered among the different classes of multisite modification networks ( with either common/separate kinase/phosphatase ) though some networks may not exhibit all symmetries ( summarized in Figure 4C ) .", "Case 1 symmetry can be broken in all the random modification networks where it exists .", "This symmetry breaking serves as a distinct mechanism for establishing directionality .", "Additionally , Case 1 symmetry breaking can also be observed in a simple ordered DSP network ( with only a single partial phosphoform ) , though only in the common kinase , common phosphatase case .", "This ordered network serves as a simpler network for understanding this symmetry breaking transparently .", "Case 2 symmetry breaking is the basis of ordering of modifications .", "While Case 2 symmetry is possible for all combinations of common/separate kinase and common/separate phosphatase , the symmetry is broken only for the different kinase and different phosphatase case .", "A combination of the flexibility afforded by the different sets of enzymes , along with sufficient nonlinearity ( due to enzymes participation in multiple complexes ) , enables this .", "Interestingly , if the dephosphorylation mechanism is processive ( rather than distributive , as assumed throughout ) , the separate kinase common phosphatase network can also give rise to Case 2 symmetry breaking , reinforcing how the interplay of processive and distributive modification can enable new behaviour ( see Suwanmajo and Krishnan , 2015 for another example ) .", "In Case 3 , the two symmetries necessarily break together .", "A distinct behaviour encountered here is the presence of symmetry breaking and oscillatory behaviour .", "In most cases , the symmetry-broken states are associated with transparent invariants which we analytically identify: these represent a behaviour reminiscent of absolute concentration robustness ( discussed below ) .", "Additionally , the symmetry breaking manifests as a supercritical pitchfork bifurcation , while in Case 3 symmetry with common kinase/common phosphatase , a subcritical pitchfork bifurcation is observed , along with possible tristability .", "Our analytical work reveals how symmetry breaking ( in all cases studied ) may be accessed in large regions of ( symmetric ) parameter space by varying total enzyme/substrate concentrations , which represent easy to manipulate experimental factors ( see Appendix 2—figure 7 ) .", "It is worth viewing the above results from a different perspective: the breaking of symmetry in ( potentially general ) biochemical networks of the ‘square’ topology ( discussed in Figure 1D ) .", "While symmetry simply imposes the restriction that the two legs of the network have kinetics which are identical , when can the symmetry actually break ?", "Our study presents multiple insights:", "( i ) firstly , a degree of nonlinearity is required , and this arises from conservation of species and sequestration of enzymes/substrates in complexes , a fundamental aspect of biochemical systems .", "All the modification networks we consider have enzymes shared between at least two enzyme-substrate complexes ( this stems from the fact that a given modification is effected by only one enzyme , and without any ordering ) and this provides the nonlinearity .", "( ii ) On the other hand , for symmetry breaking to occur , a sufficient flexibility is required in the network to be able to allow for this .", "This is clearly seen in Case 2 symmetry in modification networks ( with distributive enzyme mechanism ) , where reduced nonlinearity notwithstanding , it is only the separate kinase separate phosphatase modification network that allows symmetry to be broken .", "In general , there is a trade-off between nonlinearity and flexibility ( associated with distinct enzymes for different steps ) , but multisite modification provides many instances of sufficient combinations of both factors to realize symmetry breaking .", "These insights , bringing together basic ( bio ) chemistry and network features , are broadly relevant in biochemical networks .", "Enzyme sequestration ( and competition ) provides the key nonlinearity for generating symmetry breaking obviating the need for explicit feedback .", "Eliminating enzyme sequestration eliminates the possibility of symmetry breaking .", "Enzyme competition is a key ingredient in multisite modification , and in general this could combine with zeroth-order ultrasensitivity to generate new behaviour .", "However , the symmetry breaking we have found does not require any explicit assumption on the kinetic regime of enzymatic action ( as seen from the sufficient conditions we have obtained ) and so zeroth-order ultrasensitivity is neither necessary nor sufficient for this .", "We now discuss the relevance of our results from different vantage points .", "All of these underscore the fact that information processing is a characteristic and consequence of the modification network ( rather than an individual modification ) and that symmetry and symmetry breaking provides distinct classes of insights therein ( see Figure 4 ) .", "Multisite modification systems encountered in vivo often exhibit different degrees of ordering ranging from complete ordering of the sequence of modifications , to partial ordering , to a complete absence of ordering ( symmetric scenario ) .", "Ordering is a fundamental aspect of substrate modification and its deployment in different pathways and processes .", "In fact , ordering of modifications is key to establishing a strictly sequential logic , which is likely to be an important aspect of information processing in those cellular contexts .", "A range of studies focus on these contexts , basic principles , and the potent role in engineering multisite modification ( Kocieniewski et al . , 2012; Lyons et al . , 2013; Ramachandran et al . , 1992; Lössl et al . , 2016; Valk et al . , 2014; Kõivomägi et al . , 2013 ) .", "How ordering has emerged is however unclear , and there could be multiple contributing factors .", "Our results indicate that the basic biochemistry of multisite modification by itself provides the basis for creating an ordering by breaking symmetry .", "The biasing which emerges can itself be very significant , and with possible additional refinements , gives rise to ordering .", "This demonstrates that a key driving factor could be at the modification network level rather than at the molecular level .", "Our analysis of the different symmetry cases allows us to explore the different ways in which both ordering and directionality may be determined .", "We determine explicit conditions for the occurrence of symmetry breaking , revealing broad ranges of parameter space where this can happen .", "In the context of ordering , this , along with the demonstration of sufficiency of the conditions for symmetry breaking , demonstrates the robustness of the mechanism .", "We further point out that even if the system is not exactly symmetric an echo of such symmetry breaking may be seen , which is indicated by multiple steady states which strongly bias one pathway over another , in a manner which is not commensurate with the ( small ) differences in kinetics of the pathways ( Appendix 2—figures 5 and 6 ) .", "Given a symmetric ( Case 2 symmetry ) or close to symmetric network where different phosphoforms behave ( essentially ) the same , there are different ways in which evolution could lead to biasing of one modification pathway over the other .", "One is by effecting local changes in one of the pathways .", "Symmetry breaking allows for a distinct mechanism whereby changing one easy to manipulate parameter ( expression level of substrate ) , a significant biasing of one pathway over the other is established .", "This could be further reinforced ( if this is a desirable outcome ) by local changes in the pathway or increasing substrate amounts further ( which further accentuates the biasing ) .", "This can lead to either partial or even complete ordering subsequently .", "Thus the mechanism could be seen as an efficient way of effecting a substantial change which could be reinforced and consolidated by further tinkering .", "It can also generate different robustness characteristics .", "A similar comment applies to directionality .", "Case 3 symmetry breaking results in a combination of ordering and directionality , which ultimately manifests itself as elevated combinations of specific partial phosphoforms and unmodified/fully modified forms .", "Such a behaviour of the network ( for instance , if observed experimentally ) could easily be misinterpreted as suggestive of either some modification being inactive or there being a strong bias in the intrinsic kinetics , neither of which may be correct .", "Our results also provide important insights in the cases of larger numbers of modification sites .", "For instance , analogues of Case 2 symmetry breaking could explain both ordering and partial ordering ( some sequences of modifications ordered ) in those systems .", "Our analysis reveals the presence of ( exact ) ACR of different species in the symmetry-broken state .", "In this regard , we note that", "( i ) the relevant species ( in some cases , partial phosphoforms , and in others the fully modified or fully unmodified phosphoforms ) exhibit concentration robustness to changes in total substrate concentration and are fixed at a level corresponding to the concentration of these species at the symmetry-breaking bifurcation ( the inception of the asymmetric branch ) .", "( ii ) Depending on the network and the type of symmetry broken , this can manifest itself as ACR for pairs of species ( Case 1 and Case 2 ) .", "( iii ) In other cases ( Case 3 ) , the robustness is in the sum of concentrations of species , either exactly or approximately .", "From the above points , we see that multisite modification contains an in-built mechanism of creating robustness for clusters of species , either individually or collectively , something which represents an appealing characteristic for natural and engineered modification networks .", "It remains to be seen how this has been exploited in cells .", "( iv ) There are different ways in which ACR may be obtained ( for instance , in bifunctional enzymes Batchelor and Goulian , 2003; Krishnan et al . , 2020 ) .", "The mechanism seen here shares a feature of ACR observed in autocatalytic networks , arising from a transcritical bifurcation ( Shinar and Feinberg , 2011; Krishnan and Floros , 2019 ) as being intimately tied to a nonlinear dynamic transition arising from the biochemistry .", "In both cases , there is more than one steady state possible , and one of the steady states exhibits the ACR .", "Based on the above , a natural question is which substrates could exhibit ( exact ) ACR and whether symmetry is a prerequisite .", "We note that in the ACR we have made no assumption/restriction or invoked any particular kinetic regime for enzymatic action .", "We answer the questions ( based on analytical work: see Appendix 1 and Appendix 2—figure 9 ) relating to ACR in these terms in the ordered DSP network .", "( i ) Only Ap can exhibit ACR , and this occurs only in response to AT⁢o⁢t⁢a⁢l ( not KT⁢o⁢t⁢a⁢l or PT⁢o⁢t⁢a⁢l ) .", "( ii ) ACR necessarily requires multiple steady states , with two branches of steady states exhibiting ACR . There is another steady-state branch which does not exhibit ACR , but intersects one of the branches in what was computationally observed to be a transcritical bifurcation .", "( iii ) There is a constraint on parameters to enable this , which is weaker than the symmetry condition .", "( iv ) In the case of symmetry , the two ACR branches are symmetric and intersect with the other branch in a pitchfork bifurcation .", "As noted above , networks deviating from exact symmetry can exhibit approximate concentration robustness ( refer Appendix 2—figure 6 ) .", "Concentration robustness ( approximate ) could also be obtained in specific limiting kinetic parameter regimes .", "In ordered DSP with common kinase common phosphatase , we find that", "( i ) App and A could also exhibit concentration robustness .", "This can happen in a regime where the enzyme producing this ( from Ap ) acts in the saturated regime while the action of both enzymes on reactions not involving the species under consideration acts in the unsaturated limit ( see Appendix 1 ) .", "Here approximate ACR occurs without requiring multistability .", "( ii ) Similarly approximate ACR can occur in Ap without multistability by ( for instance ) having phosphorylation of A in the saturated regime and phosphorylation of Ap and dephosphorylation of Ap⁢p in the unsaturated limit ( or having phosphatases in excess ) .", "Similar insights can enable approximate ACR for one species in the corresponding random network ( see Appendix 1 ) .", "In contrast to such limiting regimes , absolute/approximate concentration robustness via symmetry breaking is present along with a rich repertoire of information processing characteristics .", "How does symmetry breaking in multisite modification both affect and be affected by the behaviour of a signalling network of which it is a part ?", "( i ) The importance of multisite modification stems from the fact that it confers functionality to proteins which can regulate other processes .", "Here the symmetry breaking allows for both regulation of downstream pathways as well as insulation of some downstream pathways from the effect of total upstream substrate and other upstream perturbations ( via ACR for specific substrates in the modification network ) .", "This ability to insulate some parts of a network , while not the others , is a desirable feature which can be exploited: symmetry breaking in multisite modification provides a way of realizing this exactly purely from chemistry without requiring elaborate network structures ( incorporating adaptation , feedback , etc . ) .", "( ii ) Additionally , we find examples of ‘shared ACR’ where the sum of two species concentrations is fixed .", "This represents a case where robustness is applied to a combination of pathways if the two species regulate different pathways .", "This may be relevant in multiple cell signalling contexts by directly incorporating an inbuilt trade-off between the activation of two pathways , for instance , for efficient resource allocation .", "If the two species regulate the same pathway in the same way , this translates into robustness in regulating the pathway , while allowing flexibility through the redundancy .", "( iii ) The effect of sequestration of a substrate species can be to either facilitate or make difficult the possibility of symmetry breaking .", "The sequestration of a substrate species in a downstream complex is the basis of a retroactive effect in a signalling pathway ( Ventura et al . , 2010; Del Vecchio et al . , 2008 ) .", "In the current case , this retroactive effect can help facilitate the possibility of symmetry breaking , and further that this happens in a context-dependent way .", "( iv ) Other factors associated with the network , for instance , feedback , may also significantly affect the possibility of this happening .", "These aspects need to be assessed systematically and will be studied in the future .", "Interestingly an existing study ( Krishnamurthy et al . , 2007 ) examines sequential multisite modification with two explicit feedbacks: one from the maximally modified phosphoform increasing the probability of ( every ) modification and the other from the unmodified form increasing the probability of every demodification .", "In a stochastic setting , this has been shown to result in breaking a symmetry between phosphorylation and dephosphorylation even with no enzyme sequestration .", "In contrast to this , all our studies are on the intrinsic behaviour of multisite modification and in a deterministic setting .", "Studies of multisite networks have focused on their capacity of generating oscillations ( Rust et al . , 2007; Van Zon et al . , 2007 ) , including random networks with common kinase and common phosphatase with a view to their relevance in circadian oscillators ( Jolley et al . , 2012 ) .", "A detailed computational study ( Jolley et al . , 2012 ) reveals regions of parameter space which facilitate the presence of oscillations , and the prominent regions are clustered around a symmetric network ( the Case 3 network that we have studied ) .", "What is the relevance of our analysis here ?", "Our study shows how oscillations occur in such cases , and also how ( by changing substrate amounts ) both oscillations and symmetry breaking may occur .", "We can identify different regimes based on our analysis .", "( i ) A regime where symmetry breaking is ruled out .", "Here our analysis indicates regimes where oscillations can occur without any potential interference from symmetry breaking .", "( ii ) A regime where symmetry breaking is possible , and in fact guaranteed , for some total substrate amount .", "In the latter case , we demonstrate that by varying total enzyme amounts ( easily tuneable dials ) it is possible to obtain , multistability , oscillations or a combination of such behaviour ( see Appendix 2—figure 7 ) .", "It indicates how in certain cases symmetry breaking may occur , limiting/preventing a range of oscillations .", "In particular , it indicates that oscillations do not have to be present for an indefinitely large range ( suggested in the computations of Jolley et al . , 2012 ) .", "We provide further insights with regard to oscillations .", "( iii ) The existence of long period oscillations which hover between different symmetric states is also seen ( Appendix 2—figure 2 ) .", "( iv ) We demonstrate the possibility of oscillations in networks with different kinases and phosphatases , which potentially benefit from a greater tuneability than the common kinase common phosphatase case .", "( v ) By contrast , we find that the other symmetries do not readily yield oscillatory behaviour , though further work needs to be done to study this exhaustively .", "The above points sharpen our understanding of oscillations emerging in random modification mechanisms and reinforce the theme of multisite modification as a complex information processor .", "Our analysis reveals the key features associated with symmetry breaking , which suggest multiple non-trivial signatures which could be seen experimentally: for instance , a considerable disparity in partial phosphoform behaviour , which may be incommensurate with the ( minor ) differences in kinetics in the legs of modification , characteristic patterns of ACR for specific species or groups .", "These signatures even if approximate could suggest the presence of symmetry breaking .", "On the other hand , experiments could be developed to realize this behaviour by constructing underlying modification circuits either for synthetic purposes or to probe and test the behaviour itself .", "The multiplicity of enzymes involved allows for the deployment of a broad experimental tool kit for these purposes .", "Systems mimicking Case 2 symmetry could be created by engineering different modification sites ( of similar properties ) , with modification by different isoforms of an enzyme .", "If this is also done for the demodification , then an approximate realization of a Case 2 symmetry ( different kinase different phosphatase ) can be realized .", "Alternatively , in a similar vein it may be possible to engineer a different kinase common phosphatase system with processive dephosphorylation: here the dephosphorylation could be induced to be processive ( as seen elsewhere in cellular contexts ) .", "Other approaches could involve reconstitution of components of existing systems , such as circadian oscillators .", "It is worth examining the implications and extensions of our study to a larger number of modification sites .", "Random networks lead to an exponential increase in the number of states .", "Additionally , the modifications/demodifications can be effected by common enzymes ( for all modifications ) , distinct enzymes ( for every modification ) , or a combination thereof , leading to a further combinatorial explosion in possibilities .", "Clearly direct analogues of the symmetry breaking seen here ( e . g . Case 1 and Case 2 ) can be encountered here .", "In addition , new possibilities can emerge .", "In Case 2 , for instance , in addition to the situation where all modification legs behave the same , we can have a situation where some modification legs ( or parts thereof ) are the same .", "Furthermore , not surprisingly , new behavioural characteristics can emerge .", "For instance , in the ordered triple-site phosphorylation network ( Case 1 symmetry: common kinase common phosphatase ) , we find shared robustness ( see Appendix 2—figure 8 ) , not seen in the ordered double-site modification .", "These aspects need a dedicated study of their own and will be studied in the future .", "Viewed from the perspective of information storage , symmetry breaking suggests that a symmetric double-site modification network contains a bit of information .", "We emphasize , however , that symmetric network encodes a richer set of information , such as simultaneously presenting homeostasis and multiple steady states , an observation relevant to networks of any number of modification sites .", "All in all , we have shown how basic biochemistry of multisite modification even within simple modification networks can be the basis of symmetry breaking .", "Symmetry breaking in turn can confer ordering , directionality , exact concentration robustness , and can significantly enhance the repertoire of information processing in multisite modification and regulation in signalling networks where they are present .", "The insights which arise from a structured systems study are of relevance in multiple contexts spanning the chemical and the biological , from systems biology to systems chemistry with potential in synthetic biology and the engineering of chemical systems ." ] ]

[ "Multisite modification is a basic way of conferring functionality to proteins and a key component of post-translational modification networks .", "Additional interest in multisite modification stems from its capability of acting as complex information processors .", "In this paper , we connect two seemingly disparate themes: symmetry and multisite modification .", "We examine different classes of random modification networks of substrates involving separate or common enzymes .", "We demonstrate that under different instances of symmetry of the modification network ( invoked explicitly or implicitly and discussed in the literature ) , the biochemistry of multisite modification can lead to the symmetry being broken .", "This is shown computationally and consolidated analytically , revealing parameter regions where this can ( and in fact does ) happen , and characteristics of the symmetry-broken state .", "We discuss the relevance of these results in situations where exact symmetry is not present .", "Overall , through our study we show how symmetry breaking", "( i ) can confer new capabilities to protein networks , including concentration robustness of different combinations of species ( in conjunction with multiple steady states ) ;", "( ii ) could have been the basis for ordering of multisite modification , which is widely observed in cells;", "( iii ) can significantly impact information processing in multisite modification and in cell signalling networks/pathways where multisite modification is present; and", "( iv ) can be a fruitful new angle for engineering in synthetic biology and chemistry .", "All in all , the emerging conceptual synthesis provides a new vantage point for the elucidation and the engineering of molecular systems at the junction of chemical and biological systems ." ]

[ "Proteins help our cells perform the chemical reactions necessary for life .", "Once proteins are made , they can also be modified in different ways .", "This can simply change their activity , or otherwise make them better suited for their specific jobs within the cell .", "Biological ‘catalysts’ called enzymes carry out protein modifications by reversibly adding ( or removing ) chemical groups , such as phosphate groups .", "‘Multisite modifications’ occur when a protein has two or more modifications in different areas , which can be added randomly or in a specific sequence .", "The combination of all the modifications attached to a protein acts like a chemical barcode and confers a specific function to the protein .", "Modification networks add levels of complexity above individual proteins .", "These encompass not only the proteins in a cell or tissue , but also the different enzymes that can modify them , and how they all interact with each other .", "Although our knowledge of these networks is substantial , basic aspects , such as how the ordering of multisite modification systems emerges , is still not well understood .", "Using a simple set of multisite modifications , Ramesh and Krishnan set out to study the potential mechanisms allowing the creation of order in this context .", "Symmetry is a pervasive theme across the sciences .", "In biology , symmetry and how it may be broken , is important to understand , for example , how organism develop .", "Ramesh and Krishnan used the perspective of symmetry in protein networks to uncover the origins of ordering .", "First , mathematical models of simple modification networks were created based on their basic descriptions .", "This system centred on proteins that could have phosphate modifications at two possible sites .", "The network was ‘symmetric’ , meaning that the rate of different sets of chemical reactions was identical , as were the amounts of all the enzymes involved .", "Dissecting the simulated network using a variety of mathematical approaches showed that its initial symmetry could break , giving rise to sets of ordered multisite modifications .", "Breaking symmetry did not require any additional features or factors; the basic chemical ‘ingredients’ of protein modification were all that was needed .", "The prism of symmetry also revealed other aspects of these multisite modification networks , such as robustness and oscillations .", "This study sheds new light on the mechanism behind ordering of protein modifications .", "In the future , Ramesh and Krishnan hope that this approach can be applied to the study of not just proteins but also a wider range of biochemical networks ." ]

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

[ "cell biology" ]

[ [ "Stochasticity in the abundance of cellular components is an intrinsic feature of biological systems .", "But while noise in molecular-scale processes such as gene expression and signal transduction have been examined in great detail , fluctuations have not been systematically characterized at one of the most critical scales of biological organization: compartmentalization of the eukaryotic cell into organelles .", "Two fundamental questions in systems cell biology are: how precisely does the cell control the number of a given organelle , and how do the mechanisms underlying organelle biogenesis affect this precision ?", "It has been suggested , for example , that the cell may be able to count and tightly control the number of a given organelle ( Rafelski and Marshall , 2008 ) .", "However , we lack comparisons of theoretical calculations of cell-to-cell variability in organelle abundances to experimentally measured organelle abundance distributions .", "There are four basic processes resulting in organelle abundance changes that can give rise to fluctuations in organelle number: de novo synthesis from a pre-existing membrane source , fission ( Lowe and Barr , 2007 ) , fusion ( Denesvre and Malhotra , 1996 ) , and decay such as through partitioning during cell division or autophagy ( van der Vaart et al . , 2008 ) .", "In the budding yeast Saccharomyces cerevisiae , Golgi abundance is the result of a steady-state determined by the balance of de novo synthesis and decay through maturation and dilution by cell division ( Rossanese et al . , 1999; Bevis et al . , 2002; Losev et al . , 2006; Matsuura-Tokita et al . , 2006 ) ; though modest levels of Golgi fusion have been reported ( Bhave et al . , 2014 ) decay appears to be dominated by maturation ( Losev et al . , 2006 ) .", "Vacuolar abundance is thought to be primarily determined by the balance of fission and fusion events ( Wickner , 2002 ) , though vacuoles can be generated de novo in mutant yeast strains with impaired vacuole inheritance pathways ( Banta et al . , 1988; Catlett and Weisman , 2000 ) and vacuolar membrane is generated de novo in cells to support vacuolar growth; the quantitative contribution of de novo vacuole biogenesis to vacuole copy number remains uncharacterized in physiological conditions .", "For other endomembrane organelles , however , the processes underlying abundance changes are even less clear .", "Peroxisomes , for example , are thought to increase in number by both de novo synthesis ( Hoepfner et al . , 2005; van der Zand et al . , 2012 ) and fission pathways ( Hoepfner et al . , 2001; Yan et al . , 2005; Motley and Hettema , 2007 ) .", "Mature peroxisomes do not undergo fusion events ( Motley and Hettema , 2007 ) .", "Furthermore , peroxisome abundances can be actively upregulated by culturing budding yeast cells in medium containing long chain fatty acids such as oleic acid ( Mast et al . , 2010 ) .", "We lack a quantitative understanding of whether de novo synthesis or fission dominates the generation of peroxisomes in either steady growth in glucose or during active proliferation in oleic acid .", "Here we present a stochastic model of organelle production , and combine a statistical property derived from the model , the Fano factor , with simple , experimentally-verified assumptions to yield two results .", "First , for organelles whose abundance-changing processes were previously characterized—the Golgi apparatus and vacuole—we made quantitatively accurate predictions about the size of organelle abundance fluctuations , leading to the surprising conclusion that the cell tolerates the maximum level of variability in Golgi apparatus and vacuole abundances generated by their biogenesis pathways .", "Second , for organelles where competing processes could contribute to organelle production but whose relative quantitative importance was unknown—the peroxisome—we used the model and experiment to infer that budding yeast cells switch from de novo synthesis dominated peroxisome production when grown in glucose-containing medium to fission dominated production in oleic acid-containing medium .", "Our theory of organelle biogenesis is very simple , but despite its simplicity we can gain mechanistic insight into the processes underlying organelle biogenesis .", "Though our framework suppresses details that could be relevant to organelle copy number regulation , such as when organelles share and thus compete for common biogenesis factors—as in the case of mitochondria and peroxisomes , which share fission factors in common for example—or when different processes affecting organelle copy number interact , such as the Golgi checkpoint regulating the progression of the mammalian cell cycle in which organelle biogenesis and decay would be coupled ( Sutterlin , et al . , 2002 ) , it is easily extendable to account for such effects .", "We therefore anticipate it will be a useful framework in which to analyze the pathways underlying organelle creation and destruction and subcellular structures more generally ." ], [ "To evaluate the relative importance of different biophysical pathways in shaping organelle abundances , we constructed a mathematical model of organelle abundance dynamics ( Figure 1A ) .", "As the average number of organelles is typically small , we formulated a model consisting of four coupled stochastic processes , each parameterized by an associated rate constant:10 . 7554/eLife . 02678 . 003Figure 1 . A stochastic model of organelle abundance dynamics .", "( A ) Organelle abundances are governed by four distinct biophysical processes:", "( i ) de novo synthesis ( kde novo , with units per time ) ;", "( ii ) fission ( kfission , with units per organelle per time ) ;", "( iii ) fusion ( kfusion , with units per organelle squared per time ) ; and", "( iv ) decay ( γ , with units per organelle per time ) .", "( B ) Sample trajectory generated by a Gillespie simulation of the model depicted in ( A ) in which the parameters are chosen to allow de novo synthesis to dominate over fission , fusion , and decay .", "( C ) Sample trajectory as in ( B ) , but with parameters chosen to allow fission to dominate over de novo synthesis , fusion , and decay .", "( D ) Sample trajectory as in ( B ) , but with parameters chosen to allow fusion to dominated over de novo synthesis , fission , and decay .", "( E ) Histogram of the number of organelles generated by Gillespie simulations with parameters chosen as in ( B ) .", "( F ) Histogram of number of organelles generated by Gillespie simulations with parameters chosen as in ( C ) .", "( G ) Histogram of number of organelles generated by Gillespie simulations with parameters chosen as in ( D ) .", "( H ) Analytical approximation of the variance of the organelle abundance distribution divided by the mean of the distribution , or Fano factor , as a function of the ratio of the fission to de novo synthesis rate for various values of the fusion rate; from top to bottom the curves are calculated in order of increasing fusion rates: 0 ( red ) , 0 . 25 ( orange ) , 1 ( green ) , 10 ( cyan ) , 100 ( blue ) .", "The value of γ is selected such that the mean organelle abundance is held constant for every point along these curves .", "( I ) Percent error in the Fano factor prediction from the analytical approximation compared to Fano factor calculated from Gillespie simulations of the reaction scheme .", "The percent error is computed as the de novo synthesis ( green dots ) , fission ( red dots ) , and fusion ( blue dots ) rate constants are varied to tune the mean organelle abundance from <n> ≈ 1 to <n> ≈ 4 , with the percent error between the prediction and simulation reaching close to 0% at <n> ≈ 2 . DOI: http://dx . doi . org/10 . 7554/eLife . 02678 . 00310 . 7554/eLife . 02678 . 004Figure 1—figure supplement 1 . Comparison of Fano factors calculated from simulations of model with first order decay process ( Model 1 ) with two alternative models substituting first order decay with partitioning at regular intervals . In Model 2 , first order decay is substituted for a decay process that reduces the organelle abundance by exactly one half at regular time intervals .", "In Model 3 , first order decay is substituted for a decay process that reduces the organelle abundance through binomial partitioning to daughter cells at regular time intervals with the probability of organelle inheritance to each daughter p=0 . 5 . DOI: http://dx . doi . org/10 . 7554/eLife . 02678 . 004", "( i ) de novo synthesis , in which new organelles are generated at a constant rate kde novo per time:n →kde novo n+1", "( ii ) fission , in which new organelles are generated at a rate kfission per organelle per time:n →kfissionn n+1", "( iii ) homotypic fusion , in which organelles are destroyed at a rate kfusion per organelle squared:n →kfusionn ( n−1 ) n−1", "( iv ) decay , representing an aggregation of a number of processes such as cell division , maturation , heterotypic fusion or autophagy , in which organelles are destroyed at a rate γ per organelle per time:n →γn n+1 These stochastic processes are then aggregated to yield the following master equation that describes the dynamics of organelle biogenesis and decay:dpn ( t ) dt= ( kde novo+kfission ( n−1 ) ) pn−1 ( t ) + ( γ+kfusionn ) ( n+1 ) pn+1 ( t ) − ( kde novo+kfissionn+kfusionn ( n−1 ) +γn ) pn ( t ) We performed stochastic simulations using the Gillespie algorithm ( Gillespie , 1977 ) in which de novo synthesis ( Figure 1B ) , fission ( Figure 1C ) , and fusion ( Figure 1D ) were allowed to dominate over other abundance-changing processes .", "We then built organelle abundance histograms from 10 , 000 repetitions of the stochastic simulations , which reveal that each process leaves a distinct statistical signature on the organelle abundance distribution ( Figure 1E–G ) —each process strongly affects the variance of the organelle abundance distribution .", "It is worth noting that our results are virtually unchanged if we incorporate processes mimicking partitioning of organelles at cell division ( Figure 1—figure supplement 1 ) , whether through deterministic reduction of organelle copy number by half or by probabilistic binomial partitioning to daughter cells at regular time intervals .", "To compare the variances of the distributions from different simulation regimes , we calculated the variance of the distribution divided by the mean of the distribution , also known as the Fano factor .", "We chose to work with the Fano factor , rather than alternative metrics for fluctuations such as the coefficient of variation , because it equals 1 for a Poisson distribution , and so we can readily detect the deviations from Poisson statistics that arise from fission and fusion mediated processes by comparing the observed Fano factor to 1 .", "To understand how de novo synthesis , fission , fusion , and decay affect the Fano factor for the abundance distribution of a given organelle , we analytically calculated the Fano factor for the reaction scheme depicted in Figure 1A .", "Building on earlier work from the theory of stochastic processes , Paulsson ( 2005 ) showed that the Fano factor for a given reaction scheme can be approximately calculated according to a restatement of the fluctuation dissipation theorem:σ2〈n〉=〈|δ|〉Cwhere 〈|δ|〉 is the average step size for the different possible stochastic processes contained in the reaction scheme ( i . e . , the ‘fluctuation’ component ) , and C is the adjustment that the reaction fluxes make in response to a given amount of change in the abundance ( i . e . , the ‘dissipation’ component ) :C=ΔR−/R−Δn/n−ΔR+/R+Δn/nwhere R− is the flux of organelle death events , R+ is the flux of organelle birth events .", "We refer the reader to a complete derivation of this result that is presented in great detail by Paulsson ( 2005 ) .", "Substituting the birth and death flux terms into the fluctuation-dissipation theorem and noting that since each process , whether de novo synthesis , fission , fusion or first order decay , adds or subtracts 1 organelle at a time therefore 〈|δ|〉 = 1 , we obtain: ( 1 ) σ2〈n〉=1kfusion ( 2〈n〉−1 ) +γkfusion ( 〈n〉−1 ) +γ−kfission〈n〉kde novo+kfission〈n〉where σ2 refers to the variance and <n> to the mean of the organelle abundance distribution .", "Consistent with our simulation results , the analytical expression for the Fano factor yields a quantity that increases with an increase in the ratio of the rates of fission and de novo synthesis , but decreases with an increase in the rate of fusion ( Figure 1H ) .", "Since Equation 1 is an approximation to the true Fano factor , it is important to assess the range over which it is valid .", "In Figure 1I , we compare our approximation for the Fano factor based on the fluctuation-dissipation theorem ( Equation 1 ) to Fano factors obtained by simulating the model using the Gillespie algorithm .", "Specifically , we vary either the de novo synthesis ( green dots ) , fission ( red dots ) or fusion ( blue dots ) rate constant while holding the others constant and for each combination of rate constants: ( 1 ) calculate the Fano factor using Equation 1 and mean organelle abundance; and ( 2 ) simulate the model using the Gillespie algorithm and calculate the Fano factor and mean organelle abundance from the organelle abundance distribution generated by the simulation .", "We then define the ‘percent error’ to be:percent error=calculated Fano factor−simulated Fano factorsimulated Fano factor To plot the curves presented in Figure 1I , we plotted the percent error metric as a function of the mean organelle abundance with each dot representing a set of rate constants .", "As shown in Figure 1I , no matter which rate constant is varied , if the mean organelle abundance is larger than 1 then the difference between the exact Fano factor obtained from simulation and the result of Equation 1 virtually disappears .", "Furthermore , as shown below , in physiologically relevant regimes the fluctuation-dissipation based approximation is highly accurate and in some cases reduces to the Fano factor obtained by exactly solving the master equation .", "Equation 1 can be broken down into distinct cases for different organelles , depending on which processes among de novo synthesis , fission , fusion and decay govern the abundance of a given organelle .", "Three of these cases are relevant to the organelles under study .", "In case 1 , corresponding to the Golgi apparatus , kfusion = kfission = 0 as the Golgi apparatus is only affected by de novo synthesis and decay through maturation in budding yeast ( Rossanese et al . , 1999; Bevis et al . , 2002; Losev et al . , 2006; Matsuura-Tokita et al . , 2006; Figure 2A ) .", "In this limit Equation 1 reduces to σ2/<n> = 1 , reflecting the fact that de novo synthesis and first order decay operating alone corresponds to a Poisson process; one of the hallmarks of the Poisson distribution is that its variance equals its mean , and our approximate equation for the Fano factor reduces exactly to this limit .", "Thus , we would expect that the Golgi abundance distribution should yield a Fano factor of 1 .", "To test this prediction , we performed spinning disc confocal microscopy on a budding yeast strain expressing the monomeric red fluorescent protein ( mRFP ) fused to the Golgi localized marker protein Anp1 ( Huh et al . , 2003 ) .", "Anp1-mRFP forms punctate spots ( Figure 2B ) marking the presence of individual Golgi , whose number we quantified in each cell to generate a Golgi abundance histogram from which we could calculate the Fano factor .", "We see excellent agreement between the theory and experiment , as the measured Golgi abundance distribution closely matches the Poisson distribution derived from the experimentally determined mean Golgi abundance ( Figure 2C ) and we measure a Fano factor σ2/<n> = 1 . 0 ± 0 . 1 ( Figure 2D , red bar ) , in agreement with the theoretically predicted σ2/<n> = 1 ( Figure 2D , blue bar ) .", "Interestingly , when we repeat these measurements for the late Golgi by performing confocal microscopy on a budding yeast strain expressing the green fluorescent protein ( GFP ) fused to the late Golgi marker protein Sec7 , we see virtually identical results with the late Golgi abundance distribution closely matching a Poisson distribution ( Figure 2—figure supplement 1A ) and thus yielding a measured Fano factor of σ2/<n> = 1 . 0 ± 0 . 1 ( Figure 2—figure supplement 1B ) .", "Furthermore , in order to reduce potentially confounding extrinsic sources of fluctuations due to variations in the phase of the cell cycle each cell in our population is in , we synchronized the cell cycle phases of the cells in our experiments by arresting them in S-phase of the cell cycle through treatment with 100 mM hydroxyurea .", "We see that synchronizing the cell cycle phases of the cells we examine by microscopy does not affect the Fano factors of the measured abundance distributions for the Golgi or late Golgi ( Figure 2—figure supplement 2A–D ) . 10 . 7554/eLife . 02678 . 005Figure 2 . Predicting the stochastic fluctuations in Golgi apparatus and vacuole abundances .", "( A ) Schematic depicting the biophysical processes that govern Golgi apparatus abundances .", "( B ) Spinning disc confocal microscopy images of the Golgi apparatus as visualized by the fusion protein Anp1-mRFP .", "( C ) Histograms depicting the theoretically predicted Golgi apparatus abundance distribution ( blue trace ) and experimentally measured single haploid cell Golgi apparatus abundance distribution ( red trace ) .", "N = 141 cells were analyzed .", "( D ) Bar graph depicting theoretical prediction ( blue bar ) and experimental measurement ( red bar ) of the Golgi apparatus abundance distribution Fano factor .", "( E ) Schematic depicting the biophysical processes that govern vacuole abundances .", "Error bars are ±1 standard error of the mean , estimated by bootstrapping .", "( F ) Spinning disc confocal microscopy images of the vacuole as visualized by the fusion protein Vph1 fused to green fluorescent protein ( Vph1-GFP ) .", "( G ) Histograms depicting the theoretically predicted vacuole abundance distribution ( blue trace ) and experimentally measured single haploid cell vacuole abundance distribution ( red trace ) .", "N = 95 cells were analyzed .", "( H ) Bar graph depicting theoretical prediction ( blue bar ) and experimental measurement ( red bar ) of the vacuole abundance distribution Fano factor .", "Error bars are ±1 standard error of the mean , estimated by bootstrapping . DOI: http://dx . doi . org/10 . 7554/eLife . 02678 . 00510 . 7554/eLife . 02678 . 006Figure 2—figure supplement 1 . Predicting the stochastic fluctuations in late Golgi abundances .", "( A ) Histograms depicting the theoretically predicted late Golgi apparatus abundance distribution ( blue trace ) and experimentally measured single haploid cell late Golgi apparatus abundance distribution ( red trace ) .", "N = 150 cells were analyzed .", "( B ) Bar graph depicting theoretical prediction ( blue bar ) and experimental measurement ( red bar ) of the late Golgi apparatus abundance distribution Fano factor .", "Error bars are ±1 standard error of the mean , estimated by bootstrapping . DOI: http://dx . doi . org/10 . 7554/eLife . 02678 . 00610 . 7554/eLife . 02678 . 007Figure 2—figure supplement 2 . Predicting stochastic fluctuations in Golgi , late Golgi and vacuole abundance distributions in budding yeast cells synchronized and arrested in S-phase of the cell cycle .", "( A , C , E )", "Histograms depicting the theoretically predicted Golgi apparatus ( A ) , late Golgi apparatus ( C ) , and vacuole ( E ) abundance distributions ( blue traces ) and experimentally measured single haploid cell abundance distributions ( red traces ) .", "N = 129 cells were analyzed in panel ( A ) , N = 163 cells were analyzed in panel ( C ) and N = 97 cells were analyzed in panel ( E ) .", "( B , D , F )", "Bar graphs depicting theoretical predictions ( blue bars ) and experimental measurements ( red bars ) of the Golgi apparatus ( B ) , late Golgi apparatus ( D ) , and vacuole ( F ) abundance distribution Fano factors .", "Error bars are ±1 standard error of the mean , estimated by bootstrapping . DOI: http://dx . doi . org/10 . 7554/eLife . 02678 . 007 In case 2 , corresponding to the vacuole , kde novo = γ = 0 as the vacuole is only affected by fission and fusion ( Figure 2E ) .", "In this limit , Equation 1 reduces to σ2〈n〉=1−1〈n〉 , where <n> is the mean number of vacuoles .", "Given an experimentally measured mean number of vacuoles of 2 . 1 ( Wickner , 2002; ‘Materials and methods’ ) , we make the non-trivial prediction that σ2/<n> = 0 . 5 for the vacuole distribution .", "This is a surprising and strong prediction of the model because a Fano factor of less than 1 resulting from fission and fusion processes operating together is purely due to the low numbers of vacuoles present at steady state; if <n> were much larger than 1 , then the Fano factor would approach 1 and be indistinguishable from case 1 .", "This distinction between case 1 and case 2 is a reflection of the fact that vacuole abundance , the result of a balance between fission and fusion events , follows a shifted Poisson distribution .", "In a shifted Poisson distribution , the probability of having n = 0 vacuoles is 0 because a cell can only reduce its vacuole numbers through fusion events to n = 1 vacuole , at which point the fusion rate kfusion n ( n-1 ) = 0 .", "The Fano factor for the shifted Poisson distribution can be calculated exactly and yields the same expression as Equation 1 with kde novo = γ = 0 .", "To test the predictions that vacuole abundances follow a shifted Poisson distribution with Fano factor σ2〈n〉=1−1〈n〉=0 . 5 , we visualize vacuoles by imaging a budding yeast strain that expresses the vacuolar membrane protein Vph1 fused to the green fluorescent protein ( GFP; Huh et al . , 2003 ) .", "Vph1-GFP forms discrete rings ( Figure 2F ) that we count in each cell to construct vacuole abundance distributions , as was done for the Golgi .", "We see an excellent match between theory and experiment; we observe a distribution overlapping with the theoretically predicted shifted Poisson distribution ( Figure 2G ) and measure a Fano factor σ2/<n> = 0 . 5 ± 0 . 1 ( Figure 2H , red bar ) , in agreement with the theoretically predicted σ2/<n> = 0 . 5 ( Figure 2H , blue bar ) ; we obtain the same match between theory and experiment in cells whose cell cycle phases have been synchronized to be in S-phase through hydroxyurea treatment ( Figure 2—figure supplement 2E–F ) .", "It is important to note that the close match between theory and experiment here suggests that de novo vacuole biogenesis , which is observed only in mutant strains that specifically disable vacuole inheritance , appears not to play a quantitatively significant role in affecting vacuole abundance in wild-type yeast .", "In an alternative model that allows de novo vacuole biogenesis to occur , Equation 1 reduces to σ2〈n〉=12〈n〉−1〈n〉−1−kfission〈n〉kde novo+kfission〈n〉; if we substitute the experimentally measured mean vacuole abundance of ≈2 into this expression and if we set this expression equal to the experimentally measured vacuole abundance distribution Fano factor of σ2/<n> = 0 . 5 , then solving for kde novo yields the result that kde novo ≈ 0 .", "Taken together , the cases of the Golgi apparatus and vacuole fluctuations allow us to make two conclusions .", "First , budding yeast cells tolerate the maximum level of variability generated by the biogenesis pathways governing Golgi apparatus and vacuole abundance , evidenced by the fact that our model predicted the experimental data with high quantitative accuracy without invoking any feedback control mechanisms to control the number of organelles .", "Second , as expected from theory , different biogenesis mechanisms generate differing levels of abundance fluctuations .", "At low mean organelle copy numbers , organelles governed by fission and fusion ( vacuole; Figure 2H ) inherently exhibit smaller abundance fluctuations than organelles governed by de novo synthesis and decay ( Golgi; Figure 2D ) .", "In the case of the vacuole , our fluctuation analysis also sheds light on the quantitative role played by de novo vacuole biogenesis ( Catlett and Weisman , 2000 ) .", "In particular , even though up to 50% of the vacuole membrane is generated de novo in a given cell ( Catlett and Weisman , 2000 ) , actual vacuole copy number is likely not strongly affected by de novo vacuole biogenesis , consistent with the most widely accepted model of vacuole biogenesis ( Wickner , 2002 ) .", "Thus we can use experimentally measured fluctuations in organelle abundance to make quantitative inferences about the relative contributions of different organelle biogenesis pathways for cases where the pathways are less understood .", "Given the success of the model in making predictions about the abundance distributions and magnitude of fluctuations in Golgi and vacuole abundances , in our last case we sought to use the model to infer mechanistic insight into organelle biogenesis .", "In case 3 , corresponding to the peroxisome , fusion is thought to be negligible and Equation 1 reduces to σ2〈n〉=1+kfission〈n〉kde novo ( Figure 3A ) .", "We can thus use the Fano factor to infer whether de novo synthesis or fission dominates the production of peroxisomes; this is a topic of active debate ( Hoepfner et al . , 2005; Motley and Hettema , 2007 ) .", "Specifically , in the absence of fusion , a Fano factor significantly larger than 1 indicates that fission dominates over de novo synthesis in generating an increased number of organelles , while a Fano factor close to 1 indicates that de novo synthesis dominates over fission .", "Even if kfusion >0 , a Fano factor larger than 1 still indicates that fission dominates over de novo synthesis .", "For example if fusion and first order decay reduces organelle numbers at similar rates , σ2〈n〉=2kde novo+3kfission〈n〉3kde novo+kfission〈n〉 which only grows much larger than 1 when kfission<n> >> kde novo .", "Furthermore , if de novo synthesis dominates over fission , we also expect to see that the organelle abundance distribution will closely match a Poisson distribution , as we observed for the Golgi apparatus , while if fission dominates then we expect a distribution broader than Poisson .", "Notably , peroxisomes are greatly upregulated in number when yeast cells are cultured in fatty acid-rich medium; therefore it is of interest to measure Fano factors for these organelle abundance distributions in both glucose and fatty-acid rich media . 10 . 7554/eLife . 02678 . 008Figure 3 . Inferring the dominant biophysical pathways in peroxisome biogenesis .", "( A ) Schematic depicting the biophysical processes that govern peroxisome abundances .", "( B ) Spinning disc confocal microscopy images of the peroxisome as visualized by the fusion protein YFP-PTS1-mRFP .", "( C ) Histograms depicting experimentally measured single cell peroxisome abundance distributions for haploid cells grown in 2% glucose ( red circles ) and haploid cells grown in 0 . 2% oleic acid ( dark red triangles ) .", "N = 129 cells were analyzed in glucose medium and N = 153 cells were analyzed in oleic acid medium .", "( D ) Bar graph depicting measured peroxisome abundance distribution Fano factors in glucose-rich and 0 . 2% oleic acid-rich media .", "The green dashed line indicates a Fano factor σ2/<n> = 1 , marking the boundary between de novo synthesis and fission dominated organelle production .", "Figure 3—figure supplement 1 depicts a peroxisome biogenesis model , referred to as Model 2 , alternative to the model depicted in panel ( A ) .", "Figure 3—figure supplement 2 depicts data similar to panels ( B–D ) but with Pex3-mRFP as the peroxisome marker .", "Figure 3—figure supplement 3 displays simulation results from Model 2 showing how increased pre-peroxisomal vesicle production affects the mean and Fano factor of the mature peroxisome abundance distribution .", "Figure 3—figure supplement 4 depicts the Fano factors of the Golgi apparatus and vacuole abundance distributions from cells grown in oleic acid-rich medium .", "Error bars are ±1 standard error of the mean , estimated by bootstrapping . DOI: http://dx . doi . org/10 . 7554/eLife . 02678 . 00810 . 7554/eLife . 02678 . 009Figure 3—figure supplement 1 . Incorporating pre-peroxisomal vesicle fusion into the model of de novo peroxisome biogenesis .", "( A ) Schematics of Models 1 and 2 .", "( B and C )", "Comparing the Fano factors ( C ) generated by one-step ( Model 1 ) vs vesicle fusion based peroxisome biogenesis ( Model 2 ) at equivalent mean peroxisome abundances ( B ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 02678 . 00910 . 7554/eLife . 02678 . 010Figure 3—figure supplement 2 . Measuring mature peroxisome abundance statistics using Pex3-mRFP as the peroxisomal marker .", "( A ) Spinning disc confocal image of budding yeast cells expressing Pex3-mRFP .", "( B ) Single cell peroxisome abundance distribution obtained from Pex3-mRFP expressing yeast cells cultured in glucose medium ( red circles ) and oleic acid containing medium ( maroon triangles ) .", "N = 447 cells were analyzed in glucose medium and N = 133 cells were analyzed in oleic acid medium .", "( C ) Fano factors calculated from single cell peroxisome abundance distributions depicted in panel ( B ) .", "Error bars are ±1 standard error of the mean , estimated by bootstrapping . DOI: http://dx . doi . org/10 . 7554/eLife . 02678 . 01010 . 7554/eLife . 02678 . 011Figure 3—figure supplement 3 . Predicting stochastic fluctuations in peroxisome abundance distributions in budding yeast cells synchronized and arrested in S-phase of the cell cycle .", "( A ) Histograms depicting the theoretically predicted peroxisome abundance distribution ( blue trace ) and experimentally measured single haploid cell peroxisome abundance distribution ( red trace ) .", "N = 168 cells were analyzed .", "( B ) Bar graph depicting theoretical prediction ( blue bar ) and experimental measurement ( red bar ) of the peroxisome abundance distribution Fano factor .", "Error bars are ±1 standard error of the mean , estimated by bootstrapping . DOI: http://dx . doi . org/10 . 7554/eLife . 02678 . 01110 . 7554/eLife . 02678 . 012Figure 3—figure supplement 4 . Effect of increasing pre-peroxisomal vesicle production in Model 2 on mean and mature peroxisome abundance distribution Fano factors . DOI: http://dx . doi . org/10 . 7554/eLife . 02678 . 01210 . 7554/eLife . 02678 . 013Figure 3—figure supplement 5 . Golgi and vacuole abundance distribution Fano factors obtained from cells cultured in oleic acid rich medium . Error bars are ±1 standard error of the mean , estimated by bootstrapping .", "N = 114 cells were analyzed in the case of the Golgi and N = 138 cells were analyzed in the case of the vacuole . DOI: http://dx . doi . org/10 . 7554/eLife . 02678 . 013 It is important to note , in applying our theory to peroxisome biogenesis , that these predictions are completely insensitive to whether or not the details of de novo peroxisome biogenesis , which occurs through fusion of pre-peroxisomal vesicles that bud off from the ER ( van der Zand , et al . , 2012 ) , are included in the model or not .", "The model we use to calculate Fano factors with ( Figure 3A ) uses a simplified peroxisome biogenesis process in which de novo peroxisome biogenesis proceeds as a single step of a mature peroxisome budding from the ER .", "In an alternative , more biologically detailed model ( Figure 3—figure supplement 1A ) we explicitly keep track of the two vesicle types , one bearing the peroxisomal membrane proteins ( PMPs ) Pex2 and Pex10 on their surfaces and the other the PMPs Pex13 and Pex14 on their surfaces , that fuse in order to form the peroxisome import machinery that then imports the enzymes that allow the peroxisome to carry out its metabolic functions ( van der Zand , et al . , 2012 ) .", "To check that our simplification of de novo peroxisome biogenesis does not introduce significant quantitative errors , we compare the mature peroxisome abundance statistics for the two alternative de novo biogenesis models ( one-step vs pre-peroxisomal vesicle fusion ) , isolated from any contributions from fission .", "We see that when we set the fission rate to 0 and tune the production rates in the two alternative de novo biogenesis models to produce the same mean peroxisome abundance ( Figure 3—figure supplement 1B ) , the alternative models of de novo peroxisome biogenesis produce abundance distributions whose Fano factors , σ2/<n> = 1 . 0 , are virtually identical to each other and to the value we expected from the Poisson distribution ( Figure 3—figure supplement 1C ) .", "From this result we conclude that our original simple , one-step de novo biogenesis pathway model is statistically equivalent to the more detailed model of de novo peroxisome biogenesis that explicitly tracks the creation and fusion dynamics of Pex2/10 and Pex13/14 containing vesicles .", "To visualize peroxisomes we imaged budding yeast strains containing fusions of the yeast enhanced monomeric Citrine ( yemCitrine ) protein to the peroxisome targeting signal PTS1 , consisting of the amino acids serine , lysine , and leucine at the C terminus of the protein ( Figure 3B ) ; we also repeated our peroxisome experiments using a budding yeast strain containing a fusion of the peroxisome membrane protein Pex3 with the monomeric red fluorescent protein ( mRFP; Figure 3—figure supplement 2A ) .", "The PTS1 and Pex3 reporters gave virtually indistinguishable results .", "After measuring single cell histograms of peroxisomes ( Figure 3C , Figure 3—figure supplement 2B ) , we calculate that the Fano factors for the peroxisome abundance distributions for cells cultured in glucose is σ2/<n> = 1 . 1 ± 0 . 1 ( Figure 3D , Figure 3—figure supplement 2C ) , which is also the peroxisome abundance distribution Fano factor we obtain from yeast cells arrested in S-phase of the cell cycle ( Figure 3—figure supplement 3 ) , leading us to infer that the organelle is generated primarily by de novo synthesis in glucose .", "In cells cultured in oleic acid-containing medium , however , we see a marked increase in the size of the organelle abundance fluctuations for peroxisomes ( Figure 3C , Figure 3—figure supplement 2B ) , with a measured Fano factor of σ2/<n> = 2 . 4 ± 0 . 2 ( Figure 3D , Figure 3—figure supplement 2C ) .", "Importantly , when the rates of pre-peroxisomal vesicle production and fusion are increased to match the increased mean mature peroxisome abundance observed in cells cultured in oleic acid medium in the alternative model of peroxisome biogenesis ( Figure 3—figure supplement 4A ) , the alternative model cannot explain this rise in the mature , import-competent peroxisome abundance distribution Fano factor upon transfer of cells to oleic acid containing medium ( Figure 3—figure supplement 4B ) .", "Therefore , we conclude that increased pre-peroxisomal vesicle production and fusion cannot explain the rise in the mature peroxisome abundance distribution Fano factor obtained from cells cultured in oleic acid medium .", "Instead we infer that upon transfer to oleic acid containing medium budding yeast cells primarily generate new peroxisomes by fission of pre-existing peroxisomes .", "To test whether our results are specific to peroxisomes or a more general aspect of culturing in oleic acid rich conditions , we also measured Golgi and vacuole abundance distributions in oleic acid rich medium .", "We confirm that for the Golgi apparatus the Fano factor remains virtually unchanged at σ2/<n> = 1 . 0 ± 0 . 2 while for the vacuole the Fano factor increases to σ2/<n> = 0 . 8 ± 0 . 1 respectively ( Figure 3—figure supplement 5 ) .", "This rise in the vacuole abundance distribution Fano factor is consistent with the increase in mean vacuole abundance in oleic acid cultured cells from <n> = 2 . 1 to <n> = 4 . 0 .", "Our model of organelle biogenesis makes predictions about how the magnitude of organelle abundance fluctuations will change if the mean organelle abundance is changed .", "For example , in the case of the Golgi apparatus and peroxisomes in glucose-grown yeast cells , where de novo synthesis and first order decay dominate the organelle abundance distributions , we expect that even if the mean is increased the distribution will still follow a Poisson distribution and the Fano factor will remain at σ2/<n> = 1 .", "On the other hand , for the case of the vacuole if the mean vacuole abundance is doubled from 2 to 4 , we expect that while the distribution will still follow a shifted Poisson distribution , the Fano factor , which recall follows σ2〈n〉=1−1〈n〉 for the shifted Poisson , will increase from σ2/<n> = 0 . 5 to σ2/<n> = 0 . 75 .", "We conjectured , motivated by the observation that organelle sizes scale with cellular volume and ploidy ( Weiss et al . , 1975; Chan and Marshall , 2010; Uchida et al . , 2011 ) , that diploid cells may have higher mean numbers of organelles , enabling us to experimentally evaluate our predictions of the relationship between mean and fluctuation size .", "We constructed diploid versions of the strains used for our above measurements and repeated our imaging experiments on these diploid strains with fluorescently labeled organelles .", "We found that vacuole and peroxisome abundance distributions have increased means in diploid cells compared to haploid cells; the Golgi apparatus and late Golgi notably did not yield a statistically significant increase in mean abundance in diploid cells ( Figure 4—figure supplements 1 , 2 ) .", "In the case of the diploid vacuole abundance distribution , <n> = 3 . 6 , which represents a 71% increase from the haploid value of 2 . 1 ( Figure 4A , B ) .", "The diploid vacuole abundance distribution is still well described by a shifted Poisson distribution that is calculated from the experimentally measured mean , again with no fitting parameters ( Figure 4A , blue line ) .", "Our equation for the Fano factor predicts that when the vacuole mean is <n> = 3 . 6 , the Fano factor σ2〈n〉=1−1〈n〉=0 . 7 , higher than the prediction in the haploid case of σ2/<n> = 0 . 5 .", "In diploid cells expressing the Vph1-GFP marker we measure a Fano factor of σ2/<n> = 0 . 7 ± 0 . 1 , significantly higher than the haploid value of σ2/<n> = 0 . 5 ± 0 . 1 ( Figure 4C ) , and consistent with our theoretical prediction . 10 . 7554/eLife . 02678 . 014Figure 4 . Predicting organelle abundance fluctuations in diploid cells .", "( A ) Histograms depicting the theoretically predicted vacuole abundance distribution ( blue trace ) and experimentally measured single diploid cell vacuole abundance distribution ( red trace ) .", "N = 127 cells were analyzed .", "( B and C )", "Bar charts comparing experimentally measured vacuole abundance means ( B ) and Fano factors ( C ) in haploid and diploid cells cultured in glucose-rich medium .", "( D ) Histograms depicting the theoretically predicted peroxisome distributions ( blue trace ) and experimentally measured single diploid cell peroxisome abundance distributions ( red trace ) .", "N = 154 cells were analyzed .", "( E and F )", "Bar charts comparing experimentally measured peroxisome means ( E ) and Fano factors ( F ) in haploid and diploid cells cultured in glucose-rich or oleic acid ( OA ) medium .", "Green line in panel ( F ) indicate Fano factor of 1 .", "Error bars are ±1 standard error of the mean , estimated by bootstrapping .", "Figure 4—figure supplement 1 displays the Golgi apparatus abundance distribution and its Fano factor from diploid cells grown in glucose-rich medium .", "Figure 4—figure supplement 2 depicts data similar to panels ( D–F ) but with Pex3-mRFP as the peroxisome marker . DOI: http://dx . doi . org/10 . 7554/eLife . 02678 . 01410 . 7554/eLife . 02678 . 015Figure 4—figure supplement 1 . Golgi apparatus abundance fluctuations in diploid cells .", "( A ) Single cell histogram of Golgi apparatus count ( red trace ) compared to theoretically calculated Poisson distribution based on measured Golgi apparatus mean ( blue trace ) .", "N = 77 cells were analyzed .", "( B ) Fano factor measured from Golgi apparatus abundance distribution compared to theoretical prediction from Equation 1 .", "Error bars are ±1 standard error of the mean , estimated by bootstrapping . DOI: http://dx . doi . org/10 . 7554/eLife . 02678 . 01510 . 7554/eLife . 02678 . 016Figure 4—figure supplement 2 . Late Golgi apparatus abundance fluctuations in diploid cells .", "( A ) Single cell histogram of late Golgi apparatus count ( red trace ) compared to theoretically calculated Poisson distribution based on measured late Golgi apparatus mean ( blue trace ) .", "N = 131 cells were analyzed .", "( B ) Fano factor measured from late Golgi apparatus abundance distribution compared to theoretical prediction from Equation 1 .", "Error bars are ±1 standard error of the mean , estimated by bootstrapping . DOI: http://dx . doi . org/10 . 7554/eLife . 02678 . 01610 . 7554/eLife . 02678 . 017Figure 4—figure supplement 3 . Peroxisome abundance fluctuations in diploid cells measured by Pex3-mRFP .", "( A ) Single cell histogram of peroxisome count ( red trace ) compared to theoretically calculated Poisson distribution based on measured peroxisome mean ( blue trace ) .", "N = 123 cells were analyzed .", "( B ) Mean peroxisome abundance from diploid cells cultured in glucose medium compared to haploid cells cultured in glucose medium and oleic acid medium .", "( C ) Fano factor measured from peroxisome abundance distribution from diploid cells compared to haploid cells cultured in glucose medium and in oleic acid medium .", "The green dashed line represents a Fano factor of 1 .", "Error bars are ±1 standard error of the mean , estimated by bootstrapping . DOI: http://dx . doi . org/10 . 7554/eLife . 02678 . 017 In the case of peroxisomes , the diploid organelle abundance distributions yield <n> = 6 . 8 , respectively , when cells are cultured in glucose-rich medium , representing roughly a doubling in mean abundance in each case compared to the haploid abundance distributions ( Figure 4D , E ) .", "As mentioned above , the organelle abundance distribution should follow the Poisson distribution , even with the higher mean , because the underlying biogenesis mechanisms of de novo synthesis and decay should not have changed .", "We calculated the Poisson distribution derived from the experimentally measured mean abundances and find that the predicted distributions , even with no fitting parameters , closely describe the experimentally measured abundance distributions ( Figure 4D ) .", "Finally , the Fano factor for the peroxisome abundance distribution should remain σ2/<n> = 1; a hallmark of the Poisson distribution is that the Fano factor is 1 for any mean , a fact also reflected in Equation 1 with kfusion = kfission = 0 .", "The measured Fano factor matches the theoretical prediction closely at σ2/<n> = 1 . 0 ± 0 . 1 for the peroxisome ( Figure 4F ) .", "As before , we obtain indistinguishable results for peroxisomes labeled by fusing the peroxisomal membrane protein Pex3 to mRFP ( Figure 4—figure supplement 3 ) .", "Interestingly , the Fano factors for the peroxisome abundance distribution in diploid cells grown in glucose are much smaller than the Fano factors measured in cells grown in oleic acid-rich medium ( Figure 4F ) despite the fact that their mean values are similar ( Figure 4E ) .", "This provides additional evidence that peroxisome biogenesis involves fundamentally different mechanisms in glucose-cultured cells vs oleic acid-cultured cells , with our model pointing to the less noisy de novo synthesis pathway dominating the former and the more noisy fission pathway dominating the latter .", "Finally , we tested the mechanistic prediction that peroxisomes switch from de novo synthesis dominated production in glucose-containing medium to fission dominated production in oleic acid-containing medium .", "Peroxisome fission is mediated by the proteins Vps1 , Dnm1 and its accessory protein Fis1 , with the dominant role played by Vps1 ( Kuravi et al . , 2006 ) .", "We engineered yeast strains containing fluorescently labeled peroxisomes and lacking either Dnm1 , Vps1 , or Fis1 , cultured these cells in oleic acid medium , counted the number of peroxisomes in each cell , and calculated the mean and Fano factor of the peroxisome abundance distributions for each strain ( Figure 5 ) .", "We also repeated these experiments in yeast cells bearing peroxisomes labeled by fusing Pex3 with mRFP and obtained virtually indistinguishable results ( Figure 5—figure supplement 1 ) .", "We expect that if fission dominates peroxisome proliferation in cells cultured in oleic acid medium , the mean peroxisome abundance will decrease upon deletion of Vps1 , Dnm1 and Fis1 , and the Fano factor for the peroxisome abundance distribution for the mutant strains will decrease toward σ2/<n> = 1 , the value for de novo synthesis dominated production .", "We find that the mean peroxisome abundance decreases by fourfold in vps1Δ cells , while the peroxisome abundance distribution Fano factor is reduced threefold .", "As expected , the effects in fis1Δ and dnm1Δ cells are weaker: the mean peroxisome abundance decreases by slightly less than twofold in fis1Δ cells and roughly 10% in dnm1Δ cells , while the Fano factors for fis1Δ and dnm1Δ cells are reduced by ∼30% and ∼15% respectively .", "Most importantly , the Fano factor measured for vps1Δ cells is σ2/<n> = 1 . 0 , exactly what one would expect for de novo synthesis dominated peroxisome production .", "These results strongly suggest that fission dominates peroxisome proliferation in wild-type cells cultured in oleic acid medium . 10 . 7554/eLife . 02678 . 018Figure 5 . The effect of fission factor deletions on peroxisome abundance statistics . Budding yeast cells containing peroxisomes labeled by YFP-PTS1 and lacking one of the peroxisome fission factors VPS1 , DNM1 or its accessory factor FIS1 were cultured in medium containing 0 . 2% oleic acid for 20 hr .", "Single cell peroxisome abundance distributions were measured for each of these strains .", "The Fano factors are plotted as a function of mean peroxisome abundance extracted from the single cell peroxisome distributions .", "Figure 5—figure supplement 1 shows data similar to Figure 5 but with Pex3-mRFP as the peroxisome marker . DOI: http://dx . doi . org/10 . 7554/eLife . 02678 . 01810 . 7554/eLife . 02678 . 019Figure 5—figure supplement 1 . Peroxisome abundance fluctuations in cells containing deletions of the fission factors Dnm1 , Fis1 and Vps1 measured labeling of peroxisomes by Pex3-mRFP . DOI: http://dx . doi . org/10 . 7554/eLife . 02678 . 019" ], [ "Perhaps the most surprising aspect of the comparison between our model of organelle abundances and measurements of endomembrane organelle abundance distributions is the close match between theory and experiment without the need to modify the model to take account feedback control mechanisms .", "This is in stark contrast to the expectation that cells know how to count and tightly control the number of its various organelles ( Rafelski and Marshall , 2008 ) .", "While some subcellular structures such as the nucleus and centrioles are clearly under the control of strong feedback control mechanisms that suppress fluctuations in their abundances ( Marshall , 2007 ) , the cell apparently tolerates the maximum amount of variability in endomembrane organelle abundance generated by a given set of biogenesis mechanisms .", "It must be noted , however , that while the close match between our theory and experiment without invoking any feedback control implies that any feedback control mechanisms operating on endomembrane organelle copy numbers are no more precise than the biogenesis system could achieve without feedback , we do not explicitly rule out the presence of feedback regulation of the organelle copy numbers .", "Our model is also very simple , and though we were able to obtain new insights into organelle biogenesis using the model we are likely suppressing details that could affect organelle abundance under certain conditions .", "For example , it has been recently shown that the rate of peroxisome decay via autophagy depends on the existence of a functional fission pathway ( Mao et al . , 2014 ) .", "Nevertheless , it would be trivial to extend our model to incorporate such findings; in the case of fission-dependent autophagy rates , for example , one would simply need to rewrite the decay rate to be a function of the fission rate rather than just being a simple numerical parameter .", "Such effects would of course require us to take greater care in interpreting the results of the model , but do not invalidate that model; indeed deviations from our simple model could even facilitate discovery of effects such as coupling between the different processes affecting organelle copy number and feedback control of organelle copy numbers .", "Finally , the consequences of these fluctuations in organelle abundance on the fundamental cell biological processes controlled by these organelles , ranging from secretion to metabolism , remain to be explored .", "Assessing the dependence of cell biological processes on the abundance of the organelles governing these processes requires systematic , quantitative measurements such as , for example , how the rate of secretion depends on Golgi apparatus abundance or how protein degradation rates depend on vacuole abundances .", "Our results suggest a resolution to the long-running debate over whether peroxisome biogenesis is the result of de novo synthesis or fission ( Hoepfner et al . , 2005; Motley and Hettema , 2007; Mast et al . , 2010; van der Zand et al . , 2012 ) , with our theory and measurements supporting a model in which peroxisomes are created de novo in glucose-rich conditions , but switch to primarily fission-based proliferation in fatty acid rich environments that demand peroxisomal function .", "With these quantitative tools in hand to characterize organelle abundance processes , it will be of great interest to uncover functional reasons why the cell employs de novo synthesis vs fission to proliferate organelles .", "Given that peroxisomes appear to switch from de novo synthesis to a noisy , fission dominated creation , it will be particularly interesting to measure the degree to which the abundances of peroxisomes with other metabolic organelles such as the mitochondria or lipid droplets are correlated in single cells .", "These correlation measurements can allow us to infer the design principles underlying cellular responses to fatty acid rich conditions: anti-correlations in noisy peroxisome and lipid droplet production , for example , would suggest a model in which different cells specialize in lipid metabolism vs storage , while correlated production would favor a model in which only a subset of cells specialize in responding to the environmental change .", "Along with previously developed frameworks examining variability in organelle number ( Hennis and Birky , 1984; Marshall , 2007 ) , our model can aid in examining the functional consequences of stochastic fluctuations in organelle abundance .", "Perhaps most importantly , the generality of our approach makes it amenable to analyzing the wealth of subcellular compartments and granules in prokaryotes ( Yeates et al . , 2008 ) and even eukaryotes whose biogenesis mechanisms we do not yet understand or are just discovering ( Narayanaswamy et al . , 2008 ) .", "With previous examinations of organelle number control ( Hennis and Birky , 1984; Marshall 2007 ) and our analysis of the peroxisome fission pathway as guides , we hope that our model will be used as a framework in which to interpret future genetic studies that aim to uncover the biophysical pathways responsible for the biogenesis of subcellular structures ." ], [ "All strains were taken from the collection of GFP fusion strains generated by Huh et al . ( 2003 ) .", "This collection includes one set of strains used as organelle references against which the localization of all other fluorescently tagged proteins were scored .", "Strains from this reference strain set contain the monomeric red fluorescent protein ( mRFP ) fused to a protein that localizes to a specific organelle with high reliability .", "Where possible , as was the case for endosomes , Golgi apparatus , and peroxisomes , we selected these organelle reference strains for visualization .", "In order to visualize mature , import-competent peroxisomes , we engineered a yeast strain that expressed a fusion of the peroxisome targeting signal 1 ( PTS1 ) , consisting of the amino acids serine , lysine and leucine , to the extreme C terminus of the monomeric , yeast-enhanced Citrine fluorescent protein ( yemCitrine ) .", "We used fusion PCR to generate a construct consisting of the yemCitrine-PTS1 gene flanked by the promoter of the TEF2 gene on the 5′ end and the transcriptional terminator element of the ADH1 gene on the 3′ end and integrated this construct into the yeast genome at the HIS3 locus .", "For the case of the vacuole , for which no mRFP reference strain was created , we used a strain from the library that was engineered to contain the green fluorescent protein ( GFP ) fused to the vacuolar membrane protein Vph1 .", "For glucose medium , strains were grown to mid-log phase at 30°C in standard synthetic medium containing 2% glucose and subsequently imaged .", "For oleic acid medium , strains were grown to mid-log phase at 30°C in standard synthetic medium containing 2% glucose , washed twice , and resuspended in medium containing 0 . 3% yeast extract , 0 . 6% peptone , 0 . 1% glucose , 0 . 1% Tween40 , and 0 . 2% oleic acid , cultured for 20 hr in the oleic acid rich medium , and subsequently imaged .", "Mating type a ( MATa ) yeast cells expressing green fluorescent protein ( GFP ) labeled organelles from the Huh et al . ( 2003 ) collection were grown to OD600 = 0 . 1 in complete synthetic medium .", "10 μM alpha factor were then added to the cell culture for 2 hr to arrest the cells in the G1 phase of the cell cycle .", "Following washing of the cells twice with 50 ml complete synthetic medium without alpha factor to remove the alpha factor from the culture medium , the G1 synchronized cells were transferred into medium containing 100 mM hydroxyurea to arrest the cells in S-phase .", "Following 2 hr of hydroxyurea treatment , the cells were fixed with 3 . 7% formaldehyde , washed , and imaged .", "Between N = 100–300 cells were imaged on an inverted Olympus IX-71 microscope fitted with a Perkin–Elmer spinning disc including a Yokogawa head .", "Samples were illuminated with laser light at 561 nm ( mRFP ) and 491 nm ( GFP ) and imaged on a Hammamatsu EMCCD camera .", "Cell segmentation was performed manually using ImageJ .", "To obtain the number of organelles in each cell , we split the data into two cases .", "For those organelles that appear as discrete foci ( endosomes , Golgi apparatus , peroxisomes ) individual slices of each image stack were filtered with a Gaussian blurring filter to eliminate high frequency noise in the image , followed by a Laplacian second derivative filter to sharpen edges thereby enhancing the foci .", "The filtered images were then thresholded to identify those pixels belonging to an organelle .", "Finally the organelles could be assigned to single cells using the manually segmented image .", "This analysis was carried out in MATLAB .", "All organelle identification and assignment to single cells was manually verified .", "In the second case , where the organelle ( vacuole ) appears as a discrete ring , all quantification of number of organelles per cell was done manually ." ] ]

[ "Fluctuations in organelle abundance can profoundly limit the precision of cell biological processes from secretion to metabolism .", "We modeled the dynamics of organelle biogenesis and predicted that organelle abundance fluctuations depend strongly on the specific mechanisms that increase or decrease the number of a given organelle .", "Our model exactly predicts the size of experimentally measured Golgi apparatus and vacuole abundance fluctuations , suggesting that cells tolerate the maximum level of variability generated by the Golgi and vacuole biogenesis pathways .", "We observe large increases in peroxisome abundance fluctuations when cells are transferred from glucose-rich to fatty acid-rich environments .", "These increased fluctuations are significantly diminished in mutants lacking peroxisome fission factors , leading us to infer that peroxisome biogenesis switches from de novo synthesis to primarily fission .", "Our work provides a general framework for exploring stochastic organelle biogenesis and using fluctuations to quantitatively unravel the biophysical pathways that control the abundance of subcellular structures ." ]

[ "Any cell that has a nucleus also contains various other organelles , such as the mitochondria that generate energy inside the cells .", "Like the nucleus , most of these organelles are enclosed within a membrane .", "Unlike the nucleus , however , there can be two or more copies of other types of organelles in a healthy cell .", "How do the numbers of the different organelles in a cell change ?", "The copy number for a given organelle can be increased in two ways: by the synthesis of new organelles , or the fission of an existing organelle to form two new organelles .", "Conversely , the number of organelles can also be decreased in two ways: an organelle can decay , or two organelles can fuse to form one new organelle .", "The steady state for a given organelle results from a balance of these creative and destructive processes .", "Researchers have thought for some time that cells are able to count how many organelles of a given type they contain .", "It was also thought that cells have some control over this number , but it was not known how precisely cells could control the number of organelles they contained .", "It was also not known how this level of precision was influenced by the different processes responsible for making new organelles .", "To address these issues , Mukherji and O'Shea have developed a stochastic model that treats the processes of organelle creation and destruction as if they were simple chemical reactions .", "A tool from statistical physics , known as the fluctuation-dissipation theorem , was then used to analyze this model and derive an equation that predicts how the fluctuations in organelle number depend on the rates of the processes that govern organelle number .", "Mukherji and O'Shea used this model to make predictions about various organelles in the budding yeast S . cerevisiae .", "For two of these—vacuoles and the Golgi apparatus—the processes that lead to an increase or decrease in the number of organelles are well understood .", "In both cases the model accurately predicted the level of fluctuations measured in experiments .", "Moreover , Mukherji and O'Shea found that cells exhibited the maximum predicted level of fluctuations that could be generated by the processes that either increased or decreased the number of each organelle .", "The model was also able to shed light on a long-running debate over the cellular origins of an organelle called the peroxisome .", "This organelle—which is involved in breaking down fatty acids and other compounds—has been studied much less than the Golgi apparatus and vacuoles , but there is compelling evidence that new peroxisomes are created by de novo synthesis and by the fission of existing peroxisomes .", "Mukherji and O'Shea found that fluctuations in the number of peroxisomes suggest that the production of new peroxisomes is dominated by fission when the yeast cells are grown in a medium that is rich in oleic acid: peroxisomes are metabolically active and proliferate rapidly in such a medium .", "In a glucose-rich medium , on the other hand , most new peroxisomes are produced by de novo synthesis .", "The case of the peroxisome thus highlights the possibility of extending this mathematical framework to explain the creation and destruction of organelles and other subcellular structures in a range of organisms and environments ." ]

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

[ "ecology", "epidemiology and global health", "tools and resources" ]

[ [ "Emerging and re-emerging human infectious diseases have increased in recent years .", "Around one-fourth of the 1415 pathogens known to infect humans appeared between 1940 and 2004 and their appearance has gradually increased since 1980 ( Taylor et al . , 2001; Woolhouse and Gaunt , 2007; Jones et al . , 2008; Daszak et al . , 2004 ) .", "Today , seven new pathogens appear every year and this number should reach 15–20 by 2020 ( Woolhouse et al . , 2008 ) , mostly due to the growth of human activities that increase contact with novel sources of pathogens and favor their spread worldwide ( Murray et al . , 2015 ) .", "Emerging threats mainly concern viruses , such as HIV ( Sharp and Hahn , 2011 ) , SARS-CoV and MERS-CoV ( de Wit et al . , 2016 ) , avian flu ( Alexander , 2007 ) and more recently Ebola ( Baize et al . , 2014 ) , chikungunya ( Burt et al . , 2012 ) and Zika ( Wikan and Smith , 2016 ) .", "However , disease emergence and re-emergence also concern bacteria ( e . g . Helicobacter pylori , Salmonella sp . , etc . ) and parasites ( e . g . Plasmodium knowlesi in South-East Asia ) .", "Sixty per cent of diseases emerging in humans are zoonoses and wildlife plays a key role by providing a zoonotic pool from which previously unknown pathogens may emerge ( Taylor et al . , 2001; Woolhouse and Gaunt , 2007; Jones et al . , 2008; Daszak et al . , 2004 ) .", "The case of P . knowlesi in South-East Asia is a good example .", "This parasite emerged in the human population after a transfer from Asian macaques .", "It is now considered as the fifth human malaria agent after Plasmodium falciparum , Plasmodium vivax , Plasmodium malariae and Plasmodium ovale ( Singh and Daneshvar , 2013 ) .", "Such emerging diseases constitute a massive public health issue that requires active monitoring for signs of outbreaks and rapid diagnosis of the involved pathogen .", "Therefore , it is crucial to anticipate and prevent potential epidemic and pandemic outbreaks by developing new methods for the early detection and monitoring of infectious agents in wild animal sources ( Kuiken et al . , 2005; Wolfe et al . , 2005 ) .", "However , in many cases , monitoring is limited or impossible due to our poor knowledge about the ecology of these pathogens ( i . e . where , when and how these agents circulate in the wildlife ) .", "The case of the Ebola virus is quite exemplary .", "Indeed , the exact nature of its reservoir ( s ) remains uncertain , although thousands of animals have been screened during the last 40 years ( e . g . [Marí Saéz et al . , 2015] ) .", "Nowadays , pathogen circulation in wild animals is screened using mainly two methods: bushmeat analysis or direct trapping of animals for organ and tissue collection .", "These methods are pertinent in many cases , but present some weaknesses .", "Bushmeat represents only a fraction of the fauna ( the one consumed by humans ) , whereas animal trapping can be difficult or dangerous .", "Moreover , such manipulation may be harmful for threatened and protected species .", "As a consequence , several methods were developed in the last years to study pathogen diversity from wild fauna without the need of direct contacts with animals , for example , by using fecal , urine or saliva samples ( e . g . [Santiago et al . , 2002; Prugnolle et al . , 2010; Pesapane et al . , 2013; Taberlet et al . , 2012] ) .", "However , the value of these non-invasive methods remains limited because not all pathogens can be detected and not all reservoirs can be explored by these methods ( for instance , it is difficult to collect feces or saliva of reptiles without trapping them ) .", "Therefore , new non-invasive methods are crucially needed to provide new opportunities for screening a larger range of hosts and pathogens .", "The use of hematophagous flies as ‘flying syringes’ may constitute a new approach to track and survey blood-borne pathogens in the wild ( Calvignac-Spencer et al . , 2013 ) .", "Nucleic acids ( DNA or RNA ) of vertebrate hosts or of pathogens in arthropod blood meals are preserved and detectable for several days ( Calvignac-Spencer et al . , 2013; Kent , 2009; Muturi et al . , 2011; Grubaugh et al . , 2015; Lee et al . , 2015 ) .", "For example , HIV was detected 8 days and 10 to 14 days after blood ingestion by bugs and by ticks , respectively ( Webb et al . , 1989; Humphery-Smith et al . , 1993 ) .", "Recently , the H5N1 flu virus was found viable in mosquitoes ( Barbazan et al . , 2008 ) , although its transmission by these insects is unproven ( Sawabe et al . , 2006 ) .", "Grubaugh and colleagues ( Grubaugh et al . , 2015 ) applied such an idea ( that they called ‘xenosurveillance’ ) using Anohpeles mosquitoes to estimate the diversity of viruses infecting human populations in remote areas .", "Nevertheless , blood-engorged mosquitoes are very difficult to collect in forest and often show strong host preferences ( in particular for mammals ) .", "Arthropods with more generalist blood feeding patterns would be more useful to survey pathogens from a large range of vertebrates ( including mammals , birds and reptiles ) in these highly complex ecosystems .", "Hematophagous flies ( tsetse flies , stomoxids and tabanids ) could be good candidates for this purpose since they are usually large Diptera ( length comprised between 3 and 25 mm ) and hematophagous in both sexes , with the exception of male tabanids ( Mullens , 2002 ) .", "They are easy to trap and some studies performed on tsetse flies and stomoxids showed that 20 to 40% of trapped flies are engorged with blood ( Mavoungou et al . , 2008; Simo et al . , 2012 ) .", "These flies feed on a large spectrum of vertebrate hosts , including birds , reptiles and mammals ( Muturi et al . , 2011; Clausen et al . , 1998; Muzari et al . , 2010 ) .", "The omnipresence of hematophagous flies in certain habitats and their opportunistic blood-feeding behaviour ( Muturi et al . , 2011; Muzari et al . , 2010; Späth , 2000 ) make of them compelling candidates to obtain blood meals from different vertebrate hosts for pathogen detection .", "In the present study , we investigated the possibility of using hematophagous flies as ‘flying syringes’ to explore the diversity of extant malaria parasites ( Haemosporida ) infecting wild vertebrates living in the forests of Gabon ( Central Africa ) ." ], [ "A total of 4099 hematophagous flies were caught in four national parks of Gabon during dry and rainy seasons over a cumulated sampling period of 16 weeks ( Figure 1a ) .", "Among them , six tsetse fly species , six stomoxid species and six tabanid species were identified ( Table 1 ) . 10 . 7554/eLife . 22069 . 003Figure 1 . Monitoring vertebrate haemosporidian diversity using haematophagous flies .", "( a ) Localization of the sampling sites ( red dots ) in Gabon ( Central Africa ) .", "( b ) Number of blood meals originating from the different vertebrate species .", "( c ) Position within the Cytb phylogeny of the haemosporidian Cytb sequences PCR-amplified from the blood meals of engorged flies with identified hosts ( red isolates ) and unidentified hosts ( green isolates ) .", "Black isolates: references ( Table 4 ) .", "Bootstrap values at important nodes are shown . DOI: http://dx . doi . org/10 . 7554/eLife . 22069 . 00310 . 7554/eLife . 22069 . 004Table 1 . Number and proportion of specimens captured per fly species .", "The number of engorged specimens and blood meals identified in each fly species are also indicated . DOI: http://dx . doi . org/10 . 7554/eLife . 22069 . 004Fly speciesNumber of collected specimensProportion ( % ) Number of engorged specimensNumber of identified blood mealsGlossinidae225254 . 941218423Glossina caliginea1443 . 518733G .", "fusca congolensis2105 . 1210442G .", "fuscipes fuscipes2907 . 0721493G .", "pallicera newsteadi1573 . 839737G .", "palpalis palpalis137233 . 47662218G .", "tabaniformis791 . 93540Muscidae136233 . 2394Stomoxys calcitrans2455 . 9852S .", "inornatus3348 . 1400S .", "niger niger2536 . 1742S .", "niger bilineatus2245 . 4600S .", "omega omega1974 . 8100S .", "transvittatus1092 . 6600Tabanidae48511 . 8331Ancala sp41100Atylotus sp1042 . 5300Chrysops sp1563 . 8131Haematopota sp130 . 3100Tabanus par521 . 2700Tabanus taeniola1202 . 9300Total40991001230428 Among the 4099 caught flies , 1230 ( 30% ) were engorged with blood .", "These were mostly tsetse flies ( n = 1218; 99% ) , particularly Glossina palpalis palpalis ( n = 662; 54% ) and G . fuscipes fuscipes ( n = 214; 18% ) specimens .", "The blood meal origin was successfully identified in 33% and 43% of these flies , respectively ( Table 1 ) .", "Overall , the blood meal origin was successfully identified in 428 fly samples ( 35% ) using a PCR system amplifying long fragments of Cytb ( 450 bp ) or COI genes ( 330 bp or 660 bp ) .", "Specifically , blood meals were from 20 vertebrate species , including 12 families and 8 orders ( Figure 1b and Tables 2 and 3 ) . 10 . 7554/eLife . 22069 . 005Table 2 . Number and origin of blood meals according to the fly species ( Fsp ) , park and climatic season . DOI: http://dx . doi . org/10 . 7554/eLife . 22069 . 005Number of identified blood meals by fly species ( Fsp ) Moukalaba-DoudouLopéRainy seasonDry seasonRainy seasonDry seasonTaxonomic group/Order/FamilyHost speciesN° IdentifiedFsp1Fsp2Fsp3Fsp4Fsp5Fsp1Fsp2Fsp3Fsp4Fsp5Fsp8Fsp1Fsp2Fsp3Fsp4Fsp5Fsp1Fsp2Fsp3Fsp4Fsp5Fsp6Fsp7Mammals Artiodactyla295143811711421233413 BovidaeCephalophus silvicultor65kobus ellipsiprymnus431Syncerus caffer12635711329211057128161Tragelaphus spekii9516415137694121 HippopotamidaeHippopotamus amphibius211 SuidaePotamochoerus porcus312 Carnivora1 HerpestidaeHerpestinae sp11 Primates67 HominidaeGorilla gorilla321Homo sapiens6411132221312141 Proboscidae10 ElephantidaeLoxodonta cyclotis10712Reptiles Crocodilia233 CrocodylidaeCrocodylus niloticus3Mecistops cataphractus19116Osteolaemus tetraspis11 Squamata12 PythonidaePython sebae82 VaranidaeVaranus sp4211 Testudines16 TestunidaeKinixys erosa11 PelomedusidaePelusios castaneus3111Pelusios chapini11Pelusios marani1138Birds Ciconiformes4 CiconiidaeCiconia sp4128 orders/12 families20 species428311144122164891671871611112262512Fsp1 = Glossina caliginea; Fsp2 = G . fusca congolensis; Fsp3 = G . fuscipes fuscipes; Fsp4 = G . pallicera newsteadi; Fsp5 = G . palpalis palpalis; Fsp6 = Stomoxys calcitrans; Fsp7 = S . niger niger; Fsp8 = Chrysops sp . 10 . 7554/eLife . 22069 . 006Table 3 . Number and origin of blood meals according to the fly species ( Fsp ) , park and climatic season . DOI: http://dx . doi . org/10 . 7554/eLife . 22069 . 006Number of identified blood meals by fly species ( Fsp ) La LékédiIvindoRainy seasonDry seasonDry seasonTaxonomic group/Order/FamilyHost speciesN° IdentifiedFsp1Fsp3Fsp4Fsp5Fsp6Fsp1Fsp2Fsp3Fsp4Fsp5Fsp1Fsp2Fsp3Fsp5Mammals Artiodactyla295213 BovidaeCephalophus silvicultor65kobus ellipsiprymnus4Syncerus caffer126211610881311Tragelaphus spekii95131285591 HippopotamidaeHippopotamus amphibius2 SuidaePotamochoerus porcus3 Carnivora1 HerpestidaeHerpestinae sp1 Primates67 HominidaeGorilla gorilla3Homo sapiens6441511 Proboscidae10 ElephantidaeLoxodonta cyclotis10Reptiles Crocodilia23 CrocodylidaeCrocodylus niloticus3Mecistops cataphractus192423Osteolaemus tetraspis1 Squamata12 PythonidaePython sebae8222 VaranidaeVaranus sp4 Testudines16 TestunidaeKinixys erosa1 PelomedusidaePelusios castaneus3Pelusios chapini1Pelusios marani11Birds Ciconiformes4 CiconiidaeCiconia sp418 orders/12 families20 species4282411318202015321122Fsp1 = Glossina caliginea; Fsp2 = G . fusca congolensis; Fsp3 = G . fuscipes fuscipes; Fsp4 = G . pallicera newsteadi; Fsp5 = G . palpalis palpalis; Fsp6 = Stomoxys calcitrans; Fsp7 = S . niger niger; Fsp8 = Chrysops sp .", "A trial study using a PCR system amplifying a shorter fragment ( 150 bp of the gene 16S ) to deal with potential DNA degradation in the blood meal showed a high gain of sensitivity in the determination of the origin of the blood meal .", "Thus , out of 89 previously unidentified blood meals , the host was identified for 76% ( n = 68 ) of them .", "The list of newly identified hosts is given in Figure 2 .", "This shows a high gain of sensitivity with the new PCR system . 10 . 7554/eLife . 22069 . 007Figure 2 . Number of blood meals identified using the shorter PCR system of Boessenkool et al . ( 2012 ) out of the previously unidentified 89 blood meals . DOI: http://dx . doi . org/10 . 7554/eLife . 22069 . 007 Extant malaria parasites were detected in 37 ( 8 . 7% ) of the 428 identified blood meals ( Figure 1c , red isolates ) .", "Phylogenetic analyses revealed that 29 . 7% of these parasites belonged to Plasmodium falciparum ( n = 11 , Figure 1c; group 1 ) , 8 . 1% to Plasmodium adleri ( n = 3 , Figure 1c; group 2 ) , and 8 . 1% to a recently described lineage of parasites infecting wild ungulates ( n = 4 , Figure 1c; group", "3 ) ( Boundenga et al . , 2016 ) .", "For all blood meals , the identified host represented the known natural host ( or one of the hosts ) of such parasites .", "Sequences of unknown parasite lineages or of parasites for which the hosts were not known were also obtained .", "For instance , one sequence ( Figure 1c; group", "4 ) detected in a blood meal originating from an ungulate was related to parasites previously isolated only from Anopheles mosquitoes ( Boundenga et al . , 2016 ) .", "One sequence detected in a blood meal originating from a bird was related to bat Haemosporida ( Nycteria ) , ( Figure 1c; group 5 ) .", "Finally , 18 sequences ( Figure 1c; group 6 ) that were amplified from blood meals originating from ungulates formed an independent and never described lineage related to groups 3 and 4 .", "In addition , 100 additional samples for which identification of the blood meal failed were randomly chosen for malarial parasite screening .", "This analysis showed that 7% were infected with P . falciparum ( n = 4 , group 1 ) , P . praefalciparum ( n = 1 , group 7 ) , malaria parasites of antelopes from group 6 ( n = 1 ) and parasites of tortoises ( group 8 , n = 1 ) ( Figure 1c , green isolates ) .", "For the parasite , the use of a shorter PCR system led to less conclusive results than those obtained for the host identification .", "Out of the 91 blood meals that were negative to Plasmodium with a PCR system amplifying a long Cytb fragment , only one was found positive with the new system .", "The positive individual corresponded to a Tragelaphus spekii and was infected with a parasite belonging to group 3 ( Figure 1c ) ." ], [ "Despite the significant scientific advances in the medical field , humans are still unable to predict where , when and how epidemics arise .", "Around 60% of emerging diseases in humans are of zoonotic origin .", "The progressive reduction of wild habitats will increase the contacts between humans and species that are potential reservoirs of diseases .", "We propose here a new non-invasive tool that can help identifying pathogens that circulate in wildlife before they spread in humans ." ], [ "The fly sampling was carried out in four wildlife reserves in Gabon ( Figure 1a ) : Moukalaba-Doudou National Park ( MDNP; S: 2° 26' 08\"/E: 10° 25' 18'' ) , La Lopé National Park ( LNP; S: 0° 31' 31\"/E: 11° 32' 34\" ) , La Lékédi Park ( LP; S: 1° 45' 32\"/E: 13° 03' 16\" ) and Ivindo National Park ( INP; N: 0° 30' 82\"/E: 12° 48' 20\" ) .", "Both MDNP and LNP are dominated by mature forests and mosaic forest-savannah .", "The INP is largely dominated by mature forest with some open biotopes that characterize the secondary forest .", "The LP is a private park dominated by large savannahs and some secondary forest and primary forest patches .", "Hematophagous flies were sampled during the rainy and dry seasons between 2012 and 2014 .", "In INP and MDNP , sampling was done during two years following a gradient of human activity from primary forest to villages .", "In the other parks , flies were sampled during a single year .", "Flies were collected by using Vavoua and Nzi traps ( Laveissiere and Grebaut , 1990; Acapovi et al . , 2001; Mihok , 2002; Gilles et al . , 2007 ) .", "The Vavoua trap , initially developed for the capture of tsetse flies was also successfully used for the capture of stomoxids at La Réunion Island ( Laveissiere and Grebaut , 1990; Gilles et al . , 2007 ) .", "The Nzi trap was more adapted to the capture of Glossina pallidipes and tabanids in Africa ( Acapovi et al . , 2001; Mihok , 2002 ) .", "In each park , we placed 24 traps ( 12 Vavoua and 12 Nzi ) during 2 weeks per climatic season .", "Each trap was activated from 7:00 AM to 5:00 PM .", "Freshly collected hematophagous flies were identified using a stereo-microscope and taxonomic procedure .", "The fly species ( tsetse , stomoxids and tabanids ) was determined following the determination keys of Pollock ( 1982 ) , Brunhes et al . , 1998 , Zumpt , 1973 , Garros et al . ( 2004 ) and Oldroyd ( 1973 ) , on the basis of their morphological characteristics , such as size , color , wing venation structure and proboscis .", "After species identification , engorged flies were dissected individually in a drop of Dulbecco's phosphate buffered saline solution ( 1x DPBS ) to isolate blood meals from midgut .", "Each hematophagous fly was dissected on a slide using one forceps and one scalpel that were changed each time to avoid contaminations .", "Each blood meal was transferred in a 1 . 5-ml microtube containing 50 µl of RNAlater stabilization solution ( Qiagen: Store at RT Tissue Collection ) to stabilize and protect nucleic acids of vertebrate hosts and pathogens contained in the blood meals .", "Samples were kept at ambient temperature during field session and then frozen at −80°C until DNA extraction .", "Samples were centrifuged at 15 , 000 rpm at 4°C for 10 min to remove the RNAlater solution .", "Pellets were used to extract DNA using the DNeasy Blood and Tissue Kit ( Qiagen ) according to the manufacturer’s instructions .", "Extracted DNA was eluted in 100 µl of buffer AE and stored at −20°C .", "The origin of blood meals was determined using the extracted DNA to amplify a 450 bp fragment of the cytochrome b ( Cytb ) gene using previously published primers ( Townzen et al . , 2008 ) .", "PCR amplifications were performed using a GeneAmp 9700 thermal cycler ( Applied Biosystems , USA ) with 50 µL reaction mixtures containing 4 µL template DNA , 10 mM Tris-HCl ( pH = 9 ) , 50 mM KCl , 3 mM MgCl2 , 20 pmol each primer ( 5’CCCCTCAGAATGATATTTGTCCTCA3’ and 5’CCATCCAACATCTCAGCATGATGAAA3’ ) , 200 mM dNTP and 1 U Taq polymerase .", "The thermal cycling conditions consisted of 3 . 5 min at 95°C , 40 cycles of 30s at 95°C , 50s at 58°C , and 40s at 72°C , followed by 5 min at 72°C .", "When Cytb amplification failed , a 330 bp and/or a 660 bp fragment of the cytochrome oxydase subunit I ( COI ) gene was amplified using previously described primers and protocols ( Townzen et al . , 2008 ) .", "All PCR-amplified products ( 10 µl ) were run on 1 . 5% agarose gels in TBE buffer , and positive samples were sent to Beckman Coulter Genomics ( France ) for sequencing in both directions ( forward and reverse ) after purification .", "Consensus sequences were compared with existent sequences using the NCBI nucleotide Blast search ( Altschul et al . , 1990 ) to determine the host species .", "Hosts were identified when the amplified and reference sequences showed at least 98% similarity .", "Haemosporidian parasite detection was performed in samples with identified blood meal origin and also in 100 randomly chosen samples for which blood meal origin could not be identified .", "Haemosporidian parasites were detected by PCR amplification of a portion of the Cytb gene ( ~790 bp ) using a nested PCR protocol , as previously published ( Ollomo et al . , 2009 ) .", "PCR products were checked on 1 . 5% agarose gels before shipment to EUROFINS MWG ( Germany ) for sequencing in both directions ( reverse and forward ) after purification .", "Multiple alignments of haemosporidian sequences were done using Muscle ( Edgar , 2004 ) .", "A phylogenetic tree with the haemosporidian sequences obtained in our study and a set of reference sequences was built using Maximum likelihood ( ML ) methods and phylogeny . fr ( Dereeper et al . , 2008 ) ( see Table 4 for accession numbers ) .", "The ML model used for construction of the tree was GTR ( General Time Reversible ) +Γ ( Gamma distribution ) +I ( Invariable site distribution ) . 10 . 7554/eLife . 22069 . 008Table 4 . Cytb sequences of parasites recovered in this study and of those used as references for phylogenetic analyses and their Genbank accession numbers . DOI: http://dx . doi . org/10 . 7554/eLife . 22069 . 008IsolateAccession numberAnopheles coustaniKT367855An .", "gabonensis2KT367852An .", "gabonensis279KT367853An .", "gabonensis3KT367861An .", "marshalliiKT367857An .", "mouchetiKT367864An .", "obscurus2KT367846An .", "obscurus78KT367849Cephalophus_silvicultor_1336KY631949Cephalophus_silvicultor_1368KY631947Cephalophus_silvicultor_484KY631963Ciconia_sp_445KY631985E15_Podocnemis_expansa_PeruKF049492E24_Podocnemis_expansa_PeruKF049495Gorilla_gorilla_34KY631983Gorilla_gorilla_756KY631982Gorilla_gorilla_761KY631981P_sp . _JA7_J725GU252027Haemoproteus_majorisAY099045Haemoproteus_sp . HM222472Haemoproteus_sp . _GA02CI1HM222486Haemoproteus_sp . _NA16K65HM222487Hepatocystis_sp .", "AA201_blikeJQ070951Hepatocystis_sp . JQ070884Hepatocystis_sp . _AA2012JQ070956HO11_Cephalophus_nigrofonsKT367819HO13_Cephalophus_monticolaKT367833HO613_Cephalophus_monticolaKT367836HO9_Kinixys_erosaKT367843Homo_sapiens_476KY631978Homo_sapiens_481KY631977Homo_sapiens_57KY631979Homo_sapiens_574KY631976Homo_sapiens_635KY631975Homo_sapiens_636KY631974Homo_sapiens_638KY631973Homo_sapiens_639KY631972Homo_sapiens_668KY631969Homo_sapiens_806KY631968Homo_sapiens_832KY631967Leucocytozoon_caulleryiAB302215Leucocytozoon_dubreuliAY099063Leucocytozoon_majorisFJ168563Leucocytozoon_sabrazesiAB299369M0278_Cephalophus_monticolaKT367834NG238_Kinixys_erosaKT367844NG277_Ceratogymna_atrataKT367825NY195_Cephalophus_dorsalisKT367838Nycteria_sp . _R_alc_C9_1KF159720Nycteria_sp . _R_lan_G3_1KF159690OI52_PangolinKT367818OL123_Cephalophus_monticolaKT367822OL131_Cephalophus_callipygusKT367830P_adleriHM235081P_azurophilumAY099055P_billcollinsiKP875474P_blacklockiHM235065P_cynomolgiAB444126P_falciparum_3D7AF069605P_gaboniJF895307P_gallinaceumAF069612P_gonderiJF923751P_knowlesiJQ345504P_malariaeHM000110P_ovaleGU723548P_praefalciparum_MOEBJF923761P_reichenowiKP875479P_relictumAY733090P_sp . _DAJJF923753P_vivaxKF591834P_atheruriAY099054P_giganteumAY099053P_vinckei_isolate_1KJ700853P_vinckei_isolate_2KJ700854P_yoelii_killickiDQ414658P .", "atheruriHQ712051P .", "cyclopsi_Hip_cy_L4_1_SchaerKF159674P .", "voltaicum_M_ang_G1_1_1KF159671Parahaemoproteus_sp . _bird_sp . 17GQ141581Parahaemoproteus_sp .", "_bird_sp . 19GQ141585Parahaemoproteus_vireonisFJ168561Plasmodium_sp . _birdGQ141574Plasmodium_sp . _bird_sp . _12HM222485Plasmodium_sp . _GD2_GD201GU252012Plasmodium_sp . _lineage_JA01KM598212Polychromophilus_melanipherus_haplotype_VIIIKJ131277Polychromophilus_murinus_haplotype_3HM055585Polychromophilus_sp . _Min_vil_G3_2KF159699Polychromophilus_sp . _Pip_gran_G3_1KF159714Polychromophilus_sp . _Neo_cap_G3KF159700Syncerus_caffer_1138KY631953Syncerus_caffer_1417KY631942Tragelaphus_eurycerus_1324KY631950Tragelaphus_spekii_1051KY631961Tragelaphus_spekii_1155KY631959Tragelaphus_spekii_1175KY631958Tragelaphus_spekii_1228KY631957Tragelaphus_spekii_1245KY631956Tragelaphus_spekii_1291KY631955Tragelaphus_spekii_1299KY631954Tragelaphus_spekii_1300KY631952Tragelaphus_spekii_1306KY631951Tragelaphus_spekii_1348KY631948Tragelaphus_spekii_1386KY631946Tragelaphus_spekii_1394KY631945Tragelaphus_spekii_1399KY631944Tragelaphus_spekii_1413KY631943Tragelaphus_spekii_385KY631964Tragelaphus_spekii_56KY631965U65_Podocnemis_unifilis_PeruKF049506Unknown_host_1036KY631960Unknown_host_110KY631966Unknown_host_512KY631962Unknown_host_520KY631980Unknown_host_649KY631971Unknown_host_665KY631970Unknown_host_819KY631984 Several measures were taken to avoid contaminations during our manipulations .", "Extraction of DNA was performed at the CIRMF ( Gabon ) in a laboratory working on mosquitoes .", "The room in which extraction was performed was away from the rooms in which DNA was amplified in this lab .", "DNA extracts were then sent to France at the IRD ( Montpellier ) .", "There , blood meal and Plasmodium identification was performed .", "This lab had never worked before on Plasmodium from ungulates or reptiles .", "Amplification of host DNA was never or very rarely performed in this lab .", "When the work was performed , no work on Plasmodium has been performed in this lab for almost 4 years .", "In addition , the laboratory is designed to avoid contaminations .", "Clearly defined and separated areas are devoted for each step of the PCR process: one area is devoted to the preparation of reagents ( mix PCR ) .", "Another room is dedicated to the pre-PCR manipulation ( loading of native DNA ) .", "This step is done under a cabinet to avoid contamination of the sample with DNA from the operator .", "Finally , an area is devoted to PCR-amplified DNA .", "In this area , cabinets are used to deposit the first PCR product into the reagents of the second PCR ( for nested PCRs ) .", "All cabinets are equipped with UV lamps and are always decontaminated with DNA-free solutions before and after manipulations .", "Gloves and coats are changed when moving between the areas and plugged tips are used at all steps .", "Blank controls were always incorporated at all steps of the experimental procedure and were always negative .", "Several observations confirm the authenticity of our results: ( 1 ) >80% of the hosts that were found have never been manipulated in our lab ( hosts that are not humans or non-human primates ) ; ( 2 ) the parasite always corresponded to the expected host ( antelope parasites were always found in antelopes , human parasites in humans and gorilla parasites in gorillas ) .", "Contaminations by external DNA would have lead to random association of hosts and parasites; ( 3 ) A new lineage of parasites was discovered .", "Since DNA is likely to be degraded in many of our samples , the use of PCR systems targeting fragments of shorter size might improve performance .", "To determine if this could be the case with our study system , we performed supplementary analyses using ( 1 ) a PCR system targeting a shorter fragment of the vertebrate mitochondrial DNA to identify the blood meal origin and ( 2 ) a PCR system targeting a shorter fragment of the Cytb DNA to identify the parasite .", "For the identification of the host , the PCR system used was the one amplifying a fragment of 150 bp of 16S as described in Boessenkool et al . ( 2012 ) and using the primers 16Smam1 and 16Smam2 .", "This PCR system was used on blood meals that failed to be identified using our original PCR system ( see the paragraph ‘Blood meal identification’ ) .", "A total of 89 blood meals were tested for this trial study .", "For the parasite , we designed new primer sets to amplify a shorter fragment of the Cytb gene of the parasite ( ~177 bp ) .", "This new PCR system was applied to blood meals for which the host was identified but that were negative to Plasmodium with our long PCR system ( ~790 bp , see Material and methods above ) .", "A total of 91 blood samples were tested .", "For the first round of amplifications , we used 6 μL of DNA template in a 25 μL reaction volume , containing: 12 . 5 μL of Mix PCR ( Qiagen ) , 2 . 5 μL solution Q ( Qiagen ) , and 4 pmol of each primer ( cytb1F CTCTATTAATTTAGTTAAAGCACACTT and 454R CCWGTWGCYTGCATYTATCT ) .", "Cycling conditions were 15 min at 95°C , 30 s at 94°C , 90 s at 57°C , 90 s at 72°C ( 40 cycles ) , and 10 min at 72°C .", "For the second round of amplification , we used 1 . 5 μL of the first PCR template in a 25 μL reaction volume , containing 2 . 5 μL of 10× buffer , 1 . 25 mM MgCl2 , 250 μM of each dNTP , 10 pmol of each primer ( 454F2 WAATTAYCCATGYCCATTRAA and Plas1rc CACCATCCACTCCATAATTCTC ) , and 0 . 1 unit Taq Platinum ( Invitrogen ) .", "Cycling conditions for the second round were 5 min at 95°C , 30 s at 94°C , 30 s at 50°C , 90 s at 72°C ( 35 cycles ) , and 10 min at 72°C .", "The amplified products ( 5 μL ) were run on 1 . 5% agarose gels in TAE buffer .", "The PCR-amplified products ( 177 bp ) were used as templates for sequencing .", "DNA sequencing was performed by Eurofins MWG ." ] ]

[ "About 60% of emerging infectious diseases in humans are of zoonotic origin .", "Their increasing number requires the development of new methods for early detection and monitoring of infectious agents in wildlife .", "Here , we investigated whether blood meals from hematophagous flies could be used to identify the infectious agents circulating in wild vertebrates .", "To this aim , 1230 blood-engorged flies were caught in the forests of Gabon .", "Identified blood meals ( 30% ) were from 20 vertebrate species including mammals , birds and reptiles .", "Among them , 9% were infected by different extant malaria parasites among which some belonged to known parasite species , others to new parasite species or to parasite lineages for which only the vector was known .", "This study demonstrates that using hematophagous flies as ‘flying syringes’ constitutes an interesting approach to investigate blood-borne pathogen diversity in wild vertebrates and could be used as an early detection tool of zoonotic pathogens ." ]

[ "About 60% of new infectious diseases in humans come from animals .", "Their increasing number and rapid spread are linked to increasing levels of contact between humans and wildlife , as recently highlighted by the epidemics of Zika in Brazil or Ebola in West Africa .", "To anticipate and prevent similar outbreaks in the future , it would be ideal to develop new methods for the early detection and monitoring of infectious diseases in wild animals .", "Currently , three methods are mainly used to screen wild animals for infectious disease , but these all have limitations .", "Analyses of bushmeat and game meat only investigate those animals that are eaten by humans .", "Testing the organs and tissues of trapped animals can be difficult and harmful for both the humans and animals involved .", "Collecting and examining samples of feces , urine or saliva cannot detect all diseases and can be difficult to do for some species .", "Bitome-Essono et al . now demonstrate a new method for assessing the diseases carried by wild animals: using blood-sucking flies as 'flying syringes' to collect their blood .", "During several weeks of sampling in Gabon , Central Africa , Bitome-Essono et al . trapped thousands of these flies , about a third of which were engorged with blood .", "Analyses of these blood samples revealed that they had come from 20 different species , including birds , mammals and reptiles .", "Different malaria parasites could also be detected in the blood .", "Although the study performed by Bitome-Essono et al . only focused on malaria parasites , in the future the technique could be extended to analyze a number of disease-causing microbes – including viruses , bacteria , protozoa and macroparasites – that are found in the blood of wild animals ." ]

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

[ "tools and resources", "neuroscience" ]

[ [ "To gain detailed mechanistic insight into ion channel function and pharmacology , one often studies single channels , heterologously expressed , under voltage-clamp protocols ( Yan and Aldrich , 2012; Lewis and Raman , 2013; Bosmans et al . , 2008 ) .", "Carefully designed sequences of voltage steps prepare channels in select conformational states ( Cordero-Morales et al . , 2006; Zhang et al . , 2012 ) .", "Distinct sub-states often have widely divergent affinities and kinetics of interaction with drugs .", "Knowledge of this state-dependent behavior is critical in developing models of channel function and in predicting how drugs will function in vivo .", "State-dependent dynamical measurements are typically the domain of manual or automated patch clamp .", "Functionally equivalent optical assays would open the prospect of high throughput screens with sophisticated state-dependent selection criteria; and might enable measurements in cell types or environments ( e . g . in a tissue or whole animal ) that are challenging to access with conventional methods .", "Tools for optical electrophysiology—simultaneous optical perturbation and optical readout of membrane potential—have been making inroads into neuroscience ( Emiliani et al . , 2015; St-Pierre et al . , 2014 ) , with a primary emphasis on spatially resolved measurements in vivo ( Packer et al . , 2015; Rickgauer et al . , 2014 ) or in complex cell cultures ( Hochbaum et al . , 2014 ) .", "Voltage-sensitive dyes ( VSDs ) have been applied in a wide range of physiological assays in vitro ( Parsons et al . , 1991 ) and in vivo , ( Cohen and Salzberg , 1978 ) but existing red-shifted VSDs are still excited by the wavelengths used to stimulate optogenetic actuators , leading to optical crosstalk ( Park et al . , 2014; Tsuda et al . , 2013 ) , or are not commercially available ( Huang et al . , 2015 ) .", "A combination of a blue-shifted channelrhodopsin ( CheRiff ) and a red-shifted voltage indicator protein ( QuasAr2 ) recently achieved spectrally orthogonal optical stimulation and readout ( Hochbaum et al . , 2014 ) .", "Optical electrophysiology measurements are typically semi-quantitative , at best , while ion channel assays require accurate perturbations to voltage and measurements of current .", "The optical techniques face several challenges: expression levels of optogenetic actuators and reporters vary from cell to cell; channelrhodopsins function as a conductance , not a voltage clamp; and fluorescence can only be used to measure membrane voltage , not current ( Cohen and Salzberg , 1978; Cohen et al . , 1974 ) .", "Thus it is not obvious whether one can apply optical electrophysiology as a functional surrogate for standard voltage-clamp protocols .", "Here we address this challenge by developing optical assays of the state-dependent electrophysiology and pharmacology of voltage-gated sodium ( NaV ) channels .", "We begin with electrophysiologically inert HEK cells .", "We then stably express four transgenic constructs: an inward rectifier potassium channel and a voltage-gated sodium channel imbue the HEK cells with the ability to produce regenerative electrical spikes ( Hsu et al . , 1993; Kirkton and Bursac , 2011; Park et al . , 2013 ) .", "A channelrhodopsin variant , CheRiff , triggers these spikes upon exposure to flashes of blue light .", "An Archaerhodopsin variant , QuasAr2 , enables fluorescent readout of membrane voltage via red excitation and near-infrared fluorescence .", "The QuasAr2 reporter has a ~1 ms response time and a linear response between −100 to +100 mV , providing a direct correlation of fluorescence and voltage ( Hochbaum et al . , 2014 ) .", "Brief flashes of blue light trigger sodium channel-mediated action potentials , which manifest as flashes of near infrared fluorescence .", "Steady state illumination with blue light induces steady state changes in voltage , and thereby changes in the distribution of NaV channels among substates .", "We develop stimulus and data analysis protocols that are robust to sources of cellular variation , and we compare our results to measurements by manual patch clamp .", "While whole-cell voltage clamp remains the gold standard for absolute accuracy , optical electrophysiology provides a favorable tradeoff between accuracy and throughput .", "NaV channels mediate the rising phase of the action potential and play significant physiological functions in excitable tissues .", "There are nine subtypes of NaV channels in the human genome .", "NaV channel dysfunction has been implicated in many human diseases .", "For example , loss-of-function mutations in NaV1 . 1 can cause Dravet syndrome and in NaV1 . 5 can cause Brugada syndrome ( Catterall , 2012 ) .", "The NaV1 . 7 sodium channel plays an important role in mediating pain sensation .", "Homozygous loss of function leads to congenital insensitivity to pain ( Cox et al . , 2006 ) , gain of function mutations lead to spontaneous severe pain , called erythermalgia ( Yang et al . , 2004 ) , and nucleotide polymorphisms modify sensitivity to pain in the general population ( Reimann et al . , 2010 ) .", "While recent results have suggested that the connection of NaV1 . 7 to pain may involve other signaling pathways as well ( Minett et al . , 2015 ) , there remains strong interest in finding selective blockers of this channel .", "Recent structural work has mapped an isoform-specific binding site for NaV1 . 7-specific blockers ( Ahuja et al . , 2015 ) , opening the possibility to develop new blockers via structure-guided design .", "Here we apply the Optopatch spiking HEK cell platform to study NaV1 . 7 , and we demonstrate its applicability to NaV1 . 5 also ." ], [ "We engineered a monoclonal HEK293 cell line stably expressing human NaV1 . 7 and the Optopatch constructs ( see Materials and methods ) .", "Both QuasAr2-mOrange2 and CheRiff-eGFP showed good membrane trafficking ( Figure 1B ) .", "We used manual whole-cell patch clamp measurements to characterize the performance of each component .", "Under whole-cell voltage clamp ( Vm = −60 mV ) CheRiff was activated by 488 nm light with an EPD50 ( effective power density for 50% activation ) of 20 mW/cm2 and a saturating steady-state photocurrent density of 13 . 0 ± 1 . 2 pA/pF ( mean ± s . e . m . , n = 5 cells , Figures 2A , B ) .", "As with channelrhodopsin 2 , CheRiff showed inward rectification ( Gradmann et al . , 2011 ) with a reversal potential of +4 mV , consistent with non-selective cation conductivity .", "Under voltage steps from a holding potential of -100 mV , NaV1 . 7 mediated robust inward currents with fast activation and inactivation kinetics within 10 ms and a peak current density of −61 . 4 ± 13 . 6 pA/pF at −20 mV ( mean ± SD , n = 11 cells , Figure 2C ) . 10 . 7554/eLife . 15202 . 003Figure 1 . NaV1 . 7 Optopatch Spiking ( NaV1 . 7-OS ) HEK cells .", "( A ) Genes expressed heterologously in NaV1 . 7-OS HEK cells .", "Kir2 . 1 maintains a hyperpolarized resting potential close to the K+ reversal potential .", "NaV1 . 7 imparts electrical excitability .", "CheRiff depolarizes the cells upon optical excitation and can trigger a NaV1 . 7-mediated action potential .", "QuasAr2 is excited by red light and emits near infrared fluorescence in a voltage-dependent manner .", "( B ) Epifluorescence images of QuasAr2 and CheRiff-eGFP expressed in NaV1 . 7-OS HEK cells .", "Scale bar 10 μm . DOI: http://dx . doi . org/10 . 7554/eLife . 15202 . 00310 . 7554/eLife . 15202 . 004Figure 2 . Biophysical characterization of NaV1 . 7-OS HEK cells .", "( A ) CheRiff current in a NaV1 . 7-Optopatch HEK cell .", "Membrane potential was held at −80 mV and then stepped for 2 s to −80 to +40 mV in 20 mV increments .", "During each depolarization , the cell was exposed to 5 pulses of blue light , 100 ms duration , with increasing intensity ( 1 . 7 , 18 , 50 , 79 , 93 mW/cm2 ) .", "The horizontal dashed line indicates zero current .", "( B ) I-V relation of CheRiff , under different light intensities .", "Currents were measured relative to baseline without blue light .", "Inset: Steady state photocurrent density as a function of blue light intensity , with a holding potential of −60 mV .", "( C ) Peak NaV1 . 7 current densities as a function of depolarization potential .", "Membrane potential was held at -100 mV and then stepped for 100 ms to −90 mV to + 30 mV in 10 mV increments .", "These measurements were performed prior to transient expression of Kir2 . 1 .", "Inset: currents in the 10 ms interval following each voltage step .", "( D ) I-V relationship of Kir2 . 1 expressed in NaV1 . 7-OS HEK cells .", "Membrane potential was held at -100 mV and stepped for 500 ms to −130 mV to +30 mV in 10 mV increments .", "Inset: representative Kir2 . 1 current recording .", "Red line indicates the time point ( 4 ms after voltage step ) at which the current was quantified .", "( E ) Simultaneous voltage and QuasAr2 fluorescence recording from NaV1 . 7-OS HEK cells .", "The cell was exposed to a series of blue laser pulses , 500 ms duration , with increasing intensities ( 1 . 1 , 2 . 3 , 4 . 3 , 7 . 0 , 11 , 15 , 20 , 26 mW/cm2 ) and QuasAr2 fluorescence was monitored with 640 nm excitation , 400 W/cm2 .", "Inset: overlay of the voltage and fluorescence recordings from the most intense blue pulse ( 26 mW/cm2 ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 15202 . 00410 . 7554/eLife . 15202 . 005Figure 2—figure supplement 1 . Current clamp recording of light triggered action potentials in Nav1 . 7-OS HEK cells .", "( A ) An action potential recorded via manual patch clamp from a Nav1 . 7-OS HEK cell cluster stimulated by 20 ms blue light pulse at 50 mW/ cm2 .", "The dashed line indicates the firing threshold .", "( B ) Plateau potential induced by different intensities of blue light stimulation .", "Current clamp recordings were performed on Nav1 . 7-OS HEK cell clusters stimulated with 500 ms blue light ranging from 1 . 1 to 84 mW/ cm2 .", "( C ) Membrane potential at 400 ms after onset of blue light stimulus as a function of the blue light intensity . DOI: http://dx . doi . org/10 . 7554/eLife . 15202 . 00510 . 7554/eLife . 15202 . 006Figure 2—figure supplement 2 . Relationship between Nav1 . 7 current density and spike height .", "( A ) Combined current clamp and voltage clamp protocol in the presence of 3 μM amitriptyline to prepare cells with varying NaV1 . 7 capacities .", "Initially , a current clamp protocol was applied in which a depolarizing pulse led to amitriptyline binding and complete channel block .", "After a variable recovery period , Δt , a test pulse of current induced a voltage spike whose amplitude was recorded .", "The voltage during the prepulse and recovery periods was then replayed in voltage clamp mode to induce an identical level of channel block .", "A step depolarization to -20 mV induced a spike in NaV1 . 7 current whose amplitude was recorded .", "For details see Materials and methods .", "( B ) Upper trace: current clamp recording showing voltage during 500 ms prepulse intervals , variable recovery period , and test pulses ( asterisks ) .", "Bottom left: magnified view of voltage during test pulses .", "Bottom right: magnified view of NaV1 . 7 current during test pulse under voltage clamp .", "( C ) Voltage spike amplitude as a function of NaV1 . 7 current amplitude for paired current-clamp and voltage-clamp recordings .", "Error bars represent s . e . m . of n = 8 cells . DOI: http://dx . doi . org/10 . 7554/eLife . 15202 . 00610 . 7554/eLife . 15202 . 007Figure 2—figure supplement 3 . Cell-to-cell variability in Optopatch measurements .", "( A ) Nav1 . 7-OS HEK cells in a confluent monolayer were imaged in the presence of 3 μM amitriptyline with a 60× oil immersion objective ( numerical aperture 1 . 45 ) .", "Cells were illuminated with a red laser at 400 W/cm2 .", "A blue prepulse ( 500 ms , 50 mW/cm2 ) depolarized membrane potential and allowed drug binding .", "A variable recovery period ( 40 ms to 5120 ms ) led to partial unbinding .", "A blue test pulse ( 20 ms , 50 mW/cm2 ) probed the voltage spike induced by residual NaV1 . 7 capacity .", "Traces show fluorescence of QuasAr2 from five different single cells recorded in parallel .", "( B ) Ratio of spike amplitude in test pulse to spike amplitude at the longest recovery time ( 5120 ms ) , as a function of recovery time for the five single cells shown in ( A ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 15202 . 00710 . 7554/eLife . 15202 . 008Figure 2—figure supplement 4 . Effects of intense red laser illumination .", "( A ) Heating by red laser illumination ( 635 nm , 400 W/cm2 ) .", "Temperatures were recorded with a small thermocouple not directly illuminated by the laser .", "Measurements were performed in a single well of a 384-well plate with 36 μL buffer , and in a 35 mm dish with 2 mL buffer ( error bars represent s . e . m . , n = 4 replicates ) .", "( B ) Test for photobleaching and phototoxicity .", "Nav1 . 7-OS HEK cell were illuminated with red laser ( 635 nm , 400 W/cm2 ) continuously for 10 min .", "Every one minute , the cells were stimulated with blue light ( 6 pulses of 20 ms , 5 Hz , 50 mW/cm2 ) .", "The first , sixth and the tenth spike trains are shown above .", "( C ) SNR and spike height ( ΔF/F0 ) for each spike train in ( B ) as a function of red laser illumination time .", "Error bars represent s . d . of n = 6 spikes .", "( D ) Effect of red laser intensity on SNR of QuasAr2 fluorescence spikes .", "Cells were illuminated with red laser at varying intensities and stimulated with pulses of blue light ( 8 pulses of 20 ms , 10 Hz , 50 mW/cm2 ) .", "( E ) SNR of spikes recorded in ( D ) as a function of red laser intensity .", "Error bars represent s . d . of n = 8 spikes . DOI: http://dx . doi . org/10 . 7554/eLife . 15202 . 008 We found that stable expression of Kir2 . 1 interfered with cell growth , so we expressed this channel via transient transfection .", "We call the quadruply expressing cells NaV1 . 7 Optopatch Spiking ( NaV1 . 7-OS ) HEK cells ( Figure 1A ) .", "In a bath solution containing 2 mM K+ , NaV1 . 7-OS HEK cells had a resting potential of −97 . 2 ± 2 . 2 mV ( mean ± s . e . m . , n = 7 cell clusters ) , sufficient to prime most of the NaV1 . 7 channels for activation .", "Upon voltage steps from −100 mV , Kir2 . 1 showed inward rectifying behavior ( Figure 2D ) .", "Using manual patch clamp , we quantified the effect of CheRiff activation on membrane voltage .", "Brief optical stimuli ( 20 ms , 50 mW/cm2 ) reliably triggered single spikes ( Figure 2—figure supplement 1A ) , with a firing threshold of -48 . 0 ± 1 . 2 mV , peak depolarization of +30 . 1 ± 3 . 7 mV , and spike width at half-maximum repolarization ( APD50 ) of 33 . 5 ± 3 . 3 ms ( mean ± s . e . m . , n = 5 cells ) .", "Under steady-state blue illumination , cells asymptotically approached a steady state depolarization that increased monotonically with stimulus intensity ( Figure 2—figure supplement 1B , C ) , reaching an asymptotic value of –25 . 8 ± 6 . 2 mV ( mean ± SD , n = 4 cells ) under intense illumination .", "We then performed simultaneous recordings of membrane voltage and QuasAr2 fluorescence under optical CheRiff stimulation ( Figure 2E ) .", "Cells were illuminated with continuous red light ( 640 nm , 400 W/cm2 ) to excite QuasAr2 fluorescence .", "Pulses of blue light ( 500 ms on , 1 . 5 s off , stepwise increasing intensity from 1 . 1 to 26 . 0 mW/cm2 ) were applied to activate CheRiff .", "Stimuli of intensity 15 mW/cm2 or greater induced action potentials .", "The fluorescence traces faithfully reproduced both the action potential waveforms and the subthreshold depolarizations .", "Optopatch measurements report membrane voltage , while patch clamp measurements typically control voltage and measure current .", "We thus used manual patch clamp measurements to determine the relation between voltage spike height measured in current clamp , and peak NaV1 . 7 current density measured in voltage clamp .", "We used the state-dependent binding of amitriptyline to induce varying degrees of channel block , and then either applied a current pulse and measured the voltage response , or a voltage step and measured the current response ( Materials and methods , Figure 2—figure supplement 2A , B ) .", "Figure 2—figure supplement 2C shows that the voltage spike amplitude was smoothly and monotonically related to the NaV1 . 7 current density .", "Thus optical measurements of spike height are a quantitative probe of NaV current-carrying capacity .", "High-magnification fluorescence measurements showed that each individual cell gave a graded spike amplitude as a function of NaV1 . 7 capacity ( Figure 2—figure supplement 3A ) , with an 8% standard deviation in spike height at 50% channel block ( n = 5 cells , Figure 2—figure supplement 3B ) .", "Finally , we tested for photothermal or photochemical damage from the intense red illumination used for QuasAr2 imaging .", "In a 35 mm dish containing 2 mL of imaging buffer , continuous red illumination at 400 W/cm2 ( 315 mW total power ) for 10 min induced a temperature rise <0 . 3°C ( Figure 2—figure supplement 4A ) .", "In a single well of a 384-well plate , containing 36 μL of imaging buffer , the temperature rise was 2 . 5 ± 0 . 4°C ( mean ± s . e . m . , n = 4 replicates ) in 35 s and 9 . 8 ± 0 . 6°C ( mean ± s . e . m . , n = 4 replicates ) in 10 min .", "While a 10°C rise is within the physiological range for measurements starting at room temperature ( 23°C ) , we kept all measurement protocols shorter than 35 s to avoid possibility of thermal artifacts .", "Under these same conditions ( 400 W/cm2 , 384 well plate , 36 μL buffer ) the cells continued to produce optically evoked spikes for 10 min , with little change in spike waveform ( Figure 2—figure supplement 4B ) .", "The baseline QuasAr2 fluorescence dropped by 12% in this interval ( Figure 2—figure supplement 4B ) and the spike amplitude dropped from 3 . 5 ± 0 . 04% ΔF/F to 2 . 3 ± 0 . 06% ΔF/F ( mean ± SD , n = 6 spikes , Figure 2—figure supplement 4C ) .", "The signal-to-noise ratio ( SNR , spike height/baseline noise ) dropped from 107 ± 2 to 73 ± 2 . 6 ( mean ± SD , n = 6 spikes , Figure 2—figure supplement 4C ) .", "We explored the dependence of SNR on red illumination intensity ( Figure 2—figure supplement 4D , E ) .", "At 400 W/cm2 the SNR was 99 . 8 ± 3 . 3 ( mean ± SD , n = 8 spikes ) at a 100 Hz frame rate .", "At 6 . 3 W/cm2 ( 5 mW ) the SNR was 9 . 5 ± 1 . 1 ( mean ± SD , n = 8 spikes ) at the same frame rate .", "Thus spiking HEK cells can be imaged under a wide range of conditions , without photochemical or photothermal toxicity .", "Sodium channel blockers are expected to change the firing properties of NaV1 . 7-OS HEK cells .", "Most clinically used sodium channel blockers ( e . g . lidocaine ) show use-dependent or state-dependent action .", "We stimulated NaV1 . 7-OS HEK cells with bursts of blue light ( 20 ms duration , six pulses ) at 2 , 4 , and 8 Hz .", "The optically evoked action potentials were recorded by QuasAr2 fluorescence , averaging over ~150 cells .", "In untreated cells , each stimulus evoked an action potential .", "After addition of lidocaine ( 200 μM ) , cells continued to spike faithfully at 2 Hz .", "At 4 Hz and 8 Hz , cells spiked in response to the first stimulus , but failed for subsequent stimuli , a hallmark of activity-dependent NaV block ( Figure 3 A ) . 10 . 7554/eLife . 15202 . 009Figure 3 . Mechanistic studies of NaV1 . 7 blockers .", "( A ) Approximately 150 NaV1 . 7-OS HEK cells were stimulated with pulses of blue light ( 20 ms , 50 mW/cm2 ) at increasing frequencies ( 2 Hz , 4 Hz , 8 Hz ) and their total QuasAr2 fluorescence was recorded with 635 nm excitation , 400 W/cm2 .", "In control cells ( blue ) , the fluorescence indicated spiking in response to each stimulus .", "After exposure to 200 μM lidocaine ( red ) , cells showed activity-dependent block at 4 Hz and 8 Hz , but not at 2 Hz ( red arrows ) .", "( B ) Simultaneous current clamp and QuasAr2 fluorescence recordings from a NaV1 . 7-OS HEK cell cluster ( 4 cells ) stimulated with prepulses of varying length ( 20 , 100 , 200 and 500 ms; 50 mW/cm2 ) followed by 200 ms recovery and a test pulse ( 30 ms , 50 mW/cm2 ) .", "( C , D )", "Application of the protocol in ( B ) to dose-response curves for ( C ) amitriptyline or ( D ) TTX .", "Test pulse spike amplitude was normalized to its value in the presence of the lowest tested concentration of drug ( n = 3–5 wells for amitriptyline per data-point; n = 4–6 wells for TTX per data-point; ~150 cells per well ) .", "( E ) Optical assay of NaV1 . 7 recovery from fast inactivation .", "A 500 ms prepulse ( 50 mW/cm2 ) populated the fast inactivated state and allowed drug binding .", "A variable recovery period ( 40 ms to 5120 ms ) was followed by a 20 ms test pulse ( 50 mW/cm2 ) .", "Traces show fluorescence of QuasAr2 for control cells and after addition of either 100 μM carbamazepine or 3 μM amitriptyline .", "( F ) Ratio of spike amplitude in test pulse to spike amplitude at the longest recovery time ( 5120 ms ) , as a function of recovery time .", "Carbamazepine modestly slowed recovery and amitriptyline dramatically slowed recovery ( n = 9–11 wells per curve , ~150 cells per well ) .", "( G ) Optical protocol to measure voltage-dependent NaV1 . 7 activation and inactivation .", "Cells were stimulated with 1000 ms prepulse with increasing intensity ( 1 . 7 , 3 . 6 , 6 . 3 , 9 . 8 , 14 mW/cm2 ) , immediately followed by a test pulse ( 150 ms , 14 mW/cm2 ) .", "Traces show representative fluorescence recordings of control and 100 μM carbamazepine .", "( H ) Effect of carbamazepine on activation and inactivation curves .", "Spike amplitudes were normalized to the maximum spike amplitude in the trace and were then plotted against prepulse intensity ( n = 3 wells per curve ) .", "Carbamazepine left-shifted the inactivation curve , decreasing the optically measured overlap between activation and inactivation . DOI: http://dx . doi . org/10 . 7554/eLife . 15202 . 00910 . 7554/eLife . 15202 . 010Figure 3—figure supplement 1 . Use-dependent inhibition of NaV1 . 7 by amitriptyline and TTX . Data used to produce Figures 3C , D .", "Plots show the fluorescence response evoked by the test pulse with variable duration prepulses .", "Each trace is the average of n = 3–5 wells for amitriptyline or n = 4–6 wells for TTX . DOI: http://dx . doi . org/10 . 7554/eLife . 15202 . 010 Sodium channel blockers often show complex state-dependent binding affinities and kinetics .", "Voltage clamp protocols have been developed to prepare specific states to probe these mechanisms .", "Most voltage-clamp protocols comprise a prepulse , an optional recovery interval , and a test pulse .", "The voltage during each interval can be selected to populate different states .", "We sought to recreate these protocols by programming the duration and intensity of the blue light pulses .", "First we varied the duration of the 488 nm optical prepulse from 20 ms to 500 ms , to probe state-dependent binding .", "The intensity was 50 mW/cm2 , which correspond to a depolarization to ~-30 mV .", "The cells were then given 200 ms recovery with no optical stimulus .", "The recovery interval was selected to allow drug-unbound channels to reprime .", "The test pulse ( 30 ms , 50 mW/cm2 ) probed residual excitability .", "We used the amplitude of the fluorescence spike during the test pulse as a proxy for the degree of remaining NaV1 . 7 current .", "Simultaneous fluorescence and manual patch clamp measurements showed close correspondence of the optical and electrical signals ( Figure 3B ) .", "After adjusting the fluorescence data for scale and offset relative to the voltage recording ( as in Ref . Kralj et al . , 2012 ) , the residual variations in fluorescence had an amplitude equivalent to 2 . 8 mV in a 200 Hz bandwidth .", "We then applied the measurements using optical stimulation and recording alone , without patch clamp .", "Amitriptyline , a tricyclic antidepressant , showed strong state-dependent binding with degree of channel block dependent on prepulse duration ( Figure 3—figure supplement 1A ) .", "The IC50 values of amitriptyline varied from 11 . 7 ± 1 . 6 μM at 20 ms prepulse to 1 . 6 ± 0 . 1 μM at 500 ms prepulse ( standard error of fit to Hill equation , n = 3–5 wells per data-point , Figure 3C ) .", "This state-dependent block is consistent with previous patch clamp results ( Wang et al . , 2004 ) .", "In contrast , TTX showed very modest state dependence ( Figure 3—figure supplement 1B ) .", "The optically recorded IC50 values of TTX were 126 ± 13 nM at 20 ms prepulse and 62 ± 5 . 8 nM at 500 ms prepulse ( standard error of fit to Hill equation , n = 4–6 wells per data-point , Figure 3D ) , consistent with prior findings that TTX has a slightly increased affinity for the inactivated channel ( Conti et al . , 1996; Patton and Goldin , 1991 ) .", "Some sodium channel blockers can slow channel repriming after inactivation ( Bean et al . , 1983 ) .", "We examined this effect by varying the recovery period .", "Cells were exposed to a prepulse with fixed duration of 500 ms , a variable recovery period from 40 ms to 5120 ms , and a test pulse of 30 ms . Cells treated with DMSO vehicle showed nearly complete recovery within 40 ms . Carbamazepine , a commonly used drug for the treatment of seizure and neurological pain , blocked recovery at 40 ms , but not at 80 ms . Amitriptyline had an even more dramatic effect , slowing the half-recovery time to 280 ± 36 ms , when tested at 3 μM ( mean ± s . e . m . , n = 9 wells per data-point ) .", "This result implies that amitriptyline has slow dissociation from the channel at resting membrane potential of -97 mV ( Figure 3E , F ) .", "Traditional voltage clamp protocols are flexible and precise in both time and voltage , while optical control of voltage is only semi-quantitative .", "Nonetheless , we explored stimulus intensity-dependent protocols , using the relation between steady state depolarization and illumination intensity ( Figure 2 , Figure 2—figure supplement 1A ) as a guide .", "We developed a protocol to probe separately voltage dependent activation and fast inactivation of NaV1 . 7 .", "Cells were exposed to a prepulse of 1 s duration with variable intensity from 1 . 7 to 14 mW/cm2 , corresponding to depolarizations of 84 to −47 mV .", "The blue light intensity was then stepwise increased to 14 mW/cm2 , with no intervening recovery period .", "We quantified the amplitude of the fluorescence spike at the onset of the prepulse and the test pulse .", "The former probed channel activation , and the latter probed fast inactivation .", "In conventional voltage clamp measurements , the region of overlap between activation and inactivation is called the 'window current' and is important in governing cellular excitability .", "Mutations that increase the window current have been associated with pain disorders ( Fertleman et al . , 2006 ) and cardiac arrhythmias ( Wehrens et al . , 2003 ) .", "Compounds that decrease the window current by left-shifting inactivation or right-shifting activation have therapeutic potential ( Fertleman et al . , 2006; Qiao et al . , 2014 ) .", "We examined the effect of carbamazepine on voltage-dependent activation and fast inactivation .", "Consistent with observations from traditional electrophysiology ( Fertleman et al . , 2006; Qiao et al . , 2014 ) , carbamazepine reduced the overlap between activation and inactivation by leftward shifting the fast inactivation curve without altering the activation curve ( Figure 3G , H ) .", "While optical electrophysiology is not able to quantify the window current , it can identify the qualitative mechanistic feature , the sign , and the approximate magnitude of the effect .", "Recently , a subtype specific drug binding pocket was identified in the voltage sensor of Domain IV of NaV1 . 7 ( McCormack et al . , 2013 ) .", "Significant effort has gone into developing subtype-specific blockers , due to their potential analgesic applications .", "One such compound , PF-04856264 , selectively blocks NaV1 . 7 in a state-dependent manner , with reported IC50 of 28 nM when the steady state potential is −70 mV ( McCormack et al . , 2013 ) .", "The mechanistic details of the interaction of this compound with the channel have not been characterized .", "We varied the precondition pulse length and found that even at 2 s of precondition pulse , PF-04856264 failed to inhibit the channel at 100 nM ( Figure 4A ) . 10 . 7554/eLife . 15202 . 011Figure 4 . Effect of PF-04856264 , a subtype-specific blocker , on NaV1 . 7-OS HEK cells .", "( A ) Dose-response curves for PF-04856264 when stimulated with prepulses of different durations and with different bath K+ concentrations ( n = 4 wells for each concentration ) .", "The optical protocol was as in Figure 3B , with prepulse duration specified in figure legends .", "( B ) Comparison between membrane voltage predicted by the Nernst Equation ( assuming pure K+ conductance ) and recorded by manual patch clamp , as a function of bath [K+] ( n = 4–7 cell clusters per data point ) .", "( C ) Use-dependent inhibition of spiking in NaV1 . 7-OS HEK cells by PF-04856264 , at 8 mM external K+ .", "Cells were stimulated with eight pulses of blue light ( 20 ms , 50 mW/cm2 ) at 5 Hz and 10 Hz and QuasAr2 fluorescence was monitored with 635 nm excitation , 400 W/cm2 .", "After photobleaching correction , the QuasAr2 fluorescence in the absence or in the presence of 100 nM PF-04856264 , was normalized to peak amplitude of the first spike at 5 Hz in the absence of the drug .", "Each trace was averaged from 4 wells .", "Inset: structure of PF-04856264 . DOI: http://dx . doi . org/10 . 7554/eLife . 15202 . 011 We hypothesized that the discrepancy between our measurements and literature results was due to the difference between the resting potential of our cells ( ~−97 mV ) and the steady state potential in the prior work ( −70 mV ) .", "To control the resting potential of the NaV1 . 7-OS HEK cells , we varied the extracellular K+ concentration and observed approximately Nernstian behavior , as expected for a leak current dominated by Kir2 . 1 ( Figure 4B ) .", "We elevated bath potassium from 2 mM to 8 mM , which decreased the magnitude of the resting potential to ~-70 mV .", "Under this condition and a 2 s prepulse , PF-04856264 inhibited NaV1 . 7 mediated spikes with IC50 at 43 nM .", "We further investigated use dependent inhibition of NaV1 . 7 by PF-04856264 with 8 mM bath potassium , and observed both tonic and use dependent inhibition ( Figure 4C ) .", "Our results show that binding of PF-04856264 is strongly dependent on the resting voltage even at potentials where NaV1 . 7 activation is minimal .", "Due to slow binding kinetics , sustained baseline depolarization is more effective than strong but brief depolarization at inducing binding .", "NaV1 . 7 is widely considered to be a promising target for analgesic drugs ( Dib-Hajj et al . , 2013 ) , so we sought to develop a high throughput screen based on Optopatch measurements in NaV1 . 7-OS HEK cells .", "We used the ability to optically stimulate and record to screen for activity-dependent modulators of NaV1 . 7 .", "The platform was based around a commercial inverted microscope ( Olympus IX-71 ) with an automated scanning stage and an air objective .", "Optogenetic stimulation and fluorescence imaging were performed through the objective .", "Fluorescence from a region 320 by 166 μm , comprising approximately 150 cells , was binned on the detector and digitized at 100 Hz .", "We programmed the system to record sequentially from each well in a glass-bottomed 384 well plate .", "We tested a library of 320 FDA approved drugs .", "Each well was treated with a single compound at 10 μM concentration .", "Amitriptyline ( 10 μM ) and DMSO ( 0 . 1% ) were used as positive and negative controls , respectively .", "Sixteen control wells ( 8 positive , 8 negative ) were placed at the beginning and end of the plate .", "Cells were incubated with compound for 20 min .", "Each well was then stimulated with 8 pulses of blue light , 20 ms per pulse , 10 Hz , and the binned fluorescence was recorded .", "Automated scanning of the whole plate required 20 min .", "Amitriptyline and DMSO showed robustly distinct firing patterns .", "DMSO wells showed consistent firing throughout the stimulus train .", "Amitriptyline wells showed rapid activity-dependent decrease in spike amplitude ( Figure 5A ) .", "We also observed that some compounds induced more complex spiking patterns , either suppressing alternate spikes , or leading to erratic firing responses .", "We designed two simple parameters to capture the main features of use-dependent block , an important attribute of sodium channel blockers ( McCormack et al . , 2013; Zhang et al . , 2015; Scholz , 2002 ) .", "Let Si be the height of the ith spike ( i runs from 1 to 8 ) , and let S∼i≡Si/S1 be the height of the ith spike divided by the height of the first spike ( hence S∼1=1 ) .", "We calculated the use dependence index as Γ=1−⟨Si∼⟩2−8 , where the subscripts indicate the range of spikes averaged .", "We also calculated a measure of recovery from inactivation via the standard deviation in the spike amplitude , σ=⟨ ( Si∼−⟨Si∼⟩ ) 2⟩1/2 .", "This parameter was large for wells that showed alternating or erratic spike patterns .", "Thus every well was represented by a point on a two-dimensional ( σ , Γ ) graph .", "Compound names and screening results are available in Figure 5—source data 1 .", "Remarkably , in this blinded screen , Doxepin was classified as functionally adjacent to the amitriptyline controls .", "Doxepin is a tricyclic antidepressant with structure and pharmacology very similar to those of amitriptyline ( Figure 5B ) . 10 . 7554/eLife . 15202 . 012Figure 5 . High throughput screening of a FDA-approved drug library in NaV1 . 7-OS HEK cells .", "( A ) QuasAr2 fluorescence from positive ( amitriptyline ) and negative ( DMSO ) control wells .", "Cells were stimulated with eight pulses of blue light ( 20 ms , 50 mW/cm2 ) at 10 Hz , and QuasAr2 fluorescence was monitored with 635 nm excitation , 400 W/cm2 .", "( B ) Screen results .", "The response of each well was parameterized by its use dependence index and standard deviation in spike amplitude .", "Positive controls ( red ) and negative controls ( green ) were well separated .", "Selected hits were chosen for further analysis .", "Inset: structures of amitriptyline and doxepin .", "( C ) QuasAr2 fluorescence traces of doxepin , trifluoperazine , isradipine and iloperidone recorded in the screen .", "( D ) Validation of select hits by manual electrophysiology .", "Nav1 . 7-Optopatch cells were held at −100 mV .", "A 200 ms prepulse to 0 mV allowed drug binding .", "Recovery times at −100 mV ranged from 1 ms to 256 ms . A test pulse to 0 mV , 100 ms duration , probed the degree of channel recovery .", "Blue: NaV1 . 7 current during prepulse .", "Red: NaV1 . 7 current during test pulse .", "( Prepulse and test pulse currents have been time-shifted and overlaid for easy comparison ) .", "Each compound was tested at 10 μM .", "( E ) Quantification of compound effects on NaV1 . 7 recovery from inactivation .", "The plots show ratio of current amplitude at test pulse to prepulse , as a function of recovery period ( n = 3 cells for each compound , n = 12 cells for control ) .", "( F ) Characterization of select hits from ( B ) in NaV1 . 5-OS HEK cells .", "Cells were stimulated with eight pulses of blue light ( 20 ms , 50 mW/cm2 ) at 4 Hz .", "The 4 Hz stimulus was selected because action potential width of Nav1 . 5-OS cells lasted longer than 200 ms under control conditions .", "Data analyzed and plotted as in ( B ) ( n = 4–6 wells per drug ) .", "Drug concentrations were TTX: 1 μM , PF-04856264: 1 μM , amitriptyline: 10 μM , trifluoperazine: 10 μM , isradipine: 10 μM , iloperidone: 30 μM ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 15202 . 01210 . 7554/eLife . 15202 . 013Figure 5—source data 1 . Spreadsheet containing compound names and screening results . DOI: http://dx . doi . org/10 . 7554/eLife . 15202 . 01310 . 7554/eLife . 15202 . 014Figure 5—figure supplement 1 . Fluorescence traces from NaV1 . 5-OS HEK cells with different drugs . NaV1 . 5-OS HEK cells were stimulated with eight pulses of blue light ( 20 ms , 50 mW/cm2 ) at 4 Hz , and QuasAr2 fluorescence was monitored with 635 nm excitation , 400 W/cm2 .", "TTX and PF-04856264 had little effect on channel function .", "Amitriptyline and trifluoperazine showed strong use-dependent block .", "Isradipine and iloperidone showed use-dependent block with fast recovery , leading to alternating response amplitudes . DOI: http://dx . doi . org/10 . 7554/eLife . 15202 . 01410 . 7554/eLife . 15202 . 015Figure 5—figure supplement 2 . Characterization of off-target effects via optical and manual patch assays .", "( A ) Optical assay to detect perturbations to Kir2 . 1 , CheRiff , or QuasAr2 in NaV1 . 7-OS HEK cells .", "NaV1 . 7 was blocked with 1 μM TTX .", "Cells were stimulated with increasing intensities of blue light ( 500 ms , 3 . 2 , 12 , 27 , 41 , 56 mW/cm2 ) , and QuasAr2 fluorescence was monitored with 635 nm excitation , 400 W/cm2 .", "Fluorescence with a test compound ( 10 μM amitriptyline , 100 μM carbamazepine , 10 μM trifluoperazine , 1 μM PF-04856264 , 200 μM lidocaine , 10 μM isradipine , 10 μM iloperidone ) was compared to TTX alone .", "The fluorescence changes were normalized to that of TTX only treated cells under 56 mW/cm2 stimulation .", "( B ) Mean fluorescence changes in ( A ) during the blue stimuli as a function of stimulus intensity .", "Error bars represent s . e . m . of n = 9–10 wells .", "( C ) Voltage clamp protocol to test for drug effects on CheRiff photocurrent .", "Cells were held at −80 mV and then stepped to −60 mV to +40 mV in 20 mV increments .", "During each step depolarization , a blue light pulse ( 100 ms , 0 . 5 W/cm2 ) was applied to activate CheRiff current .", "Control trace and trace after carbamazepine treatment are shown as an example .", "( D ) I-V relationship of CheRiff current under control condition ( before drug treatment ) and with test compounds at the same concentrations as in ( A ) .", "To control for cell-to-cell variations in CheRiff expression , the current amplitudes were normalized to that of control at −80 mV .", "Error bars represent s . e . m . , n = 29 cells for control , n = 3–4 cells for each compound .", "( E ) Voltage-clamp measurements to test for drug effects on QuasAr2 voltage sensitivity .", "QuasAr2 fluorescence was monitored with 640 nm excitation , 400 W/cm2 while membrane voltage was modulated as shown .", "Single cell recordings were performed before and after addition of drug or buffer control .", "Example traces show fluorescence before and after addition of buffer , lidocaine or carbamazepine .", "( F ) Mean QuasAr2 fluorescence as a function of voltage in the presence of test compounds at the same concentrations as in ( A ) .", "To control for cell-to-cell variation in QuasAr2 expression , the fluorescence changes were normalized to the pre-drug fluorescence at +40 mV .", "Error bars represent s . e . m . , n = 30 cells for control , n = 3–4 cells for each compound . DOI: http://dx . doi . org/10 . 7554/eLife . 15202 . 015 Using σ alone to distinguish positive and negative controls , the Z’ factor ( Zhang et al . , 1999 ) for the assay was 0 . 57 , within the range appropriate for a high-throughput screen ( Zhang et al . , 1999 ) .", "We identified compounds for which σ was greater than 5 standard deviations from the average of negative controls .", "The hit rate was 12 . 2% by this measure , consistent with the notion that voltage gated sodium channels are promiscuous binders ( Figure 5B ) ( Zhang et al . , 2015 ) .", "The 'hit' compounds showed diversity in their spike patterns .", "For amitriptyline , doxepin , and trifluoperazine , spike amplitude decayed monotonically throughout the pulse sequence .", "For other compounds , e . g . isradipine and iloperidone , spike amplitude alternated between even and odd stimuli , a pattern we called 'alternans' ( Figure 5C ) .", "We hypothesized that the alternans compounds had a more transient inhibitory effect on channel repriming , compared to the amitriptyline-like compounds .", "To test this hypothesis and to relate the parameters measured by the screen to more conventional electrophysiological parameters , we performed voltage clamp recordings in the presence of several hits from the screen .", "A 200 ms prepulse and a 100 ms test pulse were separated by a recovery interval varying from 1 ms to 256 ms . We measured the ratio of the peak inward sodium currents at the test and pre-pulse .", "Control cells showed half-maximal recovery in 4 . 5 ms . Cells treated with isradipine or iloperidone ( alternans compounds ) , showed half-maximal recovery in 20 ms . Cells treated with amitriptyline or trifluoperazine ( strong blockers ) showed less than 50% recovery in 256 ms ( Figure 5D , E ) .", "Thus compounds that clustered nearby in the optically measured ( σ , Γ ) graph , also showed similar effects by conventional patch clamp measurements .", "For a compound to be a safe analgesic , it should not interact significantly with other NaV channels , particularly the cardiac NaV1 . 5 channel .", "We created a NaV1 . 5-OS HEK cell line analogous to the NaV1 . 7-OS HEK cell line described above , and re-tested some hits from the screen against NaV1 . 5 ( Figure 5—figure supplement 1 ) .", "Unsurprisingly , on a ( σ , Γ ) plot , all hits arranged in a similar pattern for NaV1 . 5 and NaV1 . 7 , indicating no subtype selectivity ( NaV1 . 7-selective compounds are exceedingly rare ) .", "In contrast , both PF-04856264 ( 1 μM ) and TTX ( 1 μM ) clustered with the negative controls when tested on NaV1 . 5 , consistent with the known fact that neither of these compounds blocks NaV1 . 5 at the concentrations tested ( McCormack et al . , 2013; Zhang et al . , 2015 ) ( Figure 5F ) .", "Kir2 . 1 is not considered as a promiscuous drug binder ( Bowes et al . , 2012 ) and we are not aware of any compounds that block the conductance of channelrhodopsin or interfere with the voltage-dependent fluorescence of QuasAr2 .", "Nonetheless , it is a formal possibility that false-positive readings could arise from compounds that modulate these other components .", "We developed a simple optical test for off-target effects and applied it to a panel of seven compounds selected for diverse mechanisms of NaV1 . 7 block .", "First , we added TTX ( 1 μM ) to block NaV1 . 7 .", "We then applied steps of blue light ( 500 ms , 3 . 2 – 56 mW/cm2 ) and monitored the QuasAr2 fluorescence , which reported optically induced depolarizations without regenerative spikes ( Figure 5—figure supplement 2A ) .", "We then performed the same measurement in the presence of TTX + test compound .", "Any drug interaction with Kir2 . 1 , CheRiff , or QuasAr2 would alter the fluorescence response .", "Six of the seven tested compounds had minimal effect ( within 7% of TTX , n = 9–10 wells per compound ) .", "Carbamazepine induced a slight decrease in fluorescence signal ( 20% smaller than TTX alone , Figure 5—figure supplement 2B ) .", "We further verified the optical tests using manual patch clamp measurements .", "None of the eight tested compounds ( seven drugs and TTX ) affected CheRiff photocurrents ( Figure 5—figure supplement 2C , D ) .", "Seven of the compounds had no effect on QuasAr2 fluorescence or voltage sensitivity relative to buffer control .", "Carbamazepine reduced QuasAr2 voltage sensitivity by 17% , consistent with the all-optical assay ( Figure 5—figure supplement 2E , F ) .", "This off-target effect of carbamazepine had no effect on our optical assays of activity-dependent block ( Figure 3F and Figure 3G ) , because fluorescence spike heights were normalized to the height of the highest spike .", "A slight decrease in overall fluorescence signal is cancelled in this analysis .", "Finally , we explored whether the OS-HEK cells could be used to screen for modulators of other channel classes .", "KV4 . 3 is an A-type fast-activating voltage-gated potassium channel , active in the heart and central nervous system .", "Conventional ionic flux based optical approaches to screening for modulators of KV4 . 3 are extremely challenging because the channel is inactivated at the resting potential of HEK cells .", "We stably expressed KV4 . 3 , NaV1 . 5 and Optopatch constructs in HEK cells .", "Voltage-clamp experiments revealed robust expression of KV4 . 3 , with a maximum current density of 218 pA/pF at +40 mV ( Figure 6A ) .", "KV4 . 3 has very fast activation kinetics with a time constant τact = 0 . 69 ms at +40 mV .", "The inactivation of KV4 . 3 can be best described as a double exponential decay ( Liang et al . , 2009 ) , with τfast = 51 ms and τslow = 352 ms at +40 mV ( Figure 6A ) .", "We then transiently expressed Kir2 . 1 to prime NaV1 . 5 and KV4 . 3 and called these cells NaV1 . 5-KV4 . 3-OS cells . 10 . 7554/eLife . 15202 . 016Figure 6 . Optopatch assay of KV4 . 3 function .", "( A ) Voltage clamp recording of KV4 . 3 current in NaV1 . 5-KV4 . 3 Optopatch HEK cells .", "The bath contained 30 μM TTX to block the NaV1 . 5 current .", "Cells were held at −70 mV and then subjected to 1 s steps to −60 mV to +40 mV in 10 mV increments .", "Peak KV4 . 3 current densities were 218 pA/pF .", "( B ) NaV1 . 5-KV4 . 3-OS HEK cells were probed with simultaneous current clamp and QuasAr2 fluorescence .", "The cells were stimulated with a pulse of blue light ( 100 ms , 50 mW/cm2 ) , and QuasAr2 fluorescence was monitored with 640 nm excitation , 400 W/cm2 .", "KV activation led to a narrow action potential width , followed by KV inactivation and a return to steady-state depolarization .", "( C ) Average QuasAr2 fluorescence traces from NaV1 . 5-KV4 . 3-OS HEK cells treated with HpTx-2 ( n = 3–4 wells for each concentration ) .", "( D ) Dose-response curve of HpTx-2 on NaV1 . 5-KV4 . 3-OS HEK cells .", "Drug effect was quantified by the fluorescence at the peak repolarization ( ~40 ms after onset of stimulus ) relative to peak fluorescence intensity under 1200 nM HPTX2 treatment . DOI: http://dx . doi . org/10 . 7554/eLife . 15202 . 016 When stimulated by pulses of blue light ( 100 ms , 50 mW/cm2 ) , the presence of KV4 . 3 led to a dramatic change in the optically induced and optically recorded action potential waveform , featuring a transient fast repolarization almost reaching resting potential before a recovery toward plateau potential ( Figure 6B ) .", "We then tested the effect of heteropoda toxin 2 ( HpTx2 ) , a potent and specific blocker of channels in the KV4 family ( Zarayskiy et al . , 2005 ) .", "HpTx2 increased the action potential amplitude , consistent with its inhibition on the fast inactivated KV4 . 3 peak current; HpTx2 also increased the plateau potential amplitude when compared at the end of the 100 ms light pulse , which can be explained by its inhibitory effect on the slow inactivated KV4 . 3 current ( Figure 6C ) .", "HpTx2 showed dose-dependent blockade , with an IC50 of 252 nM , consistent with literature results ( Figure 6D ) ( Brahmajothi et al . , 1999 ) .", "However , the high rate at which test compounds blocked the NaV channel precluded use of these cells in high-throughput screening applications .", "Screening would require use of a NaV channel or a NaV channel mutant which is resistant to most drugs ." ], [ "Despite variable expression levels of optogenetic actuator and voltage indicator , we have shown that Optopatch assays can probe state-dependent pharmacology of NaV channel modulators , and can accurately report binding affinities and kinetics .", "Key to achieving this accuracy were ( 1 ) performing measurements averaged over large numbers of cells , and ( 2 ) developing stimulus and analysis protocols that were insensitive to modest variations in expression levels of the optogenetic components .", "Optical flux-based assays have been widely used in ion channel screens ( Yu et al . , 2016 ) .", "However , these assays typically only probe steady-state channel behavior .", "Flux-based assays are widely used , however , because they offer high throughput and high reproducibility .", "Recent advances in automated electrophysiology ( Dunlop et al . , 2008 ) enable control of membrane voltage in heterologous expression systems .", "Automated electrophysiology offers the advantage of direct control of voltage and measurement of current .", "However these techniques have lower throughput and higher cost than optical assays , only work on certain cell types , and can be challenging to optimize .", "Patch clamp measurements also involve a perturbation to the integrity of the cell membrane , which can lead to changes in cytoplasmic composition and artifacts from mechanosensitive channels ( Morton and Main , 2013 ) .", "The Optopatch assays developed here provide detailed and quantitative mechanistic information; are compatible with high-throughput screening; and are non-invasive .", "What are the limitations on throughput of optical electrophysiology screens ?", "Here we performed serial measurements , one well at a time .", "At a measurement time of ~3 s/well , a 384-well plate was scanned in ~20 min .", "There are no fundamental principles that prevent scaling to more densely packed wells ( e . g . 1534 well plates ) , or to parallelizing the measurements .", "While optopatch measurements require high intensity red illumination , high SNR can be obtained at lower intensities than the 400 W/cm2 we used here ( Figure 2—figure supplement 4 ) .", "Specialized instrumentation has been developed for sensitive fluorescence recording from multi-well plates ( Hempel et al . , 2011 ) , and with such instrumentation one could achieve throughputs compatible with primary screening .", "Given greater parallelism of measurement , one could also implement more complex stimulus protocols such as we developed here , while maintaining adequate throughput .", "Illumination intensities for imaging Arch-based GEVIs are typically 10 to 100-fold greater than are used for imaging GFP-based GEVIs .", "Thus it is natural to worry about phototoxicity from the red laser .", "A recent study explored phototoxicity in cultured mammalian U2OS cells ( Wäldchen et al . , 2015 ) .", "Illumination at 200 W/cm2 , λ = 488 nm for 240 s led to 100% of the cells being either dead or 'frozen'; while illumination at 5900 W/cm2 , λ = 640 nm for 240 s led to undetectable cell death .", "Our observation of good cell viability at 400 W/cm2 , λ = 640 nm is consistent with these literature results .", "In principle , the screening approaches described here could be adapted to work with a red-shifted voltage-sensitive dye .", "Fluorescence signals would be more homogeneous than with a genetically expressed indicator; one could more readily switch between cell lines; and there is a possibility that the imaging could be performed at lower illumination intensity , on conventional equipment .", "However , existing red-shifted dyes , e . g . PGH1 ( Salama et al . , 2005 ) and Di-2-ANBDQPQ ( Zhou et al . , 2007 ) still retain considerable excitation at the blue wavelengths used for channelrhodopsin activation , and these dyes are not at present commercially available .", "Finally , we consider the diversity of channels for which Optopatch-style screens may be feasible .", "Here we demonstrated assays for NaV1 . 7 , NaV1 . 5 , and KV4 . 3 .", "We previously demonstrated spiking HEK cells expressing NaV1 . 3 ( Park et al . , 2013 ) and Hsu et al . demonstrated spiking CHO cells expressing NaV1 . 2 .", "HEK cell lines expressing NaV1 . 1 through NaV1 . 8 are commercially available , and a method for heterologous expression of chimeric NaV1 . 9 was recently demonstrated ( Goral et al . , 2015 ) .", "Voltage-gated Ca2+ channels can also mediate regenerative spiking and thus are also plausible targets for the assay .", "Fast and repetitive optogenetic activation of CaV3 . 2 by channelrhodopsin2 has been achieved in HEK293T cells ( Prigge et al . , 2010 ) .", "Recently , the state dependent inhibition of CaV1 . 3 has been studied by channelrhodopsin stimulation protocols ( Agus et al . , 2015 ) .", "In principle , optogenetic activation could be applicable to other types of CaV channels .", "Delayed rectifier potassium channels such as hERG and KV7 may also be amenable to optical interrogation if co-expressed with an inactivation deficient NaV channel .", "Modulation of the potassium current would manifest as a change in the action potential duration ( Fujii et al . , 2012 ) ." ], [ "The pIRESpuro3-NaV1 . 5 and pcDNA3-KV4 . 3 plasmids were obtained from ChemCORE at Johns Hopkins University .", "The Optopatch construct contains coding sequences of CheRiff-eGFP and QuasAr2-mOrange2 , separated by a P2A self-cleaving peptide sequence .", "The entire Optopatch construct was cloned into a modified FCK lentivirus vector ( mFCK ) , in which the original CaMKII promoter was replaced by a CMV promoter .", "The Kir2 . 1 cDNA was amplified from Addgene plasmid 32 , 669 ( pENTR-L5-Kir2 . 1-mCherry-L2 ) and cloned into a pLX304 lentivirus vector that contained a blasticidin selection marker .", "The Kir2 . 1 cDNA was also cloned into pIREShyg vector using the Gibson assembly method .", "The Kv4 . 3 cDNA was amplified from pcDNA3-KV4 . 3 plasmid and then cloned into pIREShyg vector using the Gibson assembly method ( Gibson et al . , 2009 ) .", "HEK293 cells were transected with pIRESpuro3-NaV1 . 5 using TransIT-293 Transfection Reagent ( Mirus Bio ) following manufacturer’s instruction .", "After 48 hr of transfection , puromycin was added to a final concentration of 2 μg/mL .", "Cells were selected for 14 days to stabilize the expression of NaV1 . 5 .", "Surviving cells were subsequently transduced with low-titer mFCK-Optopatch lentivirus .", "After 10 days of infection , all the GFP positive cells were enriched by fluorescence activated cell sorting ( FACS ) .", "This polyclonal NaV1 . 5-Optopatch stable cell line was used to generate the NaV1 . 5-OS and NaV1 . 5-KV4 . 3 OS cells .", "To generate NaV1 . 5-OS cells , NaV1 . 5-Optopatch cells were transduced by pLX304-Kir2 . 1 lentivirus .", "After 48 hr of transduction , Kir2 . 1 expressing cells were selected by 5 μg/mL blasticidin .", "At the same time , 2 μg/mL puromycin was also included to ensure the stable expression of NaV1 . 5 .", "Cells were cultured for 14 days and then single cells were dispersed in wells of a 48 well plate .", "Monoclonal NaV1 . 5 -OS lines were screened via Optopatch measurements for robust generation of action potentials under blue laser stimulus , and corresponding QuasAr2 fluorescence transients with SNR greater than 30 .", "To generate NaV1 . 5-KV4 . 3-OS cells , NaV1 . 5-Optopatch cells were transiently transfected by pIREShyg-KV4 . 3 .", "Two days after transfection , 200 μg/mL hygromycin was used to establish the NaV1 . 5-Optopatch-KV4 . 3 monoclonal stable cell line .", "Each monoclonal cell line was optically evaluated for spiking and fast repolarization behavior after transient transfection of pIREShyg-Kir2 . 1 plasmid .", "The best monoclonal cell line ( NaV1 . 5-Optopatch-KV4 . 3 ) was further expanded and NaV1 . 5-KV4 . 3-OS cells can be reliably generated by transient transfection of Kir2 . 1 into this monoclonal cell line .", "The NaV1 . 7-OS HEK cells were generated based on a NaV1 . 7 stable cell line established by G418 selection , a kind gift from Dr . Bruce Bean at Harvard University .", "This stable cell line was transduced with Optopatch by mFCK-Optopatch lentivirus .", "After 10 days , GFP positive ( NaV1 . 7-Optopatach ) cells were enriched by FACS .", "We attempted , unsuccessfully , to further stabilize Kir2 . 1 in these NaV1 . 7-optopatach cells by using pLX304-Kir2 . 1 lentivirus transduction .", "Surviving cells after blasticidin selection were not able to fire action potentials , likely due to poor expression level of Kir2 . 1 .", "Therefore , single cells of NaV1 . 7-Optopatch cells were dispersed into a 48 well plate and each NaV1 . 7-optopatch monoclonal cell line was evaluated by transient transfection of pIREShyg-Kir2 . 1 using lipofectamine 2000 ( Invitrogen ) following manufacturer’s instruction .", "The best NaV1 . 7-Optopatch monoclonal line that produced robust spikes with corresponding high SNR QuasAr2 fluorescence was selected and further expanded .", "The transfected cells are called NaV1 . 7-OS cells .", "Cells tested negative for mycoplasma contamination .", "Absence of contamination from other cell lines was ensured by growing up cells from a single clone .", "NaV1 . 5-OS , NaV1 . 5-Optopatch-KV4 . 3 cells , and NaV1 . 7-Optopatch HEK cell lines were maintained in Dulbecco’s Modified Eagle Medium ( DMEM ) with 10% fetal bovine serum , penicillin ( 100 U/mL ) , streptomycin ( 100 μg/mL ) .", "For NaV1 . 5-OS cells , 2 μg/mL puromycin and 5 μg/mL blasticidin were included in the medium to maintain expression of NaV1 . 5 and Kir2 . 1 .", "For NaV1 . 5-Optopatch-KV4 . 3 cells , 2 μg/mL puromycin and 200 μg/mL hygromycin were included in the medium to maintain expression of NaV1 . 5 and KV4 . 3 .", "For NaV1 . 7-Optopatch cells , 500 μg/mL of G418 was included in the medium to maintain NaV1 . 7 expression .", "Electrophysiology measurements were performed in a bath solution of Tyrode’s , containing ( in mM ) : 125 NaCl , 2 KCl , 2 CaCl2 , 1 MgCl2 , 10 HEPES , 30 glucose .", "The pH was adjusted to 7 . 3 with NaOH and the osmolality was adjusted to 305–310 mOsm with sucrose .", "Filamented glass micropipettes ( WPI ) were pulled to a resistance of 4–7 MΩ and filled with internal solution containing 140 mM KCl , 1 mM MgCl2 , 10 mM EGTA , 10 mM HEPES , 3 mM Mg-ATP , pH adjusted to 7 . 3 with KOH .", "To record CheRiff and NaV1 . 7 current , NaV1 . 7-Optopatch HEK cells were replated onto 0 . 02 mg/mL poly-d-lysine coated glass-bottom dishes ( In Vitro Scientific ) at a density of ~10 , 000 cells/cm2 .", "The patch clamp recording was performed 4–8 hr after re-plating when most cells had firmly attached to the glass and were still dispersed as single cells .", "The whole cell voltage clamp recordings were acquired using an Axopatch 200B amplifier ( Molecular Devices ) , filtered at 5 kHz with the internal Bessel filter and digitized with a National Instruments PCIE-6323 acquisition board at 10 kHz .", "The series resistance and membrane capacitance were compensated , and whole cell membrane capacitance was obtained by direct reading from the amplifier .", "CheRiff mediated current was triggered by Illumination from a blue laser ( 488 nm , 50 mW , Omicron PhoxX ) that was sent through an acousto-optic modulator ( AOM; Gooch and Housego 48058–2 . 5- . 55-5W ) for rapid control over its intensity .", "The Kir2 . 1 current was recorded from NaV1 . 7-OS HEK cells by using the same configuration with 1 μM of TTX in the bath solution to block NaV1 . 7 current .", "The KV4 . 3 current was recorded from NaV1 . 5-Optopatch-KV4 . 3 cells with 30 μM of TTX in the bath solution to block NaV1 . 5 current .", "To correlate NaV1 . 7 current density with voltage spike amplitude we performed alternate single-cell current clamp and voltage-clamp measurements in the presence of 3 μM amitriptyline .", "We used cells not expressing Kir2 . 1 to avoid confound from Kir currents .", "Paired current- and voltage-clamp protocols were always performed on the same cell .", "For both protocols , an extended prepulse depolarization induced amitriptyline binding and complete channel block .", "A recovery interval at −100 mV of variable duration led to partial channel recovery .", "A test depolarizing pulse of either current or voltage then probed the response of the recovered channels .", "In the current clamp protocol , holding current , ih , was adjusted between −100 to −50 pA to attain a steady-state voltage of approximately −100 mV .", "The cell was then stimulated with a depolarizing current pulse of magnitude −0 . 5 ih for 500 ms . This current brought the steady-state voltage to ~0 mV .", "The current was then brought back to ih for a recovery period of variable duration from 40–5120 ms . Finally , the cell was stimulated with a test current pulse of magnitude -0 . 5 ih for 20 ms to induce a voltage spike whose amplitude we recorded .", "Then the cell was switched to voltage-clamp mode .", "The holding potential was −100 mV .", "To match precisely the degree of channel block in the current-clamp and voltage-clamp protocols , the voltage prepulse and recovery waveforms were copied directly from the voltage recorded during the immediately preceding current-clamp protocol .", "The test pulse comprised a 20 ms step depolarization to −20 mV .", "The inward NaV1 . 7 current at each test pulse was then measured .", "The NaV1 . 7-Optopatch monoclonal cell line was transfected with pIREShyg-Kir2 . 1 plasmid using lipofectamine 2000 following standard protocols .", "The resulting Nav1 . 7-OS HEK cells were recorded 48 hr after transfection .", "The day before recording , cells were replated onto 35 mm glass-bottom dishes ( In Vitro Scientific ) at a density of ~10 , 000 cells/cm2 .", "At the time when recording was performed , the cells formed small clusters comprising 3–4 cells .", "The whole cell current clamp recording was performed on these small clusters under the I-Clamp Normal configuration of the Axopatch 200B amplifier .", "The liquid junction potential was measured and corrected by the standard Neher method ( Neher , 1992 ) .", "Patch clamp and fluorescence imaging data were synchronized by clocking the camera with analog output from National Instruments PCIE-6323 acquisition board while using the same clock for driving patch clamp inputs and outputs .", "The imaging experiments were conducted on a home-built inverted fluorescence microscope ( Hochbaum et al . , 2014 ) .", "Briefly , QuasAr2 was excited by combined illumination from two red lasers ( 640 nm , 140 mW , Coherent Obis 637–140 LX and 640 nm , 100 mW , Coherent CUBE 640-100C ) via a polarizing beam splitter .", "The red beam was expanded and focused onto the back focal plane of a 60× oil-immersion objective ( 60x APO , NA 1 . 49 , Olympus ) .", "CheRiff was activated by Illumination from a blue laser ( 488 nm , 50 mW , Omicron PhoxX ) , which was modulated by an acousto-optic modulator receiving control signals from a National Instruments PCIE-6323 acquisition board .", "During a typical Optopatch experiment , both blue and red lasers were reflected into the sample plane by a quad-band dichroic mirror ( Di01-R405/488/561/635-25x36 , Semrock ) .", "The red laser intensity was maintained at 400 W/cm2 , while the blue laser intensity was modulated via the AOM and ranged from 1–100 mW/cm2 .", "A 710/100-nm bandpass filter ( Chroma , HHQ710/100 ) was used for QuasAr2 imaging , and a variable-zoom camera lens ( Sigma 18–200 mm f/3 . 5–6 . 3 II DC ) was used to image the sample onto an EMCCD camera ( Andor iXon Ultra 897 ) , with 512× 512pixels .", "The variable zoom enabled imaging at a range of magnifications while maintaining the high light-collection efficiency of the oil-immersion objectives .", "Data were acquired with a ROI of 128 × 128 pixels at 4 × 4–pixel binning to achieve a frame rate of 200 frames/s .", "The NaV1 . 7-Optopatch monoclonal cell line was transfected with pIREShyg-Kir2 . 1 plasmid using lipofectamine 2000 .", "A glass-bottom 384-well plate ( P384-1 . 5H-N , Cellvis ) was treated with 0 . 02 mg/mL poly-d-lysine to promote cell adhesion .", "At 24 hr after transfection , cells were replated onto the multiwell plate at a density of ~20 , 000 cells/well in 50 μL of culture medium .", "The imaging experiments were performed at 48 hr after transfection when the cells formed a confluent monolayer .", "The cells were washed with Tyrode’s solution once and then each well is filled with 30 μL of Tyrode’s solution .", "For drug additions , 6 μL drug solution at 6x target concentration was added to each well .", "Cells incubated in drug for 20 min at room temperature before imaging .", "Experiments were conducted on an inverted epi-fluorescence microscope ( Olympus IX-71 ) equipped with an automated scanning stage ( Ludl electronics MAC 6000 ) .", "Briefly , illumination from a red laser ( 635 nm , 500 mW , Dragon Lasers MRL-635-500 mW ) was expanded and focused onto the back focal plane of a 20× air objective ( NA 0 . 75 , Olympus -UPlanSApo 20x/0 . 75 ) .", "Illumination from a blue laser ( 473 nm , 50 mW , Dragon Lasers MBL-473-50mW ) was sent through an acousto-optic tunable filter ( AOTF; Gooch and Housego 48058 ) for rapid intensity modulation .", "The red illumination intensity at the sample was 400 W/cm2 .", "QuasAr2 fluorescence was filtered by a 710/100-nm bandpass filter ( Chroma , HHQ710/100 ) and collected by an EMCCD camera ( Andor iXon Ultra 897 ) .", "Data were acquired with a full camera chip of 512 × 512 pixels at 16 × 16–pixel binning to achieve a frame rate of 100 frames/s .", "Cells were plated in a 384 well plate as above .", "After the cells formed a confluent monolayer ( 48 hr after transfection ) the cells were washed with Tyrode’s solution once and then each well was filled with 20 μL of Tyrode’s solution .", "A compound library consisting of 320 FDA-approved drugs was purchased from Broad Institute at 10 mM stock concentration in DMSO and then diluted to 30 μM in Tyrode’s solution .", "10 μL of the diluted compounds were added to the cell plate ( Well A3-P22 ) to achieve a final concentration of 10 μM .", "Wells A2-H2 and A23-H23 were treated with 0 . 1% DMSO vehicle and used as negative controls .", "Wells I2-P2 and I23-P23 were treated with 10 μM amitriptyline and used as positive controls .", "After 20 min of drug incubation , the 384-well plate was placed on the microscope stage and each well was imaged serially .", "The scanning started at well A2 and ended in well P23 in a column-wise manner .", "Each well was exposed to eight pulses ( 20 ms ) of blue laser ( 50 mW/cm2 ) at 10 Hz to stimulate the firing of NaV1 . 7-OS HEK cells .", "The QuasAr2 fluorescence from each well was collected as above .", "Data were saved as a single tiff stack at the end of scanning .", "Imaging data were stored as a tiff stack and loaded into ImageJ software .", "For data acquired at high magnification ( 60× ) , a rectangular ROI surrounding the cells of interest was manually selected .", "Background fluorescence was determined by measuring the mean intensity of a nearby cell free region and was subtracted from the cell fluorescence .", "For data acquired from cell monolayers under low magnification ( 20× ) , a rectangular ROI ( 400 × 208 pixels ) covering the region with most intense laser illumination was selected .", "This ROI corresponds to a 320 μm × 166 μm area on the sample plane , containing approximately 150 cells .", "The mean intensity within this ROI was calculated for all frames of the tiff stack .", "To calculate ΔF/F0 , background fluorescence was determined by measuring the intensity from a well plated with parental HEK cells without QuasAr2 expression .", "After background subtraction , the data were further analyzed to extract spike parameters .", "Briefly , intensity traces were corrected for photobleaching by dividing the raw intensity by a median filtered copy of the intensity .", "Spike amplitude was defined as the difference between the maximum point of an action potential and the baseline .", "The use dependence index was defined as the fractional reduction of the spike amplitude averaged from the second to the eighth stimulus , compared to the initial stimulus .", "Dose-response curves were fitted with the Hill equation y=START+ ( END-START ) /[1+ ( IC50/S ) n] , where START and END are the values of the parameter at minimum and maximum drug concentration , IC50 is the drug concentration at 50% maximum effect , S is the drug concentration , and n is a measure of cooperativity .", "The Z’ factor for the screen was calculated as Z’ = 1–3 ( σp+σn ) /│ ( μp-μn ) │ , where σp is the standard deviation of the positive controls , σn is the standard deviation of the negative controls , μp is the mean of the positive controls and μn is the mean of the negative controls .", "The activation time constant of KV4 . 3 , τact , was determined by fitting the activation current trace using the equation: i ( t ) =a ( 1−e−t/τact ) 4 +", "b . The inactivation time constants , τfast and τslow of KV4 . 3 were determined by fitting the inactivation current trace using the equation: i ( t ) =a e−t/τfast+b e−t/τslow+", "c . Information on number of replicates for each experiment is given in figure legends .", "For manual patch clamp measurements , sample size was predetermined to be >5 cells , following standard practice .", "For optical electrophysiology measurements , sample size was predetermined to be >100 cells .", "These sample sizes were selected for feasibility of measurement .", "In the screen of the FDA library , one of the 32 control wells showed an anomalous spiking pattern ( visible in Figure 5A ) and was omitted from Figure 5B ." ] ]

[ "Voltage-gated ion channels mediate electrical dynamics in excitable tissues and are an important class of drug targets .", "Channels can gate in sub-millisecond timescales , show complex manifolds of conformational states , and often show state-dependent pharmacology .", "Mechanistic studies of ion channels typically involve sophisticated voltage-clamp protocols applied through manual or automated electrophysiology .", "Here , we develop all-optical electrophysiology techniques to study activity-dependent modulation of ion channels , in a format compatible with high-throughput screening .", "Using optical electrophysiology , we recapitulate many voltage-clamp protocols and apply to Nav1 . 7 , a channel implicated in pain .", "Optical measurements reveal that a sustained depolarization strongly potentiates the inhibitory effect of PF-04856264 , a Nav1 . 7-specific blocker .", "In a pilot screen , we stratify a library of 320 FDA-approved compounds by binding mechanism and kinetics , and find close concordance with patch clamp measurements .", "Optical electrophysiology provides a favorable tradeoff between throughput and information content for studies of NaV channels , and possibly other voltage-gated channels ." ]

[ "Ion channels are specialized proteins that span the cell membrane .", "When activated , these channels allow ions to pass through them , which can produce electrical spikes that carry information in nerve cells and regulate the beating of the heart .", "Researchers interested in understanding how ion channels behave often use a technique called patch clamp electrophysiology to measure the electrical current across the cell membrane .", "The technique can be used to probe if a specific drug can block an ion channel , but it is not well suited to screening lots of potential drugs because it is slow and expensive .", "A group of ion channels known as voltage-gated sodium channels play an important role in generating the electrical spikes in nerve cells .", "One subtype called NaV1 . 7 is involved in sensing pain and drugs that block NaV1 . 7 might be useable as painkillers , but only if they are specific to this channel .", "This is because there are many similar sodium channels that are important in other processes in the body .", "Zhang et al . have now developed a new light-based technique to measure how ion channels behave .", "The technique uses light to activate the channel and a fluorescent protein to report on the membrane’s voltage .", "Zhang et al . used the new technique to probe how sodium channels , in particular NaV1 . 7 , interact with drugs .", "Mammalian cells grown in the lab were engineered to produce NaV1 . 7 , a light-activated ion channel ( called CheRiff ) , and a fluorescent reporter protein .", "A flash of blue light delivered to the cells activated CheRiff , which in turn activated NaV1 . 7 .", "At the same time , the fluorescence of the reporter protein was used as a read-out of NaV1 . 7’s activity .", "Zhang et al . showed that they could reproduce many conventional electrophysiology measurements using their new light-based approach .", "Optical measurements were then used to screen 320 drugs to see whether they could block NaV1 . 7 .", "The results of the screen corresponded closely with measurements made using conventional electrophysiology .", "These results demonstrate that the new optical technique is both fast and precise enough to be used in drug discovery .", "Further studies could now ask if this optical technique can also be used to study other ion channels , such as potassium channels and calcium channels ." ]

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

[ "neuroscience" ]

[ [ "In darkness , sparse single photon signals are relayed to retinal ganglion cells via a high gain/high convergence pathway formed by rods , rod bipolar cells , amacrine AII cells , and cone bipolar cells ( Nelson , 1982; Dunn et al . , 2006 ) .", "However , rod signals can bypass rod bipolar cells and enter directly into the cone pathway ( DeVries and Baylor , 1995 ) .", "The earliest opportunity for this crossover is represented by rod–cone gap junctions ( GJs ) ( Raviola and Gilula , 1973; Tsukamoto et al . , 2001 ) .", "GJs are assemblies of channels made by the docking of pairs of connexon hemichannels on adjacent cells , each formed by six connexin subunits .", "Cones contact nearby photoreceptors mainly at the tips of thin telodendria , which emerge from their synaptic pedicles , where they express connexin isoform 36 ( Cx36 ) ( Lee et al . , 2003; O’Brien et al . , 2012 ) , while the isoform expressed by rods has not been conclusively identified ( Lee et al . , 2003; Feigenspan et al . , 2004 ) .", "The rod–cone junctional plaques are very small , each containing few connexon channels ( Raviola and Gilula , 1973 ) , and although rod signals were recorded in cat ( Nelson , 1977 ) and in macaque cones ( Schneeweis and Schnapf , 1995 , 1999; Hornstein et al . , 2005 ) , the extent to which rod–cone coupling contributes to mammalian vision remains unclear .", "On the one hand , psychophysical and electroretinographic ( ERG ) experiments , in humans , detected putative correlates of rod–cone coupling ( reviewed by Sharpe and Stockman , 1999 ) , and ganglion cell and ERG recordings , in mice lacking Cx36 , supported the view that rod–cone coupling is relevant for dim light vision ( Deans et al . , 2002; Volgyi et al . , 2004; Abd-El-Barr et al . , 2009; Seeliger et al . , 2011 ) ; on the other hand , alternative mechanisms could explain the human data ( Sharpe and Stockman , 1999 ) , and recordings in cone bipolar cells in Cx36 knockout mice suggested that rod–cone coupling plays a marginal role in rod signal flow ( Pang et al . , 2010 , 2012 ) .", "Highly relevant to this debate is the fact that rod–cone GJs appear to be dynamically regulated: measurements of the extent of tracer diffusion in the outer rodent retina from the surface of a cut made with a razor blade ( a technique referred to as ‘cut-loading’ ) ( Ribelayga et al . , 2008; Ribelayga and Mangel , 2010; Li et al . , 2013 ) , and ERG ( Heikkinen et al . , 2011 ) suggest that endogenous neuromodulators , light , and circadian clocks influence the level of coupling ( but see Schneeweis and Schnapf , 1999 ) similarly to other retinal GJs ( Lasater and Dowling , 1985 ) .", "However , cut-loading cannot discriminate between rod–rod , rod–cone and cone–cone GJs , and it does not provide information on the absolute strength of coupling , but only on its relative changes .", "Therefore , the possibility that the impact of coupling on vision is strongly context-dependent raises three pressing questions:", "( i ) what is the maximum level of rod–cone coupling ?", "( ii ) how strongly are cones influenced by rod input under these conditions ?", "( iii ) what is the level of coupling under different physiological states ?", "Here we investigated the first two of these questions in mouse , a mainstay of current retina research and one in which direct proof of rod input in cones is still lacking .", "This important gap in our knowledge is explained by the fact that mouse cones were only recently shown to be accessible for patch clamp recordings ( Cangiano et al . , 2012 ) ." ], [ "The kinetics protocol was delivered in rods as a control ( Figure 2A1 , A2 ) .", "As expected , the first dim G flash evoked a large response , the brighta G flash evoked a saturating response consisting of a fast peak and plateau ( for an analysis of the currents involved , see Della Santina et al . , 2012 ) , and dim G flash sensitivity recovered slowly after the saturating flash .", "Rod responses run down in kinetics during patch recordings ( Cangiano et al . , 2012 ) , a process that also alters their time course of recovery from saturating flashes ( Figure 2A1 , cf . black and gray records ) .", "Thus , the kinetics protocol was also delivered in a number of rods recorded in loose seal mode , as with this technique , light response kinetics can be stable for several hours ( Figure 2A2 ) .", "The time course of the response to the kinetics protocol of rods in the initial minutes of patch recordings ( n = 19 , Figure 2A1 , black records , taken before a significant amount of rundown occurred ) was similar to that in loose seal recordings ( n = 17 ) , as shown in a graph of the normalized dim flash response amplitudes ( Figure 2B ) .", "In cones , the kinetics protocol evoked a spectrum of response types depending on the cone and on the time from seal ( see below ) .", "Some cones responded only to the brighta G flash ( Figure 2C , top ) , the expected behavior for uncoupled cones ( Figure 1 ) .", "However , the large majority of cones displayed , similar to rods , responses to the first dim G flash , a plateau after the brighta G flash , and a slow recovery of the dim flash response ( Figure 2C , middle/bottom ) .", "This similarity in the time course of recovery after the bright flash emerges from comparing graphs of normalized dim flash response amplitudes during the kinetics protocol in rods ( Figure 2B ) and cones ( Figure 2D , n = 11 ) .", "This is strong evidence for the presence of rod input in cones . 10 . 7554/eLife . 01386 . 004Figure 2 . Cones express a rod-like sensitivity to dim flashes and slow recovery after bright flashes .", "( A1 )", "Response of a patched rod to the kinetics protocol ( Figure 1C ) in the first minutes after establishing the seal ( black traces ) .", "At later times , a previously described rundown of kinetics was observed ( gray traces; see Cangiano et al . , 2012 ) .", "( A2 )", "Loose seal recording showing a scaled version of the rod photovoltage in response to the kinetics protocol .", "The advantage of the loose seal approach is that no kinetics rundown takes place , even in very long recordings ( inset ) .", "( B ) Summary of rod responses to the kinetics protocol in patch ( black circles; data from the first 2 min after sealing ) and loose seal recordings ( white circles ) .", "Dim flash responses were normalized to those of the brighta flash ( bars are SEM ) .", "Rods display a large response to the first dim flash and a progressive recovery after the brighta flash .", "( C ) Responses of three cones to the kinetics protocol , representing the observed spectrum of behaviors .", "( D ) Summary of data from a subset of cones that displayed large dim flash responses .", "The time course of recovery of the dim flash response after the bright flash is comparable to that of rods .", "In panels A1 , A2 , and C , baselines were aligned to each other ( max shift 2 mV ) , and in all records ( except C/top ) , each trace was the average of several sweeps . DOI: http://dx . doi . org/10 . 7554/eLife . 01386 . 004 We observed in cones a progressive increase in dim and bright flash peak response amplitudes and in the plateau .", "The net change in their response to the kinetics protocol was a scaled version of the typical rod response ( Figure 3A ) .", "The rapid time course of this process implied that it began with the recording itself .", "It did not depend on the repeated exposure of the photoreceptor to light , since it occurred also when single deliveries of the kinetics protocol were separated by several minutes of uninterrupted darkness ( Figure 3A ) .", "A response amplitude vs flash strength graph , obtained in two time ranges in the same cone ( Figure 3B ) , highlights how cones acquired light sensitivity at intensities normally covered by rods .", "Note that collecting each full flash response curve of Figure 3B required ∼10 min , a time comparable to the time course of the spontaneous increase; therefore , different flash strengths were unavoidably delivered at different levels of progress of the phenomenon .", "Figure 4 shows , in simplified form , the evolution of dim G flash response amplitude in 50 cones .", "Its rate of increase varied greatly among cones .", "When possible we estimated the dim G flash response amplitude prior to patching , by extrapolating to time zero the values observed in the first minutes of recording ( Figure 4 , short red horizontal bars ) .", "Based on these estimates , it appears that most cones responded to dim G flashes already before patching , although response amplitudes were generally modest when compared to those expressed during the recordings .", "If the dim flash sensitivity of cones is due to rod input , then these observations imply that rod–cone junctional conductance is not hardwired , but it is modifiable in a wide range . 10 . 7554/eLife . 01386 . 005Figure 3 . Cones shift toward a rod-like phenotype during recording .", "( A ) Response of a cone to the kinetics protocol delivered at 1 , 6 , 12 and 17 min after obtaining the seal ( records are not averages; Vdark ≈ −44 mV ) .", "In this experiment , darkness was maintained between recordings .", "The net change between the first and the average of the last two records ( 12 and 17 min ) matches the response of rods ( Figure 2A ) .", "( B ) A different cone in which the kinetics protocol was delivered at close intervals for an extended time .", "Records above compare the average responses of this cone to the kinetics protocol at the beginning of the experiment , with those after >1 hr .", "The graph below shows the selective increase in sensitivity to dimmer flashes that occurred during the recording ( bars are SEM ) .", "This cone also appears in Figure 4 ( four-pointed star ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 01386 . 00510 . 7554/eLife . 01386 . 006Figure 4 . The dim flash sensitivity of cones increases during recording . Time course of the peak amplitude of the response to the dim G flash in 50 cones .", "Each curve corresponds to a different cell and is a qualitative fit to the raw data points .", "In a subset of cones , the unperturbed amplitude of the dim G flash response could be extrapolated with a reasonable degree of confidence ( red horizontal segments at time zero ) .", "Some cones were recorded with an EGTA zero Ca2+ perforated patch solution in the pipette ( blue lines; see ‘Results’ ) .", "Dashed lines represent gaps in the data resulting from the delivery of other protocols .", "Some cones also appear in other figures ( stars and triangles ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 01386 . 006 We attempted to isolate the pure cone component by saturating rods with a continuous light background of 6100 photons·µm−2·s−1 at 520 nm ( for 5 min ) based on ex vivo ERG data in mouse ( Heikkinen et al . , 2011 ) and our own rod data obtained with patch clamp and in loose seal .", "Control recordings confirmed that this particular background was , indeed , rod saturating ( Figure 5A1 ) and showed that , upon returning to darkness , rod responses to the kinetics protocol recovered to near control levels after ∼2 min ( n = 3 ) .", "This is presented in Figure 5A2 , where dim G flash , brighta G flash , and slow plateau response amplitudes are plotted normalizing them to their control values before exposure to the background .", "The same background was delivered in cones expressing prominent dim G flash responses .", "In contrast to rods , a sharp response to the brighta G flash persisted during the background , while the slow plateau disappeared as one would expect if it was generated by rods ( Figure 5B1 ) .", "The sharp peak did not represent a residual rod response , since increasing flash strength led to a marked increase in its amplitude ( Figure 5B1 , arrow ) .", "Importantly , when darkness was restored , the dim and bright flash responses recovered to near control levels in ∼2 min ( n = 3 ) , in agreement with what was observed in rods ( cf . Figure 5 B2 with A2 ) .", "This strongly suggests that the peculiar features displayed by cones—a high light sensitivity and slow kinetics—may be entirely explained by rod input fed into the cone pathway through GJs . 10 . 7554/eLife . 01386 . 007Figure 5 . Rods and cones recover from a rod-saturating background at the same rate .", "( A1 )", "Loose seal recording of a rod showing the complete suppression of its response to the kinetics protocol by a saturating light background ( 520 nm , 6100 photons·µm−2·s−1 for 5 min; dim G flashes were not delivered during the background ) , and its recovery upon returning to darkness .", "( A2 )", "Graphs summarizing the effect of the light background on this and two additional rods .", "The response amplitudes of the first dim G flash , the brighta flash , and the plateau ( 0 . 4–0 . 6 s post bright flash ) were normalized to their control values ( bars show the SEM , while their horizontal extent shows the time range of the underlying flashes ) .", "( B1 )", "Recording of a cone of high light sensitivity and slow kinetics showing the effect of the same rod-saturating background .", "In contrast to rods , a fast response component persisted in the cone during the background ( arrow; gray trace shows the effect of increasing flash strength by a factor of 3 . 7 ) .", "( B2 )", "Graphs summarizing the effect of the light background on this and two additional cones .", "The time course of recovery in cones matched that of rods .", "All records in A1 and B1 are averages obtained in the specified time ranges . DOI: http://dx . doi . org/10 . 7554/eLife . 01386 . 007 Heikkinen et al . ( 2011 ) interpreted the effects of non rod-saturating light backgrounds on the cone-driven ERG flash response as evidence that light uncouples rod–cone GJs , with a time course of minutes .", "Given our observation that the rod component in cones recovers from a prolonged rod-saturating background with a similar time course as the rods themselves , we could not confirm the conclusions of that study .", "It is possible , however , that the process leading to a progressive increase in rod–cone coupling during patch recordings might interfere with the relevant signaling pathways .", "In the presence of rod input , one should observe a shift in the spectral preference of S- and S/M-cones toward that of rods .", "The spectral protocol ( Figure 1D ) enabled us to rapidly determine the apparent spectral preference of cones for dim and brightb flashes , as well as their intrinsic spectral preference by removing any rod contribution with a rod-saturating pre-flash .", "We delivered the spectral protocol in rods and cones and quantified spectral preference by the ratio of the peak response amplitudes to G over UV light ( for the same flash strength in photons·µm−2 ) .", "As expected , rods were more sensitive to dim G than to dim UV flashes ( Figure 6A1 , A2 , arrows; same rod ) , with an estimated dim G/UV ratio of 2 . 7 ( SEM 0 . 2; n = 8 ) ( all rhodopsins have a prominent secondary absorption peak in the ultraviolet [Rodieck , 1973] , including mouse rhodopsin [Lyubarsky et al . , 1999] ) .", "For both G and UV brightb flashes , rods expressed saturating responses ( ratio of 1; Figure 6A2 , filled box ) , while they did not respond to brightb flashes delivered after a rod-saturating pre-flash ( Figure 6A1 , empty box ) .", "Figure 6A1 , A2 shows a cone exposed to the same spectral protocol delivered in rods .", "Surprisingly , while for dim flashes we observed a larger response to G than to UV light ( Figure 6A1 , A2 , arrowheads; same cone ) , the response to brightb flashes delivered after the rod-saturating pre-flash showed that this cone had an intrinsic UV preference ( Figure 6A1 , empty circle ) and was therefore a mixed S/M cone .", "We found that all cones stimulated with dim flashes displayed larger responses to G than to UV light ( n = 20 ) .", "This behavior was independent of their intrinsic spectral preference in a wide range of G/UV ratios ( Figure 6B ) ( corresponding to a wide range of M- vs S-opsin expression levels ) , again supporting our hypothesis that dim flash responses in cones are driven by rods . 10 . 7554/eLife . 01386 . 008Figure 6 . Irrespective of their dominant cone opsin , for dim flashes , cones prefer green light .", "( A1 and A2 )", "Responses of a rod and a cone to the spectral protocol ( Figure 1D ) .", "As expected , the rod was more sensitive to dim G than dim UV flashes ( arrows ) , it saturated with brightb G and brightb UV flashes ( filled box ) and did not respond after a rod-saturating pre-flash ( empty box ) .", "Similar to the rod , the cone was more sensitive to dim G flashes ( arrowheads ) .", "However , its responses after a rod-saturating pre-flash unmasked an intrinsic preference for UV light ( empty circle ) .", "( B ) Cones of widely varying intrinsic spectral preference ( corresponding to widely varying M- vs S-opsin expression levels ) display a rod-like preference for green light when tested with dim flashes .", "Spectral preference was quantified as the ratio of G and UV flash response amplitudes .", "Line and shaded areas show the mean dim flash preference ± 1 SD in rods ( four loose seal and four patch recordings ) .", "Triangular shades show the expected location in the graph of uncoupled cones .", "( C ) Both M- and S-dominant cones couple to rods , as shown by a plot of the maximum dim G flash response amplitude observed in each cone vs its intrinsic spectral preference . DOI: http://dx . doi . org/10 . 7554/eLife . 01386 . 008 An important question is whether the cones’ maximal strength of coupling to rods varies with their dominant opsin type .", "We examined this by plotting the maximum dim G flash response amplitude observed in each cone vs its intrinsic spectral preference ( Figure 6C ) .", "The graph shows that both M- and S-dominant cones could possess or acquire dim flash sensitivity during recordings .", "Performing a finer analysis under our experimental conditions would be complicated by the fact that the maximum level of coupling displayed by cones is strongly dependent upon recording duration , which varied widely .", "We also recorded from three putatively pure S-cones ( i . e . blue cones; Figure 6C , leftmost circles ) , an important sample since the immunohistochemically derived frequency of these neurons is very low ( Haverkamp et al . , 2005 ) .", "In one of these cones ( Figures 4 , 6C , 7C , black triangle ) , the seal was maintained for >85 min , but no coupling was detected .", "The remaining two cones were recorded for only 4 and 7 min and thus", "( i ) little averaging of their responses to the spectral protocol could be performed to improve signal over noise , and", "( ii ) the possible development of coupling could not be monitored .", "With these limitations in mind , in one cone , no coupling could be detected , while in the other , a dim G flash response of ∼0 . 3 mV may have been present although superimposed on a noisy baseline . 10 . 7554/eLife . 01386 . 009Figure 7 . Examples of cones with different S- vs M-opsin expression levels .", "( A ) Initially uncoupled M-dominant cone , which then develops strong coupling ( also shown in Figure 4 and Figure 6C , labeled by a black five-pointed star ) .", "( B ) Weakly coupled S/M cone with approximately equal sensitivity to G and UV light ( white five-pointed star in previous figures ) .", "( C ) Uncoupled , presumably pure S-cone ( black triangle in previous figures ) .", "All records are averages obtained in the specified time ranges .", "Bars are SEM . DOI: http://dx . doi . org/10 . 7554/eLife . 01386 . 009 Figure 7 also serves the purpose of illustrating in greater detail three cones shown in previous plots ( Figure 4 and Figure 6C , five-pointed stars and black triangle ) exhibiting different relative opsin expression levels: an M-dominant cone initially uncoupled , which then develops strong coupling ( panel A ) ; a weakly coupled S/M cone , with approximately equal sensitivity to G and UV light ( panel B ) ; an uncoupled presumably pure S-cone ( panel C ) .", "If , as we hypothesized , the cone dim flash response originates in coupled rods , while the bright flash response originates in both photoreceptors , they should have a different apparent reversal potential ( assuming the actual reversal potential EOS in rods and cones to be the same; EOS ≈ 0 mV; Luo et al . , 2008 ) .", "The reason is that the cone membrane potential at the pipette tip ( VCone ) would differ from the membrane potential of its coupled rods ( VRod ) when a current flows through the finite resistance of the GJs ( iGJ ) , a condition occurring when the cone is held depolarized via the pipette ( Figure 8A , equivalent circuit ) .", "In essence , any coupled rods would represent electrotonically distant compartments from the patch pipette .", "The prediction is that there should be a range of depolarized cone membrane potentials ( beyond EOS ) within which VRod ( DARK ) < EOS and dim and bright flash responses display opposite polarities ( Figure 8A , expected responses: bottom row ) ( note that , since homotypic Cx36 GJs decrease their conductance in the presence of large voltage differentials ( Bukauskas , 2012 ) , it is entirely possible that all one would observe when VCone > EOS is an uncoupling , that is disappearance of dim flash responses ) .", "If , on the other hand , dim and bright flash responses originated entirely in cones , their polarity should always be concordant . 10 . 7554/eLife . 01386 . 010Figure 8 . Dim and bright flash responses in cones originate from separate electrotonic compartments .", "( A left )", "Equivalent circuit of a recorded cone ( orange ) coupled to neighboring rods ( blue ) via GJs ( OS , outer segment; EOS , reversal potential of the rod/cone light sensitive conductance; GJ , gap junction; iGJ , junctional current; ipip , pipette current; VRod and VCone , rod/cone membrane potential ) .", "( A right )", "When the cone is depolarized , either in current clamp ( by constant current injection ) such that VCone ( DARK ) > EOS , or in voltage clamp such that VCone ( HOLD ) > EOS , a junctional current iGJ will flow into the rods and depolarize them beyond , at , or below EOS .", "Each of these three possible outcomes is expected to lead to the indicated different combinations of response polarities when delivering dim and bright flashes .", "( B ) A reduced version of the spectral protocol ( sequences 1–2 in Figure 1D ) was delivered with a cone recorded in current clamp in control conditions ( VCone ( DARK ) = −40 mV ) or during depolarization by constant current injection beyond the reversal potential of its light-sensitive conductance ( VCone ( DARK ) = +66 mV ) .", "While brightb flash responses reversed polarity , dim flash responses became smaller but did not reverse .", "This is not compatible with an origin of the dim and bright flash responses in the same electrotonic compartment , and matches one of the predicted outcomes for coupled cones ( panel A , VRod ( DARK ) < EOS ) .", "The moderate shift toward G in the spectral preference displayed by this cone ( second brightb flash ) could be explained by cone–cone coupling and/or by initial recovery from saturation of its coupled rods ( see ‘Discussion’ ) .", "( C ) The same experiment as in panel B but performed in voltage clamp in a different cone ( VCone ( HOLD ) = −40 mV and +60 mV ) .", "Dim flash responses could not be detected above noise when the cone was depolarized , despite the presence of large inverted brightb flash responses .", "This matches a different predicted outcome for coupled cones ( panel A , VRod ( DARK ) = EOS ) .", "The slight shift toward G in the spectral preference displayed by this cone ( second brightb flash ) is likely explained by a slow ‘bump’ in the plateau displayed after brightb flashes , present only at −40 mV ( not shown , but observed in sequence three of the spectral protocol ) .", "The outcome predicted for the case of VRod ( DARK ) > EOS in panel A was never observed .", "Records are averages of 3–4 sweeps . DOI: http://dx . doi . org/10 . 7554/eLife . 01386 . 010 A full analysis of the voltage-dependence of the photoresponses could not be carried out due to the instability of recording at positive membrane potentials and the need of acquiring several responses for averaging .", "We chose instead a more limited approach of delivering the spectral protocol in cones sensitive to dim flashes , while they are depolarized well beyond the reversal potential of their photoresponse .", "This was done both in current clamp ( CC ) and voltage clamp ( VC ) .", "In CC , we imposed VCone ( DARK ) > EOS by constant current injection .", "In all five cones tested , we observed that the brightb flash responses reversed in polarity ( i . e . , depolarizing ) and actually became larger than in control conditions .", "In contrast , four of these cones had their dim flash responses reduced in amplitude to the point of being no longer distinguishable from noise , while the remaining one retained a small hyperpolarizing response ( Figure 8B ) .", "This result suggests that in these conditions , VRod ( DARK ) was slightly below or anyway near EOS ( Figure 8A , middle and bottom rows ) .", "In VC , the photocurrent responses of a cone to the spectral protocol were recorded at holding potentials ( VCone ( HOLD ) ) of −40 mV and +60 mV .", "At the depolarized potential , bright flash photoresponses were reversed and of much larger amplitude , while the dim flash responses became undetectable ( Figure 8C ) .", "This suggests that also in this case ( VC recording ) VRod ( DARK ) was close to EOS ( Figure 8A , middle row ) .", "Again , it is possible that the absence of dim flash responses was partly due to a voltage-dependent reduction of the junctional conductance .", "As a further confirmation of the results obtained thus far , we tested the specific GJ blocker meclofenamic acid ( MFA ) , previously known to be effective at GJs containing Cx36 in AII amacrine cells ( Pan et al . , 2007; Veruki and Hartveit , 2009 ) .", "In three control rod recordings , 100 µM MFA was superfused with essentially no effect , except for a marginal reduction in light sensitivity that we attribute to the normal rundown observed during long rod recordings .", "In four cones tested , on the other hand , superfusion with 100 µM MFA markedly reduced both dim flash responses and the slow plateau after bright flashes .", "An example is shown in Figure 9A1 , where a prominent level of coupling was reached about 30 min from seal formation , after which MFA was delivered .", "The slow pharmacodynamics of the blocker observed in our recordings ( Figure 9A1 , lower plot ) confirmed a previous report in AII amacrine cells ( Veruki and Hartveit , 2009 ) .", "The significant rundown of the cone response kinetics , particularly evident toward the end of the experiment , was first described in Cangiano et al . ( 2012 ) and is unrelated to the effect of the blocker , since fast cone responses were observed when the seal was made with MFA already present in the bath ( n = 2 ) .", "Comparing graphs of response amplitude vs flash strength before and during perfusion with MFA in the same cone highlights how junctional coupling transforms the sensitivity profile of the cone making it responsive to dim flashes , thereby widening its dynamic range ( Figure 9A2 ) .", "Although control data for this graph were obtained before the cone reached its full coupling potential , a remarkable effect of the blocker on dim flash sensitivity was already visible . 10 . 7554/eLife . 01386 . 011Figure 9 . Blocking gap junctions reverts cones to their intrinsic phenotype .", "( A1 )", "Response of a cone to the spectral protocol during its spontaneous shift toward a rod phenotype ( top and middle records ) and subsequent superfusion with the GJ blocker meclofenamic acid ( MFA , 100 µM; bottom records ) .", "MFA abolished both dim flash responses and bright flash plateaus .", "Records are averages .", "( A2 )", "Response amplitude vs flash strength for the same cone as in A1 before and during perfusion with MFA .", "Note the selective reduction in dim flash sensitivity .", "Bars are SEM . DOI: http://dx . doi . org/10 . 7554/eLife . 01386 . 01110 . 7554/eLife . 01386 . 012Figure 9—figure supplement 1 . Uncoupled cones display light sensitivities comparable to those predicted from literature ( compare with Figure 1A , B , simulated curves ) .", "( A–C )", "Response profiles of three cones to G and UV flashes , recorded during superfusion with 100 µM MFA .", "The cone in panel C is also shown in Figure 9 ( six-pointed star ) .", "( D–F )", "Response profiles to G and UV flashes in three naturally uncoupled cones .", "Bars are SEM . DOI: http://dx . doi . org/10 . 7554/eLife . 01386 . 01210 . 7554/eLife . 01386 . 013Figure 9—figure supplement 2 . The spontaneous increase in coupling and MFA had a limited impact on our estimates of intrinsic cone spectral preferences . Graph of the ratio of G over UV flash response amplitudes ( brightb flashes delivered after a rod saturating pre-flash; spectral protocol ) vs dim G flash response amplitude .", "Data points from the same cone are connected by lines , with the arrow showing time progression .", "For two cones , complete data were available spanning both coupling increase and MFA superfusion ( 1–4 ) .", "Also shown are two cones sealed upon with MFA already in the bath ( isolated orange circles ) and a completely uncoupled cone in normal solution ( isolated black circle ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 01386 . 013 Uncoupled cones , including those recorded during superfusion with MFA ( Figure 9—figure supplement 1 ) , displayed markedly decreased flash sensitivities compared to those found in Cangiano et al . ( 2012 ) , and approached those predicted using published estimates from functionally rodless retinas ( Figure 1A , B , simulated curves on the right; ‘Materials and methods’ ) .", "As the experiments shown so far were performed near room temperature , we verified the occurrence of rod–cone coupling also near body temperature .", "The response to the kinetics protocol of a rod recorded in loose seal ( Figure 10A ) showed , as expected , faster kinetics near body temperature ( cf . Figure 2A2 ) .", "Nevertheless , the rod was still unable to respond to the dim G flash delivered at the earliest delay following the brighta G flash .", "In all five cones recorded near body temperature , we observed dim G flash responses ( 0 . 28 , 0 . 32 , 0 . 43 , 1 . 47 , and 1 . 70 mV ) and plateaus after brighta G flashes ( the response of 1 . 70 mV is shown in Figure 10B ) .", "In one of these cones , we tested the effect of the GJ blocker MFA ( 100 µM ) : both dim flash responses and plateaus following the brighta flash were abolished , leaving behind a fast response to the brighta flash ( Figure 10B ) .", "The flash response profile of this cone , acquired during superfusion with MFA ( Figure 10B ) , revealed that it was significantly more sensitive to G light than the dorsalmost ( i . e . , the ‘greenest’ ) simulated cones in Figure 1A .", "This was not necessarily unexpected , given that the simulated profiles:", "( i ) are based on models which predict the average ratio of S- to M-opsin expression over a large cone population , and", "( ii ) assume that all cones express the same amount of opsin ( ‘Materials and methods’ ) . 10 . 7554/eLife . 01386 . 014Figure 10 . Rod–cone coupling is also expressed near body temperature .", "( A ) Loose seal recordings at 36°C in a rod , showing its response to the kinetics protocol .", "As expected , rod recovery from the brighta flashes was faster compared to near room temperature .", "( B ) Cone recorded at 36°C displaying a rod-like phenotype in response to the kinetics protocol ( upper records ) .", "MFA abolished dim flash responses and slow plateaus after the bright flash ( lower records ) .", "The graph shows the G flash response profile of the cone during superfusion of MFA . DOI: http://dx . doi . org/10 . 7554/eLife . 01386 . 014 The progressive increase in rod–cone coupling observed in our recordings was not evoked by slicing of the retina but rather by some interaction between the recording pipette and the cone and/or its nearby rods: cones were patched not earlier than 1 hr after slicing and in some cases , after several hours , yet in most cones , initial coupling upon sealing was weak , developing rapidly thereafter ( Figure 4 ) .", "To shed light on the nature of this interaction , we examined whether the mechanism underlying the coupling process in photoreceptors is related to phenomenologically similar ones found in other systems expressing Cx36 ( Zoidl et al . , 2002; Del Corsso et al . , 2012; Veruki et al . , 2008 ) .", "Two possible drivers of the spontaneous coupling increase emerging from these studies were", "( i ) dialysis of the cell by the pipette , and", "( ii ) an increase in intracellular calcium , both occurring as a consequence of whole cell recordings ( ‘Discussion’ ) .", "However , the fact that we observed this phenomenon in perforated patch clamp recordings , a technique known to cause minimal disturbance to the intracellular environment , makes it unlikely that in mouse cones , it is caused by pipette-cell dialysis ( we could exclude an unintended rupture of the patch membrane , since the amphotericin-B present in our pipette solution would have perforated the cell membrane , depolarizing it close to 0 mV and shunting the photovoltages—effects that we indeed observed when going whole cell at the end of recordings to stain the neurons with Lucifer Yellow , LY ) .", "Nonetheless , we examined the remote possibility that amphotericin-B ( nominally 400 µM ) formed Ca2+-permeable pores ( Romero et al . , 2009 ) and that the diffusion of calcium ions from the pipette into the cone led to an opening of GJs ( Ca2+ traces are normally present in the high purity water used for intracellular solutions ) .", "In four of five cones recorded with 1 mM EGTA in the pipette ( EGTA zero Ca2+ perforated patch solution ) , we observed the same progressive increase in coupling ( Figure 4 , blue lines ) .", "These results effectively rule out an involvement of pipette Ca2+ but leave open the possibility that an increase in free [Ca2+]i in the cone , promoted by the pipette , drives the coupling process .", "We thus performed whole cell recordings ( i . e . , without amphotericin-B ) with the goal of dialyzing cones with a low Ca2+ buffered solution .", "Seals were made on cone pedicles ( as confirmed by LY staining , Figure 11E ) to ensure a rapid and effective ‘Ca2+ clamp’ on the cone side of the junctional contacts ( located on short telodendria that protrude from the pedicles; Tsukamoto et al . , 2001; O’Brien et al . , 2012 ) .", "This represented an important step , given what is known about the active compartmentalization of free [Ca2+]i in cones ( Wei et al . , 2012 ) .", "We recorded from three cones with an EGTA low Ca2+ whole cell solution ( 50 nM free [Ca2+]i; see ‘Materials and methods’ ) , of which only one had weak coupling to rods upon breaking the patch .", "In two of the three cones , rod coupling increased rapidly in the first minutes of recording ( Figure 11A ) , while in the third cone , coupling did not develop despite a stable recording for more than 1 hr ( not shown ) .", "Further , we recorded from three cones with a BAPTA low Ca2+ whole cell solution ( 50 nM free [Ca2+]i ) to rule out an involvement of fast and localized calcium transients .", "While all three cones were initially uncoupled , they rapidly increased their coupling to rods up to moderate levels ( Figure 11B ) .", "Since in all these experiments free calcium in the pedicle was buffered at very low levels compared to those normally present in darkness ( ∼2 µM spatially averaged: Szikra and Krizaj , 2006 ) , we conclude that high calcium ( on the side of the cone ) is not required for the expression of the progressive increase in coupling .", "High calcium , however , could favor coupling .", "To test this , we recorded from six cones with an EGTA high Ca2+ whole cell solution ( approx . 5 µM free [Ca2+]i ) , all uncoupled or weakly coupled upon breaking the patch .", "Among five cones that we could monitor for sufficient time , one developed strong coupling over several minutes ( Figure 11C ) , while the remaining four displayed weak coupling , which , however , did not appear to increase over time .", "Thus , no obvious favoring effect on coupling of high calcium emerged from this group of cones . 10 . 7554/eLife . 01386 . 015Figure 11 . The spontaneous increase in coupling does not require Ca2+ changes in the cone .", "( A–C )", "Three cones recorded in whole cell patch clamp using either low Ca2+ intracellular solutions ( 50 nM free [Ca2+] ) buffered with EGTA ( panel A ) or BAPTA ( panel B ) , or a high Ca2+ intracellular solution ( approx . 5 µM free [Ca2+] ) buffered with EGTA .", "All three cones expressed the same time-dependent increase in coupling to rods observed in perforated patch recordings: increased dim flash response and bright flash plateau amplitudes .", "Note that , in contrast to the perforated patch , the component of the light response originating in the cones themselves ran down rapidly ( second brightb flashes , arrows ) .", "Records are averages of 1–3 sweeps .", "( D ) Time course of rod ( continuous lines ) and cone ( dashed lines ) response components in the experiments shown in panels A–C .", "( E ) Pedicles ( cone synaptic terminals ) were targeted in all of these whole cell recordings to ensure a rapid and effective calcium clamp at the GJs , which are located on the adjacent telodendria .", "The image shows a Lucifer Yellow stain of the cone in panel B with the pipette sealed on the pedicle ( final image obtained by blending two photographs acquired on slightly different focal planes; pedicle and cell body appear larger than their actual size due to an intentional overexposure during acquisition , implemented to highlight the dim outer segment ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 01386 . 015 Figure 11D summarizes the time course of coupling for the cones shown in panels A–C , by plotting the dim G flash response amplitude .", "Also shown is the time course of the amplitude of brightb G flash responses delivered after the pre-flash .", "Note that the intrinsic responses of these cones ran down rapidly ( cf . lower records in panels A–C , arrows ) .", "This run down occurred in all low [Ca2+]i and in two high [Ca2+]i recordings .", "Interestingly , in the cones resistant to rundown , the distal portion of the neuron ( cell body and outer segment ) was only weakly stained by LY , as if a barrier to diffusion was present in the axon .", "Taken together , these experiments appear to exclude any major contribution in the coupling increase of Ca2+ , at least on the cone side of the GJs ." ], [ "Except for a small number of pure S-cones ( i . e . blue cones ) , most mouse cones reportedly express both S and M opsins , with their expression ratio varying along the dorsoventral axis of the retina ( Applebury et al . , 2000; Haverkamp et al . , 2005; Daniele et al . , 2011; Wang et al . , 2011 ) .", "Moreover , any pure M-cones would respond to UV light since M-opsin has a prominent secondary absorption peak in the near UV region ( the β-band; Govardovskii et al . , 2000 ) .", "It was thus not surprising that almost all of our recorded cones were intrinsically sensitive to both G and UV light ( Figure 6 and Figure 7 , cf . with Figure 1A , B ) .", "Our dissection and recording techniques did not allow us to identify their dorsoventral position in the retina , so that we could not directly examine possible correlations between their location and the level of maximal coupling .", "We could however conclude that both S-dominant cones ( known to be mid and ventrally located ) and M-dominant cones ( dorsally located ) are able to couple strongly to rods ( Figure 6C ) .", "As for pure S-cones , our small recorded sample is in no way sufficient to conclude that they have a reduced propensity to couple to rods , since we found uncoupled cones also within the S/M group .", "Interestingly , there is some evidence in macaque that blue cones form fewer junctional contacts with rods compared to other cone types ( O’Brien et al . , 2012 ) .", "It is important to note that the G/UV ratios obtained in our sample of cones after rod-saturating pre-flashes ( Figure 6C ) do not provide a completely unbiased representation of the relative sensitivity to G over UV light within the native cone population .", "First , while we recorded from both central and peripheral retina , it is unlikely that we obtained a uniform sampling at all eccentricities; this , combined with the dorsoventral gradients in S- and M-opsin expression ( see references above ) , will distort the observed distribution .", "Second , any differences in size or accessibility among cones would have affected their odds of being recorded ( see Cangiano et al . ( 2012 ) for rod vs cone recording bias ) .", "Third , compared to the classical approach of estimating cone spectral preference by adjusting the strengths of flashes of different wavelengths to match their response amplitudes , our use of the ratio of the peak response amplitude to moderately bright G and UV flashes of equal strength ( our G/UV ratios ) shifts values somewhat toward unity as cone saturation is approached ( we chose this approach because it was quick and avoided exposing rods to very bright light ) .", "Fourth , there could be a contribution of other cones coupled to the recorded cone , which would shift the measured G/UV ratio in one or the other direction depending on their relative expression of S- and M-opsins .", "Finally , we cannot exclude that in some experiments , coupled rods may have retained a small response to the bright flash following the saturating pre-flash , again shifting the G/UV ratio .", "These last two factors could explain the shifts in G/UV ratio observed in some cones during spontaneous coupling increase and superfusion with MFA ( Figure 9—figure supplement 2 ) , as well as in the ‘reversal’ experiment of Figure 8B .", "GJs are often documented only anatomically , leaving open the question of their functional impact on neurons .", "Moreover , since they can be actively regulated , knowledge of their full coupling potential is essential .", "Our finding that the amplitude of rod signals in cones may attain a significant fraction of their source amplitude is a measure of the importance that rod–cone GJs can have in visual processing by the cone pathway .", "Is such a strong level of coupling compatible with what is known about rod–cone GJs ?", "Each cone in mouse forms junctional contacts with an average of 32 rods ( Tsukamoto et al . , 2001 ) .", "To our knowledge , the only measurement in mammals of the transjunctional conductance was made in rod–cone pairs of the cone-dominated ground squirrel retina ( legend of supplementary Figure 6 in Li et al . , 2010 ) .", "Assuming that their average value of 121 pS for coupled pairs ( corresponding to ∼20 open homotypic Cx36 channels; Moreno et al . , 2005 ) was also representative of the mouse , multiplying it by a rod–cone convergence of 32 gives a summed junctional conductance of ∼3900 pS .", "This would imply that a 4 mV hyperpolarization in rods relative to a maximally coupled cone would be sufficient to draw from it an overall junctional current of ∼16 pA—a very large value as it is comparable to estimates of the circulating current in mouse cones in darkness ( Nikonov et al . , 2006 ) .", "Therefore , our data are compatible with the limited available evidence .", "Moreover , our results bear direct relevance to other mammals , including primates: in the area centralis of the cat retina , Smith et al . ( 1986 ) estimated a convergence of ∼48 rods on each cone , while in the peripheral retina of macaque , ∼25 rods converge on each cone ( O’Brien et al . , 2012 ) , a remarkably similar value to that in the mouse .", "We did not observe cone signals entering rods through GJs , since rods did not respond appreciably to bright cone-stimulating flashes delivered during a rod-saturating background , or following rod-saturating pre-flashes .", "Two possible explanations for this are:", "( i ) rod–cone divergence in mouse is small ( ∼1; Tsukamoto et al . , 2001 ) ;", "( ii ) coupling might not be promoted when patching on rods .", "A previous study in macaque reported a twofold speed-up of rod signals as they flow into cones through GJs ( Hornstein et al . , 2005 ) .", "We confirmed this in mouse , observing however a much less dramatic effect: in patched cones , the time to peak ( TTP ) of dim flash responses was 197 ms ( SD 35 , n = 24 ) , significantly shorter ( p<0 . 0001 ) than the 258 ms ( SD 25 , n = 23 ) of rods recorded in loose seal ( rod and cone data were from both the kinetics protocol and the spectral protocol at 24°C ) ( note that the TTP of dim flash responses in patched rods was 288 ms ( SD 51 , n = 24 ) , moderately but significantly longer ( p=0 . 02 ) than that of rods recorded in loose seal; since rods recorded in loose seal did not display a kinetics rundown , their TTP values were assumed to be representative of the unperturbed state of rods in our preparation ) .", "The considerable discrepancy between the magnitude of the speedup in macaque and mouse could be explained if macaque rods underwent a rundown of kinetics during recordings ( as it occurs in mouse ) , without this being noticed .", "As mentioned in the ‘Results’ , a progressive increase in junctional coupling as a specific consequence of patch recording was observed in other studies targeting Cx36-expressing neurons , including heterologous expression systems ( Zoidl et al . , 2002; Del Corsso et al . , 2012 ) and rat retinal amacrine AII cells ( Veruki et al . , 2008 ) .", "Veruki et al . , recording in whole cell mode , found that high resistance ( i . e . , fine-tipped ) electrodes prevent the coupling increase , suggesting a dialysis process .", "Del Corsso et al . ( 2012 ) surmised that an increase in intracellular Ca2+ triggers the coupling increase during whole cell recordings in neuroblastoma cells in vitro .", "Moreover , they reported that the coupling increase did not occur when recording in perforated patch clamp mode with amphotericin-B , again implicating pipette-cell dialysis .", "However , here we reach a different conclusion from that of the studies cited above .", "First , in cones , this phenomenon is not dependent on diffusion of Ca2+ between the cell and the pipette , as the spontaneous increase in coupling was expressed in perforated patch recordings , even in combination with EGTA in the pipette solution .", "This , therefore , excludes the possibility of a Ca2+ entry through hypothetical Ca2+-permeable pores formed by amphotericin-B in the patch membrane ( Romero et al . , 2009; see also ‘Results’ ) .", "Second , our recordings in whole cell patch clamp with buffered pipette Ca2+ levels , made in close proximity to the GJs , strongly suggest that changes in free [Ca2+]i in cones are not responsible for the expression of this phenomenon .", "Moreover , since the progressive increase in coupling was observed both when preserving the intracellular environment of the cone ( perforated patch recordings ) and when intentionally dialyzing it ( whole cell recordings ) , a general tentative conclusion may be advanced: diffusible messenger molecules in cones are not necessarily involved in the process .", "Thus , any diffusible messengers ( including Ca2+ ) could be located in the rods surrounding the recorded cone and act on the connexon hemichannels on the rod side .", "Evidence for a primary trigger mechanism can be found in a previous study on macaque ( Hornstein et al . , 2005 ) , which reported that Neurobiotin injected in cones at the end of perforated patch recordings diffused preferentially to rods located under the recording pipette , leading them to suggest that ‘the electrode might alter the coupling efficiency of the rod–cone junctions by mechanical disturbance’ .", "This observation implies that their cones also expressed a progressive increase in coupling , a process whose electrophysiological counterpart must have gone unnoticed .", "In this scenario , and taking into account the arguments given above , coupling would be promoted by stretch/deformation of the rods adjacent to the recorded cone .", "This could explain the large variability in the rate of increase and maximum level of coupling that we observed in our recordings ( Figure 4 ) , since the angle of entry and depth of the pipette in the tissue varies from experiment to experiment , likely changing its mechanical interaction with neighboring rods .", "With regard to the downstream pathways , Del Corsso et al . ( 2012 ) found in their heterologous expression system a Ca2+-mediated enhancement in the activity of calmodulin-dependent protein kinase II ( CaMKII ) , which would lead to phosphorylation of Cx36 and increased channel conductance .", "Interestingly , CaMKII was recently found to participate in the physiological regulation of Cx36 in AII amacrines ( Kothmann et al . , 2012 ) , raising the possibility that this kinase may play a role also in photoreceptors , on the rod side of rod–cone GJs ( see above ) .", "At the start of the recordings , we found most cones modestly coupled to rods relative to their full coupling potential , although we observed strong initial coupling in a limited sample ( Figure 4 ) .", "A key question that needs to be addressed is when the retina exploits the strong coupling potential detected in this study .", "Candidate regulators of rod–cone GJs in mammals are ( references in the ‘Introduction’ ) :", "( i ) the circadian synthesis and release of the endogenous retinal neuromodulators , melatonin , dopamine , and adenosine , whereby coupling would be stronger at night when melatonin levels are high; most mouse strains , including the C57BL/6 used here , have deficits in melatonin synthesis ( Roseboom et al . , 1998 ) and are thus inadequate to investigate circadian processes; it is therefore possible that the low level of coupling that we estimated for our unperturbed cones may be related to this deficit and that full blown coupling , of the strength emerging during our recordings , is recruited in melatonin-competent mouse strains at night;", "( ii ) acute light exposure , acting on the same neuromodulators and possibly also locally in the photoreceptors , which would inhibit coupling .", "Unfortunately , unresolved discrepancies persist in the literature , a major one being the lack of effect of light and dopamine on rod–cone coupling studied with patch clamp in macaque ( Schneeweis and Schnapf , 1999 ) .", "Similar discrepancies ( Hartveit and Veruki , 2012 ) exist for the GJs between AII amacrines , also containing Cx36 and thought of being modulated in a circadian and light-dependent fashion .", "While a common explanation for these conflicting pieces of evidence may eventually be found , our demonstration that rod–cone coupling can be upregulated so as to have a major impact on cones provides an important framework in which to place circadian/light-dependent modulation of these GJs in mouse .", "In fact ,", "( i ) let’s suppose that the level attained in our experiments by the fully developed coupling process had been quite smaller than what we actually observed; then this would have raised serious doubts about the physiological importance of circadian/light-dependent modulation of rod–cone coupling for mouse visual processing;", "( ii ) given however our demonstrated high level of coupling , should a future direct examination of the role of circadian rhythmicity find that only a small fraction of this coupling potential is utilized , one might then well suspect that yet unidentified physiological factors play a greater role in recruiting rod–cone GJs , or , alternatively , that these may have an important part in response to stress and injury .", "One should recollect that GJs play a dual role , for example , conduits of electrical signals and of intracellular molecules .", "Cx36 , in particular , is critically involved in neuronal responses to injury and disease ( Belousov and Fontes , 2013 ) .", "Currently , only a few studies have investigated this in the retina , with both positive ( Striedinger et al . , 2005; Paschon et al . , 2012 ) and negative results ( Kranz et al . , 2013 ) depending on the specific models tested .", "The existence of a large degree of GJ-mediated anatomical convergence from rods to cones is not peculiar of the mouse , since it has been found in other rod-dominated mammalian retinas including the peripheral retina of macaque .", "It follows that our findings on the functional impact of rod input in mouse cones may have broad relevance , supporting the possibility that rod–cone coupling plays a significant role in vision and/or in biochemical signaling between photoreceptors ." ], [ "Full field stimuli of unpolarized light were delivered by a green LED ( peak emission at 520 nm; OD520; Optodiode Corp . , Newbury Park CA ) or an ultraviolet LED ( peak emission at 365 nm; APG2C1-365-S; Roithner LaserTechnik , Vienna Austria ) mounted beside the objective turret .", "LEDs were driven by current sources commanded through the analog outputs of a Digidata 1320A ( Axon Instruments , Foster City , CA ) .", "The power density reaching the recording chamber vs LED drive was measured separately with a calibrated low power detector ( 1815-C/818-UV; Newport , Irvine , CA ) positioned at the recording chamber .", "Flash duration was in the range of 1–10 ms . Unless specified in the protocol , consecutive bright flashes were delivered at intervals of 12 s or more between each other .", "The photon flux density reaching the photoreceptors was derived from the measured power density and was likely to be overestimated to varying degrees across recorded cells due to reflection at the air–water interface and absorption by the surrounding tissue , including retinal pigment epithelium .", "Outer segments could be oriented at a variety of angles with respect to the direction of incident light .", "Rod input in cones was dissected with a kinetics protocol and a spectral protocol .", "These consisted of a mix of dim and bright flashes at 520 nm ( green , G ) or 365 nm ( ultraviolet , UV ) .", "For reference , in mouse , the absorption peaks of rhodopsin , M-opsin , and S-opsin are at 498 nm , 508 nm , and 359 nm , respectively ( Sun et al . , 1997; Yokoyama et al . , 1998 ) .", "Dim flashes ( 16 . 6 photons·µm−2 ) were sufficient to elicit a significant response in rods ( Figure 1A , B , curves on the left; our data , see legend ) but expected to be too weak to directly stimulate cones ( Figure 1A , B , curves on the right; predictions from the literature , see next paragraph ) .", "Bright flashes ( brighta = 1570 , brightb = 3140 ph·µm−2 ) were sufficiently strong to saturate rods for 1–2 s ( Figure 1A , lower graph , our data ) and expected to evoke a measurable response in cones ( UV flashes in all cones , while G flashes in most cones; Figure 1A , B , curves on the right; predictions from the literature , see below ) .", "Most mouse cones express both S and M opsins , with their relative proportions varying along the vertical axis of the retina ( M dominates dorsally , while S dominates in the mid and ventral retina ) ( Applebury et al . , 2000; Haverkamp et al . , 2005; Nikonov et al . , 2006; Daniele et al . , 2011; Wang et al . , 2011 ) .", "In Figure 1A , B , we show the predicted average flash response profiles of cones at different retinal latitudes ( 1–4: mixed S/M cones ) and that of the small but distinct population of pure S cones sparsely distributed throughout the retina .", "To generate these profiles , we: ( 1 ) used hyperbolic saturation functions ( Nikonov et al . , 2006 ) ; ( 2 ) assumed the flash sensitivity of the normalized response of S- and M-cones in Gtα−/− mice , recorded with suction pipette and without a rod-saturating background ( Table 1 in Nikonov et al . , 2006: 0 . 042% photons−1·µm2 , equivalent to a half-saturating flash strength of 2381 ph·µm−2 ) , to be valid for S- and M-cones in wild type mice; moreover , we assumed that all cones express the same total amount of opsin; ( 3 ) the typical fraction of M-opsin expressed by cones as a function of their retinal latitude ( i . e . , the average among cones within each horizontal slice of retina ) was taken as the average of the values predicted by two recently published quantitative models ( Daniele et al . , 2011; Wang et al . , 2011 ) obtained from mice in which rod responses were absent or suppressed; since these models extend to slightly different retinal latitudes ( ±2 . 5 mm and ±2 . 0 mm , respectively ) , we normalized their ranges prior to averaging; the relevant parameters of the cones shown in Figure 1 , given as %M-opsin/position , are 1: 64%/dorsalmost , 2: 24%/dorsal third , 3: 4 . 7%/ventral third , 4: 1 . 2%/ventralmost , S: 0%/ubiquitous; ( 4 ) the absorbance of M-opsin at 365 nm ( peak of the β-band ) was taken as 20% of maximum , while for S opsin , its absorbance at 520 nm was taken to be 4 log-units below maximum ( based on Govardovskii et al . , 2000 ) .", "The kinetics protocol ( Figure 1C ) was designed to detect rod input in cones by exploiting the high sensitivity of rods and their slow recovery after a saturating flash .", "A dim G test flash was delivered both before a brighta G flash and at three increasing delays following it ( 1 , 2 . 5 , 4 s ) .", "Observing in cones:", "( i ) a response to the first dim flash ,", "( ii ) a slow plateau after the bright flash , and", "( iii ) a progressive recovery of the dim flash response , would be evidence for rod coupling .", "The spectral protocol ( Figure 1D ) was designed to determine the impact of rod coupling on the spectral preference of cones and the possible relationship between cone opsin expression and coupling strength .", "The cones’ apparent spectral preference was determined by delivering G and UV flashes of the same strength ( equal number of ph·µm−2; either dim or brightb flashes ) .", "The cones’ intrinsic preference was determined by delivering G and UV brightb flashes after a G rod-saturating pre-flash .", "Data are reported as mean and SD ( standard deviation ) , SEM ( standard error of the mean ) .", "Statistical significance was assessed with the Mann–Whitney–Wilcoxon test .", "In all figures , electrophysiological records were ‘box car’ filtered with a running window of 20 ms ." ] ]

[ "Rod and cone photoreceptors are coupled by gap junctions ( GJs ) , relatively large channels able to mediate both electrical and molecular communication .", "Despite their critical location in our visual system and evidence that they are dynamically gated for dark/light adaptation , the full impact that rod–cone GJs can have on cone function is not known .", "We recorded the photovoltage of mouse cones and found that the initial level of rod input increased spontaneously after obtaining intracellular access .", "This process allowed us to explore the underlying coupling capacity to rods , revealing that fully coupled cones acquire a striking rod-like phenotype .", "Calcium , a candidate mediator of the coupling process , does not appear to be involved on the cone side of the junctional channels .", "Our findings show that the anatomical substrate is adequate for rod–cone coupling to play an important role in vision and , possibly , in biochemical signaling among photoreceptors ." ]

[ "People can see in a range of light levels—from dim moonlight to bright midday sun—because our eyes contain two types of light-sensitive cells: rods and cones .", "Rods are more plentiful than cones , and while they are sensitive at low light levels , rods can only provide grey-scale vision .", "Further , bright light can rapidly ‘dazzle’ the ability of rods to see in near-darkness , and they are slow to recover when this happens .", "In contrast , cones need bright light to function , but allow us to see in colour .", "The signals received by rods and cones are sent through the optic nerve to the brain , where they are interpreted as vision .", "However , ‘gap junctions’ that connect the rods and cones allow for electrical and chemical ‘crosstalk’ between these cells , before the signals then travel along the optic nerve .", "Furthermore , even though it is thought that the connections between rods and cones are regulated in response to light , the body’s daily rhythms and other biochemical signals , their importance for vision is not known .", "Now , Asteriti et al . have taken tissue slices from the retinas at the back of mice eyes , and measured the electrical signals generated when cones are exposed to light .", "This revealed that the rod-cone coupling is strong enough to make the cones responsive to dim light , just like rods .", "Moreover , the cones also recovered slowly after being exposed to flashes of bright light .", "When chemical inhibitors were used to block the gap junctions , the cones stopped behaving like rods and became less sensitive to dim light .", "The findings of Asteriti et al . show that rod-cone coupling is sufficient to play an important role in vision .", "The next challenge is to find out what this role is , and how it might be affected by different physiological conditions , including stress and injury ." ]

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

[ "stem cells and regenerative medicine", "tools and resources", "genetics and genomics" ]

[ [ "Comparative functional genomic studies of humans and other primates have been consistently hindered by a lack of samples ( Gallego Romero et al . , 2012 ) .", "In spite of their clear potential to inform our understanding of both human evolution and disease , practical and ethical concerns surrounding working with non-human primates have constrained the field to using a limited set of cell types collected in a non-invasive or minimally invasive manner , primarily lymphoblastoid cell lines ( LCLs ) and fibroblasts .", "Comparative studies of any other primate tissue have been limited to using post-mortem ( typically frozen ) materials , thereby precluding most experimental manipulation and yielding primarily observational insights ( see , e . g . , Blekhman et al . , 2008; Blekhman et al . 2010; Brawand et al . , 2011 ) .", "An alternative has been to use model organisms in an attempt to recapitulate inter-primate regulatory differences .", "The typical approach involves the introduction of sequences of evolutionary interest into a model system , and then searching for spatial or temporal differences in gene expression that can be ascribed to the introduced sequence ( Enard et al . , 2009; Cotney et al . , 2013 ) .", "This is a difficult and challenging approach and , perhaps as a result , there are still only a handful of well-described examples of human-specific regulatory adaptations in primates ( Prabhakar et al . , 2008; McLean et al . , 2011 ) and even fewer cases where the underlying regulatory mechanisms have been resolved ( Rockman et al . , 2005; Pollard et al . , 2006 ) .", "While these studies are useful and often informative , they also entail assumptions of functional conservation between the model system and the species of interest that may not necessarily be true ( Gallego Romero et al . , 2012 ) .", "Induced pluripotent stem cells ( iPSCs ) can provide a viable means of circumventing these concerns and limitations , at least with respect to the subset of phenotypes that can be studied in in vitro systems .", "Reprogramming somatic cell lines to a stable and self-sustaining pluripotent state ( Takahashi and Yamanaka , 2006; Takahashi et al . , 2007 ) has become routine practice for human and murine cell lines , but extension to other animals , especially non-human primates , is not yet widespread despite some exceptions ( e . g . , Ezashi et al . , 2009; Ben-Nun et al . , 2011; Nagy et al . , 2011; Marchetto et al . , 2013b ) .", "Instead , the broadest application of iPSCs to date has been the generation of lines derived from patients suffering from a variety of genetic disorders ( Cohen and Melton , 2011; Israel et al . , 2012; Liu et al . , 2012; Merkle and Eggan , 2013; Wang et al . , 2014 ) , with the dual aims of providing a deeper understanding of disease phenotypes and developing new therapeutic avenues .", "These cell lines have been shown to display in vitro properties corresponding to relevant patient phenotypes observed in vivo , both as iPSCs and when differentiated into other pertinent cell types , supporting their utility in clinical applications; more generally , these properties also highlight the tantalizing flexibility of iPSCs as a means of exploring developmental and cell lineage determination pathways .", "Thus , the development of an iPSC-based system for comparative genomic studies in primates will allow us to compare regulatory pathways and complex phenotypes in humans and our close evolutionary relatives using appropriate models for different tissues and cell types .", "This will be a powerful resource with which to examine the contribution of changes in gene regulation to human evolution and diversity .", "To demonstrate the validity of this approach , we have generated a panel of 7 chimpanzee iPSC lines that are fully characterized and comparable to human iPSC lines in their growth and differentiation capabilities ." ], [ "The chimpanzee iPSC lines closely resemble human iPSC lines in morphology ( Figure 1A; all images shown in main text are from chimpanzee line C4 . Similar images of the other lines are available as Figure 1—figure supplements 1–5 ) .", "All lines could be maintained in culture for at least 60 passages without loss of pluripotency or self-renewal capability using standard iPSC culture conditions , both on mouse embryonic fibroblast ( MEF ) feeder cells and in feeder-free conditions .", "The genomes of all our lines appeared to be cytogenetically stable; all exhibited normal karyotypes after reprogramming and more than 15 passages in culture , ruling out the presence of gross chromosomal abnormalities ( Figure 1B , Figure 1—figure supplement 1 ) . 10 . 7554/eLife . 07103 . 003Figure 1 . Characterization of chimpanzee induced pluripotent stem cell ( iPSC ) lines .", "( A ) Phase contrast image of representative chimpanzee iPSC line .", "Scale bar: 1000 μm .", "( B ) Representative karyotype from chimpanzee iPSC line after >15 passages , showing no abnormalities .", "( C ) ICC staining of iPSC lines with antibodies for pluripotency markers as indicated .", "Scale bar: 200 μm .", "( D ) Quantitative PCR testing for expression of endogenous pluripotency factors in all 7 chimpanzee iPSC lines .", "Line H20961 is a male human iPSC line generated in-house used as reference .", "( E ) PCR gel showing an absence of exogenous episomal reprogramming factors in all 7 chimpanzee iPSC lines .", "All PCRs were carried out on templates extracted from passage >15 with the exception of C3651* , which is from passage 2 .", "Fib—is a negative fibroblast control ( from individual C8861 ) prior to transfection , day 12 + is a positive control 12 days after transfection , 27 , 077 + to 27 , 082 + are the plasmids used for reprogramming . DOI: http://dx . doi . org/10 . 7554/eLife . 07103 . 00310 . 7554/eLife . 07103 . 004Figure 1—figure supplement 1 . Karyotypes for the 6 chimpanzee iPSC lines not shown in main text figures , generated after >15 passages in culture . Passage number for each line represents passages on mouse embryonic fibroblast ( MEF ) feeders plus additional passages on Matrigel . DOI: http://dx . doi . org/10 . 7554/eLife . 07103 . 00410 . 7554/eLife . 07103 . 005Figure 1—figure supplement 2 . ICC staining of the 6 chimpanzee iPSC lines not shown in main text figures with antibodies for pluripotency markers as indicated . Scale bar: 200 μm . DOI: http://dx . doi . org/10 . 7554/eLife . 07103 . 00510 . 7554/eLife . 07103 . 006Figure 1—figure supplement 3 . ICC staining showing SSEA1 expression in chimpanzee iPSC culture plates , clearly distinct from NANOG expression . DOI: http://dx . doi . org/10 . 7554/eLife . 07103 . 00610 . 7554/eLife . 07103 . 007Figure 1—figure supplement 4 . Melt curves showing a lack of exogenous reprogramming gene expression in episomally reprogrammed chimpanzee iPSCs after >10 passages . DOI: http://dx . doi . org/10 . 7554/eLife . 07103 . 00710 . 7554/eLife . 07103 . 008Figure 1—figure supplement 5 . Exogenous gene expression in retrovirally reprogrammed chimpanzee iPSCs after various passages . All values are relative to expression in a day-7-post-transfection chimpanzee fibroblast . DOI: http://dx . doi . org/10 . 7554/eLife . 07103 . 008 We confirmed nuclear expression of OCT3/4 , SOX2 and NANOG in all lines by immunocytochemistry ( Figure 1C; Figure 1—figure supplement 2 ) .", "The pluripotent cells also express the surface antigens Tra-1-81 and SSEA4 , while cells collected from the center of differentiating colonies expressed SSEA1 at levels comparable to differentiating colonies of human iPSC lines ( Figure 1—figure supplement 3 ) .", "To confirm that the observed expression of pluripotency-associated genes is of endogenous origin , we performed qPCR with primers designed to specifically amplify the endogenous OCT3/4 , SOX2 , NANOG and L-MYC transcripts ( Figure 1D; all PCR primers used in this work are listed in Supplementary file 2 ) .", "Indeed , we found no evidence of exogenous gene expression after 10 passages ( Figure 1—figure supplement 4 ) , and no traces of genomic integration or residual episomal plasmid retention after 15 passages ( Figure 1E ) .", "These observations indicate that self-renewal in our chimpanzee iPSC lines is maintained solely through endogenous gene expression .", "To confirm pluripotency and test the differentiation capabilities of our lines , we performed a number of assays .", "First , we generated embryoid bodies from all 7 chimpanzee iPSC lines and assayed their ability to spontaneously differentiate into the three germ layers by immunocytochemistry .", "All lines spontaneously gave rise to tissues from the three germ layers ( Figure 2A; Figure 2—figure supplement 1 ) .", "Second , we carried out directed differentiations to hepatocytes and cardiomyocytes in a subset of the lines using previously published protocols ( see ‘Materials and methods’ , Figure 2—figure supplement 2 and Video 1 ) .", "Third , we performed teratoma formation assays in four of the lines using Fox Chase SCID-beige and CB17 . Cg-PrkdcscidLystbg-J/Crl immunodeficient male mice .", "All four iPSC lines were capable of generating tumours in mice , and all tumours examined contained tissues of endodermal , ectodermal and mesodermal origins ( Figure 2B , Figure 2—figure supplement 3 ) .", "To confirm the chimpanzee origin of these tissues , we extracted and performed Sanger sequencing on mitochondrial DNA from the tumours ( Figure 2—figure supplement 4 ) . 10 . 7554/eLife . 07103 . 009Figure 2 . ( A ) ICC staining of differentiated embryoid bodies with antibodies for the three germ layers as indicated . Scale bar: 200 μm .", "( B ) Histological staining of teratomas derived from iPSC line C4955 , showing generation of tissues from all three germ layers .", "Scale bar: 100 μm . DOI: http://dx . doi . org/10 . 7554/eLife . 07103 . 00910 . 7554/eLife . 07103 . 010Figure 2—figure supplement 1 . ICC staining of differentiated embryoid bodies derived from the 6 chimpanzee iPSC lines not shown in main text figures , with antibodies for the three germ layers as indicated . Scale bar: 200 μm . DOI: http://dx . doi . org/10 . 7554/eLife . 07103 . 01010 . 7554/eLife . 07103 . 011Figure 2—figure supplement 2 . ICC staining of directly differentiated hepatocytes from line C2 , with antibodies as indicated . Scale bar: 200 μm . DOI: http://dx . doi . org/10 . 7554/eLife . 07103 . 01110 . 7554/eLife . 07103 . 012Figure 2—figure supplement 3 . Histological staining of teratomas derived from three additional chimpanzee iPSC lines , showing generation of tissues from all three germ layers . Scale bar: 500 μm . DOI: http://dx . doi . org/10 . 7554/eLife . 07103 . 01210 . 7554/eLife . 07103 . 013Figure 2—figure supplement 4 . Sequencing traces from teratomas generated from chimpanzee iPSC lines for the mitochondrial genes 12S ( C3649 , C4955 ) and cytb ( C8861 , C40210 ) .", "All traces show clear evidence of the presence of chimpanzee tissue in the teratoma . DOI: http://dx . doi . org/10 . 7554/eLife . 07103 . 01310 . 7554/eLife . 07103 . 014Video 1 . Calcium transient flux in and out ( GFP labelled ) and contractility of directly differentiated cardiomyocytes from chimpanzee iPSC line C7 . DOI: http://dx . doi . org/10 . 7554/eLife . 07103 . 014 Finaly , we characterized pluripotency in our lines through PluriTest , a bioinformatic classifier that compares the gene expression profiles of new lines to those obtained from a reference set of over 400 well-characterized human pluripotent and terminally differentiated lines ( Müller et al . , 2011 ) , modified to accommodate data from both species .", "All chimpanzee lines have PluriTest pluripotency scores greater than the pluripotency threshold value of 20 ( Figure 3A , Supplementary file 1 ) .", "We also calculated PluriTest novelty scores for all samples .", "In human PSCs , novelty values above 1 . 67 are suggestive of chromosomal duplications or expression of differentiation-associated genes .", "Human PSCs with high novelty scores are typically either difficult to maintain and expand in culture ( because they differentiate spontaneously at a high rate ) , or cannot be consistently differentiated to all three germ layers .", "All of our chimpanzee lines had novelty scores above the 1 . 67 threshold ( Figure 3B ) .", "However , in contrast to human PSCs with high novelty scores , our chimpanzee lines can be both easily maintained in culture and differentiated into all three germ layer lineages , as demonstrated by the embryoid body and teratoma assays detailed above .", "We thus hypothesize that the observed high novelty scores are likely driven by inter-species gene regulatory differences that the PluriTest assay , which was trained exclusively on human samples , interpreted as abnormal gene expression . 10 . 7554/eLife . 07103 . 015Figure 3 . ( A ) PluriTest pluripotency scores in the 7 chimpanzee lines and 4 human reference iPSC lines . Purple circles denote chimpanzees; yellow squares , humans .", "( B ) PluriTest results after removal of probes not mapping to the chimpanzee genome .", "All samples in the top left quadrant are human and have satisfactory pluripotency and novelty scores .", "Samples in the top right quadrant correspond to our chimpanzee iPSC panel , and have consistently high pluripotency yet high novelty scores . DOI: http://dx . doi . org/10 . 7554/eLife . 07103 . 01510 . 7554/eLife . 07103 . 016Figure 3—figure supplement 1 . The effects of probe sub-setting in PluriTest pluripotency score calculations . Lighter shades indicate pluripotency scores before the removal of probes not mapping to the chimpanzee genome , darker shades indicate pluripotency after probe removal .", "Purple circles denote chimpanzees; yellow squares , humans . DOI: http://dx . doi . org/10 . 7554/eLife . 07103 . 016 To better examine gene expression and regulatory differences between human and chimpanzee iPSCs , we generated genome-wide RNA-sequencing and DNA methylation data ( see ‘Materials and methods’ ) from all chimpanzee iPSC lines , as well as from 7 human iPSC lines also generated and validated in our laboratory .", "While all of the chimpanzee iPSCs were derived from fibroblast cell lines ( Supplementary file 1 ) , the human iPSCs were derived from both fibroblasts and immortalised LCLs from Caucasian and Yoruba individuals ( see Supplementary file 1 for additional details ) .", "We designed the comparative study this way in order to demonstrate that regulatory differences between human and chimpanzee iPSCs cannot be explained by technical differences due to culturing conditions or the cell type of the somatic precursor cells used for reprograming .", "To prevent biases due to genetic divergence between the two species , we chose to restrict our gene expression analyses to a curated set of genes with one-to-one orthology between humans and chimpanzees ( Blekhman et al . , 2010; Blekhman , 2012 ) .", "Following assessment of quality control metrics ( see ‘Materials and methods’ ) , we obtained normalised RPKM estimates for 12 , 171 genes that were expressed in at least 4 iPSC lines from either one of the species ( see ‘Materials and methods’ ) .", "We similarly restricted our DNA methylation analyses to a set of 335 , 307 high quality probes with a high degree of sequence conservation between humans and chimpanzees ( as in Hernando-Herraez et al . , 2013; see ‘Materials and methods’ ) .", "To examine broad patterns in the data , we used principal component analysis ( PCA ) .", "We observed clear and robust separation of human and chimpanzee iPSC lines along the first principal component ( PC ) in both the gene expression and DNA methylation data ( Figure 4A , B; regression of PC1 by species; p < 10−13 for the expression data; p < 10−12 for the DNA methylation data ) .", "Within the human samples , PC2 appears to be driven by ethnicity , as we observe all Caucasian samples consistently clustering together despite their different cell types of origin ( p = 0 . 005 for the association between PC2 and human ethnicity in the expression data , p = 0 . 044 in the DNA methylation data ) . 10 . 7554/eLife . 07103 . 017Figure 4 . Principal component ( PC ) analysis plots of data from the iPSCs .", "( A ) Principal component analysis ( PCA ) generated from expression data of 12 , 171 orthologous genes .", "( B ) PCA generated from DNA methylation data measured by 335 , 307 filtered probes . DOI: http://dx . doi . org/10 . 7554/eLife . 07103 . 01710 . 7554/eLife . 07103 . 018Figure 4—figure supplement 1 . Volcano plot showing the distribution of DE genes between iPSCs of chimpanzee and human origin . DOI: http://dx . doi . org/10 . 7554/eLife . 07103 . 01810 . 7554/eLife . 07103 . 019Figure 4—figure supplement 2 . Density plots of log2 FC change values amongst DE genes for the main comparisons presented in the text . The area bounded by the grey lines represents log2 FC changes with an absolute magnitude <2 . DOI: http://dx . doi . org/10 . 7554/eLife . 07103 . 019 We then analysed regulatory differences between the species by first focusing on the gene expression data .", "At an FDR of 1% , we identified 4609 genes ( 37 . 9% ) as differentially expressed ( DE ) between the iPSCs of the two species ( Supplementary file 3; see ‘Materials and methods’ for details ) .", "The majority of DE genes do not exhibit large inter-species fold-change differences in expression levels ( Figure 4—figure supplements 1 , 2 ) .", "An analysis of functional annotation of the DE genes reveals that no Gene Ontology Biological Process terms ( GO BP; Ashburner et al . , 2000 ) are significantly overrepresented among these genes at an FDR of 5% ( Supplementary file 4 ) , although we identified 123 overrepresented terms if we limit our analysis to the 546 genes with absolute log2 fold-change difference >2 ( Supplementary file 4 ) .", "Additionally , we tested for concordance between our list of DE genes and a list of 2730 genes that were previously classified as DE between human and non-human primate iPSC lines ( Marchetto et al . , 2013b ) .", "Given our stringent approach to consider orthologous genes , only 2081 ( 76% ) genes could be analysed across the two studies .", "Of these , 1495 genes are detectably expressed in our lines , and 1079 ( 72 . 2% ) are classified as DE between the species in both data sets ( a highly significant enrichment; χ2 p < 10−16 ) .", "Expression trends within these DE genes are in the same direction in both data sets in 1060 of cases ( 98 . 24% ) .", "Next , we used a similar approach to identify differentially methylated ( DM ) probes and regions between the iPSCs of both species ( see ‘Materials and methods’ ) .", "We identified 63 , 791 probes that are DM between the two species at an FDR of 1% , 26 , 554 of which have a mean intergroup β difference ≥0 . 1 , our arbitrary effect size threshold for retaining probes for DM region ( DMR ) identification and downstream analyses .", "Of these , 10 , 460 probes could be further grouped into 3529 regions of 2 or more DM probes within 1 kb , which we designated DMRs; ( Supplementary file 5 ) ; the numbers of probes and regions identified as DM at a range of mean interspecies β thresholds are given in Supplementary file 6 .", "In order to consider the DNA methylation and gene expression data jointly , we focused on a subset of 2348 DMRs that could be associated with a single Ensembl gene .", "Overall , these DMRs were associated with 2141 genes , of which 1350 were also detectably expressed in the iPSCs , and 558 ( 41 . 3% ) were classified as DE between the species , a slightly higher proportion than expected by chance alone ( p = 0 . 1 ) .", "We further classified the DMRs as either ‘promoter’ , ‘genic’ or ‘mixed’ depending on their position relative to annotated gene transcripts ( see ‘Materials and methods’ ) .", "The overall set of DMRs , as well as genic DMRs , are significantly associated with 4 and 79 GO BP terms respectively ( FDR < 5% ) , including terms related to neurogenesis and skeletal system development .", "Enrichment of several terms related to neurogenesis and skeletal system development is likewise marginally significant amongst promoter and mixed DMRs ( Supplementary file 7 ) .", "However , the subset of inter-species DE genes that are also associated with DMRs are not significantly enriched with annotation for any GO BP or MF terms .", "We used ChIP-seq to characterize the genome-wide distribution of two types of histone modifications ( H3K27me3 and H3K27ac ) in three of our chimpanzee iPSCs ( see ‘Materials and methods’ ) .", "We compared the chimpanzee data to histone modification data from three human iPSC lines from the Roadmap Epigenomics project ( Figure 5 ) .", "To do so , we downloaded raw sequence files from GEO and processed data from both species using the same pipeline ( see ‘Materials and methods’ ) .", "We identified ChIP-seq peaks using MACS or RSEG , as appropriate , and accounted for differences in genome sequence between the species as well as for incomplete power to identify peaks across species ( see ‘Materials and methods’ ) .", "To relate the ChIP-seq data to genes ( and integrate over data from all peaks that are in proximity to a given gene ) , we then generated enrichment ChIP scores for a set of previously defined 26 , 115 orthologous transcription start sites ( TSSs , from Zhou et al . , 2014 ) .", "The enrichment score ( see ‘Materials and methods’ for details , also Supplementary file 8 ) , reflects the ratio of mapped ChIP-seq read counts across all peaks within a 4 kb window centred on an orthologous TSS , relative to the genome-wide read count average after adjusting expectations based on the input control sample .", "We chose to classify as ‘enriched’ any region where the mean enrichment score across all three individuals in the species was larger than 1 .", "This cut-off is arbitrary , but we confirmed that our qualitative results are robust by additionally testing enrichment cut-offs of 2 , 5 , and 10 . 10 . 7554/eLife . 07103 . 020Figure 5 . Overlap of H3K27me3 and H3K27ac signal between chimpanzee and human iPSCs at orthologous transcription start sites ( TSSs ) .", "( A ) H3K27me3 enrichment near all genes with an orthologous TSS .", "( B ) H3K27me3 enrichment near 2910 genes previously identified as bivalent in human PSCs .", "( C ) H3K27ac enrichment near all genes with an orthologous TSS .", "( D ) H3K27ac peaks near 14 known pluripotency master regulators with orthologous TSSs . DOI: http://dx . doi . org/10 . 7554/eLife . 07103 . 02010 . 7554/eLife . 07103 . 021Figure 5—figure supplement 1 . Density plots of H3K27ac enrichment scores at orthologous TSSs in the entire data set and at 3572 genes enriched only in chimpanzee iPSCs . DOI: http://dx . doi . org/10 . 7554/eLife . 07103 . 02110 . 7554/eLife . 07103 . 022Figure 5—figure supplement 2 . Density plots of mean RPKM in chimpanzee iPSCs in all 12 , 171 genes with expression data and in the subset of 1737 genes with expression data and H3K27ac signal enrichment solely in chimpanzee iPSCs . DOI: http://dx . doi . org/10 . 7554/eLife . 07103 . 02210 . 7554/eLife . 07103 . 023Figure 5—figure supplement 3 . H3K27ac peaks observed in at least 1 chimpanzee or human iPSC , as identified by MACS at 22 known pluripotency master regulators . In the case of all three genes that differ between this figure and Figure 5D—KLF5 , NR5AD and SMAD1—processed enrichment signal after accounting for orthology is weak , and falls very close to our normalised enrichment score threshold of 1 , explaining the difference between the two . DOI: http://dx . doi . org/10 . 7554/eLife . 07103 . 023 Using this approach , we first examined genome-wide patterns of H3K27me3 enrichment in chimpanzee and human iPSCs .", "Overlap across the two species is considerably higher than expected by chance ( Figure 5A , χ2 p < 10−16 ) , but it is somewhat unclear how to interpret this observation with respect to the expectation that human and chimpanzee iPSCs would have similar pluripotency potential .", "We thus focused on a set of 3913 genes ( Li et al . , 2013 ) previously annotated as bivalently modified in human PSCs—that is , genes known to be associated with both high H3K4me3 and H3K27me3 , indicative of a ‘poised’ or ‘primed’ state ( Bernstein et al . , 2006 ) .", "We expect the vast majority of these genes to also be associated with similar modifications in chimpanzee iPSCs .", "Only 2910 of the known bivalent genes were associated with clear orthologous TSSs and could be tested using our comparative H3K27me3 ChIP-seq data .", "Of these , 306 were not associated with the modification in either species , whereas of the 2604 genes that were associated with H3K27me3 in at least one species , 2368 ( 90 . 1% ) were enriched for H3K27me3 in both species ( Figure 5B , χ2 p < 10−16 ) .", "We then examined H3K27ac enrichment patterns in both species .", "This mark is indicative of active promoters and gene transcription .", "Overall , we find good agreement between human and chimpanzee genes enriched for H3K27ac , with 95 . 8% human genes associated with the mark also enriched in chimpanzees ( Figure 5C ) .", "However , there is a clear excess of genome-wide H3K27ac signal in chimpanzee iPSCs relative to humans , possibly due to an overall more sensitive ChIP enrichment in the chimpanzee samples ( Figure 5—figure supplements 1 , 2 ) .", "We proceeded by focusing on a list of 22 core pluripotency transcription factors ( taken from Ng and Surani , 2011; Orkin and Hochedlinger , 2011 ) , where we expect to find H3K27ac signal shared across the two species at a higher rate than in the genome-wide data , given the role of these factors in maintaining pluripotency .", "Due to our stringent requirements for establishing orthology , we were initially able to examine data from 14 of those genes; 11 of which were associated with H3K27ac in both species ( Figure 5D ) —one of the discrepancies is REX1 ( also known as ZFP42 ) , which we discuss further below .", "We extended our analysis to include the full set of 22 pluripotency transcription factors regardless of orthology , by testing solely for absence or presence of signal peaks identified by MACS ( i . e . , without considering enrichment scores; see ‘Materials and methods’ ) .", "We again found a high overlap in H3K27ac enrichment across species , with 15 of the 22 genes associated with H3K27ac enrichment in both species ( including the three master regulators of pluripotency , OCT4 , SOX2 , and NANOG; Figure 5—figure supplement 3 ) .", "Of the remaining 7 genes , one ( DAX1 ) was not found to be associated with H3K27ac in either species , four genes ( ESSRB , KLF2 , KLF4 , and KLF5 ) were associated with H3K27ac only in chimpanzee ( although this observation may reflect incomplete power to detect peaks in the human data ) , and only two genes ( ZFX and REX1 ) were associated with H3K27ac in human but not in chimpanzee iPSCs .", "In order to further consider inter-species differences in the core pluripotency regulatory network , we examined expression levels in our chimpanzee and human iPSCs in the same list of 22 core pluripotency TFs described above .", "Expression values in all iPSC lines are shown in Figure 6A ( see also Figure 6—figure supplement 1 ) .", "Given the stringency of our interspecies analysis approach with respect to unique read mapping , we are unable to calculate RNA-seq-based expression estimates for six of these TFs , including OCT4 or NANOG , both of which have multiple pseudogenes that can confound mapping algorithms ( however , as shown in Figure 1D , our qPCR results demonstrate that expression of those 2 genes is similar amongst all chimpanzee iPSC lines , and marginally higher than in our human iPSC control line ) .", "Of the 16 TFs with expression data for iPSCs from both species , 4 ( E2F1 , ESRRB , SALL4 and REX1 ) are DE between human and chimpanzee iPSCs at an FDR of 1% .", "Of these , ESRRB and REX1 are associated with absolute inter-species expression log2 fold-changes >1 .", "However , because ESRRB is expressed at very low levels across all samples ( mean RPKM across all 14 samples = 0 . 47 ) , we focused our subsequent analyses on REX1 , which is expressed at low or undetectable levels in 6 of our 7 chimpanzee iPSCs ( mean RPKM = 0 . 667 ) , but at high levels in all human iPSC lines ( mean RPKM = 180 . 58 ) and a single chimpanzee iPSC , C6 ( Figure 6A ) .", "Our DNA methylation data is consistent with this gene expression pattern: all 10 probes located in the 5′ UTR or up to 1500 bp upstream from the REX1 TSS are highly methylated in the six chimpanzee lines ( mean β across all promoter probes = 0 . 87 ) , but exhibit intermediate or low levels of DNA methylation in all of the human iPSC lines and the REX1-expressing C6 line ( Figure 6B ) ; the entire region is a DMR ( Supplementary file 5 ) .", "Consistent with these findings , REX1 is also differentially enriched for H3K27ac signal in the two species—we identified no H3K27ac peaks at the REX1 TSS in the three chimpanzee lines , which did not include C6 ( Figure 5D , Figure 5—figure supplement 3 ) . 10 . 7554/eLife . 07103 . 024Figure 6 . REX1 may be dispensable for pluripotency in chimpanzee iPSCs . In both panels REX1-expressing chimpanzee iPSC line is coloured red , significant interspecies differences are indicated along the left-hand side , and purple boxes indicate chimpanzee lines , yellow boxes indicate human lines .", "( A ) Expression values of 16 core pluripotency transcription factors in all human and chimpanzee iPSC lines .", "( B ) Methylation status of 13 CpG sites associated with REX1 in all human and chimpanzee iPSCs .", "Location of the probe relative to the gene sequence is indicated along the right hand side .", "( C ) Fraction of differentially expressed ( DE ) genes in multiple categories downstream of REX1 in human and mouse ESCs .", "1: Genes associated with any Gene Ontology term that contains the words ‘ectoderm’ , ‘mesoderm’ or ‘endoderm’ .", "2: CNS development genes are associated with GO:0007417 or any of its offspring .", "3: cardiovascular system development genes are associated with GO:0072358 or any of its offspring .", "4: hepatobiliary system development genes are associated with GO:0055123 or any of its offspring .", "( D ) Expression levels of 34 genes associated with GO:0006096 , glycolysis , in all human and chimpanzee iPSC lines .", "All reported p-values were calculated after excluding C6 . DOI: http://dx . doi . org/10 . 7554/eLife . 07103 . 02410 . 7554/eLife . 07103 . 025Figure 6—figure supplement 1 . Expression values of 15 core pluripotency transcription factors in all human and chimpanzee iPSC lines . The data used to generate this figure are identical to those used to generate Figure 5A except that expression levels of REX1 are not included in the calculation .", "REX1-expressing chimpanzee iPSC line C6 is coloured red , significant interspecies differences are indicated along the left-hand side , and purple boxes indicate chimpanzee lines , yellow boxes indicate human lines . DOI: http://dx . doi . org/10 . 7554/eLife . 07103 . 02510 . 7554/eLife . 07103 . 026Figure 6—figure supplement 2 . Expression levels of REX1 in human , chimpanzee and bonobo iPSC lines generated in this study and in Marchetto et al . ( 2013b ) .", "Data for this figure were jointly normalised . DOI: http://dx . doi . org/10 . 7554/eLife . 07103 . 02610 . 7554/eLife . 07103 . 027Figure 6—figure supplement 3 . Plot of PluriTest pluripotency scores vs normalised REX1 intensity in 73 human iPSC lines derived in-house . DOI: http://dx . doi . org/10 . 7554/eLife . 07103 . 02710 . 7554/eLife . 07103 . 028Figure 6—figure supplement 4 . Methylation status of 13 CpG sites associated with REX1 in chimpanzee and human iPSCs from this study and human PSCs from Ziller et al . ( 2011 ) .", "REX1-expressing chimpanzee iPSC line is coloured red; location of the probes relative to the gene sequence is indicated along the right hand side . DOI: http://dx . doi . org/10 . 7554/eLife . 07103 . 028 The REX1 genes codes for a transcription factor present in all placental mammal species , which has long been established as a marker of pluripotency in human and mouse PSCs ( Brivanlou et al . , 2003 ) .", "Multiple publications have suggested that this gene plays an important role in maintaining pluripotency and inhibiting differentiation into the three primary tissue germ layers ( Masui et al . , 2008; Scotland et al . , 2009; Son et al . , 2013 ) , with multiple mechanisms of action having been proposed .", "However , REX1-knockout mouse ESC lines can give rise to chimeric animals , and homozygous F2 REX1 null mice are viable ( Masui et al . , 2008 ) , suggesting that REX1 may not be indispensable for murine pluripotency .", "In humans , loss of REX1 expression in ESCs following shRNA knockdown has been associated with a rapid loss of pluripotency , as well as a decrease in glycolytic activity and a lack of observable mature mesodermal structures in teratoma formation assays ( Son et al . , 2013 ) .", "To determine the consequences of a lack of REX1 expression in chimpanzee iPSCs , we considered gene expression data from all human iPSC lines and the 6 chimpanzee iPSC lines that do not express REX1 .", "We asked whether there is an excess of DE genes among those thought to be directly regulated by , or downstream of , REX1 ( Figure 6C , D; see ‘Materials and methods’ ) , but failed to find enrichment in all categories except for genes associated with GO term BP:0006096 , glycolysis , where 19 of 34 testable genes were DE at an FDR of 1% between the two species ( p < 0 . 01 from 100 , 000 permutations ) .", "The direction of this effect ran contrary to previous reports , however , with genes highlighted by Son et al . ( 2013 ) as downregulated following REX1 knockdown , such as PGAM1 or LDHA , having significantly higher expression in chimpanzee iPSCs than in human iPSCS ( Figure 6D ) .", "Furthermore , the REX1-expressing line C6 is not an outlier amongst the other chimpanzee iPSC lines ( Figure 6D ) , suggesting that the observed inter-species regulatory differences cannot be attributed to differences in REX1 expression between the species .", "We note that both the teratomas and EBs generated from chimpanzee iPSC lines that do not express REX1 gave rise to mature structures from all three germ layers similar to those observed in REX1-expressing line C6 ( Figure 2—figure supplements 1 , 3 ) .", "Furthermore , and consistent with our observations , REX1 is either absent or expressed at low levels in one replicate of either of the two retrovirally reprogrammed bonobo ( Pan paniscus , sister species to chimpanzees ) iPSC lines generated by Marchetto et al . ( 2013b ) , although it is expressed in both replicates of both chimpanzee iPCS lines from the same group ( Figure 6—figure supplement 2 ) .", "Together , these findings suggest that that the variable loss of REX1 expression in chimpanzee and bonobo iPSCs does not impair pluripotency , and that its regulatory functions of in humans may be being fulfilled in chimpanzee iPSCs by other regulatory mechanisms .", "We collected RNA-sequencing data from all cell lines used to generate both the chimpanzee and human iPSCs ( Supplementary file 6 ) .", "Following quality control and normalisation steps , we obtained RPKM values for 13 , 147 genes across all 28 iPSC and precursor samples ( see ‘Materials and methods’ ) .", "We also obtained DNA methylation profiles from all samples at the same 335 , 307 probes described above .", "PCA of both data sets show that the first PC was significantly associated with tissue type in both data sets ( p < 10−27 for the expression data; p < 10−17 for the DNA methylation data; see Figure 7 and Supplementary file 9 ) , while human and chimpanzee samples are separated by species along PC2 ( p = 0 . 001 for the expression data; p < 10−4 for the methylation data ) .", "However , given the absence of chimpanzee LCLs in our dataset , it is not possible to determine whether the separation is driven by tissue type , species , or both . 10 . 7554/eLife . 07103 . 029Figure 7 . Relationships of iPSCs to their precursors .", "( A ) PCA of gene expression data from all iPSCs and their precursor cell lines .", "( B ) Neighbour-joining tree of Euclidean distances between all samples generated based on the gene expression data .", "( C ) PCA of DNA methylation data from all iPSCs and their precursor cell lines .", "( D ) Neighbour-joining tree of Euclidean distances between all samples generated based on the DNA methylation data . DOI: http://dx . doi . org/10 . 7554/eLife . 07103 . 02910 . 7554/eLife . 07103 . 030Figure 7—figure supplement 1 . Boxplots of methylation beta values at 335 , 307 probes across all samples . Plots are colored by tissue type: light blue: chimpanzee iPSCs; dark blue: human iPSCs; light orange: chimpanzee fibroblasts; dark orange: human fibroblasts; turquoise: human lymphoblastoid cell lines ( LCLs ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 07103 . 03010 . 7554/eLife . 07103 . 031Figure 7—figure supplement 2 . Boxplots of methylation beta values across all samples , grouped by potency and genomic features . Boxes are colored by tissue type: light blue: chimpanzee iPSCs; light orange: chimpanzee fibroblasts .", "( A ) By methylation feature; ( B ) By relative position . DOI: http://dx . doi . org/10 . 7554/eLife . 07103 . 03110 . 7554/eLife . 07103 . 032Figure 7—figure supplement 3 . Venn diagrams showing overlap in interspecies differences before and after reprogramming .", "( A ) Overlap in DE genes between chimpanzee and human fibroblasts , and chimpanzee and human fibroblast-derived iPSCs .", "( B ) Overlap in differentially methylated ( DM ) probes between chimpanzee and human fibroblasts , and chimpanzee and human fibroblast-derived iPSCs . DOI: http://dx . doi . org/10 . 7554/eLife . 07103 . 03210 . 7554/eLife . 07103 . 033Figure 7—figure supplement 4 . Venn diagram showing overlap of genes identified as DE between iPSCs of the two species when we normalize the iPSC data independently and alongside data from the precursors . DOI: http://dx . doi . org/10 . 7554/eLife . 07103 . 03310 . 7554/eLife . 07103 . 034Figure 7—figure supplement 5 . Venn diagram showing overlap of probes identified as DM between iPSCs of the two species under the full and reduced limma models . DOI: http://dx . doi . org/10 . 7554/eLife . 07103 . 03410 . 7554/eLife . 07103 . 035Figure 7—figure supplement 6 . Normalized XIST expression values in 7 chimpanzee and human iPSCs . Circles denote chimpanzee iPSCs , squares indicate human iPSCS . DOI: http://dx . doi . org/10 . 7554/eLife . 07103 . 03510 . 7554/eLife . 07103 . 036Figure 7—figure supplement 7 . Quantile-normalized methylation beta values at 8210 X-chromosome probes in 7 chimpanzee iPSCs and 7 human iPSCs . The colour bar beneath the dendrogram indicates sex of the individuals: purple: female; yellow: male .", "Sample names ending with _FB indicate fibroblast lines used to generate the corresponding iPSC line , samples ending with _LCL indicate LCL lines used to generate the corresponding iPSC line . DOI: http://dx . doi . org/10 . 7554/eLife . 07103 . 03610 . 7554/eLife . 07103 . 037Figure 7—figure supplement 8 . Normalized methylation beta values at 168 assayable probes known to be subject to parental imprinting effects , from Ma et al . ( 2014 ) .", "Sample names ending with _FB indicate fibroblast lines used to generate the corresponding iPSC line , samples ending with _LCL indicate LCL lines used to generate the corresponding iPSC line . DOI: http://dx . doi . org/10 . 7554/eLife . 07103 . 037 Overall , chimpanzee iPSCs have significantly higher levels of DNA methylation compared to the somatic lines they were generated from ( p < 10−15; Figure 7—figure supplement 1 ) , an observation that extends to all genomic features we tested ( Figure 7—figure supplement 2 ) ; similar observations have been previously made in human PSCs ( Bock et al . , 2011; Nazor et al . , 2012 ) .", "Remarkably , both DNA methylation and gene expression levels in iPSCs are relatively homogeneous within species , far more so than in their corresponding precursor cells ( Figure 6B , D; p < 10−14 when comparing overall pairwise distances within all chimpanzee iPSCs and within all chimpanzee fibroblasts in the methylation data; p < 10−9 for the same comparison in the gene expression data ) .", "DNA methylation levels in iPSCs also have significantly reduced coefficients of variation relative to their precursor lines ( range of CVs for chimpanzee iPSCs = 0 . 78–0 . 80 , for chimpanzee fibroblasts = 0 . 87–0 . 90; p < 10−06 ) .", "We observed the same pattern in the human data , although in this case the multiple somatic origins of the cell lines of origin contribute to the higher level of variation .", "We then performed analyses of gene expression and DNA methylation differences in the combined iPSC and somatic precursor dataset .", "First , we carried out a comparison of the iPSCs and the precursor cells within each species ( see ‘Materials and methods’ ) and classified 9235 genes as DE between chimpanzee fibroblasts and the corresponding iPSCs .", "In humans the number of DE genes is 7765 if we consider all iPSC lines and their somatic precursors , 8087 if we only consider those derived from LCLs ( n = 5 ) , and 5489 if we only consider those derived from fibroblasts ( n = 2; Supplementary file 10 ) .", "Similarly , we identified 18 , 029 DMRs between chimpanzee fibroblasts and iPSCs , and 12 , 078 DMRs between all human somatic precursors and all human iPSCs ( Supplementary files 11 , 12 ) .", "No GO categories are significantly overrepresented in any of these data sets .", "Next , we focused on a comparison of inter-species differences in gene expression and DNA methylation levels across cell types .", "Following joint normalisation and modelling of data from all samples ( see ‘Materials and methods’ ) , we classified 5663 genes as DE between the chimpanzee precursor fibroblasts and the collection of human precursor LCLs and fibroblasts , as well as 84 , 747 DM probes and 9107 DMRs ( always at an FDR of 1% ) .", "Most of these regulatory differences , however , reflect variation across cell types rather than across species ( 6324 genes and 70 , 312 probes are DE or DM between the human fibroblasts and LCLs , respectively ) .", "We thus considered only data from the fibroblast precursors in the two species .", "Only 2 of the human iPSCs were reprogrammed from fibroblasts , leading to a loss in power; we were nonetheless able to identify 1236 DE genes and 25 , 456 DM probes between human and chimpanzee fibroblasts , and 1118 DE genes and 16 , 392 DM probes between the corresponding iPSCs of the two species .", "None of these gene sets were significantly enriched for functional annotations using GO BP terms .", "Although the overlap of inter-species DE genes and DM probes between the iPSCs and the precursors is considerable ( 13 . 6% of DE genes and 11 . 8% of DM probes ) , a large number of regulatory differences are only observed between the iPSC lines of the two species ( Figure 7—figure supplement 3 ) .", "This observation is robust with respect to different approaches to normalising and modelling the data ( Figure 7—figure supplement 4 ) , strongly suggesting that many of the differences we observe between our chimpanzee and human iPSC lines may be intrinsic features of the pluripotent state in these two species ." ], [ "iPSCs have the potential to transform our understanding of the biology of non-model organisms and facilitate functional comparative studies .", "To this end , we have generated a panel of 7 fully characterized chimpanzee iPSCs .", "All lines are capable of spontaneously giving rise to the three tissue germ layers in vitro and in vivo and meet all currently established criteria for pluripotency .", "The chimpanzee iPSC lines provide a tantalising avenue for investigating how changes in gene expression and regulation underlie the architecture of complex phenotypic traits in humans and our closest living relatives ( Gallego Romero et al . , 2012; Marchetto et al . , 2013a ) .", "In particular , we believe that through the use of directed differentiation protocols , functional studies could be performed in cell types where strong a priori hypotheses support a role for selective pressure underlying inter-species divergence ( e . g . , liver , heart , kidney [Blekhman et al . , 2008 , 2010] ) .", "In that sense , we hope that this panel of cell lines will be a useful tool to researchers interested in overcoming current limitations of comparative studies in primates .", "To that purpose , all chimpanzee iPSC described in this publication the panel are available fully and without restrictions to other investigators upon request to the corresponding authors .", "Other groups have previously generated pluripotent stem cells from primates ( Liu et al . , 2008; Chan et al . , 2010; Tomioka et al . , 2010; Wu et al . , 2010; Ben-Nun et al . , 2011; Deleidi et al . , 2011; Okamoto and Takahashi , 2011; Wu et al . , 2012; Hong et al . , 2014; Wunderlich et al . , 2014 ) .", "Indeed , a recent publication ( Marchetto et al . , 2013b ) reported the generation of two chimpanzee and two bonobo ( P . paniscus ) iPSC lines through the use of retroviral vectors .", "However , in the course of our work we have found that retroviral vector silencing in chimpanzee iPSCs was not as stable as in human iPSC lines generated at the same time using the same method ( see ‘Materials and methods’ and Figure 1—figure supplement 5 ) .", "Our use of episomal vectors circumvents this problem , and more broadly the problems of both random exogenous gene reactivation and disruption of the host genome through retroviral integration ( Sommer et al . , 2012 ) .", "More generally , while the sum total of primate PSC generation efforts so far has resulted in a sizable number of lines being established from various donors and species , these have been generated through various reprogramming protocols and source cell types .", "We have generated iPSCs from a panel of seven individuals using a consistent protocol and cell type of origin .", "Given the panel size , it is powerful enough to robustly detect inter-species differences in gene expression , splicing and regulation .", "The fact that our panel contains both female and male lines also allows for future studies of sex-specific differences in gene expression in various cell types .", "Indeed , we have previously shown that this can be accomplished using as few as six individuals from each species ( Blekhman et al . , 2010 ) .", "Beyond its future applications , however , our panel has already yielded insights into the pluripotent state in chimpanzees and humans .", "On the one hand , both at the transcriptional and epigenetic level , our iPSCs are remarkably homogeneous both within and between species , significantly more so than their precursors cells .", "This finding aligns with our current understanding of the reprogrammed pluripotent state as a complex , highly regulated state ( Jaenisch and Young , 2008 ) , deviations from which are likely to result in loss of pluripotency and lineage commitment .", "Additional support for this notion was provided by the strong overlap in H3K27me3 signal between the two species , especially in known bivalent genes .", "It is remarkable that we have been able to observe this considerable conserved chromatin signature despite the obvious confounding technical batch effect in these comparative data .", "On the other hand , we were also able to identify over 4500 genes that are DE between human and chimpanzee iPSCs , as well as over 3500 DMRs between the two species .", "These numbers are greater than what has been previously observed in comparisons of other tissues across humans and chimpanzees with similar sample sizes ( Blekhman et al . , 2008 , 2010 ) .", "We believe that the reasons for this difference are likely to primarily stem from increased power to detect DE genes and DMRs in our sample relative to previous work .", "Given the small amount of intra-species variability we observed in RNA-seq and methylation relative to other tissues , we expect to have greater power to detect small , statistically significantly inter-species differences that would have been missed by studies that consider more variable tissue samples .", "This notion is supported by the fact that the fraction of genes with log FC < 2 we detect as DE between human and chimpanzee iPSCs is greater than in other comparison we have performed with any other tissue ( Figure 4—figure supplement 2 ) .", "Though small in magnitude , we expect that a subset of these regulatory differences may be biologically relevant ( e . g . , we find that inter-species regulatory differences in methylation levels are enriched in regions associated with developmental processes; Supplementary files 4 , 5 ) .", "We specifically highlighted an inter-species difference in REX1 expression levels .", "This gene is considered an indispensible pluripotency marker in human and mouse PSCs , but our observations suggest that it may not be the case in chimpanzees .", "Although only one chimpanzee iPSC line expresses REX1 , we were unable to identify any systematic differences between our human and chimpanzee iPSCs that would indicate a reduction in pluripotency .", "We also examined REX1 expression levels in 73 human iPSC lines generated in-house from Caucasian individuals using the Illumina HT12v4 array ( Figure 6—figure supplement 3 ) .", "All lines had PluriTest pluripotency scores >20 , yet 3 of 73 lines ( 4 . 1% ) showed levels of REX1 expression that were indistinguishable from background signal , suggesting that REX1 may not be expressed in these individuals despite their high pluripotency scores .", "We also examined methylation status at the REX1 locus in previously published human ESCs and iPSCs from Ziller et al . ( 2011 ) , and found that although all ESC lines examined exhibited consistent levels of low methylation at the REX1 promoter , human iPSC lines analysed in exhibited either hemi- or hyper-methylated REX1 promoter regions ( Figure 6—figure supplement 4 ) .", "In the absence of publicly available REX1 expression data from either of the hiPSC lines with hypermethylated promoters we cannot be certain that the gene is not expressed in these lines , but the combination of these findings with our observations above and previous literature suggest that REX1 may be important in regulation and maintenance of pluripotency in ESCs , but not necessarily so in iPSCS .", "Additionally , in chimpanzees , the REX1 gene has undergone multiple deletions and insertions relative to the human sequence , most significantly a 647 bp insertion in its first intron , and two insertions in the 3′ UTR region of approximately 300 bp each that may disrupt the local regulatory landscape; the gene has also been duplicated , with a second copy retrotransposed into chromosome 14; none of these changes are shared with gorillas or orang-utans .", "Although it is currently unclear whether some or all of these changes are also present in the bonobo , these findings might explain why we observed low or no REX1 expression more frequently in chimpanzees than in humans , and suggests that the gene may not be necessary for maintaining pluripotency in the Pan lineage .", "PSCs have been used to study developmental pathways in vitro ( e . g . , Paige et al . , 2012; Rada-Iglesias et al . , 2012; Wamstad et al . , 2012; Xie et al . , 2013 ) .", "Although optimization of existing differentiation protocols will likely be necessary for application in the chimpanzee system , our panel of iPSCs makes it possible to carry out comparative developmental studies between humans and chimpanzees , and firmly test the hypothesis that changes in gene regulation and expression , especially during development , underlie phenotypic differences between closely related species , especially primates ( Britten and Davidson , 1971; King and Wilson , 1975; Jacob , 1977; Carroll , 2005 , 2008 ) .", "In addition , we should be able to recreate and test the effect of inter-species regulatory changes in the correct cell type and species environment , enabling studies that cannot otherwise be performed in humans and non-human primates .", "The use of panels of iPSCs including lines from both humans and non-human primates will thus allow us to gain unique insights into the genetic and regulatory basis for human-specific adaptations ." ], [ "All biopsies and animal care were conducted by the Yerkes Primate Research Center of Emory University under protocol 006–12 , in full accordance with IACUC protocols .", "Skin punch biopsies ( 3 mm ) were rinsed in DPBS containing Primocin ( InvivoGen , San Diego , California ) and penicillin/streptomycin ( Pen/Strep , Corning , Corning , New York ) and manually dissected into 10–15 smaller pieces .", "The tissue was digested in 0 . 5% collagenase B ( Roche , Indianapolis , Indiana ) for 1–2 hr until cells were released from the extracellular matrix .", "Dissociated cells were pelleted by centrifugation at 250×g , and the supernatant was spun a second time at 700×g to pellet any cells that had not been completely released from the extracellular matrix .", "Cell pellets were resuspended in a 1:1 mixture of α-MEM and F12 ( both from Life Technologies , Carlsbad , California ) supplemented with 10% FBS ( JR Scientific , Woodlawn , California ) , NEAA , GlutaMAX ( both from Life Technologies ) , 1% Pen/Strep , 64 mg/l L-ascorbic acid 2-phosphate sesquimagnesium salt hydrate ( Santa Cruz Biotech , Dallas , Texas ) and Primocin .", "Cells were plated in a single well of a 6-well plate coated with 4 µg/cm2 human fibronectin ( BD Sciences , Franklin Lakes , New Jersey ) and 2 µg/cm2 mouse laminin ( Stemgent , San Diego , California ) .", "Cultures were grown at 5% CO2/5% O2 until confluent and then split using 0 . 05% trypsin .", "For routine passaging cells were cultured at 5% CO2 and atmospheric oxygen in primate fibroblast media , which is the same as plating media but does not contain F12 base media .", "We initially attempted to generate lines by retroviral transduction through transfection with pMXs- vectors encoding the human OCT3/4 , SOX2 , KLF4 , L-MYC and NANOG sequences ( Addgene plasmids 17 , 217 , 17 , 218 , 17 , 219 , 26 , 022 and 18 , 115 ) as well as vectors encoding the MSCV-VSV . G envelope protein ( Addgene plasmid 14 , 888 ) and MSCV gag-pol ( Addgene plasmid 14 , 887 ) .", "15 μg of each vector was transfected into 293FT cells ( Life Technologies ) using Lipofectamine 2000 ( Life Technologies ) as directed by the manufacturer .", "We collected virus-containing supernatant from the 293FT cells 48 and 72 hr after transfection and immediately used this viral media to transduce chimpanzee fibroblasts , alongside 10 μg/ml of polybrene ( H9268 from Sigma Aldrich [St Louis , Missouri] ) .", "To aid viral penetration , we centrifuged the cells at 1800 RPM for 45 min following each transduction .", "24 hr after the second transduction , we replaced the viral media with A-MEM + 10% FBS , NEAA and Glutamax .", "Transduced fibroblasts were allowed to recover for a further 2 days and then seeded on γ-irradiated , CF-1-derived MEF at a density of 10 , 000 cells/cm2 , and maintained in hESC media ( DMEM/F12 supplemented with 20% KOSR , 0 . 1 mM NEAA , 2 mM GlutaMAX , 1% Pen/Strep , 0 . 1 mM BME and 25 ng/ml human bFGF ) supplemented with 0 . 5 mM valproic acid ( Stemgent ) until day 14 .", "We obtained iPSCs from 5 chimpanzees by using this protocol .", "Yet , when we performed quality control and pluripotency checks on these lines we found that the exogenous transfected genes were still expressed ( Figure 1—figure supplement 5 ) .", "Pluripotency in these lines could not be maintained exclusively through endogenous expression .", "We discarded all 5 lines and proceeded with a different reprograming strategy as detailed below .", "Fibroblasts were grown at 5% CO2/atmospheric O2 in primate fibroblast media until 70–80% confluence and released by trypsinisation for transfection .", "1 . 5 × 106 cells were transfected with 1 . 5 μg per episomal vector containing the following genes: OCT3/4 , SHp53 , SOX2 , KLF4 , LIN28 , and L-MYC ( Addgene plasmids 27 , 077 , 27 , 078 , 27 , 080 and 27 , 082; [Okita et al . , 2011] ) .", "To boost the initial retention of vectors following transfection , 3 μg of in vitro transcribed ARCA capped/polyadenylated EBNA1 mRNA was cotransfected with the vectors ( see below ) .", "Transfected cells were seeded at 15 , 000/cm2 on tissue culture plates precoated with 1 μg/cm2 vitronectin ( Stemcell Technologies , Vancouver , Canada ) .", "Cells were grown in Essential 8 media ( made in house as previously described in Chen et al . ( 2011 ) ) without TGFβ1 , supplemented with 0 . 5 mM sodium butyrate ( NaB , Stemgent ) and 100 nM hydrocortisone ( Sigma Aldritch ) .", "Hydrocortisone was used between days 1–12 , or until cell density exceeded >70% confluence .", "At day 12 , cells were detached using TrypLE ( Life Technologies ) and replated at a density of 5000 cells/cm2 on cell culture dishes precoated with 0 . 01 mg/cm2 ( 1:100 ) of hESC-grade Matrigel ( BD Sciences ) and grow in Essential 8 media without TGFβ1 or NaB .", "Colonies began to form at days 18–22 and were picked between days 24–30 onto dishes coated with γ-irradiated CF-1 derived MEF and subsequently grown in hESC media ( as described above ) supplemented with 100 ng/ml human bFGF ( Miltenyi Biotech , Teterow , Germany ) .", "Clones were routinely split using Rho-associated kinase ( ROCK ) inhibitor Y27632 ( Tocris , Minneapolis , Minnesota ) at a concentration of 10 μM .", "Cells were migrated to 1:100 hESC Matrigel ( BD Sciences ) and maintained on Essential 8 media after a minimum of 15 passages on MEF .", "Feeder free cells were passaged using EDTA-based cell release solution as in Chen et al . ( 2011 ) .", "To generate a template for in vitro transcription , an EBNA1 template was designed using the wild type HHV4 EBNA1 as a reference sequence ( NCBI accession YP_401677 . 1 ) .", "The reference sequence was modified by replacing the GA repeat region and domain B ( amino acids 90–375 ) with a second , tandem , chromatin-binding domain ( domain A , amino acids 27–89 ) , similar to what was done by Howden et al . ( 2006 ) .", "The nuclear localization signal ( amino acids 379–386 ) was removed and replaced with the sequence GRSS .", "Using the amino acid sequence as the starting template , the corresponding DNA sequence was generated by reverse translation and optimized for expression in human cell lines using Genscript's OptimumGene codon algorithm .", "This sequence was synthesized by Genscript ( Piscataway , New Jersey ) and provided in the pUC57 cloning vector; the EBNA1 coding sequence was subcloned into pcDNA3 . 1+ ( Life Technologies ) using the restriction enzymes BamHI and HindIII .", "Capped and poly ( A ) mRNA transcripts were generated using the mMESSAGE mMACHINE T7 ULTRA kit ( Life Technologies ) with 1 μg of BamHI linearized pcDNA3 . 1+EBNA1 as the template .", "The plasmids encoding the wild type and modified EBNA1 sequences have been deposited to Addgene as plasmid ID#s 59 , 199 and 59 , 198 for the wild type and modified sequences respectively .", "iPSC colonies were cultured on MEF for 4–6 days and fixed using PBS containing 4% PFA ( Santa Cruz Biotech ) for 15 min at room temperature .", "After rinsing with PBS , fixed cells were blocked and permeabilised for 1 hr in PBS containing 0 . 3% triton and 5% BSA .", "Primary antibodies: OCT3/4 ( SC-5279 ) , SOX2 ( SC-17320 ) , NANOG ( SC-33759 ) , SSEA-4 ( SC-21704 ) , and Tra-1-81 ( SC-21706 ) , all from Santa Cruz Biotech , were diluted 1:100 in blocking solution .", "Fixed cells were incubated with the primary antibody solution overnight on a rocker at 4°C .", "After washing out the primary antibody solution , fixed cells were incubated with secondary antibodies ( labeled with either Alexa-488 or Alexa-594 , 1:400 , Life Technologies ) diluted in blocking for 1 hr on a rocker at room temperature .", "Nuclei were counterstained using 1 μg/ml Hoechst 33 , 342 ( ThermoFisher Scientific , Waltham , Massachussets ) .", "All fluorescence imaging was conducted using an AMG EVOS FL ( Life Technologies ) .", "RNA was extracted using Qiagen RNA miniprep columns from cell pellets collected from fibroblasts , day 7 post transfection and feeder free ( Matrigel and Essential 8 ) iPSC lines at passage 10 or higher for both the retroviral and episomal reprogrammings; 1 μg of total RNA was reverse transcribed using the Maxima first strand cDNA synthesis kit ( ThermoFisher Scientific ) .", "Quantitative PCR was performed using a 1:96 dilution of cDNA and SYBR Select master mix ( Life Technologies ) with both forward and reverse primers at a concentration of 0 . 2 μM .", "Data was collected and analysed using the Viia7 ( Life Technologies ) .", "Primer sequences are shown in Supplementary file 2 , exogenous gene expression melt curves are shown in Figure 1—figure supplement 5 .", "Colonies growing on MEF were detached using Dispase/Collagenase IV ( 1 mg/ml each; both from Life Technologies ) in DMEM/F12 and grown as a suspension culture on low adherent plates using hESC media without bFGF .", "After 1 week of suspension growth , cells were transferred to 12 or 24-well plates coated with 0 . 1% gelatin and grown in DMEM supplemented with 20% FBS , 0 . 1 mM nonessential amino acids , 2 mM GlutaMAX , 1% Pen/Strep and 64 μg/ml L-Ascorbic acid 2-phosphate sesquimagnesium salt hydrate .", "Embryoid bodies were grown for 1–2 weeks prior to fixation and immunofluorescence staining .", "Cultures were fixed and stained as described above using the following antibodies: AFP ( 1:200 , SC-130302 , Santa Cruz Biotech ) , FOXA2 ( 1:200 , SC-6554 , Santa Cruz Biotech ) , α-smooth muscle actin ( 1:1500 , CBL171 , EMD Millipore , Billerica , Massachussets ) and MAP2 ( 1:200 , sc-20172 and sc-74420 , Santa Cruz Biotech ) .", "To test for genomic integration and residual retention of episomal plasmids , each iPSC line was migrated to feeder free conditions and grown beyond passage 15 on hESC-qualified Matrigel ( 1:100 dilution , BD Sciences ) coated plates in Essential 8 media ( Life Technologies ) .", "DNA was extracted from feeder free cultures using DNeasy Blood and Tissue Kits ( Qiagen , Valencia , California ) .", "PCR was performed using 100 ng of genomic DNA , an annealing temperature of 72°C and 25 cycles using primers designed to amplify a region common to all episomal vectors used ( Supplementary file 2 ) .", "Genomic DNA ( 100 ng ) isolated from day 7 cultures , and 1 pg of each episomal vector were used as positive controls .", "PCR products were run on a 1% agarose gel and visualised using ethidium bromide .", "After 15 passages on MEF and hESC media , cells were migrated to 1:100 hESC Matrigel ( BD Sciences ) and maintained on Essential 8 media for upwards of 6 passages .", "Feeder-free adapted cells were sent to Cell Line Genetics Inc ( Madison , WI ) for karyotyping as described in Meisner and Johnson ( 2008 ) .", "In vivo developmental potential of the reprogrammed cell lines was examined .", "Monolayer iPSCs from three chimpanzee lines were grown on Matrigel ( 1:100 ) in E8 medium ( Life Technologies ) and collected by EDTA treatment ( Life Technologies ) .", "Cells were counted and resuspended at a ratio of 1:1 cell volume to Matrigel and kept on ice until the injection .", "6-week-old CB17 . Cg-PrkdcscidLystbg-J/Crl immunodeficient male mice were obtained ( Charles River Laboratories , Wilmington , Massachussets ) and approximately 1 million iPSCs for each clone were injected into the testis-capsule .", "After 5–8 weeks teratomas were isolated , weighed , measured , dissected , and fixed in 10% formalin .", "The specimens were embedded in paraffin , stained with hematoxylin and eosin , and analyzed by a histopathologist .", "All animal work was conducted under the approval of the Institutional Care and Use Committee of UCSD ( Protocol# S09090 ) .", "In addition , live feeder free iPSC cultures maintained in Essential 8 media on Matrigel iPSCs from C4955 ( passage 15 + 7 ) were provided to Applied Stem Cell Inc . ( Menlo Park , CA ) for teratoma analysis as previously described ( Chen et al . , 2012 ) .", "DNA was extracted from frozen teratoma tissue using DNeasy Blood and Tissue Kits ( Qiagen ) .", "For teratomas derived from individual C4955 , core sections were isolated from FFPE embedded teratomas tissue using a 3 mm dermal punch tool; DNA was extracted from core samples using a QIAamp DNA FFPE Tissue Kit ( Qiagen ) .", "PCR was performed using universal mitochondrial primers ( [Kocher et al . , 1989] Supplementary file 2 ) amplifying cytochrome b ( Cytb , chimpanzee reference sequence NC_001643:bp 14 , 233–14 , 598 ) or the 12S ribosomal gene ( 12S , NC_001643:bp 484–915 ) with 250–500 ng of genomic DNA as the starting template .", "Two-step PCR was conducted with an annealing temperature of 50°C for 1 min and an extension step at 72°C for 4 min for a total of 30 cycles .", "DNA was purified using a Wizard SV gel and PCR Clean-up kit ( Promega , Madison , Wisconsin ) ; dye terminator cycle sequencing was conducted by the University of Chicago Comprehensive Cancer Center using 60 ng of purified PCR template and 4 μM of either the forward or reverse primer .", "Alignment to the chimpanzee , human ( NC_012920 ) and mouse ( NC_005089 ) reference sequences was accomplished using CLC Main Workbench 6 . 9 ( Qiagen ) and MUSCLE ( Edgar , 2004 ) .", "In order to demonstrate that chimpanzee iPSCs can be directly differentiated into other cell types , we differentiated C2 iPSC into hepatocytes and C7 into cardiomyocytes using the published protocols of Cheng et al . ( 2012 ) and Lian et al . ( 2013 ) respectively , with the following modifications: In both cases we plated iPSCs at 0 . 35 × 106 cells/cm2 in 0 . 44 ml/cm2 and cultured them in Essential 8 media 24 hr prior to initiating all differentiations .", "To increase hepatocyte differentiation efficiency , 1 μM of sodium butyrate was added during the first 24 hr of differentiation .", "After 24 days of differentiation , cells were immunostained as described above with a primary antibody for albumin ( 1:200 , A6684 , Sigma Aldrich; Figure 2—figure supplement 2 ) .", "After 10 days of differentiation , differentiated C7 cultures were enriched for cardiomyoctes by culture in RPMI based media without glucose supplemented with 5 mM sodium DL-lactate for 10 days as described previously ( Tohyama et al . , 2013; Burridge et al . , 2014 ) .", "After day 20 purified cardiomyocytes were cultured in media lacking glucose supplemented with 10 mM galactose ( Rana et al . , 2012 ) .", "After 25 days of cardiac differentiation , we characterized calcium flux in and out of iPSC-derived cardiomyocytes by treating cultures with 5 μM Fluo-4 AM ( F-14217 , Life Technologies ) for 15 min , washing cultures once and imaging them with an AMG EVOS FL microscope ( Video 1 ) .", "RNA from passage ≥15 iPSCs was extracted using the Qiagen RNeasy kit according to the manufacturer's instructions .", "Quality of the extracted RNA was assessed using an Agilent ( Santa Clara , California ) Bioanalyzer 2100 ( RIN scores for all samples ranged from 9 . 9 to 10 ) , and RNA was processed into biotinylated cRNA and hybridized to the HT12v4 array using standard Illumina ( San Diego , California ) reagents as directed by the manufacturer .", "Arrays were scanned using an Illumina HiScan , and data processed using Illumina's GenomeStudio software .", "Using these data , we carried out PluriTest as previously described ( Müller et al . , 2011 ) .", "Additionally , we mapped all detected HT12v4 probe sequences ( n = 46 , 297 ) to the chimpanzee ( panTro3 ) genome using BWA 0 . 6 . 3 ( Li and Durbin , 2009 ) .", "Probes that mapped to a single genomic location with no mismatches were retained ( n = 21 , 320 , 46 . 2% of all probes ) for the analysis that was restricted only to the chimpanzee lines .", "When we considered data from human and chimpanzee iPSCs together , without excluding probes based on sequence matches to the chimpanzee genome , all chimpanzee lines in the panel had pluripotency scores slightly below the pluripotency threshold ( Figure 3—figure supplement 1 , lighter points ) .", "However , low pluripotency scores could stem from differences in our ability to estimate gene expression levels in the chimpanzee compared to the human due to attenuated hybridization caused by sequence divergence ( Gilad et al . , 2005 ) .", "Indeed , when we subset the array to retain only those detected probes that map to the chimpanzee genome with no ambiguity or mismatches , all chimpanzee lines have pluripotency scores greater than the pluripotency threshold value of 20 ( Figure 3—figure supplement 1 , darker points ) .", "50 bp single-end RNA sequencing libraries were generated from RNA extracted from 7 chimpanzee and 7 human iPSC lines using the Illumina TruSeq kit as directed by the manufacturer , as well as from their precursor fibroblast or LCL cell lines .", "All iPSC samples were multiplexed and sequenced on four lanes of an Illumina HiSeq 2500; while the precursor cell lines were multiplexed and sequenced on six lanes of the same sequencer .", "We generated a minimum of 28 , 010 , 126 raw reads per sample ( Supplementary file 6 ) , and confirmed the raw data were of high quality using FastQC ( available online at http://www . bioinformatics . babraham . ac . uk/projects/fastqc/ ) .", "We mapped raw reads to the chimpanzee ( panTro3 ) or human ( hg19 ) genome as appropriate using TopHat 2 . 0 . 8 ( Trapnell et al . , 2009 ) , allowing for a maximum of 2 mismatches in each read .", "Due to the relatively poor annotation of the chimpanzee genome and to prevent biases in expression level estimates due to differences in mRNA transcript size and genetic divergence between the two species , we limited the analysis to reads that mapped to a list of orthologous metaexons across 30 , 030 Ensembl genes drawn from hg19 and panTro3 , as in Blekhman et al . ( 2010 ) .", "Following mapping , gene level read counts were generated using featureCounts 1 . 4 . 4 as implemented in Subread ( Liao et al . , 2013 ) .", "Due to mapping biases between human and chimpanzee ribosomal proteins and pseudogenes , we removed all genes associated with the Gene Ontology Cellular Compartment category ‘ribosome’ ( GO:0005840 , n = 141 ) and all annotated pseudogenes in Ensembl release 65 ( n = 3170 , December 2011 , the oldest available archival version of Ensembl ) from the data at this point .", "We considered two normalization approaches in our analysis .", "In one instance , we examined only RNA-sequencing data from chimpanzee and human iPSCs , and retained 12 , 171 genes with at least 4 observations in one of the two species of log2 CPM > 1 .", "CPM were then loess normalized by species within individuals with voom ( Law et al . , 2014 ) .", "As the orthologous genes are not constrained to be the same length in both species , we computed RPKM for each gene before carrying out any inter-species comparisons .", "We then used the R/Bioconductor package limma 3 . 20 . 3 ( Smyth , 2004 ) to test for differential expression in our RNA-seq data , with a model that included only a species effect .", "Finally , we tested for an enrichment of GO categories amongst DE genes using the R package topGO 2 . 16 . 0 ( Alexa et al . , 2006 ) .", "These normalised values were used only to identify genes DE between iPSCs of the two species .", "For the dataset containing RNA-sequencing data from iPSCs and their precursors , we again only retained 13 , 147 genes with at least 4 observations in one of the four groups ( chimpanzee iPSCs , chimpanzee precursors , human iPSCs or human precursors ) of log2 CPM > 1 .", "Gene counts were then loess normalised within individuals by tissue , after correcting for the lack of independence within different tissues from the same individual , through the function corfit .", "As above , we then computed species-specific RPKM values , and used limma and topGO to test for differential expression and GO category enrichment , respectively .", "In this instance , we used a model design with 6 parameters for the main effect ( chimpanzee iPSC , human LCL-derived iPSC , human fibroblast-derived iPSC , chimpanzee fibroblast , human LCL and human fibroblast ) and no additional covariates .", "To confirm that our conclusions are robust with respect to the choice of normalization procedure , in both cases , we also tried a variety of other normalization schemes , including correcting for %GC content as in Risso et al . ( 2011 ) , none of which had a substantial effect on the final results ( Supplementary file 6 ) .", "Finally , we built neighbor joining trees using Manhattan distances calculated from RPKM values at all 13 , 147genes using the nj function in the R library ape ( Paradis et al . , 2004 ) .", "All analyses were performed at a false discovery rate ( Benjamini and Hochberg , 1995 ) threshold of 1% unless otherwise noted , using R 3 . 1 . 0 ( R Development Core Team , 2013 ) and Bioconductor 2 . 14 ( Gentleman et al . , 2004 ) .", "To analyze DNA methylation , we extracted DNA from all chimpanzee and human iPSC lines described above , as well as from the source fibroblast or LCLs .", "In all cases , 1000 ng of genomic DNA were bisulphite-converted and hybridized to the Infinium HumanMethylation450 BeadChip at the University of Chicago Functional Genomics facility as directed by the manufacturer .", "Since the probes on the array were designed using the human reference genome , we followed the approach described in Hernando-Herraez et al . ( 2013 ) to compare humans and chimpanzees .", "We retained those probes that had either a perfect match to the chimpanzee reference genome , or had 1 or 2 mismatches in the first 45 bp but no mismatches in the 3′ 5 bp closest to the CpG site being assayed .", "We also removed all probes that contained human SNPs ( MAF ≥0 . 05 ) or chimpanzee SNPs ( MAF ≥0 . 15 ) within the last 5 bp of their binding site closest to the CpG being assayed .", "Within each individual , probes with a detection p > 0 . 01 were excluded .", "This resulted in the retention of 335 , 307 autosomal probes , and an additional 8210 X chromosome probes , which we normalized and analyzed separately by sex .", "In all cases we performed a two-color channel signal adjustment , quantile normalization and β-value recalculation as implemented in the lumi package ( Du et al . , 2008 ) .", "Because the HumanMethylation450 BeadChip contains two assay types which utilize different probe designs , we performed a BMIQ ( beta mixture quantile method ) normalization ( Teschendorff et al . , 2013 ) on the quantile-normalized autosomal data set .", "We did not perform this step on the X chromosome data , due to its methylation patterns .", "We built neighbor joining trees using Manhattan distances at all 335 , 307 probes using the nj function as above .", "In order to identify DM probes we used an identical approach to that described above for the identification of DE genes .", "First , we identified probes that were DM between the iPSCs of both species using limma by using a reduced data set and model containing only data from the iPSCs themselves .", "Then , we fit a linear model to the data using limma with 6 parameters corresponding to the 6 tissue/species combinations in the data , classifying probes as DM at an FDR of 1% .", "As with the expression data , the reduced model has more power to identify DM probes between the two iPSC groups than the full model; however , there is great concordance between the two sets of results ( Figure 7—figure supplement 5 ) .", "We excluded all probes with mean β inter-group differences <0 . 1 in order to group DM probes into DMRs , which we define as 2 or more DM probes separated by <1 kb , with the additional requirement that the effect be in the same direction in all DM probes within the region .", "Finally , to examine the content of these DMRs , we used annotation files for the HumanMethylation450 Bead Chip provided by the manufacturer and discarded all DMRs associated with either multiple or no genes .", "We tested for enrichment of GO BP categories amongst the genes contained in the DMRs by using the R package topGO 2 . 16 . 0 ( Alexa et al . , 2006 ) , using as a background set all genes in which it is theoretically possible to detect DMRs .", "ChIP-seq assays were performed as previously described ( Schmidt et al . , 2009 ) , with slight modifications .", "Specifically , approximately 60 million iPSCs from three chimpanzee individuals ( C2 , C5 and C7 ) were cross-linked with 1% formaldehyde for 10 min .", "Cells were lysed and chromatin sheared with a Covaris S2 ( settings: 4 min , duty cycle 10% , 5 intensity , 200 cycles per burst in 4 6 × 16 mm tubes per individual ) .", "H3K27ac- and H3K27me3-enriched regions were isolated using 5 μg of either H3K27ac antibody ( ab4729 , Abcam , Cambridge , MA , USA ) or H3K27me3 antibody ( 07–449 , Millipore , Billerica , MA , USA ) .", "ChIP and input DNA from each individual were end-repaired , A-tailed and ligated to Illumina Truseq sequencing adapters before 18 cycles of PCR amplification .", "200–300 bp DNA fragments were selected for sequencing .", "Input libraries were multiplexed and sequenced on one lane of an Illumina HiSeq2500 using the rapid run mode , ChIP libraries were multiplexed and sequenced on three lanes of an Illumina HiSeq2500 using the rapid run mode .", "For comparison purposes , we downloaded ChIP input , H3K27ac and H3K27me3 data from 3 human iPSC lines ( iPS 6 . 9 , iPS-18a , and iPS . 20b , all of them release 5 ) generated by the Roadmap Epigenomics Consortium ( Roadmap Epigenomics Consortium et al . , 2015 ) from the NIH GEO database ( Supplementary file 6 ) .", "Human and chimpanzee samples were mapped to either hg19 or panTro3 using BWA 0 . 7 . 9 ( Li and Durbin , 2009 ) ; reads that mapped outside chromosomes 1–22 + X were discarded , as were reads that did not map uniquely to a single genomic region with less than 2 mismatches , or reads that were marked by Picard ( http://picard . sourceforge . net ) as originating from PCR duplicates .", "After mapping and filtering , we used MACS 1 . 4 . 4 ( Zhang et al . , 2008 ) and RSEG 0 . 4 . 4 ( Song and Smith , 2011 ) to identify peaks in the H3K27ac and H3K27me3 data respectively .", "Our analyses in this section follow those of Zhou et al . ( 2014 ) .", "Briefly , for MACS , we specified an initial p-value threshold of H3K27ac , 0 . 001 , and used each line's ChIP input file for comparison .", "For RSEG , we used the ‘rseg-diff’ function to compare H3K27me3 enrichment against each individual's ChIP input file , with the recommended 20 maximum iterations for hidden Markov model training .", "We then filtered enriched regions or peaks identified by either program by retaining only those that overlapped a previously defined set of 200 bp orthologous windows ( Zhou et al . , 2014 ) , where at least 80% of bases are mappable across species using liftOver .", "We define mappability as the ability of each 20 bp kmer beginning in that window to be uniquely mapped to the genome .", "To ensure that sequence divergence did not confound our analyses , we mapped each identified region or peak in humans to the chimpanzee genome , and vice versa , using liftOver , and excluded regions and peaks where 80% or greater of bases in the enriched peaks or regions failed to align to the other genome .", "To further minimise the number of false positive results in our interspecies comparison ( due to incomplete power ) , we applied a two-step cutoff ( Cain et al . , 2011 ) to the list of enriched regions and peaks .", "For H3K27ac , we retained all peaks that were identified with a first , stringent cutoff of FDR < 5% in one species and a , second , relaxed cutoff of FDR < 15% in the other , as in Zhou et al . ( 2014 ) .", "Because RSEG does not report FDR values for enriched regions , we used each region's domain score , which is the sum of the posterior scores of all bins within the domain , and set a first , stringent cutoff of 20 in one species , and a second , relaxed threshold demanding only that the region be classified as ‘enriched’ by RSEG , without a specific score requirement .", "Having done this , we integrated data from multiple peaks ( when present ) to generate a gene-level metric of ChIP signal in each individual .", "Specifically , we computed an enrichment score for each histone mark in each individual in a set of previously defined 26 , 115 orthologous TSSs ( Zhou et al . , 2014 ) by dividing RPKM values at each TSS at gene i for either mark minus RPKM values in TSS at gene i for ChIP input , all of it over the genome-wide average RPKM for either mark minus the genome-wide average RPKM for ChIP input .", "Given the way in which we have defined this enrichment score , a score >0 indicates those genes where we detected more histone mark reads than input reads , while a score >1 indicates a gene with an excess of histone mark reads greater than what we would expect given the genome-wide distribution .", "Because 8 of the 22 genes in the list of pluripotency master genes used to generate Figure 5D do not have clearly defined orthologous TSSs , we also examined whether MACS identified peaks in the 2 kb ± TSS for all 22 genes and their orthologous position in the chimpanzee genome , identified solely through liftOver—that is , without taking into account whether there is evidence for a TSS at that position in the chimpanzee genome .", "To generate Figure 5—figure supplement 3 , we simply asked how many of the 22 genes had at least 1 peak at an FDR < 5% in at least 1 individual in either species , regardless of orthology and sequence conservation .", "We note that since different labs produced the human and chimpanzee data , we expect a considerable technical batch effect to be completely confounded with species annotation .", "Given this study design , we expect the technical batch effect to result in the appearance of inter-species differences; yet , our goal is to demonstrate similarity across species .", "Thus , our conclusions ( of high overlap across species ) , are conservative with respect to the technical batch effect .", "To examine the possible consequences of reduced expression of REX1 in chimpanzee iPSCs , we retrieved genes that responded to a REX1 knockdown in mESCs from Supplementary files 2 , 3 of Scotland et al . ( 2009 ) and converted Affymetrix MG 430 2 . 0 probe IDs to ENSM and ultimately to orthologous ENSG identifiers using Biomart release 66 ( to control for deprecated identifiers ) .", "Because Masui et al . ( 2008 ) ; Scotland et al . ( 2009 ) ; Son et al . ( 2013 ) have highlighted REX1's function in controlling cell cycle progression , glycolysis and cellular differentiation , we additionally retrieved genes associated with these terms to generate Figure 6C as follows: The core set of pluripotency TFs are those described by Orkin and Hochedlinger ( 2011 ) and Ng and Surani ( 2011 ) .", "Cell cycle and glycolysis categories contain all genes associated with GO BP:0007049 and BP:0006096 respectively , whereas cell fate contains genes associated with any GO term that contains the words ‘ectoderm’ , ‘mesoderm’ or ‘endoderm’ .", "We also examined individual examples of cell fate differentiation: CNS development genes are associated with BP:0007417 or any of its offspring; cardiovascular system development genes are associated with BP:0072358 or any of its offspring; hepatobiliary system development genes are associated with BP:0055123 or any of its offspring .", "Confidence intervals around the null hypothesis were generated independently for each category from 100 , 000 permutations in R . Finally , to compare our data with a previously published set of chimpanzee and bonobo iPSC lines ( Marchetto et al . , 2013b ) , we downloaded fastq files from GEO ( Series GSE47626 ) and mapped only the first mate from all reads using the same approach as above , but allowing 4 mismatches in the entire 100 bp read .", "We normalised expression estimates jointly with our own cell lines to generate Figure 6—figure supplement 2 , and used all data points from a given species , irrespective of origin , to generate boxplots .", "To generate figure 6—figure supplement 3 , we took Illumina HT12v4 array data from 73 human iPSC lines generated in house and calculated Pluritest pluripotency and novelty scores as above , using the full probe set .", "Independently , we normalised and background-corrected the raw array intensities using the lumiExpresso function in lumi and extracted expression values for all 73 human iPSC lines at the single array probe associated with REX1 .", "In order to examine REX1 methylation levels in other human PSCs , we obtained Infinium HumanMethylation450 BeadChip for the lines reported in Ziller et al . ( 2011 ) from the authors , and normalised it jointly with our own data as described above .", "We then extracted normalised methylation β levels at the 13 probes that map to REX1 in both chimpanzees and humans to generate Figure 6—figure supplement 4 .", "Finally , we assessed two broad indicators of stability in our chimpanzee lines .", "All iPSC lines derived from female chimpanzees , and 3 of 4 lines derived from human females , show strong evidence for elevated expression of XIST relative to male lines ( FDR-adjusted p = 0 . 0010; Figure 7—figure supplement 6 ) and maintenance of X-chromosome inactivation during pluripotency .", "X-chromosome methylation patterns in females corroborate these observations , with the majority of probes mapping to the X-chromosome in our data being either hemimethylated ( 0 . 2 < β < 0 . 8 ) or hypermethylated ( β ≥ 0 . 8 ) in females but not in males ( Figure 7—figure supplement 7 ) .", "We also used a list of 168 imprinted probes from Ma et al . ( 2014 ) to check for maintenance of genomic imprinting after reprogramming .", "We find that the majority of imprinted loci remain hemimethylated following reprogramming in both human and chimpanzee iPSC lines ( Figure 7—figure supplement 8 ) .", "However , we identify two sets of probes that are consistently hypermethylated in pluripotent lines but were hemimethylated in their precursor cells .", "The first cluster contains 5 probes that are hypermethylated across both chimpanzee and human iPSCs; these probes are associated with the genes KCNK9 , ANKRD11 and MKRN3 .", "The second cluster is comprised of 21 probes that are hypermethylated in all human iPSCs but only 2 chimpanzee iPSCs in our data , and is associated with the gene PEG3-ZIM2 , which has been previously shown to be abnormally methylated in both hESCs and hiPSCs ( Lund et al . , 2012 ) .", "All novel RNA-sequencing , DNA methylation and ChIP-seq data are available at the GEO under SuperSeries number GSE61343 .", "Additionally , a table with p-values for all hypothesis testing performed using the methylation data ( by probe ) is available on the Gilad lab website ( http://giladlab . uchicago . edu/Data . html ) .", "All chimpanzee iPSC lines described in this publication are available fully and without restrictions to other investigators upon request to the corresponding authors ." ] ]

[ "Comparative genomics studies in primates are restricted due to our limited access to samples .", "In order to gain better insight into the genetic processes that underlie variation in complex phenotypes in primates , we must have access to faithful model systems for a wide range of cell types .", "To facilitate this , we generated a panel of 7 fully characterized chimpanzee induced pluripotent stem cell ( iPSC ) lines derived from healthy donors .", "To demonstrate the utility of comparative iPSC panels , we collected RNA-sequencing and DNA methylation data from the chimpanzee iPSCs and the corresponding fibroblast lines , as well as from 7 human iPSCs and their source lines , which encompass multiple populations and cell types .", "We observe much less within-species variation in iPSCs than in somatic cells , indicating the reprogramming process erases many inter-individual differences .", "The low within-species regulatory variation in iPSCs allowed us to identify many novel inter-species regulatory differences of small magnitude ." ]

[ "Comparing the genomes of different species can reveal how they are related to one another .", "Such comparative studies can also reveal how genomes are modified in species-specific ways to regulate gene activity .", "The genomes of humans and chimpanzees are very similar in sequence .", "It is therefore likely that differing patterns of gene regulation underlie many of the differences observed between the two species .", "However , only a few kinds of chimpanzee cell that can be grown in the laboratory are available for research; this lack of samples has limited the ability of researchers to perform such comparative studies .", "One way around this problem is to use induced pluripotent stem cells ( or iPSCs ) .", "IPSCs are created by exposing mature cells—for example , skin cells—to conditions and molecules that convert them into an embryonic-like state .", "This state—called ‘induced pluripotency’—allows the cells to be coaxed into becoming many different cell types that can be grown in the laboratory .", "But it is more difficult to establish high quality iPSCs from chimpanzees than it is from humans or mice .", "Gallego Romero , Pavlovic et al . have now addressed this problem by creating iPSCs from skin cells taken from seven healthy chimpanzees .", "These cell lines were then analysed and compared to each other and to seven iPSC lines created from human cells .", "The chimpanzee iPSC lines were found to be much more similar to each other than the mature cells that were used to make them .", "Similar results were also observed for the human iSPCs , which likely reflects the conserved changes that take place when the genomes of mature cells are reprogrammed to pluripotency .", "This high level of similarity between iPSCs from different individuals of the same species allowed Gallego Romero , Pavlovic et al . to discover many subtle differences in gene regulation between chimpanzees and humans .", "For example , over 4500 genes were found to be expressed differently in human and chimpanzee iPSCs , and over 3500 genomic regions had different patterns of certain DNA modifications that can help to regulate gene expression .", "These newly created chimpanzee iPSC lines represent a valuable resource for comparative studies of gene regulation .", "In the future , this resource could help researchers to identify further differences in gene regulation between closely related primate species ." ]

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

[ "ecology", "evolutionary biology" ]

[ [ "An individual’s health , survival , and fertility can be profoundly shaped by its early life environment ( Uller et al . , 2013 ) .", "For example , in humans , low early life socioeconomic status predicts increased risk of mortality and many measures of poor health in adulthood ( Naess et al . , 2004; Beebe-Dimmer et al . , 2004; Kittleson et al . , 2006; Smith et al . , 1998; Frankel et al . , 1999; Lidfeldt et al . , 2007; van de Mheen et al . , 1998; Kuh et al . , 2002; Galobardes et al . , 2004 ) .", "Similarly , several studies of wild mammals and birds ( Lea et al . , 2015; Douhard et al . , 2014; Nussey et al . , 2007; Pigeon and Pelletier , 2018; Herfindal et al . , 2015; Balbontín and Møller , 2015; Millon et al . , 2011 ) find that adult fecundity is reduced in animals that experienced adverse early life environments , and some have also found an effect of early life adversity on adult survival ( Nussey et al . , 2007; Pigeon and Pelletier , 2018; Herfindal et al . , 2015; Tung et al . , 2016 ) .", "If the effects of early adversity extend to the descendants of exposed individuals , the epidemiological and evolutionary impact of these effects would be further amplified .", "However , in humans , evidence that intergenerational effects stem directly from parental experience is mixed , as studies have produced somewhat contradictory results ( Veenendaal et al . , 2013; Painter et al . , 2008; Kaati et al . , 2007; Pembrey et al . , 2006 ) .", "For example , a study of the historical Överkalix population in Sweden identified strong , contrasting effects of grandparents’ exposure to early-life food scarcity on grand-offspring survival , depending on small differences in the age at which the grandparent was exposed to scarcity ( Pembrey et al . , 2006 ) .", "Similarly , two studies of a population that was exposed in utero to the Dutch hunger winter ( a famine that resulted from a German blockade of the Netherlands during the winter of 1944–1945 ) found contradictory , sex-specific intergenerational effects , in one case suggesting an intergenerational effect that depended only upon the mother’s early experience ( Painter et al . , 2008 ) , and in the other case an effect that depended only upon the father’s early experience ( Veenendaal et al . , 2013 ) .", "Compelling evidence for intergenerational effects of early adversity faced only in the parental generation comes from numerous laboratory studies of short-lived animals , which find strong relationships between a female’s early life environment and the body size of her offspring ( Huck et al . , 1986; Alonso-Alvarez et al . , 2007; Helle et al . , 2012; Goerlich et al . , 2012; Taborsky , 2006; Beckerman et al . , 2003; Saastamoinen et al . , 2013; Vijendravarma et al . , 2010; Fischer et al . , 2003; Jobson et al . , 2015; reviewed in Burton and Metcalfe ( 2014 ) ; but see Bowers et al . , 2017 for a rare example in the wild on house wrens ) .", "These findings provide important evidence that intergenerational effects of early adversity can occur .", "However , these studies do not address whether intergenerational effects of early adversity , independent of parent-offspring environmental correlations , occur in natural populations of long-lived animals .", "And while a few studies of short-lived captive animals have demonstrated a relationship between a female’s early environment and her offspring’s survival or reproduction ( Huck et al . , 1987; Naguib et al . , 2006; Marcil-Ferland et al . , 2013 ) , the ecological validity of these findings has yet to be verified by studying intergenerational fitness effects in a population of wild and/or long-lived animals .", "In wild populations , animals are exposed only to natural , unmanipulated levels of early adversity , and are also subject to any social factors which might mitigate or aggravate the influence of those early adverse events .", "Addressing whether the effects of early adversity in one generation affect reproduction or survival in the next is challenging because of the difficulties of linking high-quality data on early adversity in one generation to health and survival outcomes in the next .", "Here , we overcome these challenges by taking advantage of a prospective longitudinal dataset from a natural primate population: the baboons of the Amboseli ecosystem in southern Kenya ( Alberts and Altmann , 2012 ) .", "This dataset includes 45 years of individual-based data on early adversity , and real-time observations of later-life survival outcomes for hundreds of subjects with known maternities and grand maternities .", "Moreover , unlike many human populations , we do not observe inter-generational transmission of adverse conditions; that is , offspring of females who experienced early life adversity are not more likely to experience early life adversity themselves ( except in the case of inheritance of low social rank , see below ) , allowing us to avoid this common confound in human societies .", "To test for intergenerational effects of early adversity , we focused on early adversity experienced by female baboons who later became mothers , and whose offspring were also in our dataset .", "We asked whether the early adversity experienced by these females ( ‘maternal early adversity’ ) predicted the survival of their juvenile offspring in the next generation , after controlling for the early adversity directly experienced by the offspring themselves .", "We considered five types of early adverse conditions ( Table 1 ) , based on previous work in our study population that demonstrated effects of these conditions on a female baboon’s own adult survival ( Tung et al . , 2016 ) .", "These included:", "( i ) maternal death during development ( 0–4 years of age ) , which indicates the loss of an important source of social support , physical protection , and nutrition ( Altmann , 1980; Lea et al . , 2014 ) ,", "( ii ) being born to a low-ranking mother , which influences growth rates and age at maturation ( Charpentier et al . , 2008; Altmann and Alberts , 2003a; Altmann et al . , 1988 )", "( iii ) being born into a large social group ( and thus experiencing high density conditions and high levels of within-group competition ) ( Lea et al . , 2015; Charpentier et al . , 2008; Altmann and Alberts , 2003b )", "( iv ) being born during a drought , which reduces fertility in adulthood ( Lea et al . , 2015; Beehner et al . , 2006 ) , and", "( v ) experiencing the birth of a close-in-age younger sibling , which may reduce maternal investment received during development ( Altmann et al . , 1978 ) .", "Importantly—and in contrast to research on humans ( Felitti et al . , 1998 ) —sources of early adversity are not strongly correlated in our population , which allows us to measure the independent effects of different sources of adversity ( Supplementary file 1 Table S1 ) ." ], [ "Our full multivariate Cox proportional hazards model for offspring survival ( Supplementary file 1 Table S2 ) included all nine early adverse conditions ( five for mothers and four for offspring ) .", "We found strong negative effects of two characteristics of the mother’s early life environment on their offspring’s survival during the first 4 years of life: maternal loss ( hazard ratio = 1 . 48 , p=0 . 006 ) and presence of a close-in-age younger sibling ( HR = 1 . 39 , p=0 . 03 ) .", "Following backwards model selection ( performed by removing the parameters with the highest p values until only predictors with a p-value<0 . 05 remained ) , these two characteristics remained the only significant maternal early life predictors of offspring survival ( Table 2 , Figure 1 , along with two conditions in the offspring’s early life environment: see below ) .", "Adding maternal age , offspring sex or interactions between maternal age or offspring sex and sources of maternal adversity did not improve the fit of the model ( Supplementary file 1 Tables S3-S5 ) .", "In sum , offspring whose mothers experienced early maternal loss experienced a 48% higher probability of dying throughout the first four years of life than unaffected offspring , and offspring whose mothers had a close-in-age sibling experienced a 39% higher probability of dying than unaffected offspring .", "This effect is striking especially considering that a median of 7 . 0 and 8 . 0 years separated the offspring’s own birth from the mother’s experience of maternal loss or birth of a close-in-age sibling , respectively .", "A similar pattern holds if mothers , rather than offspring , are treated as the unit of analysis: mothers who experienced early adversity have lower average offspring survival than mothers who did not ( see Figure 1c ) .", "Notably , previous work in our population found that these two sources of adversity—maternal loss and the presence of a close-in-age younger sibling during early life—are also sources of mortality risk once females reach adulthood , and in fact are the two strongest predictors of adult survival among six different early-life conditions considered ( Tung et al . , 2016 ) .", "Hence , early-life conditions that are especially adverse for females when they reach adulthood also negatively affect the survival of their offspring .", "Both the full and reduced models of offspring survival also included two conditions in the offspring’s early life environment as significant predictors of juvenile survival .", "Specifically , maternal loss experienced by the offspring and low maternal rank during the offspring’s juvenile period had strong negative effects on offspring survival ( Supplementary file 1 Table S2 , maternal death: Hazard Ratio = 1 . 95 [1 . 51–2 . 54] , p=5×10−7 , low maternal rank: Hazard Ratio = 1 . 43 [1 . 05–1 . 94] , p=0 . 025 ) .", "Thus , maternal loss in the offspring’s generation had a stronger effect on offspring survival ( nearly doubling offspring mortality risk ) than maternal loss in the mother’s generation .", "In contrast , the effect of having a low-ranking mother , which was associated with a 43% increase in offspring mortality risk , was comparable in its effect size to the two significant predictors from the maternal generation ( maternal loss and close-in-age sibling for the mother , 48% and 39% increase in offspring mortality , respectively ) .", "Thus , two adverse conditions in a mother’s early life had as large or larger of an impact on her offspring’s survival than all but one adverse condition experienced by the offspring directly .", "The strong effect of the mother’s death on offspring survival prior to four years ( Table 2 ) is unsurprising at first consideration: the most obvious explanation for this effect is that offspring depend upon their mothers , so that if the mother dies the offspring is also likely to die at the same time or die subsequently .", "Indeed , this sequence of events does occur in our population: of the 32 offspring that were alive and less than eight months old when their mother died , 31 ( 97% ) died before reaching one year of age .", "However , offspring death could also precede maternal death if it acts as a harbinger of the mother’s death , as opposed to a consequence of it .", "In this scenario , offspring mortality risk is increased because their mothers are in poor condition and hence unable to provide adequate care or resources to the offspring .", "This hypothesis therefore proposes an alternative causal chain from poor maternal health to offspring death , which would occur while the mother is still alive .", "To examine whether this phenomenon occurs in our study population , we modeled offspring survival to age 2 years ( halfway through the juvenile period ) as a function of maternal death during years 2–4 after an offspring’s birth ( i . e . , the two years that followed the offspring survival period modeled in the response variable ) .", "In this analysis , we considered only the subset of offspring in our dataset whose mothers survived the entire first two years of the offspring’s life , and for whom we were able to evaluate the four significant predictors of offspring survival identified above and in Table 2 ( N = 671 ) .", "Our results were striking: offspring were less likely to survive during the first two years of life if their mothers died 2–4 years after their birth .", "In other words , these offspring were more likely to die even when their mother was still present ( hazard ratio = 1 . 50 [1 . 01–2 . 23] , p=0 . 045 ) .", "To test for a role of maternal early adversity in this effect , we next partitioned our analysis of offspring survival to age two based on whether the mother experienced either maternal loss or a close-in-age younger sibling ( i . e . , either or both of the two maternal early life conditions that significantly predicted their offspring’s survival; Table 2 ) .", "We found that , among offspring whose mothers experienced either or both of these two adverse events ( N = 247 ) , maternal death in years 2–4 after the offspring’s birth significantly predicted reduced offspring survival to age 2 years ( Figure 2a , hazards ratio = 1 . 78 , 95% CI = [1 . 05–3 . 01] , p=0 . 034 ) .", "Maternal death in the same period did not , however , predict reduced offspring survival when mothers had not experienced maternal loss or a close-in-age younger sibling ( N = 424; Figure 2b , hazard ratio = 1 . 21 , 95% CI = [0 . 7–2 . 2] , p=0 . 53 ) .", "Hence , the pattern we observed when analyzing the full data set of offspring that survived to age 2 ( N = 671 ) is completely driven by the offspring of mothers who experienced substantial early adversity .", "This finding is consistent with the hypothesis that maternal early life adversity results in compromised maternal condition in adulthood , which in turn results in both earlier death for adult females and a reduction in their ability to successfully raise offspring towards the end of their lives ( i . e . , a maternal effect on the offspring generation ) ." ], [ "We have demonstrated that adverse environmental conditions during the early life of a female baboon , which are already known to negatively affect both her survival ( Tung et al . , 2016 ) and her reproduction ( Lea et al . , 2015 ) in adulthood , also reduce the survival of her offspring .", "Importantly , this effect is independent of the environment experienced by those offspring themselves ( Figure 1 ) .", "The reduction in offspring survival is likely linked to reductions in maternal viability: mothers that experienced early life adversity are significantly less able to successfully raise offspring born near the ends of their lives , while the same is not true for mothers that did not experience early life adversity ( Figure 2 ) .", "Together , these findings support the hypothesis that early life adversity produces constraints during development that lead not only to reduced adult survival and lifetime reproductive success ( Tung et al . , 2016 ) but also to a reduced ability to successfully raise those offspring that are produced ( Figure 2a ) .", "We did not identify any sex-specific intergenerational effects of maternal early adversity .", "The results reported here help to fill a key gap in the literature concerning the intergenerational effects of early life adversity on survival .", "Human studies have yielded inconsistent results on this topic thus far when maternal and offspring environments are not correlated: different studies on the same populations have reported contradictory sex-specific effects on health ( Veenendaal et al . , 2013; Painter et al . , 2008 ) or have found that small differences in the age at which subjects’ parents or grandparents were exposed to adversity can lead to a reversal in the direction of these effects ( Kaati et al . , 2007; Pembrey et al . , 2006 ) .", "Among studies in non-human animals , several studies in fish ( Donelson et al . , 2008; Venturelli et al . , 2010 ) , reptiles ( Warner and Lovern , 2014 ) , birds ( Blomqvist et al . , 1997; Ridley , 2007 ) , and ungulates ( Cameron et al . , 1987; Théoret-Gosselin et al . , 2015; Keech et al . , 2000; Clutton-Brock et al . , 1987; Clutton-Brock et al . , 1984 ) have found that parental body condition at the time of offspring birth influences offspring survival , but none have linked parents’ early adverse experiences to offspring survival .", "Additionally , while previous studies have identified effects of parental early adversity on offspring traits in a limited number of captive , short-lived systems ( Burton and Metcalfe , 2014; Huck et al . , 1987; Naguib et al . , 2006 ) , ours is the first to link parental early adversity to an important component of offspring fitness in a wild , long-lived animal .", "Our findings help to explain the persistence of health deficits across generations ( Aizer and Currie , 2014; Kane et al . , 2018; Cnattingius et al . , 2012 ) , by revealing that in long-lived primates , the early life experiences of mothers have important implications for offspring health and survival .", "Recent studies in humans have demonstrated that conditions experienced by mothers during pregnancy ( e . g . , low SES , psychosocial stress , mood dysregulation , prenatal smoking ) can affect HPA axis regulation ( Thayer and Kuzawa , 2014; Entringer et al . , 2009 ) and birthweight ( Aizer and Currie , 2014; Kane et al . , 2018 ) in her offspring .", "These and other maternal characteristics present during pregnancy are influenced not only by mothers’ experiences in adulthood , but also by the long-term effects of environmental conditions experienced in mothers’ early lives ( Kane et al . , 2018; Kuzawa , 2005 ) .", "Our findings therefore motivate future work to test for comparable intergenerational fitness effects of early adversity in humans and other non-human animals .", "Our findings are consistent with the hypothesis that early adversity results in intergenerational developmental constraints ( Lea et al . , 2015; Grafen , 1988; Monaghan , 2008; Lea et al . , 2017a ) and are not consistent with an intergenerational predictive adaptive response hypothesis ( Monaghan , 2008; Gluckman et al . , 2005; Herman et al . , 2014 ) .", "Rather than being buffered against the effects of maternal loss , those offspring that experienced maternal loss and whose mothers had also experienced maternal loss were more likely , not less likely , to die , as compared to offspring that experienced maternal loss but whose mothers did not .", "Thus , individuals in the offspring generation experience constraints not only as a result of their own early environment , but also as a result of their mothers’ developmental histories , including events that occurred years before their own conception .", "Our results are consistent with the hypothesis that a female’s condition at the time of her offspring’s conception and/or birth reflects her previous experiences , and that her condition thereby influences the development and survival of her offspring ( Kuzawa , 2005; Kuzawa , 2017; Lea et al . , 2017b ) .", "Our study is unable to definitively identify the mechanism by which effects of early adversity may be transmitted from parent to offspring .", "However , our finding that reduced offspring survival appears to be partially mediated by reduced maternal viability suggests that the mode of transmission is most readily explained as a classic parental effect , in which early life adversity affects the phenotypic quality of the mother during adulthood , and in turn affects her offspring’s development ( Mousseau and Fox , 1998a; Mousseau and Fox , 1998b; Russell and Lummaa , 2009; Badyaev and Uller , 2009 ) .", "Recently , intergenerational transmission of adversity has been discussed as a potential consequence of inherited epigenetic changes ( Heard and Martienssen , 2014 ) .", "While we cannot exclude this possibility , our results are a reminder that simpler mechanisms—in this case , a classic maternal effect—may be a more parsimonious ( albeit non-mutually exclusive ) explanation .", "Notably , the importance of both maternal death and a close-in-age younger sibling suggest that maternal investment may be key to understanding the intergenerational developmental constraints we observed .", "Both maternal death and the presence of a close-in-age sibling suggest a possible reduction in the amount of maternal investment that the mothers in our analysis received during their early life .", "Maternal loss , even after weaning , may affect a developing primate’s ability to learn to forage , to avoid social harassment , and to receive social benefits , such as grooming , that are linked to health ( King , 1994; Janson and van Schaik , 1993; Akinyi et al . , 2013; Walters , 1987; Altmann , 1998; Ezenwa et al . , 2016 ) .", "Having a close-in-age sibling likely predicts a relatively early weaning event , which may reflect less maternal provisioning than would occur with more delayed weaning and a longer birth interval ( Hinde and Milligan , 2011; Mattison et al . , 2015; Silk , 1988 ) .", "Thus , we hypothesize that mothers who lost their own mothers or had a close-in-age sibling suffered reduced energetic and social input from their mothers , which subsequently led to lifelong developmental constraints .", "Additionally , these females may not have had adequate time to learn from their mothers how to provide high quality maternal care later in life .", "While we do not routinely collect detailed data on maternal care as part of long-term monitoring , the results reported here motivate targeted analyses of how maternal adversity relates to differences in maternal care and style in future work ." ], [ "The Amboseli Baboon Research Project is a long-term longitudinal study of wild baboons living in and around Amboseli National Park , Kenya .", "A detailed description of the study system can be found elsewhere ( Alberts and Altmann , 2012 ) .", "Researchers have continuously collected behavioral , environmental , and demographic data from the population since 1971 .", "All subjects are visually recognized , and near-daily censuses allow us to precisely document the timing of demographic events , including the birth and death of study individuals .", "Critical to this study , we have continuously collected near-daily measures of group size , daily rainfall levels ( beginning in 1976 ) , and monthly calculations of social dominance rank ( Hausfater , 1975 ) .", "In our analyses of offspring survival , we included all individuals who met two criteria:", "( i ) they lived in social groups that fed exclusively on wild foods rather than having their diet supplemented with human-sourced refuse; and", "( ii ) we were able to evaluate each of the five sources of maternal early life adversity and four sources of offspring early life adversity outlined below .", "Although transmission of paternal early adversity may also occur in our population , we did not consider it here because we knew paternal identities for only a subset of our study subjects and had early life data on only a subset number of fathers .", "Our analysis ultimately relied on data spanning more than four decades , from 1976 to 2017 .", "Previous work in the Amboseli population defined six binary indicators of early life adversity and considered a single index of cumulative adversity based on the sum of these indicators ( Tung et al . , 2016 ) .", "This cumulative adversity index is a strong predictor of adult lifespan: females that experienced high levels of early life adversity ( i . e . , a greater number of adverse early life conditions ) but still survived to adulthood lived dramatically shorter lives compared to females that did not experience early adversity ( Tung et al . , 2016 ) .", "In addition to the five sources of early adversity discussed above , this previous analysis also considered early social connectedness ( social integration versus social isolation ) as a sixth source of adversity ( Tung et al . , 2016 ) .", "Social connectedness data are missing for some mothers who were born relatively early in the long-term study .", "To maximize our sample size , we therefore did not include measures of social connectedness in this analysis .", "Our operational definitions for each source of adversity mirrored those used by Tung et al . ( 2016 ) for the remaining five conditions , except that here we employed measures of proportional rather than ordinal dominance rank ( i . e . , rank measured as a proportion of females that the focal individual dominates , rather than her ordinal rank number ) .", "We also built an index of cumulative maternal adversity , but because that model did not fit the data better than our reduced multivariate model ( in contrast to the results for adult female survival; Tung et al . , 2016 ) we report the multivariate model in the main text .", "The alternative model based on cumulative maternal adversity is presented in Supplementary file 1 Table S6 .", "We built a mixed effects Cox proportional hazards model of offspring survival during the first four years of life using the R package coxme ( Therneau , 2012; R Development Core Team , 2018 ) .", "The response variable in our model was the age at which offspring death occurred ( if at all ) during the first 4 years of life .", "We considered offspring survival to age four as the key survival period of interest because it roughly corresponds to the end of the juvenile period for baboons ( Charpentier et al . , 2008 ) .", "Offspring that survived beyond age four were treated as censored individuals who survived until at least age 4 .", "In our models of offspring survival as a function of maternal viability ( Figure 2 ) , we altered the first model to predict survival during the first two years of life as a function of maternal survival during years 2–4 after offspring birth ( see Supplementary file 1 Table S7 for model syntax ) .", "Datasets presented in this article can be downloaded from Dryad using the following Digital object identifier ( DOI ) : 10 . 5061/dryad . 4hc8k1r ( Zipple et al . , 2019 ) ." ] ]

[ "Early life adversity can affect an individual’s health , survival , and fertility for many years after the adverse experience .", "Whether early life adversity also imposes intergenerational effects on the exposed individual’s offspring is not well understood .", "We fill this gap by leveraging prospective , longitudinal data on a wild , long-lived primate .", "We find that juveniles whose mothers experienced early life adversity exhibit high mortality before age 4 , independent of the juvenile’s own experience of early adversity .", "These juveniles often preceded their mothers in death by 1 to 2 years , indicating that high adversity females decline in their ability to raise offspring near the end of life .", "While we cannot exclude direct effects of a parent’s environment on offspring quality ( e . g . , inherited epigenetic changes ) , our results are completely consistent with a classic parental effect , in which the environment experienced by a parent affects its future phenotype and therefore its offspring’s phenotype ." ]

[ "Experiences early in life can have lasting effects on the health and survival of humans and other creatures .", "Whether early hardships can also influence the wellbeing of the next generation is less clear .", "One previous study with captive hamsters suggested that adversity early in the life of a mother may indeed shorten how long her offspring will live .", "But hamsters only live for a few years and much less is known about the possibility for intergenerational effects in animals with longer lifespans .", "This is partly because such studies are time-consuming and thus more difficult to complete .", "Over the past 45 years , scientists have collected data on generations of baboons living in and around the Amboseli National Park in southern Kenya .", "Baboons live in social groups with a strict hierarchy , and individuals can live for up to 30 years in the wild .", "Previous research has shown that early life adversity – such as being orphaned or simply having a low-ranking mother – can shorten the lifespan of female baboons even if they make it to adulthood .", "It was unclear , however , whether these ill effects could be passed on to the next generation .", "Now , Zipple et al . have used the wealth of data about the Amboseli baboons to find the answer .", "After taking into account any adversity that each baboon experienced directly , Zipple et al . showed that juvenile baboons whose mothers were orphaned before reaching adulthood were 44% more likely to die young than juveniles whose grandmothers survived during their mother’s early years .", "Baboons whose mothers had a close-in-age younger sibling were also 42% more likely to die early as compared to those whose mothers did not , perhaps because the younger sibling competed with the mother for access to maternal care .", "The analysis suggests that early life adversity in female baboons can have intergenerational effects .", "More studies are needed to determine if this is also true of humans .", "If it is , such a result may help explain the persistence of poor health outcomes across generations and shed light on how best to intervene to interrupt this transmission ." ]

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

[ "physics of living systems" ]

[ [ "The collective migration of eukaryotic cells plays crucial roles in processes such as wound healing , tumor progression , and morphogenesis , and has been the focus of extensive study ( Haeger et al . , 2015 ) .", "The collective effects are typically associated with cell–cell interactions , such as long-range interaction mediated by secreted chemicals or short-range stable cohesive interaction mediated by adhesion molecules .", "However , the study of self-propelled particles in physics has revealed that motile elements which lack such activities may nonetheless give rise to dynamic collective motion , such as a traveling band ( Chaté et al . , 2008; Ginelli et al . , 2010; Ohta and Yamanaka , 2014; Solon et al . , 2015 ) , mediated by a relatively simple transient short-range interaction , such as alignment interaction ( Marchetti et al . , 2013; Vicsek et al . , 1995; Vicsek and Zafeiris , 2012 ) .", "The emergence of such collective motions of self-propelled particles , such as formations of clusters and traveling bands , has been observed in a wide variety of systems , ranging from animal flocks ( Ballerini et al . , 2008 ) , bacteria swarms ( Wioland et al . , 2013; Zhang et al . , 2010 ) , and cell assemblies ( Szabó et al . , 2006 ) to biopolymers and molecular motors ( Butt et al . , 2010; Schaller et al . , 2010; Sumino et al . , 2012 ) .", "For cell assemblies of eukaryotic cells , higher order organized movements have been also reported for migrating cells confined in circular micropatterns ( Doxzen et al . , 2013; Segerer et al . , 2015; Wan et al . , 2011 ) or spheroids ( Chin et al . , 2018 ) .", "For some of these systems , the connection between a macroscopic collective behavior and the microscopic dynamics of its constituents has been established .", "For the traveling band formation of biopolymers and molecular motors , local physical interactions among constituent elements effectively works as an alignment interaction , which induce the collective motion ( Schaller et al . , 2010; Sumino et al . , 2012; Suzuki et al . , 2015 ) .", "In the case of eukaryotic cells , however , the connection between macroscopic traveling band formation ( Kuwayama and Ishida , 2013 ) and microscopic short-range cell–cell interactions remains unclear .", "In particular , quantitative characterization of the traveling band formation and genetic analysis to reveal responsible cell-cell interaction have not been performed yet .", "The social amoeba Dictyostelium discoideum is a model organism for the study of collective cell migration .", "The coordinated movement of cell population is achieved by individual chemotactic motion to the cAMP gradient , which is formed in a self-organized way .", "However , a mutant cell that lacks chemotactic activity to cAMP still exhibits an organized coordinated motion that is probably mediated by cell-cell contacts ( Kuwayama and Ishida , 2013 ) .", "Here , we demonstrate that this coordinated motion is a spontaneous polar order formation which phase-separates with a disordered background .", "We further show that this polar order formation is attributable to the tail-following behavior among the migrating cells , called contact following locomotion ( CFL ) .", "We find that the polar ordered phase caused by CFL has an internal structure .", "An agent-based model with CFL further reveals that this internal structure is characteristic of the CFL-induced polar order formation .", "Thus , we establish the link between the collective behavior and the cell-cell interactions .", "Our findings open new possibilities that the concept of self-propelled particles contributes to the understanding of a highly orchestrated biological event of migrating cells in multicellular systems ." ], [ "In the present study , we investigated collective cellular motion in a mutant strain of Dictyostelium discoideum , known as “KI cell , ” which lacks all chemotactic activity ( Kuwayama and Ishida , 2013; Kuwayama et al . , 1993 ) , and thus does not form a cell aggregate under starvation conditions .", "Wildtype Dictyostelium discoideum forms an aggregate as a result of chemotaxis mediated by a self-secreted extracellular chemoattractant .", "Under starvation conditions , KI cells spread on a non-nutrient agar plate show a segregation of cell density , which propagates as bands in around six hours ( Kuwayama and Ishida , 2013 ) , when the cell density is within a particular range ( 1 . 0×105 cells cm-2 to 4 . 0×105 cells cm-2 ) ( Video 1 ) .", "Initially , the traveling bands propagate in random directions with high orientational persistence .", "When two bands collide , they appear to pass through each other , retaining their shapes ( Figure 1a left ) ( Kuwayama and Ishida , 2013 ) .", "However , over time , the propagation directions gradually become aligned , probably due to weak reorientation of propagation direction as an effect of collisions .", "Finally , the bands are arranged almost periodically in space with a spatial interval of about 1 mm ( Figure 1a right , b ) .", "To determine the mechanism underlying this collective cellular motion , we conducted high-magnification observations .", "At around 16 hours after cells were spread on an agar plate , a punched-out section of the agar plate was placed upside down on the glass slide , such that the monolayer of cells was sandwiched between agar and glass ( Figure 1—figure supplement 1a ) .", "These cells formed a high-density area that moved as a band in low-density area for long periods of time with high orientational persistence ( Figure 1c , d and Video 2 ) .", "Whereas the cells in high-density area are packed without extra space , and thus the cell density is similar across different samples ( Figure 1—figure supplement 1c ) , the size W of the band along the propagation direction showed a broad distribution , ranging from W=200 µm to 700 µm ( N=10 ) ( Figure 1—figure supplement 1b ) .", "In contrast , the traveling speed vb=0 . 5±0 . 03 µm/s ( N=10 ) was consistent among different bands , independent of size W ( Figure 1—figure supplement 1b ) .", "To study the relationship between these collective behaviors and the migration of individual cells , we next performed cell-tracking analysis .", "Cellular movements were recorded by tracing the motion of fluorescent microbeads that were incorporated into the cells by phagocytosis .", "Figure 2a shows typical trajectories of individual KI cells .", "The distribution of migration speeds indicates that cell migration speed inside the band is slightly faster than that outside the band ( Figure 2b ) .", "The average migration speeds of individual cells inside and outside the band were vin=0 . 38±0 . 14 μm s-1 and vout=0 . 30±0 . 16 μm s-1 , respectively .", "The migration direction of the cells inside the band was distributed around the direction of band propagation ( 176 . 2 degrees , Figure 2c ) , although the fluctuation around the average direction is relatively large ( standard deviation was 42 . 7 degrees ) .", "In contrast , the migration direction outside the band was distributed almost uniformly ( Figure 2c ) .", "The mean squared displacement ( MSD ) inside the band was proportional to t2 for more than 103 s ( Figure 2d ) .", "In contrast , the MSD outside the band exhibited a transition at around 100 s .", "from a persistent motion proportional to t2 , to a random motion proportional to t , which indicates that this motion can be described as a persistent random motion with no preferred direction ( Figure 2d ) .", "This observed directional randomness reflects the effects of cellular collisions , as well as its intrinsic nature of single cells .", "In sum , cells inside the band exhibit directionally persistent motions , whereas cells outside move randomly .", "We then compared the average cell speed inside the band vin and the band propagation speed vb , ( Figure 2e ) , and found that the band propagates faster than the cell migration speed for all samples investigated .", "This implies turnover of cells in the band , and that the band is continuously assembled at the front of the band and disassembled at the back .", "Thus , it is the cell density profile that shows propagation as a band ( Kuwayama and Ishida , 2013 ) .", "Such turnover of cells is also evident from the individual trajectories shown in Figure 2a and f , where the trajectories started in a low-density region entered a band ( high-density region ) at its front , and then left the band from its back .", "To quantitatively characterize the multicellular movement , we introduce the local polar order parameter , φn , t=vit/viti∈ℒn , obtained from the instantaneous cell velocity vi ( t ) , where ℒn is the nth domain along the direction of band propagation ( see Materials and methods ) .", "In the high-density region that propagates as a band , φn , t reaches around 0 . 8 , while φn , t in the low-density area remained below 0 . 4 ( Figure 3a ) .", "Thus , the high-density region is polar-ordered phase , which propagates in the low-density disordered phase .", "The polar order parameter of the band showed intersample variability , and was distributed from 0 . 6 to 0 . 85 ( Figure 3b ) .", "We found that the order parameter of band was positively correlated with the width of band W ( Figure 3b ) .", "The polar order phase is not completely homogeneous with respect to migration direction , but exhibits heterogeneity; this is related to the underlying assembly mechanism .", "This heterogeneity can be visualized in the velocity field obtained by optical flow , in which the direction of cell migration can be distinguished by color ( Figure 3c and Video 3 ) .", "The size-dependent squared local order parameter ⟨φl2", "( s ) ⟩ ( see Materials and methods ) shows a logarithmic decay with area S ( Figure 3e ) , indicating that this heterogeneity is not spatially uncorrelated .", "Within the band ( Figure 3c bottom ) , the migration direction was widely distributed from about 145 to 210 degrees ( a black line in Figure 3f; the mean is 178 . 1 degrees and the standard deviation is 31 . 2 degrees ) .", "The probability density functions ( pdf ) of the migration direction obtained for the four regions ( Figure 3c bottom ( i–iv ) ) show peaks at different directions ( Figure 3f ) , indicating the presence of two subpopulations; one in which the migration direction is ~160 degrees ( regions ( ii ) and ( iv ) ) and another in which it is ~190 degrees ( regions ( i ) and ( iii ) ) .", "These two subpopulations are also recognized in Figure 3c ( bottom ) as the regions with dark blue and light green colors , respectively , forming stripes .", "These two types of stripes extend perpendicular to the direction of band propagation , and are alternately arranged .", "The typical width of the stripe was around 125 μm , as determined by the analysis of autocorrelation function ( Figure 3—figure supplement 1a ) .", "The kymograph in Figure 3d shows the temporal evolution of the velocity field along the line PQ in Figure 3c , indicating that the stripes ( light green and dark blue ) are almost immobile , suggesting that the same cells experience the two stripes sequentially .", "In a reference frame co-moving with the band , cells move from the front to the end , since the speed of traveling band is faster than the speed of cells ( Figure 2e , see also Figure 3—figure supplement 1b ) .", "During this relative motion of cells from the front to the end of a band , they move downward in a stripe , and then enter the next stripe moving upward direction ( Figure 3—figure supplement 1b ) .", "We also tested if the similar behavior can be seen in the single cell trajectories analyzed in Figure 2 .", "As shown in Figure 3—figure supplement 4 , although the density of tracked cell is quite sparse , we found that the migration directions indicated by colors also exhibit heterogeneity with light green and dark blue ( Figure 3—figure supplement 4a ) .", "The temporal average of migration direction at a given x position indicates that the direction is distributed from about 150 to 210 degrees ( Figure 3—figure supplement 4b ) , which is consistent with the optical flow analysis ( Figure 3f ) .", "Furthermore , the trajectories move through regions with different colors , implying that cells change their migration direction following the flow direction in the regions ( Figure 3—figure supplement 4a ) .", "From the trajectories used in Figure 3—figure supplement 4a , the cell migration speed in the x-direction was 0 . 28±0 . 09 μm/s , while the width of stripes was estimated to be around 50 to 100 μm .", "Thus , the time scale that cells pass across a stripe is about 200-400 s , if the stripe is almost immobile as we have shown above .", "The time interval that cells travel across the traveling band is obtained as 1120 s .", "Thus , cells pass several different stripes during the time interval that cells stay in the traveling band .", "These analyses illustrate that the polar order phase possesses an internal structure with respect to the migration direction .", "The formation of a polar-ordered phase with an internal structure is ultimately related to the microscopic interactions between individual cells , which are short-ranged .", "In the low-density region , cells are not completely isolated , but rather are often associated with each other , migrating in single files ( Figure 4a and Video 4 ) .", "This tail-following behavior has been described for wild-type Dictyostelium cells within aggregation streams ( Dormann et al . , 2002 ) .", "We call this behavior 'contact following locomotion' ( CFL ) .", "In low-density assay , when two cells collide , they either form CFL ( Figure 4b and Video 5 ) or not ( Figure 4—figure supplement 1a ) .", "To quantitatively characterize CFL , we measured the duration of cell–cell contact after two cells collide .", "During the formation of CFL , the typical cell-to-cell distance is given by da=24 μm .", "We measured the time interval during which the distance is less than da from the time series of the distance between two cells ( Figure 4—figure supplement 1b ) .", "As shown in Figure 4c , in half of the cases , cell–cell contact persists for more than 300 s .", "To determine whether cells that form contacts for >300 s exhibit CFL or side-by-side behavior , we measured the average angle a of the angles a1 and a2 , which are the angles of the velocity vectors v1 and v2 with respect to the vector connecting the two cell centers d , respectively ( Figure 4d ) .", "In almost 60% of all cases , the angle a is 0–30 degrees ( Figure 4e ) that corresponds to CFL .", "To determine whether CFL is responsible for the collective behavior of KI cells , we sought a mutant cell that lacks CFL activity .", "A knockout mutant that fails to express the cell–cell adhesion molecule TgrB1 exhibits reduced CFL activity ( Fujimori et al . , 2019 ) .", "TgrB1 is known to mediate cell–cell adhesion via a heterophilic interaction with its partner TgrC1 ( Hirose et al . , 2011; Hirose et al . , 2015; Li et al . , 2015; Fujimori et al . , 2019 ) .", "We first assessed whether the tgrB1 null mutant forms propagating bands .", "As in the control case , under starvation conditions , we spread the tgrB1 null mutant cells on a non-nutrient agar plate at a cell density of 2 . 0 to 3 . 0×105 cells cm-2 ( see Materials and methods ) .", "However , neither segregation of cell density nor propagating bands appeared ( Videos 6 and 7 ) .", "We then quantitatively characterized the formation of cell–cell contacts .", "We found that in 80% of all cases , cell–cell contact is disrupted before 300 s ( Figure 4f ) , and that only 10% of cells established CFL ( Figure 4—figure supplement 1d ) .", "In particular , in half of all cases , the cell–cell distance becomes larger than da in 120 s , indicating that these cells failed to establish cell–cell contact .", "Thus , our analyses illustrate that in the tgrb1 null mutant , CFL is nearly absent .", "Since TgrB1 is a protein that can mediate cell-cell adhesion as well as contact-dependent signaling , it is reasonable that the tgrB1 null mutant cell does not show CFL .", "This analysis suggests that the reduced ability to perform CFL can be linked to the defect of tgrB1 mutant in the formation of traveling band formation , although we cannot exclude other effect that could explain the phenotype of this mutant .", "For instance , if there are some changes in the locomotive activity of individual cell due to the mutation of tgrB1 , it could also affect the formation of traveling band .", "Thus , we next compared the locomotive activities between control cells and tgrB1 null mutants .", "The velocity auto-correlation functions C ( Δt ) of the isolated single cells showed similar behaviors ( Figure 4—figure supplement 1c ) , indicating that locomotive activities were comparable between KI cells and the tgrB1 null mutant cells .", "The above analyses suggest that the difference in the cellular scale behavior between control KI cell and tgrb1 mutant cell is the ability of CFL , and we thus conclude that CFL is essential for the segregation of cell density and the formation of propagating bands .", "The collective motion of KI cells induced by the CFL interaction can be modeled by an agent-based simulation ( Hiraiwa , 2019 ) .", "In the model , particle i at position ri self-propels at a constant velocity v0 in the direction of its own polarity qi subjected to white Gaussian noise .", "Thus , without interactions , the particles exhibit a persistent random walk ( Hiraiwa et al . , 2014 ) .", "The effect of CFL is introduced so that polarity qi orients to the location of the adjacent particle j , when particle i is located at the tail of particle j ( parameterized by ζ ) .", "In addition to this effect , the particles interact with each other through volume exclusion interaction , adhesion , and the effect of polarity qi orienting toward the direction of its velocity vi=dri/dt ( parameterized by α ) .", "For a fixed parameter set ( α=0 . 4; see Materials and methods ) , without CFL ( ζ=0 ) , the collective behaviors did not form ( Figure 3—figure supplement 2 ) .", "In contrast , with CFL ( ζ≥0 . 1 ) , a polar-ordered phase appeared as a propagating band in the background of disordered phase ( Figure 3g and Video 8 ) .", "The speeds of the traveling band and particles within the band were 0 . 96 and 0 . 9 , respectively , relative to the speed of isolated particles , indicating that the band is dynamic with assembly in the front and disassembly in the tail , consistent with our experimental results .", "From the spatial pattern shown in Figure 3gh , in which the migration direction is indicated by color code , heterogeneity in the migration direction is recognized within the polar-ordered phase .", "In the simulation , we studied the pdf of migration direction in regions , whose size is comparable to that in Figure 3c ( ( i ) – ( iv ) ) , and found that the pdf exhibited peaks at different directions ( Figure 3i ) , similar to our experimental results ( Figure 3f ) .", "To determine whether this formation of internal structure is a characteristic of propagating bands induced by CFL , we studied a propagating band formed by increasing alignment effect α without CFL ( ζ=0 ) , and found that the pdfs of migration direction exhibit peaks at closely similar positions , indicating that the migration direction in the ordered phase is more homogeneous ( Figure 3—figure supplement 3 ) .", "Thus , the formation of internal structure appears to be a characteristic of the collective behavior induced by CFL .", "The size-dependent squared local order parameter ⟨φl2", "( s ) ⟩ ( see Materials and methods ) also shows the characteristic decay with a logarithmic dependence on area S ( Figure 3e ) , as observed experimentally ." ], [ "In this study , we report that a mutant of Dictyostelium cell that lacks all chemotactic activity exhibits spontaneous segregation into polar ordered solitary band ( Kuwayama and Ishida , 2013 ) .", "This pattern formation is attributable to the cell-cell interaction called contact following locomotion ( CFL ) ( Figure 4 ) .", "The agent-based model that includes CFL reproduces the observed macroscopic behaviors ( Figure 3g ) .", "Thus , we establish a link between the microscopic cell-cell interactions and the macroscopic polar pattern formation .", "We showed that the width of band is distributed widely from W=200 µm to 700 µm ( Figure 1—figure supplement 1b ) , and found the positive correlation between the width and the order parameter within the band ( Figure 3b ) .", "The local cell density within the band is similar across different samples ( Figure 1—figure supplement 1c ) , suggesting that the local cell density may not be a relevant factor for the increase in the order parameter .", "We speculate that if the correlation in the migration direction is gradually decorrelated from the front to the end of the band , bands with lower order parameters will be more prone to larger decorrelation in the migration direction .", "Consequently , we expect that the stronger the polar order , the wider the band width W . One characteristic behavior of the present polar pattern formation is the formation of internal structure , which consists of subpopulations with transversal motions ( Figure 3c , d , f .", "From the numerical simulation result , this formation of subpopulation was not seen in the model without CFL ( Figure 3—figure supplement 3d , e ) .", "Thus , the internal structure is a characteristic of CFL induced polar pattern formation .", "A population of cells enters the band at its front with directional alignment induced by CFL in random direction .", "During the relative movement of these cells from the front to the end of band , the migration direction may not be dampened completely to the direction of band propagation probably due to the directional persistence induced by CFL .", "In this way , subpopulations with respect to the migration direction are formed when CFL is present .", "A full analysis of this mechanism remains to be a future topic .", "In this paper , we mainly focused on the behavior of single solitary band .", "We studied the traveling band , which was well separated from other bands .", "Thus , all properties of single solitary band studied in this paper is independent of interaction between different bands .", "In some area , the traveling bands are arranged almost periodically in space with a spatial interval of about 1 mm ( Figure 1b ) .", "How bands interact with each other to reach a periodic spacing and whether the interval is independent of band width W are to be investigated .", "Wildtype Dictyostelium discoideum usually aggregates through chemotaxis to form a hemispherical mound with a central tip region that regulates the formation of slug-like multicellular structure ( Williams , 2010 ) .", "It has been suggested , however , that other mechanism also involves in the formation of aggregate , such as contact following ( Dormann et al . , 2002; Fujimori et al . , 2019; Shaffer , 1962; Umeda and Inouye , 2002 ) .", "In fact , whereas the KI cell alone does not form the multicellular structure , KI cells are able to spontaneously migrate to the central tip region transplanted from a wildtype slug and undergo normal morphogenesis and cell differentiation; this is not observed in mutant KI cells lacking TgrB1 ( Kida et al . , 2019 ) , suggesting that TgrB1-dependent CFL without chemotaxis allows KI cells to spontaneously migrate in slug .", "Furthermore , in wildtype cells , the chemical guidance cue has been shown to cease during the multicellular phase , which suggests that an alternative mechanism induces collective cell migration in the multicellular body ( Hashimura et al . , 2019 ) .", "We propose that polar order formation induced by CFL plays an important role in late-stage morphogenesis in this organism .", "Contact following locomotion , or chain migration , have been reported in other cell types ( Li and Wang , 2018 ) .", "The macroscopic behaviors reported in this paper may thus be found in other systems as well ." ], [ "1 mL of Klebsiella aerogenes suspended in 5LP medium ( 0 . 5% Lactose , 0 . 5% bactopeptone 211677 , Optical density = 0 . 1 ) was spread on the 9 cm 5LP plate ( 0 . 5% Lactose , 0 . 5% bactopeptone 211677 , 1 . 5% agar ) , 5LP medium dried , the non-chemotactic Dictyostelium discoideum , KI mutant cells were inoculated on the plate .", "The KI cells were incubated for about five days at 21°C .", "After cultivation , the KI cells and Klebsiella on the plate were collected with a phosphate buffer ( PB ) .", "To remove the Klebsiella , the suspension was centrifuged and discard as much of the supernatant liquid as possible by aspiration , then clean PB was added .", "After repeating this process two times , the number of cells was counted using a hemacytometer .", "The washed KI cells were spread ( cell density = 5 . 0 × 105 cells/cm2 ) on a 9 cm non-nutrient agar plate ( 1 . 5% agar ) to cause starvation .", "After drying of the PB , the plate was scanned every 15 min using a film scanner ( V850 , EPSON ) .", "The brightness in scanner images is inversely correlated with cell density ( Takeuchi et al . , 2014 ) .", "For Figure 1a and b , the original images were inverted with color that depends on time points .", "The KI cells were spread ( cell density = 2 . 0 to 3 . 0 × 105 cells/cm2 ) on the non-nutrient agar plate and incubated at 21°C for around 16 hr .", "A punched-out piece of the agar plate was placed upside down on the glass slide , and the travelling bands between the agar and glass was observed by phase contrast imaging .", "For Figures 1 and 2 , 3a , b and 4 , the images are taken every 15 s , using an inverted microscope ( TiE , Nikon , Tokyo Japan ) equipped with camera ( iXon+ , Andor Technology ) and a 20x phase-contrast objective .", "For Figure 3c–f , the images are taken every 3 s , using an inverted microscope ( TiE , Nikon , Tokyo Japan ) equipped with camera ( DS-Fi3 , Nikon , Tokyo Japan ) and a 20x phase-contrast objective .", "For the tracking analysis shown in Figure 2 and Figure 3 and 1 μL of the PB including 3% fluorescent microbeads ( ex:441 , em:486 , 1 . 0 μm , Polysciences , Inc ) was spread at the same time with the KI cells .", "The trajectories of the microbeads were automatically tracked by using the ParticleTracker 2D , a plugin for Image J ( National Institutes of Health , USA ) .", "To eliminate the trajectories of the microbeads that was not internalized by the KI cells , if |vcell| was slower than 0 . 25 μm/s for 300 s continuously , we excluded such trajectories .", "For the analysis shown in Figure 2 , we used the trajectories longer than 1 hr .", "The number of trajectories analyzed in Figure 2b–d was N = 35 with 2044 time points for the cells inside of the band and 9095 time points for the cells outside of the band .", "For Figure 2e , we performed the same experiment for 10 times .", "The numbers of trajectories that last for more than 1 hr in the 10 samples were N = 35 , 19 , 34 , 12 , 13 , 17 , 19 , 16 , 18 , 7 .", "The MSD ( Figure 2d ) was calculated using the formula below . MSD∆t=1N ( T-∆t ) ∑i=1N∑tT-∆t{rit+∆t-rit}2 , where Δt , T , and N means a time interval , final time , and number of the trajectory , respectively .", "To obtain the local polar order parameter φn , t shown in Figure 3a , the picture shown in Figure 1c was divided into n sections with width Δx ( µm ) , and the order parameter was calculated in each section at each time from the trajectories obtained by the tracking analysis .", "The local order parameter φ is defined asφn , t= 1N ( n ) ∑i ∈ ℒ ( n ) vi ( t ) vi ( t ) , where ℒn is the set of cells that satisfy ( n-1 ) Δx≤xi≤nΔx , N ( n ) is number of the cells in ℒ ( n ) , vi and xi are the velocity and x-position of i-th fluorescent microbeads , respectively .", "In this study , n = 14 and Δx = 119 µm .", "To obtain the order parameter φ of the traveling band used in Figure 3b , we first obtained φ in the band region at each time step .", "Then , we took average it over all time steps .", "Optical flow analysis was performed based on the Gunnar-Farneback method using OpenCV library .", "In the optical flow analysis , the displacement of each pixel in the original pictures are characterized by coloring based on the HSV ( ‘hue’ , ‘saturation’ , ‘value’ ) representation .", "The ‘hue’ varies depending on angular variation of each pixel .", "In this study , a ‘saturation’ and ‘value’ of the processed images via optical flow was fixed to 150 and 255 , respectively .", "The sequential images of the traveling band used for this analysis were taken every 3 s .", "To characterize the internal structures of the traveling band , the size-dependent squared local order parameter φl2S is introduced ( Figure 3e ) .", "To obtain the size-dependent squared local order parameter , we first calculate the squared polar order parameter φl2S within a ROI of size S , which is defined asφl2 ( S ) =1S2∑x , y∈ROIcos⁡Θx , y2+∑x , y∈ROIsin⁡Θx , y2where Θx , y=hue× ( 360/255 ) indicates the angular variation of the pixel at position ( x , y ) .", "The value of hue was obtained from the optical flow analysis ( Figure 3c ) .", "Then , φl2S is averaged over the entire area to obtain φl2S .", "If Θx , y is a random number without spatial correlation , as the increase of the area S , φl2S is expected to decay in proportion to S-1 .", "Firstly , we divided the x-t plane into the lattice with the interval of 20 µm ( for x axis ) and 60 s ( for t axis ) .", "We then collect trajectories that pass through each lattice .", "In each lattice , the angle of migration direction was averaged .", "The obtained average angle in each lattice is shown with the color indicated in the color bar .", "Then , the traveling band region in x-t plane was divided as shown in Figure 3—figure supplement 4b right .", "The temporal average of migration direction was taken at a given x position , which was plotted in Figure 3—figure supplement 4b left .", "Because the band show propagation in x-direction , autocorrelation function of transverse motion Csin is defined using y-component of motion asCsin ( ∆x ) =1Y ( X-∆x ) ∑y=1Y∑x=1X-∆xsinΘ ( x , y ) sinΘ ( x+∆x , y ) , where ∆x is pixel interval along the x-axis .", "Csin was plotted after that unit of ∆x is converted to the length .", "The gene disruption construct for tgrB1 was synthesized by a polymerase chain reaction ( PCR ) -dependent technique ( Kuwayama , 2002 ) .", "Briefly , the 5-flanking region of the construct was amplified with two primers , 5-CAACAGGTGGAGACTTCGGG-3 and 5- GTAATCATGGTCATAGCTGTTTCCTGCAGGCCAGCAGTAATAGTTGGAG-3 .", "The 3-flanking region of the construct was amplified with primers , 5- CACTGGCCGTCGTTTTACAACGTCGACGAGAACTGTTGATTCTGATGG-3 and 5- CTTGGTCCTGAACGAACTCC-3 .", "The bsr cassette in the multicloning site of pUCBsr Bam ( Adachi et al . , 1994 ) was amplified using the primer pair 5-CTGCAGGAAACAGCTATGACCATGATTAC-3 and 5-GTCGACGTTGTAAAACGACGGCCAGTG-3 , both of which are complementary to the two underlined regions , respectively .", "The three amplified fragments were subjected to fusion PCR that produced the required gene-targeting construct .", "The gene-targeting constructs were cloned using a TOPO TA cloning kit for sequencing ( ThermoFisher Scintific MA , USA ) .", "The linear construct was amplified by PCR using the outermost primers up to 10 µg and transformed into KI-5 cells .", "The KO clones were selected by genomic PCR using the outermost primers .", "tgrB1 KO KI-5 cell ( NBRP ID: S90519 ) is available in National BioResource Project Cellular slime molds ( https://nenkin . nbrp . jp ) .", "The tgrb1 null cells were cultured in HL5 medium ( 1 . 43% Proteose Peptone 211684 , 0 . 72% Yeast Extract212750 , 1 . 43% Gulcose , 0 . 05% KH2PO4 , 0 . 13% Na2HPO412H2O ) at 21 degrees Celsius .", "After reaching confluent , cells on the bottom were peeled off and collected , then washed two times with a centrifuge and PB .", "Next , the tgrb1 null cells were transferred on the 1/3 SM plate ( 0 . 33% Gulcose , 0 . 33% bactopeptone 211677 , 0 . 45% KH2PO4 , 0 . 3% Na2HPO4 , 1 . 5% agar ) with Klebsiella suspension , and incubated for around two days at 21°C .", "After , through the wash and count , the tgrb1 null cells were spread on the non-nutrient agar plate , after which the plate was scanned every 15 min using the film scanner .", "The KI cells and tgrb1 null cells for the collision assay were scraped from the traveling bands and surface of the plate , respectively .", "The scraped cells were placed on the non-nutrient agar and sandwiched with the glass .", "After around one hour incubation at 21°C , binary collisions of two cells were observed by microscopy and recorded every 15 s .", "The motion of the cells was tracked manually using the Manual Tracking , a plugin of Image J . Here , collision was defined as the contact of pseudopods .", "We collected the data from three and four independent experiments for the KI cells and the tgrb1 null cells , respectively .", "The total numbers of collision events are 136 ( KI cells ) , and 156 ( tgrb1 null cells ) .", "Firstly , the migrations of the KI and tgrb1 null mutant cells were recorded every 20 s for 60 min .", "Here , to extract an intrinsic locomotive activity of the cells , interactions with other cells , wall , and etc . were eliminated .", "Using obtained trajectories of cells that migrate with the velocity v , the velocity autocorrelation function C ( τ ) was calculated .", "C ( τ ) is described with the form ofC ( Δt ) =1N ( T-τ ) ∑i=1N∑tT-τ{vit+τ-vit}2 , where τ , t , T , and N means a time interval , time , final time , and number of the trajectory , respectively ( Figure 4—figure supplement 1c ) .", "The collective motion of KI cells induced by the CFL interaction can be modeled by an agent-based simulation .", "In the model , self-propelled particle i at position ri moves at a constant velocity v0 in the direction of its own polarity qi subjected to white Gaussian noise .", "Thus , without interactions , the particles exhibit persistent random walk ( Hiraiwa et al . , 2014 ) .", "Collective motion can be modeled by assuming particle-particle interactions ( Hiraiwa , 2019 ) .", "We firstly assume that the particles interact with each other through volume exclusion ( parameterized by β ) and adhesion ( parameterized by γ ) .", "We also assume the feature that the polarity of each particle orients to the direction of its velocity vi=dri/dt ( parameterized by α ) ; it is known that this assumption can effectively give rise to the alignment interaction between the particles when it is combined with the volume exclusion effect ( Li and Sun , 2014 ) .", "( Therefore , we simply refer to this feature as alignment effect in the main text . )", "As the main focus of this article , we incorporate CFL into this model by assuming the particle-particle interaction by which polarity qi orients to the location of the adjacent particle j when particle i is located at the tail of particle j ( parameterized by ζ ) .", "The equation of motion for the particle i are then given by ( 1 ) dridt=v0qi|qi|−β∑j∈N ( i ) Rrj−ri|rj−ri|2+γ∑j∈N ( i ) rj−ri|rj−ri| ( 2 ) dqidt=Iqi ( 1−|qi|2 ) +Ci+αvi|vi|+ξiwhere the second and third terms on the right-hand side of Equation 1 are the effects of volume exclusion and adhesions , respectively .", "Here , 𝒩 ( i ) is a set of particles that are contacting with the particle i , that is the particle j∈𝒩 ( i ) satisfies |rj-ri|≤R .", "On the right hand side of Equation 2 , the first term shows the self-polrization , the third term gives the effect that the polarity orients to the velocity direction vi/|vi| , the last term is white Gaussian noise with ⟨ξi⟩= ( 0 , 0 ) and ⟨ξi ( t ) ⋅ξj ( t' ) ⟩=σ2δijδ ( t-t' ) , and the second term Ci describes the CFL , parameterized by ζ , given by ( 3 ) Ci=ζ2∑j∈N ( i ) rj−ri|rj−ri| ( 1+qi|qi|⋅rj−ri|rj−ri| ) .", "Here , when the polarity of particle j , qj , and the vector from particles i to j , rj-ri , are in the same orientation , the maximum following effect is exerted on particle i to the direction of particle j .", "Such a situation is expected when particle i is located in the tail of particle j with respect to the polarity qj .", "In contrast , when particle i is located in the front of particle j , Ci almost vanishes .", "The simulation is implemented within a square box of size L with periodic boundary condition .", "For all simulations , we used fixed parameter values except ζ and α , given by v0=1 . 0 , β=1 . 0 , R=1 . 0 , γ=1 . 20 , and σ2=0 . 4 .", "For I in Equation 2 , we consider the situation where I is infinitely large , so that q was projected onto the unit vector q=1 for the numerical simulation .", "The density of particles per unit area ρ is given ρ=1 .", "The number of particles n is n=80 , 000 ( Figure 3g-i and Figure 3—figure supplement", "3 ) and n=10 , 000 ( Figure 3—figure supplement 2 ) .", "Firstly , we selected only the ROIs in the vicinity of the band front in the following way: We define a ROI as being within the bands if the particle density is higher than 1 . 16 , which corresponds to the 2D dense packing fraction of disks , ~0 . 91 .", "Using this definition , we define the ROI as being vicinity of the band front if the ROI is within the band at the last Fana frames whereas it is out of the band at the frames between the last Fana+Fwait+Fout and the last Fana+Fwait .", "In other words , Fana means the number of frames to be analyzed and must be within the band , Fout means the number of frames to determine the band front ( i . e . the frames in which the ROI must be still out of the band assuming that the band travels only in one direction ) , and Fwait means the number of the waiting frames ( i . e . the frames which are not used at all ) between these frame sets .", "The results of this algorithm for CFL-induced ( ζ=0 . 1 , α=0 . 4 ) and alignment-induced ( ζ=0 . 0 , α=1 . 0 ) bands are shown in Figure 3—figure supplement 3a and b , respectively .", "Here , we used the following sets of the parameters for our analysis in this article: Fana=55 , which corresponds to the time window in the analysis of experimental data .", "Fwait=76 , with around which the band front can propagate across one ROI .", "Fout=5 , which has been empirically determined .", "The duration between each frame is dt=0 . 2 in the unit of time of our numerical simulation .", "Secondly , using these near-front ROIs , we calculate the histograms of migration direction ( dri ( t ) /dt ) /|dri ( t ) /dt| for each ROI using all the Fana frames ( t ) and the particles ( i ) in it at each frame .", "Then , we plot only the histograms for the ROIs which have the top eight and nine peak probability densities for CFL-induced and alignment-induced bands , respectively .", "The results are plotted in Figure 3—figure supplement 3c and d , respectively .", "One can find the clear difference in these histograms between the CFL-induced and alignment-induced bands .", "The peak position and height for the CFL-induced band have large varieties , whereas those for alignment-induced band are less distributed .", "Furthermore , the peaks for the CFL-induced band are much higher than those for alignment-induced band .", "Figure 3i of the main text and Figure 3—figure supplement 3e plot three typical histograms from Figure 3—figure supplement 3c and d , respectively ." ] ]

[ "Biophysical mechanisms underlying collective cell migration of eukaryotic cells have been studied extensively in recent years .", "One mechanism that induces cells to correlate their motions is contact inhibition of locomotion , by which cells migrating away from the contact site .", "Here , we report that tail-following behavior at the contact site , termed contact following locomotion ( CFL ) , can induce a non-trivial collective behavior in migrating cells .", "We show the emergence of a traveling band showing polar order in a mutant Dictyostelium cell that lacks chemotactic activity .", "We find that CFL is the cell–cell interaction underlying this phenomenon , enabling a theoretical description of how this traveling band forms .", "We further show that the polar order phase consists of subpopulations that exhibit characteristic transversal motions with respect to the direction of band propagation .", "These findings describe a novel mechanism of collective cell migration involving cell–cell interactions capable of inducing traveling band with polar order ." ]

[ "The cells of animals and many other living things are able to migrate together in groups .", "This collective cell migration plays crucial roles in many processes in animals such as forming organs and limbs , and healing wounds .", "A soil-dwelling amoeba called Dictyostelium discoideum – or just Dicty for short – is commonly used as a model to study how groups of cells migrate collectively .", "Individual Dicty cells may live alone but sometimes many cells come together to form a larger mobile structure called a “slug” .", "Chemical signals coordinate how the cells collectively migrate to form the multicellular slug .", "Mutant Dicty cells that lack these chemical signal processes can still move together as a band that travels across a surface .", "This movement resembles a type of collective motion that has previously been observed in physics experiments using self-propelled particles .", "However , it remains unclear how this collective behavior works .", "Hayakawa et al . have now combined genetics , cell biology and computational approaches to study how groups of the mutant Dicty cells migrate together .", "The experiments showed that the traveling band is dynamically maintained by cells joining or leaving , and that this turnover is caused by simple interactions between the cells known as “contact following locomotion” .", "Contact following locomotion has been also reported in mammalian cells so the findings of Hayakawa et al . may aid research into how animals develop and how errors in cell migration may lead to diseases .", "Further studies are required to find out whether other cells showing contact following locomotion also travel in a band ." ]

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

[ "cell biology" ]

[ [ "Extensive studies have shown the importance of precise cell fate decisions in many life activities , such as cell cycle entry in response to environmental changes and pattern formation during embryonic development ( Xiong and Ferrell , 2003; Gregor et al . , 2007; Balázsi et al . , 2011 ) .", "However , little is known about how cells utilize the information of the input signal to make robust and reliable decisions especially in cases of noisy and time-varying signals .", "We address this issue by using the Start transition in budding yeast ( Saccharomyces cerevisiae ) as a model system .", "Start is a major cell cycle checkpoint in budding yeast ( corresponding to the restriction point in mammalian cells ) , which decides whether or not the cell should make the irreversible commitment to the next round of division ( Hartwell et al . , 1974 ) .", "The environmental and internal conditions are sensed and passed to the Start signal Cln3 ( Gallego et al . , 1997; Polymenis and Schmidt , 1997; Hall et al . , 1998; Parviz et al . , 1998; Menoyo et al . , 2013 ) .", "As a G1 cyclin , Cln3 triggers the Start transition by activating a downstream positive feedback loop composed of the repressor Whi5 , the transcription factor SBF/MBF and the cyclin Cln1/2 ( Skotheim et al . , 2008; Charvin et al . , 2010 ) ( Figure 1A upper panel ) .", "The Start checkpoint coordinates cell growth and cell division and is thought to control the cell size under different growth conditions ( Johnston et al . , 1977; Jorgensen and Tyers , 2004 ) .", "However , the mechanisms of the coordination and control have not been fully elucidated . 10 . 7554/eLife . 03977 . 003Figure 1 . G1 length is inversely proportional to the average Cln3 . ( A ) Schematic of the Start regulatory network in wild-type ( upper panel ) and in strains used in this study ( lower panel ) .", "( B ) G1 length is defined as the time interval during which Whi5 resides in the nucleus ( Figure 1—figure supplement 1 ) .", "Whi5 localization is schematically shown in red .", "( C ) Population-averaged G1 length at different IPTG concentrations for cells carrying IPTG-induced CLN3 as the Start signal ( Strain YCT2002 ) .", "Error bars represent standard deviation .", "( D ) Composite bright-field and fluorescence images for cells carrying GFP-GFP-CLN3* and WHI5-tdTomato ( Strain YCT2003 ) under full induction ( 2 mM IPTG ) .", "White arrows indicate the beginning of G1; yellow arrows indicate the timing of Start transition .", "( E–H )", "Time courses of the GFP-GFP-Cln3* ( green ) and nuclear Whi5-tdTomato ( red ) fluorescent intensities in a representative single cell at each different IPTG concentration .", "The empty squares and circles denote the raw data of Cln3 and nuclear Whi5 fluorescence , respectively; the green and red lines are the smoothing splines of the raw data; purple boxes show the G1 duration .", "( I ) G1 length vs the average Cln3 fluorescent intensity in G1 .", "Each dot represents a measurement from one cell cycle event; error bars indicate standard deviation .", "Color groups represent data collected from each of the corresponding IPTG concentrations .", "( J ) G1 length vs the average Cln3 fluorescence in log–log scale ( empty circles are calculated from I ) .", "<Cln3> represents the average Cln3 fluorescence in G1; T0 and Cln3c are fitted from I as described in ‘Materials and methods’ .", "The solid line is the best linear fit of the binned data ( filled squares ) .", "Error bars indicate standard deviation . DOI: http://dx . doi . org/10 . 7554/eLife . 03977 . 00310 . 7554/eLife . 03977 . 004Figure 1—figure supplement 1 . Definition of G1 length .", "( A ) Nuclear Whi5-tdTomato intensity in a representative single cell; open circle shows the raw data; solid red line shows the spline smoothing .", "( B ) The first derivative of the smoothed data; pink shade area denotes the G1 duration . DOI: http://dx . doi . org/10 . 7554/eLife . 03977 . 00410 . 7554/eLife . 03977 . 005Figure 1—figure supplement 2 . The promoter GlacSpr is repressed by LacI and induced by IPTG .", "( A ) The sequence of GlacSpr .", "( B ) The dose–response curve of GlacSpr .", "The transcriptional activity of GlacSpr in the OFF state is a little higher than GAL1pr in glucose , as the result , GlacSpr is not tight enough to completely shut off the cyclin activity of the wild-type Cln3 .", "The cln3Δ bck2Δ cells carrying GlacSpr-CLN3 are viable in the absence of IPTG with prolonged G1 phase . DOI: http://dx . doi . org/10 . 7554/eLife . 03977 . 00510 . 7554/eLife . 03977 . 006Figure 1—figure supplement 3 . The homologous structure of Cln3-Cdk1 complex . Cln3 is shown in dim grey and Cdk1 in light grey .", "The Cln3 in this model only contains amino acid 70-312 .", "The hydrophobic patch is indicated in red , while the interface to Cdk1 is indicated in green .", "The mutation sites we screened by alanine substitution are shown in sticks .", "Among those sites , D76 , D79 , K106 , R108 , D137 , K138 , K163 , D166 , K168 , R170 , K357 , and K359 are in the clustered charge residues; M107 , R108 , L110 , and I111 are in the hydrophobic MRAIL patch; H80 , Y81 , K163 , F164 , D166 , E194 , and W203 are on the interface of Cln3-Cdk1 complex . DOI: http://dx . doi . org/10 . 7554/eLife . 03977 . 00610 . 7554/eLife . 03977 . 007Figure 1—figure supplement 4 . Cell size and GFP brightness of selected Cln3 mutants under full induction . DOI: http://dx . doi . org/10 . 7554/eLife . 03977 . 00710 . 7554/eLife . 03977 . 008Figure 1—figure supplement 5 . Size control is maintained with the mutant Cln3 , Cln3* .", "( A ) The correlation between birth size and GFP-GFP-Cln3* intensity under full induction , Pearson Coefficient is 0 . 75 .", "( B ) The correlation between birth size and G1 length under full induction , Pearson Coefficient is −0 . 51 ( YCT 2003 ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 03977 . 00810 . 7554/eLife . 03977 . 009Figure 1—figure supplement 6 . The correlation between G1 length and average Cln3 fluorescence intensity in mother and daughter cells ( YCT2003 ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 03977 . 00910 . 7554/eLife . 03977 . 010Figure 1—figure supplement 7 . The correlation between G1 length and average Cln3 fluorescence intensity with different Cln3 signals: CLNR108A ( YCT2003 ) and CLN3D166A ( YCT2004 ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 03977 . 01010 . 7554/eLife . 03977 . 011Figure 1—figure supplement 8 . The correlation between G1 length and average Cln3 fluorescence intensity with different G1 length markers: WHI5-tdTomato ( YCT2003 ) and MCM-mCherry ( YCT2010 ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 03977 . 01110 . 7554/eLife . 03977 . 012Figure 1—figure supplement 9 . G1 length is inversely proportional to averaged Cln3 intensity without deducting asymptotes . Open circle denotes raw data in log–log scale; closed square denotes the binned data; solid line is the best linear fit of the middle part; error bars are standard deviation . DOI: http://dx . doi . org/10 . 7554/eLife . 03977 . 012 It was proposed that Cln3 concentration in the nucleus ( or total Cln3 abundance ) increases with cell size and the Start transition is triggered when Cln3 level reaches a critical threshold ( Chen et al . , 2004; Jorgensen and Tyers , 2004 ) .", "There are several issues with this model .", "For instance , it is unclear how nuclear Cln3 concentration is coupled to cell size .", "Furthermore , both the CLN3 mRNA and the Cln3 protein turn over very fast ( with a few minutes half-lives [Cross and Blake , 1993; Tyers et al . , 1992; Yaglom et al . , 1995] ) at very low abundance ( a few copies of the mRNA [McInerny et al . , 1997; Arava et al . , 2003] and 100–200 copies of the protein [Tyers et al . , 1993] ) , enabling it to rapidly respond to the environmental and cellular condition changes .", "This would imply considerable noise and fluctuations in the Cln3 profile .", "If the Start transition were triggered by the instantaneous Cln3 concentration passing above a threshold ( Chen et al . , 2004; Jorgensen and Tyers , 2004 ) , the decision would be rather stochastic and could be unreliable .", "In this study , by quantitatively measuring an inducible Cln3 mutant and simultaneously monitoring the timing of Start transition in single cells , we address the question of what information in the Cln3 profile the cell is using to make the Start decision .", "We found that the Start transition is triggered by the time integration of Cln3 activity on phosphorylated Whi5 .", "Furthermore , cells modulate Whi5 concentration in different nutrient conditions .", "Cln3 and Whi5 together control G1 length through the time integration mechanism to coordinate cell division with cell growth .", "The time integration strategy can reduce noise and minimize uncertainty in the decision and may be widely implied in decision making systems at the cellular level ." ], [ "To quantitatively investigate how Start transition is triggered by Cln3 , we first integrated a copy of inducible CLN3 onto the genome in a strain lacking the native CLN3 and BCK2 and fused the endogenous WHI5 with the red fluorescent protein tdTomato .", "The G1 length TG1 is defined as the time interval between Whi5 nuclear entry in late mitosis and its exclusion from the nucleus at the Start transition ( Taberner et al . , 2009 ) ( Figure 1B and Figure 1—figure supplement 1 ) , which is a measure for how long the cell waits to make the Start decision .", "Cln3 level under a synthetic inducible promoter GlacSpr ( Figure 1—figure supplement", "2 ) was controlled by titrating the inducer IPTG .", "The cells were grown in a microfluidic chip and monitored by time-lapse microscopy .", "We found that in both mother and daughter cells G1 length is prolonged as IPTG concentration decreases , which is consistent with the previous findings that increased Cln3 dosage shortens G1 length ( Di Talia et al . , 2007 ) and suggests a negative correlation between G1 length and Cln3 level ( Figure 1C ) .", "The low abundance and short half-life of the wild-type Cln3 make its detection in single cells extremely difficult ( data not shown ) .", "To better quantitate the observed negative correlation between G1 length and Cln3 level , we screened for Cln3 mutants with lower activity and longer half-life based on a homolog modeling of the Cln3-Cdk1 complex ( Figure 1—figure supplement 3 ) .", "Lower activity requires higher Cln3 concentration to pass Start and longer half-life allows more fluorescent proteins to mature before the tagged Cln3 is degraded , thus making the mutant Cln3 detectable in single cells .", "The fluorescent intensity and CDK activity of the GFP-GFP-Cln3 mutants were carefully examined .", "The desired mutants should fulfill three criteria:", "1 ) the fluorescent brightness of the mutant can be quantitatively measured;", "2 ) under full induction , the mutant can rescue the physiological function of the endogenous Cln3 in cln3Δ cells ( Figure 1—figure supplement 4 ) ;", "3 ) the cell cycle of cln3Δ bck2Δ strain can be arrested when the mutant is shut off .", "Two successful mutants , CLN3R108A and CLN3D166A , were obtained from the screening .", "Since the fully induced mutants do not change cell cycle behaviors such as the doubling time and cell size , we consider them faithful replacements of wild-type CLN3 under our experimental conditions .", "Unless otherwise specified , the mutant CLN3R108A ( denoted CLN3* ) was used in following experiments ( Figure 1A lower panel and D ) .", "Interestingly , we found that under full induction , the nuclear concentration of GFP-GFP-Cln3* is positively correlated and G1 length is negatively correlated with birth size ( Figure 1—figure supplement 5 ) , which implies that size control is maintained with the mutant Cln3 ( Di Talia et al . , 2007 ) .", "It was proposed that Cln3 responds to size through the upstream open reading frame ( uORF ) of its mRNA ( Polymenis and Schmidt , 1997 ) and/or ER-associated proteins Whi3 and Ydj1 ( Wang et al . , 2004; Vergés et al . , 2007 ) .", "However , in our construct , the 5′ leader of Cln3 mRNA was removed , and we did not observe any ER-like localization of Cln3 protein .", "The promoter activity does not correlate with size either ( data not shown ) .", "The result suggests that there may be other regulation mechanism coupling Cln3 concentration to cell size .", "When the inducible promoter is repressed to different extent by reducing IPTG concentration , the correlation between Cln3 concentration and cell size is disrupted and promoter activity becomes the dominant controller of Cln3 concentration .", "In this work , we leave the question of how Cln3 is coupled to size and focus on how Start transition is triggered by a controllable Cln3 concentration .", "Using Cln3* as the signal to trigger Start , we again observed a negative correlation between G1 length and Cln3 signal strength in single cells ( Figure 1E–H ) : when the signal is strong , the cell passes Start sooner; when the signal is weak , the cell waits for a longer time .", "In Figure 1I , we plot G1 length vs the average Cln3 in G1 for many single cells .", "The data shows an inverse-like correlation .", "Similar results were obtained when mother–daughter cell types were distinguished , CLN3D166A was used as the signal , or G1 length is defined by the MCM marker ( Liku et al . , 2005 ) ( Figure 1—figure supplement 6–8 ) .", "All of these results strongly suggest that the inverse-like correlation between G1 length and Cln3 signal strength is an intrinsic property of the Start transition .", "To get a quantitative understanding , we plot log ( TG1 − T0 ) against log ( <Cln3> − Cln3c ) in Figure 1J , where T0 is the horizontal asymptote representing the minimum G1 length ( time from Whi5 nuclear entry to cytokinesis ) and Cln3c is the vertical asymptote representing the minimum Cln3 concentration for cell to eventually pass Start .", "The slope of the linear fit is very close to −1 ( R2 = 0 . 91 ) ( Figure 1J and Figure 1—figure supplement 9 ) .", "Thus G1 length is inversely proportional to the average Cln3 concentration in G1 , which can be expressed as , ( 1 ) TG1−T0=A<Cln3>−Cln3c , where A is the constant of proportionality .", "Equation ( 1 ) can be rewritten into an integral form: ( 2 ) ∫T0TG1 ( Cln3 ( t ) −Cln3c ) dt=A .", "The above equation implies that the Start transition is triggered ( at t = TG1 ) when the time integration of the Start signal is above a threshold ( A ) .", "To investigate the potential benefit of having signal integration during the cell cycle commitment , we simulated the mRNA expression and protein level of Cln3 with a stochastic algorithm ( Gillespie , 1976 ) .", "Most parameters in the model were derived from published papers ( Figure 2—source data 1 ) .", "Due to the low abundance and short half-life , the Cln3 profile fluctuates considerably ( Figure 2A ) .", "We compared two hypothetical models of Start triggering: the Instantaneous Model in which the Start transition is triggered once the Cln3 level reaches a certain threshold , and the Integration Model in which the Start is triggered once the integration of Cln3 level over time reaches a certain threshold ( Figure 2A ) .", "The Instantaneous Model leads to a large variability in G1 length even in identical cells ( no extrinsic noise from cell-to-cell variability ) and under a constant environment ( Figure 2B ) .", "When extrinsic noise is added in the model , the G1 variability of the Instantaneous Model is as large as 92% , which is significantly larger than experimental observations for WT cells in previous and this studies ( Di Talia et al . , 2007; Skotheim et al . , 2008; Charvin et al . , 2008; Ferrezuelo et al . , 2012 ) ( CVs are no more than 50% , Figure 2C ) .", "In contrast , since the G1 length in the Integration Model depends on the integration of Cln3 level within a time window , a considerable amount of noise is averaged out .", "As a result , the variability in G1 length is much smaller ( Figure 2A–C ) and is consistent with the experimental data ( Figure 2C ) .", "Furthermore , the shape of the G1 length distribution generated by the Integration Model is more similar to that of the experimental data .", "While the Instantaneous Model leads to a G1 length distribution with a long tail; this means that a significant fraction of the cells would have prolonged G1 lengths , which could be disadvantageous for the population .", "The conclusion holds the same when considering the nuclear volume increase during cell growth ( Figure 2—figure supplement", "1 ) and using a wide range of parameters . 10 . 7554/eLife . 03977 . 013Figure 2 . The integration of Cln3 reduces the variability of G1 length .", "( A ) Representative Cln3 profiles in single cells .", "Each color represents simulation of a single cell .", "In the Instantaneous Model , Start is triggered at the time TG1 ( vertical blue dash line ) when Cln3 profile hits a threshold ( here set to 150 ) for the first time .", "In the Integration Model , Start is triggered at the time TG1 ( vertical red dash line ) when the integration of Cln3 ( the area under the Cln3 curve as indicated by shadow ) reaches a threshold ( here set to 1900 ) .", "The two thresholds are chosen to generate the same average TG1 ≈ 20 min in both models .", "In generating Cln3 profiles , both intrinsic noise and extrinsic noise are used ( Elowitz et al . , 2002; Raser and O'Shea , 2004 ) .", "( B–C )", "The distributions of TG1 in the Instantaneous ( blue ) and Integration ( red ) models , respectively , with intrinsic molecular noise only ( B ) and with both intrinsic and extrinsic noise ( C ) .", "The parameters to generate this figure are specified in Figure 2—source data 1 .", "The G1 length distribution from experimental data ( Strain YCT2001 ) is shown in ( C ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 03977 . 01310 . 7554/eLife . 03977 . 014Figure 2—source data 1 . Meaning , value and reference of the parameters to generate Figure 2 . DOI: http://dx . doi . org/10 . 7554/eLife . 03977 . 01410 . 7554/eLife . 03977 . 015Figure 2—figure supplement 1 . Stochastic simulation of Cln3 profile and Start triggering process with nucleus volume increase .", "( A ) Representative Cln3 concentration profiles in single cells .", "Each color represents simulation of a single cell .", "( B–C )", "The distributions of TG1 in the Instantaneous ( blue ) and Integration ( red ) models , respectively , with intrinsic molecular noise only ( B ) and with both intrinsic and extrinsic noise ( C ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 03977 . 01510 . 7554/eLife . 03977 . 016Figure 2—figure supplement 2 . The Instantaneous Model fails with the Cln3 profiles measured in experiment .", "( A ) Schematic plot of the test .", "The test is considered a pass if the timing of Start is near the timing of Cln3 peak by the specified tolerance value .", "The Cln3 profiles are from the real data .", "Open circles denote the raw data; solid lines are the smoothing splines .", "( B–C )", "Test failure percentage .", "Grey bars indicate the failure percentage and red bars indicate the percentage of cells whose Cln3 peak value is more than 20% larger than Cln3 at Start , for all cells ( B ) and cells with G1 longer than 30 min ( C ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 03977 . 01610 . 7554/eLife . 03977 . 017Figure 2—figure supplement 3 . Measuring the memory length through Whi5 nuclear entry when Cdk1 is inactivated .", "( A ) Dynamics of nuclear Whi5 intensity in a representative single cell ( YCT2016 ) .", "50 μM 1-NM-PP-1 was added to the medium to inhibit Cdk1 activity at 30 min .", "Open circles are the raw data; solid line is the smoothing spline .", "( B ) Fitting Whi5 dephosphorylation rate and the memory length from Whi5 nuclear entry when Cdk1 is inactivated .", "Open circles are from A . Solid line is the least squares best fit of the equation Whi5 ( t ) = A − B × exp ( −t/τ ) , where 1/τ denotes Whi5 dephosphorylation rate and τ is the memory length ( Supplementary file 1A ) .", "( C–D )", "The distributions of the memory length in mother ( C ) and daughter ( D ) cells , respectively .", "The red solid lines are Gaussian envelops of the distributions . DOI: http://dx . doi . org/10 . 7554/eLife . 03977 . 017 We tested the Instantaneous Model with the measured Cln3 profiles .", "If the Instantaneous Model were true , cells should pass Start at or near the peak of Cln3 profile during G1 .", "However , we found that in near 80% cells , the timing of Cln3 peak is different from the timing of Start ( Figure 2—figure supplement 2 ) .", "Thus , it is very unlikely that Start is triggered by the instantaneous Cln3 concentration .", "Integration of Cln3 profile would imply an accumulation of Cln3-Cdk1 kinase activity over time .", "We next proceeded to identify the integrator , that is , on what molecule this kinase activity is being accumulated .", "The Start network is composed of two modules: Cln3-Cdk1 phosphorylating Whi5 as the triggering module and the positive feedback loop as the switching module ( Figure 1A ) .", "The two modules are coupled via Whi5 .", "The triggering module initiates Start by reducing Whi5 concentration in the nucleus .", "The switching module sets a threshold for the nuclear Whi5 concentration , Whi5c , below which the switch will be flipped .", "Thus , G1 length is the time needed for the nuclear Whi5 concentration to drop below Whi5c .", "We constructed a mathematical model for the kinetics of Whi5 phosphorylation ( Supplementary file 1A ) .", "The model can reproduce the observed experimental data and make several predictions .", "The basic feature of the integration mechanism can be captured by a simplified version of the model as below .", "In the simplified model , we assume there is only one phosphorylation site on Whi5 , which is phosphorylated by Cln3-Cdk1 and dephosphorylated by some basal phosphatase .", "We further assume both enzymes operate at saturation ( see Supplementary file 1A on how the assumption can be justified and/or relaxed ) .", "Then the rate change of the phosphorylated Whi5 , Whi5P , can be expressed by the following equation: ( 3 ) dWhi5Pdt=k1⋅Cln3−k2 , where k1 is the catalytic rate of Cln3-Cdk1 on Whi5 , and k2 is the rate of the phosphatase times the phosphatase concentration .", "The concentrations of unphosphorylated and phosphorylated Whi5 fulfill a mass equation: ( 4 ) Whi5+Whi5P=Whi5tot .", "By our definition , G1 starts from Whi5 nuclear entry , when Cdc14 is the major phosphatase that is responsible for Whi5 dephosphorylation ( Taberner et al . , 2009 ) .", "Cln3 cannot initiate the export of nuclear Whi5 until Cdc14 is inactivated at cytokinesis ( Shou et al . , 1999; Visintin et al . , 1999 ) .", "We define the time from Whi5 nuclear entry to Cdc14 inactivation as T0 .", "We assume Whi5P ( t = T0 ) = 0 , thus Whi5tot is the nuclear Whi5 concentration when the phosphorylation reaction becomes dominant .", "At the Start transition t = TG1 , the nuclear Whi5 concentration drops to Whi5c , which is the critical Whi5 threshold determined by the positive feedback loop ( Supplementary file 1A ) , so that Whi5P ( t = TG1 ) = Whi5tot − Whi5c .", "By integrating Equation ( 3 ) from T0 to TG1 , we have: ( 5 ) Whi5tot−Whi5c=∫T0TG1 ( k1Cln3−k2 ) dt .", "Equation ( 5 ) can be reformulated as: ( 6 ) ∫T0TG1 ( Cln3−Cln3c ) dt=A , where A = ( Whi5tot − Whi5c ) /k1 and Cln3c = k2/k1 .", "We thus identified Whi5 as the integrator: the number of phosphorylated Whi5 , Whi5P , accumulates with Cln3-Cdk1 activity and the increase of Whi5P causes a decrease in the nuclear Whi5 concentration .", "The Start transition happens when the nuclear Whi5 concentration drops below Whi5c , or the accumulation of Whi5P reaches a threshold .", "In the case where the phosphatase operates in the linear region , Equation ( 3 ) becomes ( 7 ) dWhi5Pdt=k1⋅Cln3−k2Whi5P .", "In this case , there is a window of ‘memory’ of length 1/k2 , beyond which the integration effect is erased .", "It is difficult to measure the window of memory or the memory length in G1 directly .", "Because most Whi5 is dephosphorylated and resides in the nucleus in G1 phase , we could not directly measure Whi5 dephosphorylation rate in G1 .", "Thus , we measured Whi5 nuclear entry right after G1 by inhibiting Cdk1 activity with a strain bearing a cdc28-as1 allele ( Bishop et al . , 2000 ) .", "The average half time of Whi5 nuclear entry , which is an estimate of the memory length in the mathematical model ( Supplementary file 1A ) , is 13 . 7 min in mother cells and 10 . 6 min in daughter cells , respectively ( Figure 2—figure supplement 3 ) .", "Note that this memory length is comparable to the average G1 length ( from cytokinesis to Start ) in daughter cells in SD medium , which is 13 . 6 min in our experiment .", "In poor nutrient conditions , when G1 length is prolonged , Whi5 dephosphorylation rate and the memory length might be further adjusted .", "The mathematical model of Whi5 kinetics makes several predictions .", "The first is that there is a linear relation between the integration threshold A and the total Whi5 concentration Whi5tot ( a more detailed ODE model predicts that Cln3c will also change with Whi5tot [Supplementary file 1A] ) .", "The second is that increasing the phosphatase activity k2 reduces memory length and increases Cln3c .", "The third is that reducing Whi5c by weakening the positive feedback loop increases the integration threshold A ( the ODE model predicts that Cln3c will also change when perturbing the loop strength [Supplementary file 1A] ) .", "Note that Cln3 half-life by itself has no effect on the integration dynamics; and that reducing the Cln3-Cdk1 catalytic efficiency k1 ( as in Cln3* ) increases Cln3c and A proportionally , requiring an increased Cln3 concentration to pass Start .", "We verified the model's predictions by perturbing the Start network .", "First , the integration effect would have difficulty to manifest itself without the integrator .", "Indeed , our experiment shows that in whi5Δ strain , most cells pass Start with minimal G1 length ( Figure 3—figure supplement 1A ) .", "We further checked to what extent the inverse correlation holds by plotting G1 length and Cln3 intensity in log–log scale ( Figure 3A ) .", "The slope of the linear fit is much larger than −1 , suggesting that the inverse proportionality is severely compromised .", "Second , the integration threshold A should increase with the total Whi5 concentration .", "This means that at the same Cln3 concentration cell waits longer time in G1 with higher Whi5 dosage .", "The prediction is consistent with the previous finding that Whi5 overexpression significantly increased the percentage of the cells in G1 phase ( Costanzo et al . , 2004 ) .", "We quantitatively verified the effect of the total Whi5 concentration on the integration threshold A with a strain containing two copies of WHI5 ( Figure 3—figure supplement 1B and Figure 3B ) .", "The inverse correlation shifts upward in log–log scale , which represents larger A . Remarkably , the positive correlation between A and Whi5 concentration can also be seen at the single-cell level ( Pearson correlation coefficient = 0 . 53 , Figure 3C ) .", "In Cln3-TG1 correlations , the variation of G1 is high when Cln3 concentration is low .", "At least part of the variation is due to the variance in Whi5tot concentration ( and thus the variance in A ) .", "After normalizing by Whi5tot , inverse correlations in both 1X and 2X WHI5 strains are more converged and collapse to the same curve ( Figure 3—figure supplement 2 ) .", "Third , the model predicts that increasing the phosphatase activity will not only increase Cln3c but also cause deviation from the inverse correlation by shortening the memory length .", "It is known that overexpressing the phosphatase CDC14 affects the nuclear accumulation of Whi5 and increases the percentage of the cells in G1 phase ( Visintin et al . , 1998; Stevenson et al . , 2001 ) .", "Thus , we tested the prediction in a CDC14 overexpressing strain .", "We observed that Cln3c increases and the inverse proportionality is compromised ( Figure 3—figure supplement 1C and Figure 3D ) .", "Finally , the model predicts that weakening the strength of the positive feedback loop will both reduce Whi5c ( thus increase A ) and increase Cln3c .", "We measured G1 length vs Cln3 intensity in cln1Δ and cln2Δ strains and observed that Cln3cs increase and the inverse correlations shift upward in log–log scale as predicted ( Figure 3—figure supplement 1D , E and Figure 3E , F ) .", "The correlation in cln2Δ shifts more than in cln1Δ , suggesting that Cln2 contributes more to the positive feedback loop .", "This is consistent with the fact that the expression level of Cln2 is about three times higher than Cln1 and with the previous finding that Cln2 is more potent in the Start transition ( Huh et al . , 2003; Tyers and Futcher , 1993; Tyers et al . , 1993 ) . 10 . 7554/eLife . 03977 . 018Figure 3 . Whi5 acts as the integrator of Cln3-Cdk1 activity . Correlations between G1 length and the average Cln3 fluorescence in log–log scale in ( A ) cln3Δ bck2Δ whi5Δ strain ( Strain YCT2008 ) ( blue ) , ( B ) cln3Δ bck2Δ 2XWHI5 strain ( Strain YCT2007 ) ( red ) , ( D ) cln3Δ bck2Δ CDC14 overexpressing strain ( Strain YCT2009 ) ( purple ) , ( E ) cln3Δ bck2Δ cln1Δ strain ( Strain YCT2013 ) ( green ) , ( F ) cln3Δ bck2Δ cln2Δ strain ( Strain YCT2014 ) ( pink ) and ( G ) cln3Δ BCK2+ strain ( YCT2015 ) ( wine ) , in comparison with in cln3Δ bck2Δ strain ( Strain YCT2003 ) ( black ) .", "Data are also plotted in linear scale in Figure 3—figure supplement 1 .", "<Cln3> represents the average Cln3 fluorescence in G1; T0 and Cln3c are fitted from Figure 3—figure supplement 1 as described in ‘Materials and methods’ .", "The solid lines are linear fits of the binned data ( filled squares ) .", "Error bars indicate standard deviation .", "Cln3c , T0 , Slope , Y Intercept and R2 of the linear fit for each strain are summarized in ( H ) .", "( C ) The integral A ( calculated as TG1 mutiplied by average Cln3 intensity in G1 ) vs Whi5tot fluorescence intensity in cln3Δ bck2Δ ( black ) and cln3Δ bck2Δ 2XWHI5 ( red ) strains .", "Each dot represents a measurement from one cell cycle event; error bars indicate standard deviation ρ signifies Pearson correlation coefficient . DOI: http://dx . doi . org/10 . 7554/eLife . 03977 . 01810 . 7554/eLife . 03977 . 019Figure 3—figure supplement 1 . Correlations between G1 length and the average Cln3 fluorescence in ( A ) cln3Δ bck2Δ whi5Δ strain ( Strain YCT2008 ) ( blue ) , ( B ) cln3Δ bck2Δ 2XWHI5 strain ( Strain YCT2007 ) ( red ) , ( C ) cln3Δ bck2Δ CDC14 overexpressing strain ( Strain YCT2009 ) ( purple ) , ( D ) cln3Δ bck2Δ cln1Δ strain ( Strain YCT2013 ) ( green ) , ( E ) cln3Δ bck2Δ cln2Δ ( Strain YCT2014 ) ( pink ) and ( F ) cln3Δ BCK2+ strain ( YCT2015 ) ( wine ) , in comparison with in cln3Δ bck2Δ strain ( Strain YCT2003 ) ( black ) .", "Each dot represents a measurement from one cell cycle event; error bars indicate standard deviation; solid lines are simulation results from the ODE model ( Supplementary file 1B ) .", "Note we didn't fit the result of cln3Δ BCK2+ strain , because the mechanism of Bck2 related Start triggering is not clear . DOI: http://dx . doi . org/10 . 7554/eLife . 03977 . 01910 . 7554/eLife . 03977 . 020Figure 3—figure supplement 2 . Variation of G1 length in low Cln3 region is due to the variance in Whi5tot .", "( A ) The Cln3-TG1 correlation indicating Whi5tot intensity of each cell by colors .", "( B ) Whi5tot distribution of cells with Cln3 intensity less than 300 .", "Red line is the Gaussian envelop .", "( C ) Average G1 length binned by Whi5tot intensity of cells with Cln3 intensity less than 300 .", "( D ) G1 length verse Cln3 intensity divided by Whi5tot intensity in 1XWHI5 ( YCT2003 ) and 2XWHI5 ( YCT2007 ) strains . DOI: http://dx . doi . org/10 . 7554/eLife . 03977 . 020 We also investigated how the correlation between Cln3 and G1 length is affected by Bck2 , which triggers the Start transition in the absence of Cln3 ( Wijnen , 1999 ) .", "As shown in Figure 3—figure supplement 1F , the Cln3-TG1 correlation of BCK2+ strain loses the long G1 tail at low Cln3 concentration .", "And in log–log scale the inverse correlation only holds to certain range .", "As Cln3 concentration decreases , the mean G1 length stops increasing at about 29 min .", "The result suggests that in wild-type cells , Bck2 acts as a bypass Start trigger to prevent a too long G1 phase when Cln3 concentration is too low .", "In budding yeast , the G1 checkpoint Start coordinates cell division with growth ( Hartwell et al . , 1974; Jorgensen and Tyers , 2004 ) .", "Under different nutrient conditions , cells spend different time growing in G1 before passing Start ( Ferrezuelo et al . , 2012 ) .", "However , it is not clear how this coordination is achieved .", "In light of our findings ( Equation ( 5 ) , Figure 3B , C ) , it is suggestive that the cell could modulate the integration threshold A , and thus the G1 length , by tuning Whi5tot .", "Indeed , we found that in suboptimal nutrient conditions , the Cln3-TG1 correlations shift towards right with higher Whi5tot intensity and A ( Figure 4—figure supplement 1 ) .", "Similar shift was observed with the MCM marker as a measure of G1 length ( Figure 4—figure supplement 3 ) .", "However , we did not observe any increase of MCM intensity in suboptimal nutrient condition , nor any correlation between A and MCM intensity , suggesting that the nutrient regulation on Whi5 and the Whi5 regulation on A are specific .", "We further quantified Whi5tot intensity in a series of nutrient conditions in WT cells .", "Surprisingly , Whi5tot increases as growth rate decreases and G1 length is accordingly prolonged in both mother and daughter cells ( Figure 4A–C ) .", "It means that in poor nutrient conditions , the Start threshold is actually higher rather than lower as proposed by previous studies ( Jorgensen and Tyers , 2004; Schneider et al . , 2004; Turner et al . , 2012 ) , although the cell size may appear to be smaller . 10 . 7554/eLife . 03977 . 021Figure 4 . Cells modulate Whi5tot concentration to coordinate cell division with nutrient conditions .", "( A ) Whi5tot intensity is negatively correlated with growth rate in various nutrient conditions ( Strain YCT2001 ) .", "( B–C )", "G1 length is positively correlated with Whi5tot intensity for mothers ( B ) and daughters ( C ) ( Strain YCT2001 ) in different nutrient conditions .", "Each color represents one nutrient condition as in A . Each dot in A–C is calculated from all cells in that nutrient condition .", "( Single-cell data are presented in Figure 4—figure supplement 4 , 5; statistic results are summarized in Figure 4—source data", "1 ) Black straight lines are guide lines; error bars indicate standard deviation . DOI: http://dx . doi . org/10 . 7554/eLife . 03977 . 02110 . 7554/eLife . 03977 . 022Figure 4—source data 1 . ( A ) The growth rates and Whi5tot intensities in different nutrient conditions .", "( B ) The Whi5tot intensities and G1 lengths in different nutrient conditions . DOI: http://dx . doi . org/10 . 7554/eLife . 03977 . 02210 . 7554/eLife . 03977 . 023Figure 4—figure supplement 1 . The correlations between Cln3 concentration , Whi5tot intensity and G1 length in suboptimal nutrient conditions .", "( A , D and G )", "The correlations between G1 length and the average Cln3 fluorescence in 2% raffinose ( A ) , limited nitrogen ( D ) and 0 . 05% glucose ( G ) , comparing with in 2% glucose ( Strain YCT2003 ) .", "Each dot represents a measurement from one cell cycle event; error bars indicate standard deviation .", "( B , E and H )", "The correlations in A , D and H are plotted in log–log scale in ( B ) , ( E ) and ( H ) , respectively ( open circles ) .", "The solid lines are linear fits of the binned data ( filled squares ) .", "Error bars indicate standard deviation .", "Cln3c , T0 , Slope , and Y Intercept are the fitting parameters as described in ‘Materials and methods’ and summarized in ( J ) .", "Note the linear fit for 2% raffinose is not very meaningful , because its Cln3 only varies in a small range .", "( C , F and I )", "The corresponding correlations between the integral A and Whi5tot fluorescence intensity in 2% raffinose ( C ) , limited nitrogen ( C ) and 0 . 05% glucose ( F ) , comparing with in 2% glucose .", "Each dot represents a measurement from one cell cycle event; error bars indicate standard deviation .", "Each nutrient condition was shown in a separate panel in Figure 4—figure supplement 2 . DOI: http://dx . doi . org/10 . 7554/eLife . 03977 . 02310 . 7554/eLife . 03977 . 024Figure 4—figure supplement 2 . The correlations between the integral A and Whi5tot fluorescence intensity in 2% glucose ( A ) , 2% raffinose ( B ) , limited nitrogen ( C ) and 0 . 05% glucose ( D ) ( Strain YCT2003 ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 03977 . 02410 . 7554/eLife . 03977 . 025Figure 4—figure supplement 3 . The inverse correlations in raffinose comparing with in glucose with the MCM marker as a measure of G1 length ( YCT2010 ) .", "( A and B )", "The Cln3-TG1 correlations with the MCM marker as a measure of G1 length in glucose and raffinose in linear ( A ) and log–log scale ( B ) .", "The solid lines in B are linear fits of the binned data ( filled squares ) .", "Cln3c , T0 , Slope , and Y Intercept are the fitting parameters as described in ‘Materials and methods’ and summarized in ( D ) .", "( C ) The correlation between A and MCM-mCherry fluorescence in glucose and raffinose .", "Error bars indicate standard deviation . DOI: http://dx . doi . org/10 . 7554/eLife . 03977 . 02510 . 7554/eLife . 03977 . 026Figure 4—figure supplement 4 . The correlation between growth rate and Whi5tot fluorescence intensity in single cells in different nutrient conditions ( YCT2001 ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 03977 . 02610 . 7554/eLife . 03977 . 027Figure 4—figure supplement 5 . The correlation between Whi5tot intensity and G1 length in single cells in different nutrient conditions in mother ( A ) and daughter ( B ) cells ( YCT2001 ) .", "Legend is the same as in Figure 4—figure supplement 4 . DOI: http://dx . doi . org/10 . 7554/eLife . 03977 . 02710 . 7554/eLife . 03977 . 028Figure 4—figure supplement 6 . The adaptation of Whi5 and G1 length after nutrient transitions ( YCT2001 ) .", "( A–E )", "The raw data of Whi5 dynamics and G1 length from representative single cells in constant 2% ( A ) , constant 0 . 01% ( B ) , 0 . 01–2% ( C ) and 2–0 . 01% ( D–E ) glucose media; 0 . 01% glucose period is marked as yellow and 2% glucose period is marked as green , respectively .", "( F–I )", "Whi5tot intensity and G1 length adapt with time after nutrient transitions; yellow squares are steady states in 0 . 01% glucose whereas green diamonds are steady states in 2% glucose; time 0 is the timing of nutrient transition; G1 beginning time is the time when a G1 phase ( not necessarily the first G1 phase ) begins after nutrient transition .", "There is no data for daughter cells in 2–0 . 01% glucose condition , because only few daughter cells budded in the first 300 min after transition . DOI: http://dx . doi . org/10 . 7554/eLife . 03977 . 028 We also monitored the adaptation of Whi5tot intensity and G1 length after nutrient change .", "When the cells were switched from low glucose to high glucose medium , both Whi5tot intensity and G1 length decreased gradually and reached the same level as in constant high glucose medium after several cell cycles ( Figure 4—figure supplement 6A–C , F , G ) .", "Contrarily , when cells were switched from high glucose to low glucose medium , Whi5tot increased fast and underwent an overshoot before it reached a steady level ( Figure 4—figure supplement 6D , H ) .", "G1 length increased even faster and its overshoot was also more pronounced than Whi5 ( Figure 4—figure supplement 6D , I ) .", "Surprisingly , we found when switched from high to low glucose , if the cell had not started budding , G1 phase could reinitiate ( Figure 4—figure supplement 6E ) .", "This result could not be explained by current knowledge of Start and must involve further changes in CDK and phosphatase activities ." ], [ "Cellular decision making is a fundamental problem with broad implications , ranging from development , cell-fate determination , stress response , to cell cycle control and signaling .", "A decision-making process in a cell usually consists of", "( i ) sensing ( of external and/or internal signals and cues ) ,", "( ii ) information processing ( of the sensed information ) , and", "( iii ) actuating ( e . g . , turning on a switch ) .", "Previous works have been mainly focused on the first and the last steps .", "Even in many cases where the molecular players and the circuitry are mapped out , how cells process information to make the best decision is largely unknown .", "This is intrinsically a quantitative question and is especially important when the signals are noisy and with uncertainties .", "Information processing is the brain of decision making; understanding this step is critical to understand the rationale and the strategy the cell adopts to make better decisions .", "We address this question with a well-studied model system: the budding yeast Start checkpoint .", "Using a quantitative single cell assay with a controllable and quantifiable Cln3 signal , we discovered that it is not the instantaneous value of the Cln3 concentration , but rather the integration of the concentration over time , that triggers the switch .", "Cln3 is a sensor that senses , with a fast response time , the instant information/condition relevant to making cell cycle commitment .", "We found that the instant Cln3 activity is memorized on phosphorylated Whi5 , and the memory length is about 10 min in SD medium .", "This implies that the cell uses information within a time window of the past in order to get an assessment of the future .", "This strategy of signal integration averages out noise and fluctuations and minimizes error and uncertainty in decision making .", "Signal integration has been seen in decision-making behaviors of animals ( Bowman et al . , 2012; Bogacz et al . , 2006 ) .", "Our work shows that it is also adopted at the cellular level , suggesting a general strategy that may be widely implemented in decision-making and signaling systems ( Li et al . , 2010; Di Talia and Wieschaus , 2012; Doncic and Skotheim , 2013 ) .", "Whi5 ( more precisely , the phosphorylated Whi5 ) serves as the integrator in the system , that is , the physical memory onto which the integration is being recorded .", "We found that cells modulate Whi5 level to set different integration thresholds in different nutrient conditions .", "Whi5 level is higher in poor nutrients , thus cells are more cautious and wait for longer time to make the decision .", "We also found that daughter cells always have higher Whi5tot intensity and steeper slope between Whi5tot and G1 length than mother cells ( Figure 4D , E ) .", "The finding suggests that besides the daughter-specific Cln3 suppression by Ace2 ( Laabs et al . , 2003 ) , Whi5 contributes to G1 delay in daughter cells as well .", "It is also consistent with the previous finding that Whi5 is required for the different behaviors of mother and daughter cells under low metal stress ( Avraham et al . , 2013 ) .", "Although Cln3 and Whi5 determine G1 length together , they have very different response time .", "While Cln3 responds to external and internal cues rapidly , Whi5 adjusts in a much longer time scale and reinforces the change of G1 length ( Figure 4—figure supplement 6 ) .", "The system could utilize the different response times of the sensor and the integrator to develop more complex strategies when adapting to environmental fluctuations and changes .", "The coordination of cell growth with division is often posed as a ‘size control problem’ .", "Previous studies on size control were more focused on Cln3's response to environmental changes ( Gallego et al . , 1997; Polymenis and Schmidt , 1997; Hall et al . , 1998; Parviz et al . , 1998; Jorgensen and Tyers , 2004 ) .", "It was proposed that the Start threshold is lowered in poor nutrient conditions , based on the conventional ( instantaneous ) model and the observation that the abundance of Cln3 is lower in these conditions ( Jorgensen and Tyers , 2004; Schneider et al . , 2004; Turner et al . , 2012 ) .", "However , our finding on Whi5 modulation reveals a different scenario – the Start threshold is in fact raised in poor nutrients and the longer G1 length is a result of a longer integration time of Cln3 .", "Cell cycle is coupled to growth rate by both Cln3 and Whi5 .", "A full understanding of size control may need to take into account the combined action of Cln3 dynamics , Whi5tot concentration and the integration mechanism ." ], [ "Standard methods were used throughout .", "All strains in this study are congenic W303 .", "W303-1A ADE2+ was made by integrating ADE2 fragment amplified from the 4741 genome at the ADE2 locus of W303-1A .", "The KAN-MX6 , NAT-MX6 , and LEU2 fragments flanking with homologous sequence to the target gene ( 40 bp ) for deletion were amplified from the plasmid pFA6-KAN-MX6 ( Longtine et al . , 1998 ) , pFA6-NAT-MX6 ( Goldstein and McCusker , 1999 ) , and pRS305 ( Sikorski and Hieter , 1989 ) , respectively .", "The WHI5-tdTomato constructs were made by digesting pCT2001 with HindIII , integrating at the WHI5 locus and losing the CaURA3 marker between the two TEF1 terminators .", "The inducible CLN3 constructs were made by digesting pCT2002 , pCT2003 , pCT2004 , or pCT2005 with PmeI and integrating at the HIS3 locus .", "The ADH1pr-HTB2-CFP construct was made by digesting pCT2006 with XbaI and integrating at the TRP1 locus .", "The ADH1pr-MCM-mCherry , ADH1pr-MCM-GFP , additional WHI5pr-WHI5-tdTomato and GPDpr-CDC14 constructs were made by digesting pCT2007 with NdeI , pCT2008 with BstBI , pCT2009 with NdeI and pCT2010 with NcoI , respectively and integrating at the URA3 locus .", "All the constructs and deletion strains were verified by PCR .", "All the strains used in this study are summarized in Supplementary file 2A .", "The plasmid pCT2001 was constructed as following: first replaced the ADH1ter between AscI and BglII on the plasmid pNI8 , a kind gift from Jonathan Weissman ( UCSF ) , with the TEF1ter amplified from the same plasmid; then replaced the mCherry fragment between PacI and AscI with the tdTomato fragment amplified from pRS304-tdTomato; next the 300 bp upstream of the WHI5 stop codon amplified from the W303 genome was inserted between HindIII and PacI sites; finally the 300 bp downstream of the WHI5 stop codon amplified from the W303 genome was inserted by using ClaI , along with an additional HindIII site at the 3′ end .", "Each inducible Cln3 plasmid contains three transcription units: the lactose transporter , the constantly expressed LacI and the inducible promoter controlled Cln3 signal .", "The STE5pr ( −602 to −1 of STE5 ) amplified from the W303 genome , the LAC12 amplified from pKR1B-LAC4-1 ( Sreekrishna and Dickson , 1985 ) , and the CYC1ter amplified from pGREG506 ( Jansen et al . , 2005 ) were inserted into pRS304 ( Sikorski and Hieter , 1989 ) with SacI-SpeI-SalI-XhoI to make the transcription unit of the transporter ( each fragment was sequentially inserted between two restriction sites ) .", "The GPDpr amplified from pRS424-GPD ( Mumberg et al . , 1995 ) , the LacI gene amplified from the Escherichia coli genome and the CYC1ter were inserted into pGREG506 with NotI-HindIII-EcoRI-SacI to make the transcription unit of LacI .", "The inducible promoter GlacSpr , which essentially is the ADH1pr ( −700 to −1 of ADH1 ) with two LacI binding sites ( one on each side of the TATA box ) , was obtained by overlapping PCR ( Figure 1—figure supplement 2 ) .", "GFP was amplified from pNT10 , a kind gift from Jonathan Weissman ( UCSF ) .", "Venus was amplified from pVenus-N1-NPY ( Nagai et al . , 2002 ) .", "A 11-amino-acid linker optimized for yeast ( Ala-Ala-Ala-Gly-Asp-Gly-Ala-Gly-Leu-Ile-Asn- ) was introduced to the C terminal of the fluorescent proteins by PCR primers .", "The wild-type CLN3 was amplified from the genome , while mutants were constructed by overlapping PCR .", "The transcription unit of Cln3 signal GlacSpr-CLN3-CYC1ter was constructed by inserting the corresponding fragments sequentially into pGREG506 with ClaI-BamHI-SalI-XhoI; GlacSpr-GFP-GFP-CLN3R108A-CYC1ter and GlacSpr-GFP-GFP-CLN3D166A -CYC1ter were constructed into pGREG506 with ClaI-BamHI-EcoRI-NotI-SalI-XhoI; GlacSpr-Venus-Venus-Venus-CLN3R108A-CYC1ter was constructed into pGREG506 with ClaI-BamHI-EcoRI-SphI-NotI-SalI-XhoI .", "Then , the transcription units of LacI and Cln3 were subcloned into pNH603 ( a kind gift from Wendell A . Lim lab , designed to make sure single integrant ) with SacI-NotII and ClaI-XhoI respectively .", "Finally , the transcription unit of transporter was amplified by PCR and inserted into the plasmid with SacI site to accomplish the inducible Cln3 plasmids pCT2002-pCT2005 .", "The pCT2006 plasmid was constructed by inserting the ADH1pr amplified from the genome , the HTB2 without stop codon amplified from the genome , the CFP with linker at the N-terminal amplified from BBa_E0020 ( iGEM Registry ) and the CYC1ter into pRS304 with SacI-NotI-SpeI-SalI-XhoI .", "The pCT2007 and pCT2008 plasmids were constructed by inserting the ADH1pr , the MCM marker amplified from pML103 ( Liku et al . , 2005 ) , GFP ( for pCT2007 ) , or mCherry ( for pCT2008 ) with linker at the N terminal and the CYC1ter into pRS306 ( Sikorski and Hieter , 1989 ) with SacI-NotI-NotI-SalI-KpnI .", "The pCT2009 plasmid was constructed by replacing the mCherry fragment between PacI and AscI on pNT8 with tdTomato , inserting the upstream 1430 bp of the WHI5 stop codon ( including WHI5pr and WHI5 coding sequence ) amplified from the genome into HindIII-PacI and subcloning the HindIII-BglII fragment of the resultant plasmid into pRS306 .", "pCT2010 was constructed by inserting the GPDpr , the CDC14 amplified from the genome and the CYC1ter into pRS306 with SacI-BamHI-SalI-XhoI .", "All the plasmids were verified by sequencing on both strands and summarized in Supplementary file 2B .", "All the inverse correlations were measured in Synthetic Dextrose ( SD ) medium containing 2% ( wt/vol ) glucose , 1× amino acid ( AA ) dropout , 6 . 7 g/L yeast nitrogen base ( YNB ) without amino acid , 100 mg/L leucine , 20 mg/L histidine , 20 mg/L tryptophan , 20 mg/L adenine , and 20 mg/L urea , unless otherwise indicated .", "The formula of 1× amino acid dropout is 20 mg/L arginine , 20 mg/L methionine , 30 mg/L tyrosine , 30 mg/L isoleucine , 30 mg/L lysine , 50 mg/L phenylalanine , 100 mg/L glutamic acid , 100 mg/L aspartic acid , 150 mg/L valine , 2 g/L threonine and 4 g/L serine .", "The limited nitrogen medium contains 2% ( wt/vol ) glucose , 0 . 05 mM ammonium sulfate , 6 . 7 g/L yeast nitrogen base ( YNB ) without ammonium sulfate and amino acid , 100 mg/L leucine , 20 mg/L histidine , 20 mg/L tryptophan , 20 mg/L adenine , and 20 mg/L urea ( Gallego et al . , 1997 ) .", "The limited carbon media have similar ingredients as the SD medium except for various carbon sources or glucose concentrations .", "All nutrients were purchased from Sigma–Aldrich , St . Louis , MO , except that glucose was purchased from Ameresco , Solon , OH .", "IPTG ( Isopropyl β-D-1-thiogalactopyranoside ) was purchased from Sigma–Aldrich , St . Louis , MO and dissolved to make 0 . 5 M stocks .", "conA was purchased from Sigma–Aldrich , St . Louis , MO and dissolved to make 1 mg/mL stocks .", "1-NM-PP-1 was purchased from Merck Millipore , Billerica , MA and dissolved in DMSO to make 5 mM stocks .", "We constructed the homologous structure of Cln3 based on the cyclin A chain ( chain B ) in 3DDQ ( PDB ID ) .", "Two other structures , the cyclin D3 chain ( chain B ) in 3G33 ( PDB ID ) and the cyclin E1 chain ( chain B ) in 1W98 ( PDB ID ) , were chosen for reference as well .", "The amino acid sequences of those proteins were aligned by the 3D-Jury server ( http://meta . bioinfo . pl/ ) ( Ginalski et al . , 2003 ) , and then the alignment result was used to model the 3D structure of Cln3 in Rosetta CM ( comparative modeling , v37268 ) ( Chivian and Baker , 2006 ) .", "Similar method was used to construct the homologous structure of Cdk1 , with the Cdk2 chain ( chain A ) in 1VYW ( PDB ID ) as the template .", "The complex model of Cln3 and Cdk1 was prepared by Rosetta Docking ( Wang et al . , 2007 ) , taking the complex structure of Cdk2-cyclin A ( PDB ID: 1FIN ) as the template .", "The contacts or residues in the complex models were optimized by Rosetta Relax with backbones fixed .", "The importance of the interface residues was evaluated by using the fixed-backbone alanine scanning protocol in Rosetta ( Kortemme and Baker , 2002 ) .", "To screen for the desired Cln3 mutants , we selected 12 single amino acid sites in clustered charged residues ( Miller et al . , 2005 ) .", "Three more sites were chosen from the conserved MRAIL hydrophobic patch involved in substrate reorganization ( Schulman et al . , 1998 ) .", "Five more mutation sites on the interface of Cln3-Cdk1 complex were suggested by in silico alanine scanning .", "Finally , we selected 20 single-amino-acid sites in total for alanine scanning experimentally .", "Mutants were constructed by site-directed mutagenesis PCR .", "The mutation sites were summarized and labeled on the homologous structure of Cln3 in Figure 1—figure supplement", "3 . To check the brightness and the activity of the Cln3 mutants , the wild-type and mutant Cln3s were fused with two tandem GFPs on the N terminus and expressed by the synthetic inducible promoter GlacSpr .", "Each construct was integrated at the HIS3 locus of the cln3Δ strain YCT2011 .", "The GFP brightness and cell size under full induction was quantified by fluorescent microscopy .", "Only mutants with significantly higher GFP intensity than wild-type were shown in Figure 1—figure supplement", "4 . We further deleted BCK2 in those strains and investigate their cell division without IPTG under microscopy .", "The shut-off of cell cycle was considered tight if less than 5% cells budded in 6 hr .", "Similar molds and methods as described previously ( Tian et al . , 2012 ) were used to fabricate the microfluidic chips .", "Medium was fed through the main channel by auto-controlled syringe pump ( TS-1B , Longer Pump Corp . , Baoding , China ) .", "The flow rate was 66 . 6 μL/hr .", "In the nutrient switching experiments , both the tubing and the syringe providing the medium were changed within 3 min , to make sure the transition is as fast as possible .", "Except for the measurement of Whi5 dephosphorylation rate , cells were grown in the microfluidic chip to maintain the constant IPTG concentration and nutrient condition .", "Temperature of the microfluidic chip was kept at 30°C with a stage top incubator ( INU-TIZHB-F1 , Tokai Hit Co . , Ltd . , Fujinomiya-shi , Japan ) .", "Time lapse movies were collected with epi-fluorescence microscopy using a Nikon Ti-E inverted microscope equipped with the objective lens Plan Apo VC 100×/1 . 40 Oil DIC N2 , the motorized XY stage and the Perfect-Focus System ( Nikon Co . , Tokyo , Japan ) .", "Images were acquired every 3 min with an Andor iXon3 897 EMCCD ( 512 × 512 , 16 μm , Andor Technology Ltd . , Belfast , UK ) and Lambda SC shutter controllers ( Sutter Instrument , Novato , CA ) .", "NIS Elements AR v3 . 2 ( Nikon Co . , Tokyo , Japan ) was used to automate image acquisition and microscope control .", "There is no significant photo-toxicity or perturbations of cell cycle time caused by the detection of either GFP-GFP-Cln3 or Whi5-tdTomato or both .", "There is no significant photo-bleaching in the GFP or tdTomato channel ( data not shown ) .", "The brightness of the mercury lamp on different days was normalized by fluorescent reference slides ( Fluor-REF , Microscopy Education , Microscopy & Imaging Place Inc . , McKinney , TX ) .", "To measure Whi5 dephosphorylation rate , cells were immobilized on glass bottomed dish by conA .", "Temperature was kept by ZILCS incubation chamber ( Tokai Hit Co . , Ltd . , Fujinomiya-shi , Japan ) .", "Cells were monitored for 30 min and then another 2 hr after adding 50 μM 1-NM-PP-1 to the medium .", "Time lapse movie was collected with a UltraVIEW VoX Laser Confocal Imaging System ( PerkinElmer , Watham , MA ) and a CSU-X1 spinning disk confocal ( Yokogawa , Tokyo , Japan ) on a Nikon Ti-E inverted microscope equipped with the APO TIRF 100X OIL NA 1 . 45 objective lens , the motorized XY stage and the Perfect-Focus System ( Nikon Co . , Tokyo , Japan ) .", "Whi5-tdTomato fluorescence was excited with the 561 nm 50 mW laser line and collected by the appropriate filters .", "Images were acquired every 3 min by a Hamamatsu C9100-13 EMCCD ( Hamamasu Photonics K . K . , Hamamatsu City , Japan ) camera .", "At each time point , 5 Z-series optical sections were collected with a step size of 1 . 6 μm , using a NanoScanZ 400 μm Piezo focusing drive ( Prior Scientific , Cambrige , UK ) .", "Maximum projections of Z stacks were performed with ImageJ .", "Cell segmentation and tracing were based on bright field images and automatically accomplished by the MATLAB custom software cellseg as described previously ( Lau , 2009; Yang et al . , 2013 ) .", "Daughters were counted in once the bud can be recognized by the software ( usually in early or middle mitosis ) .", "We quantified the mean intensity of the brightest 5 × 5 Whi5-tdTomato pixels in one cell as nuclear Whi5 concentration .", "G1 length was defined as the time interval between the local maximum and minimum of its first derivative within certain time frame ( as shown in Figure 1—figure supplement 1 ) .", "In strains bearing the MCM marker , G1 length was defined similarly except for using the MCM marker .", "The MCM marker is an artificial marker generally reporting CDK activity .", "It enters the nucleus 2 min prior to and exports 5 min later than Whi5 ( data not shown ) .", "Whi5tot intensity was taken as the maximum of nuclear Whi5 intensity during one G1 phase .", "Since Cln3 localizes in the nucleus , similar to Whi5 , we quantified the mean intensity of the brightest 9 × 9 GFP-GFP-Cln3* pixels in one cell as Cln3 concentration , which had been verified to be a good proxy ( data not shown ) .", "The brightest 3 × 3 pixels were deducted from the brightest 9 × 9 pixels to eliminate the artifact of aggregates .", "Cell size was taken as cell area in two dimensional .", "Cells were assumed grow exponentially in G1 .", "Growth rate fitted by more than six data points and with standard error smaller than 0 . 02 were used in the final statistics .", "Export of the fluorescent intensities and cell area was automatically accomplished by cellseg with minor revision .", "Following analysis was accomplished by the MATLAB custom software analyzestart , which was developed before ( Lau , 2009; Yang et al . , 2013 ) and modified for the purpose of this study .", "The integral A in Figures 3C , 4B , Figure 4—figure supplement 1D , G , I , 2 , 3B was calculated as Cln3 intensity multiplied by G1 length .", "Whi5 dephosphorylation rate was fitted by the equation Whi5 ( t ) =A−B⋅exp ( −t/τ ) , where 1/τ is the Whi5 dephosphorylation rate and τ is the memory length; A denotes Whi5tot concentration; B denotes Whi5tot − Whi5 ( t = 0 ) ( Supplementary file 1A ) .", "Cln3-TG1 correlations in log–log scale were fitted by the following criteria:T0 was set as 5 min for strains with the Whi5 marker ( Di Talia et al . , 2007 ) or 12 min for strains with the MCM marker . Except for whi5Δ and BCK2+ strains , Cln3c was fitted in the range of min_Cln3-200 to min_Cln3 .", "For each strain , min_Cln3 is the lowest average Cln3 intensity we observed that can pass Start . In whi5Δ strain , Cln3c was set as 0 . In cln3Δ BCK2+ strain , Cln3c was set as the same value as cln3Δ bck2Δ strain . Data points in log–log scale were binned according to their Cln3 intensity . Only bins with more than 10 data points were used in fitting . Median of Cln3 intensity and G1 length within each bin was used in fitting to avoid the effect of outliers .", "For cln3Δ bck2Δ strain in 2% glucose , we adopted the least square fit .", "For other strains or growth conditions , we adopted the fit whose slope is closest to −1 .", "For cln3Δ BCK2+ strain , there was no good linear fit for the whole Cln3 intensity region , thus we only fitted the high Cln3 part .", "Stochastic simulation of the Cln3 profile was done using Stochastic Simulation Algorithm ( SSA ) converted from the Ordinary Differential Equations ( Gillespie , 1976 ) :d[mRNAcln3]dt=a1−D1⋅[mRNAcln3]d[ProteinCln3]dt=a2⋅exp ( α⋅t ) ⋅[mRNAcln3]−D2⋅[ProteinCln3]Initial state:[mRNAcln3]=Int ( a1D1⋅r ) , [ProteinCln3]=Int ( a1⋅a2D1⋅D2⋅r ) Int ( x ) ={n|n∈ℤ , |x−n|≤0 . 5 The meaning , value and reference of the parameters to generate Figure 2 are summarized in Figure 2—source data", "1 . We adopted flowing assumptions in generating Cln3 profile:Constant transcription rate of CLN3 gene . Constant degradation rate of Cln3 mRNA and protein . Exponential growth of cell volume . Ribosome number ( reflected by translation rate ) is proportional to cell volume . Cln3 transcription gets suppressed at early G1 ( r ratio of full capacity ) to mimic the Ace2 suppression in daughter cells . Nuclear volume is constant thus Cln3 abundance directly reflects Cln3 nuclear concentration .", "In the model , we simply simulated Cln3 mRNA and protein numbers instead of concentrations .", "In Instantaneous Model , G1 starts from zero time point with Cln3's initial state .", "TG1 is the time when ProteinCln3 first exceeds Instantaneous_threshold .", "In Integration Model G1 starts from zero time point with Cln3 initial state .", "TG1 is the time when the integration of Cln3 ( ∫​ProteinCln3dt ) surpasses Integration_ threshold .", "Instantaneous_threshold was chosen as 150 , while Integration_threshold was chosen as 1900 to generate average 19 min TG1 .", "Extrinsic noise was simulated by randomizing the model parameters around their nominal values within a certain percentage range .", "Extrinsic noise of 20% CV was applied to all parameters ( D1 , a1 , D2 , a2 , α , r , Instantaneous_threshold and Integration_threshold ) within this model .", "For the robustness of our conclusion , we checked our model with different parameter sets of {D1 , a1 , D2 , a2 , α} varying in all possible ranges and found that the Integration Model does generate a TG1 distribution with much closer resemblance to experimental result than the Instantaneous Model under all circumstances .", "We also considered the case that nuclear volume increases with cell volume in Figure 2—figure supplement", "1 . In the simulation , nuclear volume was assumed to be proportional to cell volume and the initial nuclear volume was set as 2 . 9 fL ( Jorgensen et al . , 2007 ) .", "Cln3 concentration equals to Cln3 protein number divided by nuclear volume .", "Instantaneous_threshold was set as 1 . 86 nM , and Integration_threshold was set as 10 . 6 nM*min .", "Other equations and parameters used in the simulation were kept the same as Figure", "2 . An explicit model of the whole Start network was constructed .", "Simulation results in , Figure 3—figure supplement 1A–E were produced by this model .", "Multiple phosphorylation of Whi5 was taken into account as well as the positive feedback loop .", "The equations and parameters are listed in Supplementary file 1B ." ] ]

[ "Cell fate decisions are critical for life , yet little is known about how their reliability is achieved when signals are noisy and fluctuating with time .", "In this study , we show that in budding yeast , the decision of cell cycle commitment ( Start ) is determined by the time integration of its triggering signal Cln3 .", "We further identify the Start repressor , Whi5 , as the integrator .", "The instantaneous kinase activity of Cln3-Cdk1 is recorded over time on the phosphorylated Whi5 , and the decision is made only when phosphorylated Whi5 reaches a threshold .", "Cells adjust the threshold by modulating Whi5 concentration in different nutrient conditions to coordinate growth and division .", "Our work shows that the strategy of signal integration , which was previously found in decision-making behaviors of animals , is adopted at the cellular level to reduce noise and minimize uncertainty ." ]

[ "Budding yeast and other single-celled organisms can reproduce by dividing to produce two daughter cells .", "The timing of the cell division is critical because if the cell is still small when it divides , the resulting daughter cells may not be big enough to survive .", "In budding yeast , the irreversible decision to divide—known as the ‘Start’ checkpoint—is only made once a cell reaches a certain size and is triggered by a protein called Cln3 .", "This protein controls the activity of another protein called Whi5 , which normally prevents the cell from dividing by switching off particular genes .", "Cln3 adds phosphate groups to Whi5 to make ‘phosphorylated Whi5’ , which allows the genes involved in cell division to be switched on .", "It is commonly believed that the level of Cln3 reflects the size of the cell and the nutrient conditions .", "Therefore , one model of cell division proposes that the cell passes the Start checkpoint when the level of Cln3 reaches a threshold value .", "However , levels of the Cln3 protein in cells can naturally fluctuate , and computer simulations based on this model showed that this would not produce reliable decisions on when to divide .", "So how do cells manage to distinguish noise from the genuine signals that indicate it is the right time to divide ?", "To address this question , Liu et al . studied yeast cells containing an artificial version of the gene encoding the Cln3 protein whose levels could be adjusted by adding a particular chemical .", "This revealed that cells with higher levels of Cln3 passed through the Start checkpoint sooner than cells that had lower levels of Cln3 .", "The observation suggests that cells add up the amount of Cln3 present over a period of time to see if this reaches the threshold needed for the Start checkpoint .", "This could be possible if , instead of sensing Cln3 levels directly , the cell senses the accumulation of phosphorylated Whi5 .", "To test this idea , Liu et al . carried out additional experiments and found that the decision to pass the Start checkpoint only occurs when the amount of phosphorylated Whi5 reaches a certain threshold .", "The cells are able to coordinate their growth and division under different nutrient conditions by altering the threshold of phosphorylated Whi5 .", "When the nutrient supply is poor , more phosphorylated Whi5 needs to be accumulated to allow the cell to pass the Start checkpoint .", "In this way , cells adjust when they divide according to nutrient conditions .", "Similar strategies may be found in other signaling or decision-making systems ." ]

[ "Introduction", "Mathematical model", "Results", "Discussion" ]

[ "physics of living systems", "computational and systems biology" ]

[ [ "Nuclear Pore Complexes ( NPCs ) are biological 'nanomachines' that conduct all the transport between the nucleus and the cytoplasm in eukaryotic cells .", "NPCs participate in a vast number of regulatory processes in the cell , as well as pathological conditions such as viral disease and cancer ( Dickmanns et al . , 2015 ) .", "Transport through the NPC is fast , highly selective and robust with respect to molecular noise and structural perturbations .", "Transport of relatively small cargoes up to several nanometers in size , or approximately 30–40 kD , occurs by pure diffusion , without specific interactions with the NPC constituents .", "By contrast , transport of larger macromolecules , such as import of transcription factors and export of mRNA particles is tightly controlled by the NPC .", "For efficient transport , macromolecules larger than several nanometers in size must be shuttled through the NPC by soluble nuclear transport proteins from a highly conserved family , known as Karyopherins ( Kaps ) in yeast or Importins/Transportins in vertebrates .", "Remarkably , despite its high selectivity and efficiency , the NPC does not consume metabolic energy during transport and does not possess an obvious 'gate' opening or closing during transport ( Terry and Wente , 2009; Wente and Rout , 2010; Stewart , 2007; Feldherr and Akin , 1997; Mohr et al . , 2009 ) .", "The spatial organization of the NPC and its transport mechanism are unique .", "The passageway through the nuclear envelope of about 35–50 nm in diameter and 50-80 nm in length is formed by a structural scaffold that comprises multiple proteins of a combined size of ∼150 MDaltons .", "This passageway is lined by a set of ∼200 intrinsically disordered polypeptide chains , collectively known as 'FG nups' due to the large numbers of Phenylalanine-Glycine ( FG ) repeats in their sequence ( Terry and Wente , 2009; Wente and Rout , 2010 ) .", "Although the actual sequences of the FG nups can vary widely among different species , the overall structure , organization and the transport mechanism of the NPC are conserved ( Terry and Wente , 2009; Hülsmann et al . , 2012; Schmidt and Görlich , 2015 ) .", "As the key component of the NPC transport mechanism , the FG nups set up the permeability barrier that prevents free passage of large macromolecules and serve as a template for the transient binding of the cargo-carrying transport proteins .", "NPCs are also remarkably resilient with respect to structural perturbations: many of the FG nups can be genetically deleted without impairing cell viability and without major effect on transport ( Strawn et al . , 2004; Popken et al . , 2015; Feldherr et al . , 2002; Hülsmann et al . , 2012 ) .", "Cargo-carrying transport proteins bind to the FG nups through multiple , yet relatively weak contacts .", "This binding is crucial for selective transport: interfering with it decreases the transport efficiency , or abolishes the transport altogether .", "Conversely , particles or molecules that normally cannot penetrate the NPC can be transported after chemical modifications that enables the to interact directly with the FG nups ( Wente and Rout , 2010; Bayliss et al . , 1999; 2000; Naim et al . , 2009; Kim et al . , 2013; Kumeta et al . , 2012 ) .", "NPC geometry and architecture are schematically illustrated in Figure 1 . 10 . 7554/eLife . 10785 . 003Figure 1 . Schematic illustration of spatial arrangements of the FG nups in the NPC and in vitro models . Left: schematic rendering of the NPC geometry ( not to exact scale ) .", "The vertebrate FG nucleoporins discussed in this paper ( Nup62 , Nup98 and Nup153 ) and their approximate locations within the NPC are highlighted in color ( Chatel et al . , 2012; Krull et al . , 2004; Chug et al . , 2015 ) .", "Other FG nups are not shown .", "Yeast NPC has an overall similar architecture but smaller dimensions .", "Vertebrate FG nucleoporins discussed in the paper have yeast analogues: Nsp1 is analogous to Nup62 , while Nup100 and Nup116 are analogous to Nup98 in their sequence and the biophysical and functional properties .", "Right upper panel: schematic depiction of one typical in vitro experimental setup of a grafted FG nup layer in equilibrium with a solution of transport proteins .", "Right lower panel: some FG nups , such as Nup98 , phase separate at high concentration and form a dense phase in equilibrium with a dilute solution . DOI: http://dx . doi . org/10 . 7554/eLife . 10785 . 003 Full understanding of the NPC transport still remains elusive , and various hypotheses remain unsettled .", "The consensus is that the binding of the transport proteins to the FG nups enables them to overcome the permeability barrier .", "The strength of this binding controls the transport selectivity and efficiency .", "Hence , transport proteins can be informally viewed as 'glorified enzymes' that lower the free energy barrier for the translocation through the NPC .", "Basic models that describe the transport as facilitated diffusion through the FG nup medium , modulated by the interactions with the FG nups , provide a good explanation of the selectivity of the NPC even in the presence of large amounts of molecular noise ( Wente and Rout , 2010; Zilman et al . , 2007; 2010; Frey and Görlich , 2007; Fernandez-Martinez and Rout , 2012 ) .", "The overall veracity of these general principles has been demonstrated by creation of artificial nanochannels and nanomaterials that mimic NPC function and recapitulate many of its transport properties ( Zilman et al . , 2007; 2010; Frey and Görlich , 2007; Schmidt and Görlich , 2015; Zilman , 2009; Jovanovic-Talisman et al . , 2009; Kowalczyk et al . , 2011; Caspi et al . , 2008; Jovanovic-Talisman et al . , 2014 ) .", "Various models of the mechanistic involvement of the FG nups in transport have been proposed .", "In the 'virtual gate' model , the permeability barrier arises due to the entropic repulsion from the fluctuating FG nup chains ( Zilman et al . , 2007; Lim et al . , 2007; Rout et al . , 2003 ) .", "In a related idea , an entropically stabilized FG nup 'brush' can be collapsed by the transport proteins thus opening the transport passageway ( Lim et al . , 2006; 2007; 2008 ) .", "In another scenario , the permeability barrier arises from a gel-like network , stabilized by the hydrophobic interactions between the FG repeats .", "Transport proteins disentangle this gel via their binding to the FG repeats thereby allowing their passage through the pore ( Hülsmann et al . , 2012; Frey and Görlich , 2007; Frey et al . , 2006 ) .", "More complex models have been proposed that take into account the sequence inhomogeneity and local molecular properties of the FG nups , their possible spatial localization and dynamics ( Kim et al . , 2013; Patel et al . , 2007; Yamada et al . , 2010; Peters , 2009; Mincer and Simon , 2011; Cardarelli et al . , 2012; Ma et al . , 2012; Solmaz et al . , 2013; Lowe et al . , 2015 ) .", "It is likely that the majority of the effects invoked in all these models contribute to the NPC transport mechanism to some degree .", "In particular , the FG nups possess various degrees of intra- and inter-chain 'cohesiveness' that can lead to formation of single and multi-chain aggregates ( Schmidt and Görlich , 2015; Frey et al . , 2006; Patel et al . , 2007; Yamada et al . , 2010; Hough et al . , 2015; Milles and Lemke , 2011 ) .", "One major contribution to FG nup cohesiveness is believed to arise from the weak binding of the hydrophobic FG repeats to each other .", "However , intrinsically disordered proteins are notoriously prone to aggregation and the cohesiveness can have multiple sources , including electrostatic , π-π and π-charge interactions as well as non-specific interactions between the non-FG parts of the chains ( Uversky , 2002; Song et al . , 2013; Borg et al . , 2007; Milles et al . , 2013 ) .", "Cohesiveness of individual FG nups correlates with the ratio of the numbers of the hydrophobic to charged residues in their sequence ( Yamada et al . , 2010 ) .", "However , the relative importance of the cohesiveness and its specific role in the transport mechanism are still under debate .", "To add to the complexity of the system , the transport proteins are present in large numbers within the NPC , and can strongly affect FG nup conformations and dynamics ( Zilman et al . , 2007; 2010; Zilman et al . , 2009; Jovanovic-Talisman et al . , 2009; Yang and Musser , 2006;Lowe et al . , 2015 ; Kapinos et al . , 2014; Schoch et al . , 2012; Milles et al . , 2013 ) .", "However , several controversies persist with respect to their contributions to the architecture and the function of the NPC .", "Specifically , it is still under debate whether the transport proteins swell or compact assemblies of FG nups ( Kapinos et al . , 2014; Wagner et al . , 2015; Eisele et al . , 2010; 2013 ) .", "Understanding the spatial organization and the collective dynamics of the FG nups and the transport proteins during the translocation processes is hindered by the scarcity of experimental methods and insufficient measurement accuracy to assess them in vivo on the relevant time ( several milliseconds ) and length ( several nanometers ) scales ( Cardarelli et al . , 2012; Ma et al . , 2012; Yang and Musser , 2006; Yang et al . , 2004; Grünwald et al . , 2011; Dange et al . , 2008 ) .", "Consequently , computational and theoretical approaches - from atomistic to highly coarse grained - have become increasingly important in the investigations of the possible configurations of the FG nups and the transport dynamics within the NPC ( Zilman et al . , 2007; 2010; Mincer and Simon , 2011; Opferman et al . , 2013; Osmanović et al . , 2013a; 2013b; Tagliazucchi et al . , 2013; Moussavi-Baygi et al . , 2011a; 2011b; Ghavami et al . , 2014; Ando et al . , 2014; Gamini et al . , 2014 ) .", "However , parameterizations of such models still remain difficult because of the sheer complexity and diversity of the FG nups , exacerbated by conflicting or non-existent measurements of the relevant parameters .", "The existing parameterizations differ significantly in their physical assumptions and outcomes ( Tagliazucchi et al . , 2013; Ghavami et al . , 2014; Ando et al . , 2014; Gamini et al . , 2014 ) .", "As a case in point , even the radii of gyration of the FG nups and their interaction affinities with the transport proteins are still under debate ( Yamada et al . , 2010; Kapinos et al . , 2014; Tetenbaum-Novatt and Rout , 2010; Isgro and Schulten , 2005; Eisele et al . , 2010 ) .", "For further progress , it is imperative to establish the most pertinent physical features and variables controlling the FG nup conformations induced by the transport proteins .", "Recent in vitro studies provide the basis for systematic understanding of the conformations of the assemblies of the FG nups with transport proteins in order to relate their molecular properties to their nanomechanical behavior ( Hülsmann et al . , 2012; Frey and Görlich , 2007; Schmidt and Görlich , 2015; Lim et al . , 2007; Frey et al . , 2006; Kapinos et al . , 2014; Wagner et al . , 2015; Eisele et al . , 2010; 2013 ) .", "In one typical experimental setup , an FG nup assembly is grafted to a rigid surface in the presence of varying concentrations of the transport proteins ( see Figure 1 ) .", "The FG nup conformations are inferred from the measurements of layer height .", "Even this relatively simple experimental setup resulted in conflicting observations .", "Depending on the experimental conditions and the measurement technique , the transport proteins can either increase or decrease the layer height , or in some cases cause no measurable change .", "However , despite the variability between different FG nups and transport proteins , general behavior motif is emerging from these experiments .", "Typically , at low concentrations , the transport proteins do not affect the layer height .", "This is followed by a partial layer collapse and height decrease , accompanied by accumulation of the transport protein in the layer .", "Increasing the concentration further reverses the collapse and eventually leads to the swelling of the layer .", "Virtually all studied mixtures of the FG nups and transport proteins regardless of the species of origin or the natural localization in the NPC exhibit this general pattern of behavior although the degree of collapse and swelling may vary ( Lim et al . , 2006; 2007; Kapinos et al . , 2014; Wagner et al . , 2015; Eisele et al . , 2010; 2013 ) .", "Related patterns of behavior are observed in experiments with bulk solutions of FG nups mixed with various transport proteins .", "At sufficiently high concentrations , the FG nups form a dense phase which either absorbs or excludes the transport proteins , depending on the size of the latter and their interaction strength with the FG nups see Figure 1 .", "The behavior is very general and is observed in a wide range or FG nups from different species ( Hülsmann et al . , 2012; Frey and Görlich , 2007; Schmidt and Görlich , 2015; Hough et al . , 2015 ) .", "The generality of these behaviors - despite the large number of molecular factors affecting the FG nup behavior - suggests that it might be understood in terms of a small number of core organizing principles .", "In this paper we develop a coarse grained theory of the transport proteins-FG nup assemblies that captures only the essential physical features:", "( i ) the flexible nature of the FG nups ,", "( ii ) their potential cohesiveness and", "( iii ) the attractive interactions with the transport proteins .", "The model is investigated using mean field theory supported by coarse-grained simulations ( Opferman et al . , 2012; Opferman et al . , 2013 ) .", "Our approach is inspired by the successes of the simplified theories in explaining the properties of highly chemically complex and diverse materials in polymer science and soft condensed matter ( Doi and Edwards , 1998; Flory , 1953; de Gennes , 1979 ) .", "Systematic comparison of the model predictions with extensive experimental data shows that the model captures and explains the observed behavior in different regimes .", "The model suggests a resolution of some of the apparent conflicts in the experimental results and proposes how to reconcile the outstanding controversies regarding the relative importance of the cohesive and the entropic effects for FG nup behavior and NPC selectivity .", "The model also sheds light on the long standing discrepancies in the measurements of the binding affinities of transport proteins to FG nups .", "The model identifies the key physical variables controlling the conformational behavior of FG nup-transport protein assemblies and suggests experimental ways of manipulating them .", "This essential theoretical framework can be systematically developed in the future with additional molecular and structural details .", "Beyond the NPC , the results of the model are interesting in a broader context of intrinsically disordered proteins and nanotechnological applications ( Stuart et al . , 2010; Tagliazucchi and Szleifer , 2015; Coalson et al . , 2015 ) .", "Many aspects of the unfolded protein behavior are still puzzling , and the conceptual and computational frameworks - many of which are built on the foundation of polymer physics - are currently being developed ( Uversky , 2002; van der Lee et al . , 2014; Tcherkasskaya et al . , 2003; Das et al . , 2015; Sherman and Haran , 2006 ) .", "The model described here demonstrates the power of such approaches on a concrete example of an important family of intrinsically disordered proteins ." ], [ "To further simplify the discussion and establish the key variables controlling the experimentally observed behaviors , we assume that the monomer density is uniform throughout the layer .", "In reality , the monomer density inside the layer decays away from the grafting surface .", "We also assume that the entropic elasticity of the chains is described by the Gaussian model ( Alexander , 1977 ) .", "This simple approximation cannot be used to predict the exact layer height , but it is a qualitatively good treatment for the moderately cohesive chains and moderate concentrations of the transport proteins of interest in this paper , because the cohesiveness causes layer compaction , as shown below ( Opferman et al . , 2012; 2013; Halperin et al . , 2011; Alexander , 1977; Zhulina et al . , 1991; Lai and Halperin , 1992 ) .", "The overall conclusions of this paper do not depend on this approximation ( see Figure 2—figure supplement 1 ) .", "The discussion is further simplified by normalizing the average layer height h by the chain length L , introducing a new variable h¯=h/L .", "With these , the free energy per unit area of a layer of chains grafted at a distance a from each other becomes ( Alexander , 1977; de Gennes , 1980 ) ( 3 ) F⁢ ( h¯ , ψ , ϕ ) /A=k⁢T⁢Ll3⁢σ¯⁢h¯22⁢lb+h¯⁢f⁢ ( ψ , ϕ ) , where σ¯= ( l/a ) 2 is the grafting density of the chains normalized by the average monomer cross-section l-2 ( l≡v01/3 ) .", "The layer height related to the monomer density through the condition σ¯⁢lb=ψ⁢h¯ which expresses the fact that the total number of the monomers in the layer is constant , h⁢l3⁢ψ=N⁢σ .", "At this level of approximation , the equilibrium layer height is found by the minimization of the free energy over h¯ and the transport protein concentration ϕ under the constraints that the chemical potential of the transport proteins in the layer and the osmotic pressure in the layer are equal to those in the outside solution of volume fraction c .", "Importantly , because L factorizes out of the free energy expression in Equation ( 3 ) , the resulting equilibrium values of h¯ and ϕ are independent of the chain length L . The chemical potential and the osmotic pressure of the outside dilute solution - assumed to be ideal - are μc=kB⁢T⁢ln⁡ ( c ) and πc=kB⁢T⁢c/v .", "This procedure has been described in detail and verified by coarse-grained brownian dynamics simulations in Opferman et al . ( 2012 ) , Opferman et al . ( 2013 ) .", "Finally , it is important to keep in mind that the calculated properties are equilibrium average values .", "On the molecular scale , the polymers are highly dynamic and their individual conformations fluctuate on the microsecond time scale .", "In a bulk solution where the chains are not grafted to a surface but are freely floating in solution , the entropic stretching ( first term in Equation ( 3 ) ) is replaced by the translational entropy of the chains , so that the mean field free energy per unit volume is Doi and Edwards ( 1998 ) , Flory ( 1953 ) , de Gennes ( 1979 ) ( 4 ) f⁢ ( ψ , ϕ ) =1N⁢ψlnψ+1v¯⁢ϕlnϕ+ ( 1-ψ-ϕ ) ln ( 1-ψ-ϕ ) + ( 1v¯-1 ) ⁢ ( 1-ϕ ) ⁢ln ( 1-ϕ ) +1v¯⁢χ⁢ψ⁢ϕ+12⁢χc⁢r⁢ψ2 .", "This free energy becomes unstable for sufficiently large interaction parameters |χ| or |χc⁢r| , leading to a phase separation where a dense phase of transport proteins mixed with the FG nups coexists with a very dilute solution .", "The compositions of the dense and the dilute phases are determined from the equality of the osmotic pressures and the chemical potentials of the transport proteins and the FG nups in the coexisting phases ( de Gennes , 1979; Zilman and Safran , 2002; Morse , 1969 ) see Appendix for details .", "The model can be used to calculate the dimensions of individual FG nup molecules in solutions .", "Due to the thermal motion , each flexible chain dynamically samples multiple spatial conformations that on average occupy a volume of size R , which can be found through minimization of the free energy 3⁢R22⁢N+43⁢π⁢R3⁢f⁢ ( ψ ) over R with the condition 43⁢π⁢R3⁢ψ=N; the first term represents the entropic elasticity of chain conformations in space , while the second term describes the intra-chain interactions where f⁢ ( ψ ) is the free energy of Equation ( 2 ) with ϕ=0 .", "Slightly different expressions for the free energy can be used , all leading to qualitatively the same results ( de Gennes , 1979; Sherman and Haran , 2006; Sanchez , 1979 ) .", "As shown below , the overall qualitative predictions of the theory are robust with respect to the choice of model parameters within a physically feasible range .", "However , quantitative or semi-quantitative comparison with the experimental data requires a specific choice of the molecular parameters b and v0≡l3 .", "The approximate volume of a transport protein molecule can be calculated from its molecular mass and the average protein density ρ≃1 . 2-1 . 5 g/cm3 .", "For Karyopherin-β⁢1 ( molecular mass ≈97-103 kD , depending on the attached tag ) , v≃120-140 nm3 and for NTF2 , the transport protein specialized for the import of RanGDP into the nucleus ( molecular mass ≈33 kD ) , v≃35-45 nm3 .", "The estimates of the 'monomer' size are somewhat less well defined .", "The average distance between two adjacent amino acids on a polypeptide chain is≈0 . 36-0 . 38 nm and the side chain size varies in the range ∼0 . 3-0 . 6 nm ( Levitt , 1976; Zamyatnin , 1972; Quillin and Matthews , 2000 ) .", "However , their effective size can be modulated by the bound ions present within the Debye screening length ( of the order of ∼0 . 5-1 nm at the experimental salt concentrations ) or bound denaturant molecules .", "Thus , for comparison with experiments , the realistic monomer size lies within a range b ≈ 0 . 4-1 . 6 nm and its volume v0 ≈ 0 . 12-1 nm3; the upper limit corresponds to a 'monomer' composed of about four amino acids .", "Finally , the exact structure of the dense phase and its maximal molecular packing fraction are unknown ( Frey and Görlich , 2007; Schmidt and Görlich , 2015; Milles et al . , 2013 ) .", "For the conversions between the theoretical volume fractions and the experimentally measured concentrations , we have assumed the maximal packing fraction z=0 . 625 in the dense phase , typical of the random close packing of dense molecular assemblies which normally lies in the range of z=0 . 5-0 . 7 ( Nolan and Kavanagh , 1992 ) ." ], [ "To establish whether the model captures the basic biophysical characteristics of the FG nup assemblies , we first apply it to the case of an FG nup layer without transport proteins .", "In this case the transport protein density is ϕ=0 , and the free energy per area A of Equations ( 2 ) and ( 3 ) is minimized over h¯ to obtain the equilibrium average layer height .", "We emphasize again that once the layer height has been re-scaled by the polymer contour length L , the theoretical predictions become independent of L . This will be important in the analysis of the experimental data .", "The conclusions of the model are summarized in Figure 2 , which shows that the FG nup layer height h decreases with the grafting distance a , because the steric repulsion between the polymers - that maintains the chains being stretched on average - is higher at lower grafting distances .", "The theory predicts that the layer height is monotonically decreasing with the cohesion strength .", "This is expected because the cohesiveness favors more compact conformations with more favorable contacts while the entropic elasticity term favors more diffuse conformations .", "In polymer physics parlance , increasing the cohesiveness is similar to changing the solvent 'quality' from 'good' to 'bad' ( Osmanović et al . , 2013a; Eisele et al . , 2013; de Gennes , 1979; Milner et al . , 1988; Halperin et al . , 2011; Lai and Halperin , 1992; Zhulina et al . , 1991; Peleg et al . , 2011; Moh et al . , 2011 ) .", "The model also shows that sufficiently strong cohesion not only decreases the layer height , but shifts the behavior into a qualitatively different regime .", "The FG nup internal conformation and cohesiveness can be characterized by the scaling exponent g that describes the dependence of the height h on the grafting distance a , h ~ a-g .", "For an ideal non-cohesive , purely entropically stabilized polymer brush , g=2/3 .", "Cohesiveness increases the value of the exponent towards g=2 , in which regime the layer effectively behaves as a material of constant density with ψ independent of h ( de Gennes , 1979; Milner et al . , 1988; Zhulina et al . , 1991; Moh et al . , 2011 ) .", "Nevertheless , even in the highly cohesive regime , the layer has significant 'free space' occupied by the solvent ( calculated as 1-ψ ) . 10 . 7554/eLife . 10785 . 004Figure 2 . Cohesion makes FG nup layers more compact: theoretical predictions . Layer height h/L normalized by the chain length as a function of the normalized grafting distance a/l for increasing cohesiveness ( χc⁢r varies from χc⁢r = 0 to χc⁢r = -1 . 5 ) .", "For any value of χc⁢r , the curve is well approximated by the dependence h ~ a-g .", "The inset shows that the exponent g increases from 2/3 to 2 as the absolute value of the cohesion strength |χc⁢r| .", "DOI: http://dx . doi . org/10 . 7554/eLife . 10785 . 00410 . 7554/eLife . 10785 . 005Figure 2—figure supplement 1 . Effect of density non-uniformity on the model predictions . The continuous lines show the model predictions assuming step density profile , for χc⁢r = 0 ( blue ) , χc⁢r = -1 ( green ) and χc⁢r = -1 . 8 ( red ) ; the corresponding predictions without the step function profile assumption , calculated with SCFT are shown in dots of the same color ( Opferman et al . , 2012; 2013 ) .", "Despite small numerical differences , the trends predicted by the two models are the same .", "Similarly , the step density profile does not affect the pertinent theory predictions in the presence of nanoparticles in relevant regime ( Opferman , 2014 ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 10785 . 005 Behavior of the FG nup layers reported in Kapinos et al . ( 2014 ) , Schoch et al . ( 2012 ) , Wagner et al . ( 2015 ) follows the general predictions of the theory .", "In particular , examination of the data shows that the height of FG nup layers in the absence of transport proteins decays faster than a-2/3 with the grafting distance a , but slower that a-2 , as shown in Figure 3 .", "The accuracy of the experimental measurements does not allow one to quantitatively differentiate between different FG nups based on the scaling exponent g .", "However , interpreted in light of the theoretical model , the data strongly indicate the presence of significant cohesion in all FG nups .", "Importantly , Nsp1 segments of different lengths fall within the same family of curves , once normalized by their length , in accord with the theoretical predictions .", "These results indicate that the salient physical mechanisms responsible for the behavior of grafted FG nup layers are adequately captured by the model . 10 . 7554/eLife . 10785 . 006Figure 3 . FG nup layer height depends on the grafting distance: theory vs . experiment . The dots are the experimentally measured layer heights from Kapinos et al . ( 2014 ) and Wagner et al . ( 2015 ) normalized by the FG nup length .", "Red , blue , green and black: grafted layers of Nup62 , Nup98 , Nup153 and Nsp1 , respectively; gray dots belong to a short Nsp1 segment .", "Solid line: h ~ a-2/3 is the ideal brush ( χc⁢r = 0 ) behavior obtained from the model .", "Dotted line: h ~ a-2 is the behavior of a strongly collapsed brush with χc⁢r = -2 . 5 .", "All the FG Nups lie between these two regimes , indicating a significant amount of cohesion for all FG nups; the dashed line is for χc⁢r = -0 . 8; the dashed-dotted line is for χc⁢r = -1 . 4 .", "To enhance the contrast , inset shows the same data with the height h normalized by the ideal brush height .", "b = 1 . 52 nm , l = 1 nm . DOI: http://dx . doi . org/10 . 7554/eLife . 10785 . 006 We now compare the theoretical predictions with the experimental data in the presence of transport proteins .", "Although at a first glance experimentally observed responses of different FG nups to the addition of transport proteins appear rather different ( Kapinos et al . , 2014; Eisele et al . , 2010 ) , closer inspection shows that most FG nups exhibit the same general pattern of behavior .", "With progressive addition of the transport proteins , the layer height decreases to some extent followed by recovery and eventual swelling .", "The initiation of the collapse is correlated with the penetration of the transport proteins into the layer .", "We have recently shown that this behavior is expected on very general grounds for polymer layers infiltrated with nanoparticles ( Opferman et al . , 2012; 2013 ) .", "This common behavior is shown in Figure 4 , which renders the experimental data from Kapinos et al . ( 2014 ) , Wagner et al . ( 2015 ) . 10 . 7554/eLife . 10785 . 007Figure 4 . Characteristic responses of FG nup layers to the transport proteins: experimental results . Upper panel: change in the layer height relative to the unperturbed layer as a function of the transport protein concentration in the outside solution .", "Lower panel: number of the transport proteins in the layer per unit length of the FG nup chain .", "Each line corresponds to a different run with a different initial layer height and grafting distance .", "Different colors correspond to different FG nups , which all exhibit qualitatively similar behavior .", "Color coding .", "Red , blue , green and black: Karyopherin-β⁢1 interacting with Nup62 , Nup98 , Nup153 and Nsp1 , respectively; magenta: NTF2 interacting with Nsp1 .", "The corresponding average grafting distances are ∼2 . 5 nm , ∼4 nm , ∼4 . 5 nm , ∼3 . 7 nm .", "The data are from Refs .", "( Kapinos et al . , 2014; Wagner et al . , 2015 ) DOI: http://dx . doi . org/10 . 7554/eLife . 10785 . 00710 . 7554/eLife . 10785 . 008Figure 4—figure supplement 1 . Nup214 . Experimentally obtained normalized height of Nup214 layer vs . Kap-β concentration in the solution .", "The inset shows the number of adsorbed Kap-β molecules per monomer as a function of their concentration in solution .", "Data from Ref . ( Kapinos et al . , 2014 ) .", "Behavior of Nup214 qualitatively obeys the same 'bi-phasic' pattern as the rest of the studied FG nups and lies within the landscape of behaviors predicted by the model .", "However , it is plotted separately because the experimental data is sparse at low concentrations and is highly variable at high concentrations .", "Multiple reasons can possibly account for this behavior , analysis of which lies outside the scope of the present work . DOI: http://dx . doi . org/10 . 7554/eLife . 10785 . 008 Intuitively , the penetration of an individual transport protein into the layer is determined by the balance between the energetic ( enthalpic ) gain of creating more contacts with the FG nups and the entropic cost of displacing and crowding the FG nup chains .", "If the overall free energy change upon insertion of one transport protein into the layer is negative , it will typically penetrate the layer .", "Otherwise , the penetration is exponentially suppressed , although there still will be some particles in the layer .", "For a single particle of radius R , at low particle concentration , the entropic cost of penetrating an ideal polymer brush layer can be estimated as ≃α⁢k⁢T⁢R2⁢ψ , where the prefactor α depends on the grafting density and the degree of cohesiveness ( Halperin et al . , 2011; Egorov , 2012; Milchev et al . , 2008 ) .", "On the other hand , a rough estimate for the energetic/enthalpic gain contacts is ≃-ϵ⁢n⁢ψ , where n is the number of the interaction sites on the protein and ϵ is the energy per contact .", "Thus , for ϵ<α⁢k⁢T⁢n/R2 the entropic repulsion dominates , and one does not expect significant penetration into the layer .", "In principle , this entropic repulsion from the flexible polymer layer is sufficient for creating the permeability barrier for non-binding molecules .", "It is crucial to bear in mind that the polymer chains are not static but highly fluctuating entities .", "It is the entropy of these molecular motions that is responsible for the penetration barrier; thinking of a polymer layer as a static entity with some amount of free space can lead to erroneous conclusions .", "The barrier could be enhanced by other effects , such as , for instance , inter-chain cohesion .", "Thus , at low concentrations the transport proteins penetrate the layer if their attractive interaction with the FG nups is strong enough .", "However , they do not cause significant conformational changes - essentially occupying the available empty space inside the layer .", "At higher densities of the transport proteins , or higher interaction strengths , the number of transport proteins in the layer increases , and collective effects start to play a role ( Opferman et al . , 2013; Halperin and Kröger , 2011; Kim and O’Shaughnessy , 2006 ) .", "Further addition of the transport proteins causes a cooperative conformational transition of the FG nups leading to either collapse or swelling of the layer , correlated with the accumulation of the transport proteins inside the layer .", "The magnitude of the collapse and swelling depend on the transport protein size , concentration , interaction strength with the FG nups , the grafting density , and the cohesion strength .", "Typical height responses to transport protein concentration are shown in Figure 5 .", "One important conclusion of the theory is that the FG nup response to the addition of the transport proteins ( for instance , 'swelling' vs . 'collapse' ) is not an intrinsic property of an FG nup , but can be modulated by the grafting density , transport protein size and the interaction strength .", "The overall repertoire of predicted behavior as a function of parameters is shown in the 'phase diagrams' in Figures 6 and 7 .", "The model captures the general salient features of the experimental observations .", "In particular , at the same grafting distance and the cohesion strength , the collapse is more pronounced for a smaller protein such as NTF2 , in accord with the experimental observations ( Wagner et al . , 2015 ) .", "Similarly , higher grafting distance results in more collapse - because the entropic repulsion of the transport proteins by the chains is lower at lower chain density .", "Different experimental results can be placed in the different parts of the 'phase diagram' , potentially explaining the observed discrepancies .", "Notably , the theory predicts a high degree of collapse at large grafting distances , in agreement with the experiments of ( Lim et al . , 2007 ) .", "Another interesting prediction of the model is that changing the grafting distance affects small and large transport proteins in a different way .", "For large ones ( e . g . Karyopherin ) , increasing the grafting distance enhances the penetration into the layer because it reduces the repulsive barrier due to lower monomer density in the layer .", "By contrast , for small transport factors ( such as NTF2 ) , for which the repulsion is less important , increasing the grafting distance decreases the penetration into the layer because it reduced the density of available binding sites in the layer . 10 . 7554/eLife . 10785 . 009Figure 5 . Layer collapse and swelling: effect of cohesion and of the grafting distance . Upper panel: Theoretical curves show that FG nup cohesion can convert layer collapse to swelling .", "The cohesion strengths are χc⁢r = 0 , -0 . 4 , -0 . 8 , -1 . 1 for a = 5 nm and χ = -550 .", "Lower panel: Increasing grafting distance increases the magnitude of the layer compaction .", "The lines correspond to model predictions for a = 3 , 4 , 5 , 6 nm for χ = -530 and χc⁢r = -1 .", "The insets show that the fraction of free space in the layer , calculated as 1 - ϕ - ψ , decreases with the addition of the transport proteins .", "b = 1 nm , l = 0 . 67 nm in both panels . DOI: http://dx . doi . org/10 . 7554/eLife . 10785 . 00910 . 7554/eLife . 10785 . 010Figure 6 . The 'phase diagram' of predicted behaviors: conformational transitions of the layer . The grayscale color denotes the degree of layer compaction , hm⁢i⁢n/h0 , relative to the unperturbed layer ( color legend is on top ) .", "The colored contour lines indicate the corresponding bulk concentration cm⁢i⁢n at which the minimal layer height is achieved ( legend on the right side ) .", "There is no layer swelling above the dashed line ( up to 1 μM transport protein concentration ) .", "Upper panel: v¯= 125 , roughly corresponding to Karyopherin-β⁢1; Lower panel: v¯ = 40 , roughly corresponding to NTF2 .", "The overall phase diagram topology is similar in both cases , but for smaller protein the collapse is more pronounced and occurs at lower interaction strengths χ .", "In both panels b = 1 . 52 , l = 1 nm , corresponding to the 'monomer' size of roughly four amino acids . DOI: http://dx . doi . org/10 . 7554/eLife . 10785 . 01010 . 7554/eLife . 10785 . 011Figure 6—figure supplement 1 . Model predictions are robust with respect to the monomer size estimate . We have performed extensive sensitivity analysis of the model with respect to the choice of the molecular parameters .", "It is illustrated in the phase diagram as a function of the grafting distance a and the interaction strength χ with a different parameter choice , l = 0 . 75⁢ nm , b = 1 . 4⁢ nm , v = 124⁢ nm3 , χc⁢r = -1 .", "The topology of the phase diagram is the same as in Figure 6 , and the same qualitative trends are observed in both cases , with three regions of behaviour: 'collapse only' , 'collapse and swelling' , and 'swelling only'; the agreement with the experimental data is also of a similar quality . DOI: http://dx . doi . org/10 . 7554/eLife . 10785 . 01110 . 7554/eLife . 10785 . 012Figure 7 . The 'phase diagram' of predicted behaviors: amount of transport protein in the layer . The grayscale color denotes the degree of layer compaction , hm⁢i⁢n/h0 , relative to the unperturbed layer ( color legend is on top ) .", "The colored contour lines show the amount of adsorbed proteins in the layer per chain monomer .", "Higher degree of collapse is correlated with higher accumulation of the proteins in the layer .", "Upper panel: v¯=125 , roughly corresponding to Karyopherin-β⁢1; Lower panel: v¯=40 , roughly corresponding to NTF2 .", "In both panels b = 1 . 52 , l = 1 nm . DOI: http://dx . doi . org/10 . 7554/eLife . 10785 . 012 Can this simplified theory be qualitatively or semi-quantitatively related to the experimental data for realistic values of parameters ?", "Due to the experimental accuracy limitations and large uncertainly in the known values of all the parameters , the fitting of the parameters to the data is not unique and has limited information content .", "Rather , we focus on clearly distinguishable trends , such as the difference between the behavior of Karyopherin-β⁢1 vs . NTF2 on the Nsp1 layer , reported in Wagner et al . ( 2015 ) .", "These measurements , performed on the same FG nup in approximately same range of grafting distances , allow us to examine the effects of the transport protein size and the binding strength , unconfounded by other factors .", "Karyopherin-β⁢1 and NTF2 have significantly different sizes and binding strengths .", "NTF2 is ∼ 4 times smaller in volume and has only two binding sites , while Karyopherin-β⁢1 can have up to ten specific FG-binding sites and a significantly larger surface area with potentially much larger number of non-FG interactions with the FG nups ( Bayliss et al . , 1999; 2000; 2002; Isgro and Schulten , 2005; Liu and Stewart , 2005 ) .", "We focus on one clearly discernible difference in the experimental behavior: significant penetration of NTF2 starts at higher concentrations but causes stronger compaction of the layer compared to Karyopherin-β⁢1 .", "Comparison of the theoretical predictions with the experimental results is shown in Figure 8 in the range of concentrations where direct comparison is possible .", "Because of the relatively large uncertainty in the measurements of the absolute layer height , the grafting distance ( see Figure 4 ) and the binding strengths , the theoretical predications are shown for a range of values approximately corresponding to the experimental ones .", "The model reproduces the observed differences in the behavior of Karyopherin-β⁢1 and NTF2 on Nsp1 at physically plausible values of the parameters in the regime of its validity .", "It might also explain why no significant change in the layer height ( or very limited swelling ) was observed upon addition of transport proteins by other experimental groups ( Eisele et al . , 2010; 2012 ) : the behavior of both NTF2 and Karyopherin-β⁢1 can be easily shifted into the swelling regime by relatively small changes in the grafting distance , cohesion or interaction strength , the latter of which can be modulated by small changes in the pH or salt and denaturant concentrations .", "Other patterns revealed by the experimental data shown in Figure 4 , such as the dependence of the maximal compaction concentration on the grafting distance , are also qualitatively explained by the theory .", "Quantitative comparison of these features requires more analysis of the data and more detailed approximations , including the sparse and high transport protein density regimes , and will be presented elsewhere . 10 . 7554/eLife . 10785 . 013Figure 8 . Comparison of the theoretical predictions with the experimental data in the layer geometry . Theoretical predictions for the range of the parameter values approximately corresponding to the experimental ones for Nsp1 layers infiltrated by Karyopherin-β⁢1 and NTF2 .", "Upper panel: layer height vs bulk concentration of the transport protein .", "Red: Karyopherin-β⁢1 , Blue: NTF2 .", "Lower panel: amount of adsorbed transport protein in the layer as a function of the concentration in the solution .", "Red: Karyopherin-β⁢1 , Blue: NTF2 .", "The shaded regions correspond to 3 . 5 < a < 4 nm and -73 < χ < -63 for NTF2 and -185 < χ < -175 for Karyopherin- β⁢1 .", "For all lines , b = 1 . 52 , l = 1 nm and χc⁢r = -1 .", "The insets show the corresponding experimental data from Wagner et al . ( 2015 ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 10785 . 01310 . 7554/eLife . 10785 . 014Figure 8—figure supplement 1 . Model predictions are robust with respect to the monomer size estimate . We have performed extensive sensitivity analysis of the model with respect to the choice of the molecular parameters .", "As an illustration , predictions of the theory for l = 0 . 75⁢ nm , b = 1 . 4⁢ nm , v = 124⁢ nm3 , χc⁢r = -1 are compared with the experimental data from Kapinos et al . ( 2014 ) ; the agreement is of a similar quality . DOI: http://dx . doi . org/10 . 7554/eLife . 10785 . 014 The chosen values of b and l correspond to the 'monomer' size of approximately four amino acids - roughly the size of one FG patch , reflecting the fact that FG nup interaction with the transport proteins requires this particular local sequence of amino acids ( Bayliss et al . , 2000; 2002 ) ; we emphasize that our model does not correspond to a randomized amino acid sequence of the FG nups ( Tagliazucchi et al . , 2013; Ghavami et al . , 2014 ) .", "The interaction parameter roughly χ corresponds to the attractive part of the second virial coefficient of the interaction between the transport proteins and the monomers , so that χ≃n⁢ϵk⁢T⁢eϵ/k⁢T , where n is the number of available binding sites on the protein , and ϵ is the average binding energy of one site ( Doi and Edwards , 1998; Pathria , 1996 ) .", "Taking into account the possible non-FG interactions , n ~ 6-14 for Karyopherin and n ~ 2-5 for NTF2 .", "This translates to ϵ ~ 2 . 5-3⁢k⁢T for the chosen parameter values , which is a reasonable estimate for the average energies of the weak hydrophobic and electrostatic interactions in question .", "These numbers also agree with recent estimates using other methods ( Kapinos et al . , 2014; Tu et al . , 2013 ) .", "Similarly , assuming that the main contribution to cohesiveness comes from the FG-FG interactions , the value of χc⁢r=-1 corresponds to ϵc⁢r≃2⁢k⁢T .", "These values will guide our analysis of bulk solutions of FG nups and transport proteins in the next section .", "Importantly , the general agreement between the theory and experiment is robust with respect to parameterization choices ( see Figure 6—figure supplement 1 ) .", "The model also sheds light on another long-standing controversy in the field - the high variability among different experimental groups of the measured affinities of the transport protein binding to the FG nups , with values of the measured dissociation constant ranging from several nanomolars to several micromolars ( Bayliss et al . , 1999; Kapinos et al . , 2014; Eisele et al . , 2010; Pyhtila and Rexach , 2003; Tetenbaum-Novatt et al . , 2012 ) .", "As can be seen in Figure 8 , due to the collective effects and the conformational changes during binding , the adsorption curves are not well described by the standard Langmiur curve typically used to quantify the interaction in binding assays ( Kapinos et al . , 2014 ) .", "An attempt to fit it with one or a combination of Langmiur isotherms would lead to different results depending on the concentration range and the grafting density ( Schmidt and Görlich , 2015 ) .", "The model developed here provides fundamental physical reasons for the discrepancies in experimental measurements .", "The fundamental physical considerations underlying the behavior of the surface assemblies of FG nups manifest themselves also in the behavior of mixtures of the FG nups and the transport proteins in bulk solutions .", "Such mixtures were systematically studied in Schmidt and Görlich ( 2015 ) over a wide range of FG nups from different species ( recombinantly expressed in bacteria ) , systematically varying the size of transport protein-cargo complexes .", "It was found that even in the absence of transport proteins , solutions of Nup98 FG nucleoporin phase separate into a dense phase with a very high protein concentration , in equilibrium with a very dilute solution .", "Importantly , unlike the previously reported 'gels' of Nup98 and other FG nucleoporins ( Hülsmann et al . , 2012; Frey and Görlich , 2007 ) , these phases form via an equilibrium phase separation mechanism , allowing comparison with our model .", "Upon addition of cargo-carrying transport proteins bound to such FG nup solutions , the transport protein-cargo complexes either penetrate the dense phase , or stay predominantly in solution , depending on their size and the interaction strength with the FG nups .", "These results are summarized in Figure 9 .", "In this section , we show that the observed patterns naturally follow from the minimal model of this paper . 10 . 7554/eLife . 10785 . 015Figure 9 . Partitioning of transport proteins into dense FG nup phase: summary of experimental results . Experimentally , partitioning of the transport proteins ( TP ) complexes with various cargoes into the dense FG nup phase depends on the cargo size and the overall interaction strength of the complex with the FG nups .", "Both Importin-β ( vertebrate homologue of Kap-β⁢1 ) and NTF2 penetrate the dense phase , but the Importin-β with either medium ( IBB-GFP ) or large ( IBB-MBP-GFP ) cargo does not .", "However , the very large complex of four Importin-β complexed with four ZsGreen proteins partitions into the dense phase .", "The results are for the dense phase of TtNup98 of Tetrahymena Thermophila recombinantly expressed in bacteria , adapted from Schmidt and Görlich ( Schmidt and Görlich , 2015 ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 10785 . 015" ], [ "Mechanistic understanding of transport through the Nuclear Pore Complex is hindered by the complexity of the NPC organization and the absence of experimental methods for directly probing FG nup conformations and dynamics during transport .", "One has to rely on the interpretation of indirect measurements of the FG nup properties in vitro , which are partially incomplete or conflicting .", "On the theoretical side , the progress is hampered by the lack of a universally accepted comprehensive theory of intrinsically disordered proteins that is able to incorporate all the factors dictating the physico-chemical and nanomechanical properties of the FG nups .", "The theoretical model developed here provides a rigorous physical framework for organizing the known phenomenology of the observed FG nup behaviors and suggests ways to reconcile the apparently contradictory experimental findings and models of transport .", "The model relies on the minimal number of key physical concepts and variables to describe the FG nups and their interactions with the transport proteins , integrating the multitude of molecular details into a small number of variables , thus avoiding the over-fitting problem , inherent to models with multiple unknown parameters .", "It has been already suggested that the biophysical properties and dimensions of individual FG nups can be , to a large extent , captured in one phenomenological parameter , the hydrophobic to charged amino acid content ratio , paralleling our cohesiveness strength χc⁢r ( Yamada et al . , 2010 ) .", "Our model extends these concepts into the regime of multiple chain assemblies interacting with transport proteins .", "We have found that this limited set of concepts is sufficient to qualitatively , and even semi-quantitatively , explain the experimentally observed patterns and trends .", "We expect that explicit inclusion of several important factors such as the spatial distributions of ions , discrete nature of the binding sites on the transport proteins , non-linear elasticity of the chains , potential heterogeneity of the FG nup sequence , and more careful modeling of the microscopic structure of the FG nups assemblies will lead to better quantitative agreement .", "Additional sources of discrepancy are the possible length polydispersity of the FG nups , transport protein aggregation , and inherent biases of the experimental techniques ( for instance , in the SPR method , for technical reasons the measurement of the layer height lags behind in time after the measurement of the protein adsorption ( Wagner et al . , 2015 ) .", "The theory provides clear experimental predictions for the variation of the properties of FG nup-transport protein assemblies with the experimental conditions such as the grafting density .", "Such measurements would allow further systematic refinement of the model .", "The analysis of this paper shows that both entropic ( 'brush-like' ) and entalpic/cohesive ( 'gel-like' ) effects naturally cooperate in determining the spatial structures of the assemblies of FG nucleoporins with the transport proteins , and suggests how the 'brush' and the 'gel' concepts can be reconciled .", "Although the overall qualitative predicted behavior of the surface layers is similar for cohesive and non-cohesive chains alike , quantitative comparison with experiments was only possible by assuming a certain amount of cohesiveness for the studied FG nups .", "Our analysis indicates that all FG nups likely possess some degree of intra- and inter-chain cohesiveness , although only for some of them it is sufficiently strong to cause aggregation in bulk solutions .", "The model also shows that the different classes of 'extended' and 'collapsed' FG nups ( Yamada et al . , 2010 ) can be accounted for by different values of the cohesiveness χc⁢r .", "Another puzzling observation of the resilience of the NPC transport with respect to the deletion of large numbers of FG nups might be attributed to the fact that the permeability and the selectivity of the FG nup assemblies are relatively insensitive to the grafting density in a significant range - at lower grafting densities , the neighboring chains simply expand , maintaining selective permeability properties of the layer ( Popken et al . , 2015 ) .", "Finally , relatively weak interaction energies of the FG nups among themselves and with the transport proteins inferred from our analysis are consistent with the high local molecular mobility inside the dense aggregates observed experimentally in Schmidt and Görlich ( 2015 ) , Hough et al . ( 2015 ) .", "The success of the theory relies on the very robust physical mechanisms underlying it .", "Attractive interactions between long flexible filaments and compact objects , such as folded proteins , cause their surface assemblies to attain more compact conformations at low concentrations of the transport proteins and swollen conformations at higher concentrations .", "Similarly , in bulk solutions of ungrafted chains , inter-chain interactions and the interactions with transport proteins lead to phase separation and formation of a dense phase at sufficiently high concentrations .", "These behavior motifs are always expected irrespective of the nature of the interactions on the molecular scale .", "In this sense , all such systems lie in the same 'universality class' ( de Gennes , 1979 ) .", "The ability of the model to capture the behavior of different FG nups in different geometries under a variety of experimental conditions makes it a useful tool for the development of further , more refined , models .", "Finally , the analysis of this paper underscores the importance of always considering the presence of the transport factors when thinking about NPC architecture .", "The theory also sheds light on the discrepancies in the experimental measurements of the binding affinities of the FG nups to transport proteins .", "Depending on the experimental procedure , the values of the measured dissociation constants range from several nanomolars to several micromolars .", "Moreover , some of these measured affinities appear to be inconsistent with the observed transport times in the millisecond range ( Yang and Musser , 2006; Yang et al . , 2004; Tetenbaum-Novatt and Rout , 2010; Tetenbaum-Novatt et al . , 2012; Tu et al . , 2013; Denning et al . , 2003; Ma , 2010 ) .", "Our theory shows how these discrepancies might stem from the fundamental statistical thermodynamics of the transport protein-FG nup interaction .", "First , as shown in this paper , penetration of the transport proteins into an FG nup layer is a cooperative process , not described well by a single Langmiur isotherm typically used in the interpretation of binding assays .", "Second , penetration of the transport proteins into the layer is determined not only by enthalpic but also entropic effects and therefore the measured effective affinity does not directly reflect the interaction energies .", "Finally , even the purely enthalpic part of the interaction can vary with the experimental conditions , because the average number of monomers available to bind to a transport protein depends on the monomer concentration , which in turn depends on the layer height and grafting distance ( Kapinos et al . , 2014; Schoch et al . , 2012; Tu et al . , 2013; Sethi et al . , 2011 ) .", "All this highlights the fact that the classical characterization of inter-molecular interactions by a single affinity value is not informative for complex multivalent interactions of spatially extended objects such as the FG nups .", "It is also worth bearing in mind that a 1000-fold difference in the dissociation constant translates into ≈7⁢k⁢T difference in the effective interaction strength - a relatively small difference that can be easily influenced by many factors .", "The results of this paper have important implications for the behavior of the Nuclear Pore Complex and design of bio-molecular sorters based on the same principles .", "One has to be careful in making inference about the NPC properties based on the in vitro results because the detailed features of the actual spatial morphologies of FG nup assemblies in the channel-like geometry of the NPC are likely to differ from flat and bulk geometries .", "Analysis of this paper establishes the pertinent parameters that constrain possible scenarios and guide future model building .", "Recent work on coarse grained models of flexible chains in channel geometries allows us to gauge the implications of the findings in the flat and bulk geometries for FG nup morphologies within the NPC ( Osmanović et al . , 2013a; Coalson et al . , 2015; Peleg et al . , 2011 ) .", "Behavior of non-cohesive or weakly cohesive chains in relatively wide pores - wider than the natural height of the planar layer - is expected to be qualitatively similar to that of flat layers .", "In this case , the chains form a relatively dense layer along the inner surface of the channel , and increase in cohesiveness compacts the layer towards the walls .", "Addition of transport proteins causes either collapse or swelling of this surface layer , analogously to the behavior in the flat geometry .", "In the other limit of strongly cohesive chains in relatively narrow channels , the monomers of the chains accumulate at the pore center in a plug-like shape .", "It is still unknown in what regime the NPC lies , and further computational and experimental work is required .", "These findings are also interesting in the more general context of intrinsically disordered proteins , where physics concepts are often invoked to organize and explain the experimental observations ( Uversky , 2002; van der Lee et al . , 2014; Das et al . , 2015 ) .", "Although the qualitative behavior motifs predicted by the theory and borne out by the experiments are very general , the specific quantitative features such as the exact height or the degree of compaction are rather sensitive to the parameter values , such as the grafting distance , the density and the interaction strength .", "This has been already noted in other computational theories ( Popken et al . , 2015; Osmanović et al . , 2013a; Tagliazucchi et al . , 2013; Gamini et al . , 2014 ) .", "This raises a question - how the Nuclear Pore Complex functioning remains so invariant across species despite the large variations in sizes and spatial organization .", "Similarly puzzling in this light is the ability of the NPC to maintain its function despite large structural perturbations ( Strawn et al . , 2004; Hülsmann et al . , 2012 ) .", "One possible solution to this puzzle is that the NPC is exquisitely ”fine tuned” in a sense that it works correctly only when all the molecular details are right - the FG nup sequence and localization , local pH , ionic strength and concentrations of other molecules - and these conditions are maintained by the cellular homeostasis .", "On the other hand , the results of this model suggest another possibility - that the NPC is 'robust' in a sense that any structure with the approximately right physical properties will function nearly optimally , which could explain the NPC resilience with respect to structural damage and re-arrangements .", "This provides an interesting example of functional conservation in the absence of sequence conservation .", "The importance of this question goes beyond the NPC and arises in the discussion of many cellular machines and networks ( Alon et al . , 1999; Bialek , 2012 ) ." ] ]

[ "Nuclear Pore Complexes ( NPCs ) are key cellular transporter that control nucleocytoplasmic transport in eukaryotic cells , but its transport mechanism is still not understood .", "The centerpiece of NPC transport is the assembly of intrinsically disordered polypeptides , known as FG nucleoporins , lining its passageway .", "Their conformations and collective dynamics during transport are difficult to assess in vivo .", "In vitro investigations provide partially conflicting results , lending support to different models of transport , which invoke various conformational transitions of the FG nucleoporins induced by the cargo-carrying transport proteins .", "We show that the spatial organization of FG nucleoporin assemblies with the transport proteins can be understood within a first principles biophysical model with a minimal number of key physical variables , such as the average protein interaction strengths and spatial densities .", "These results address some of the outstanding controversies and suggest how molecularly divergent NPCs in different species can perform essentially the same function ." ]

[ "Animal , plant and fungal cells contain a structure called the nucleus , inside which the genetic material of the cell is stored .", "For the cell to work properly , certain proteins and other molecules need to be able to enter and exit the nucleus .", "This transport is carried out by pore-like molecular “devices” known as Nuclear Pore Complexes , whose architecture and mode of operation are unique among cellular transporters .", "Nuclear Pore Complexes are charged with a daunting task of deciding which of the hundreds of molecules it conducts per second should go through and which should not .", "Small molecules can pass freely through Nuclear Pore Complexes .", "However , larger molecules can only pass through the pore efficiently if they are bound to specialized transport proteins that interact with the proteins – called FG nucleoporins – that line the pore .", "A unique feature of the FG nucleoporins is that , unlike typical proteins , they do not have a defined three-dimensional structure .", "Instead , they form a soft and pliable lining inside the Nuclear Pore Complex passageway .", "Exactly how interacting with transport proteins affects the structure and spatial arrangements of the FG nucleoporins in a way that allows them to control transport is not well understood .", "This is in part because existing experimental techniques are unable to study the structures of the FG nucleoporins in enough detail to track how they change during transport .", "The complexity and the diversity of the FG nucleoporins also make them difficult to model in detail .", "Vovk , Gu et al . have developed a theoretical model that is based on just three basic physical properties of the FG nucleoporins – their flexibility , their ability to interact with each other , and their binding with the transport proteins .", "Future work can refine the model by incorporating further molecular details about the interactions between FG nucleoporins and transport proteins .", "The predictions made by this simple model agree well with experimental results in a wide range of situations – from single molecules to complex spatial assemblies .", "They also explain why some of the experimental results appear to contradict each other and suggest how several outstanding controversies in the field can be reconciled .", "Because the model invokes only fundamental physical principles of FG nucleoporin assemblies , it shows that some of their general properties do not depend on the exact conditions .", "In particular , this might shed light on why Nuclear Pore Complexes in different organisms perform essentially the same function , although the details of their molecular structure may differ .", "This also suggests how the FG nucleoporins can be manipulated to build artificial devices based on the same principles ." ]

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

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

[ [ "People skillfully combine acquired knowledge , and sensory feedback , a combination that is typically modeled using Bayesian statistics ( Körding , 2007; Angelaki et al . , 2009 ) .", "This framework effectively captures behavior in numerous tasks broadly corresponding to perceptual decision-making ( Ernst and Banks , 2002; van Beers et al . , 1999; Fetsch et al . , 2011; Drugowitsch et al . , 2014; Acuna et al . , 2015 ) , or online movement control ( Wolpert et al . , 1995; Körding and Wolpert , 2004; Izawa and Shadmehr , 2008; Crevecoeur et al . , 2016 ) .", "Although perceptual decision-making and sensorimotor control are often considered different phenomena , they cannot really be dissociated in the real world – we need to use the same brain for movement and perception ( Cisek , 2012; Wolpert and Landy , 2012 ) .", "Perceptual decision-making and sensorimotor behaviors may thus be linked .", "A salient case of crosstalk between perception and sensorimotor behavior is saccadic suppression: visual acuity is reduced around the time of a saccade .", "It is often assumed that this mechanism maintains stable perception of our surroundings ( Wurtz , 2008 ) .", "However , the behavioral and neural dynamics of saccadic suppression are difficult to explain if it were purely related to compensating for shifts in the retinal image induced by saccades and by the need to maintain perceptual stability .", "Indeed , previous work has shown that saccadic suppression is controlled centrally , and typically lasts for >100 ms even for saccadic movements of ~50 ms ( Ibbotson and Krekelberg , 2011 ) .", "Furthermore , simulating the displacement of the retinal image without a saccade does not elicit similar suppression as during real saccades ( Diamond et al . , 2000; Thiele et al . , 2002 ) .", "As well , the reduction of visual acuity was reported to selectively impact the magnocellular pathway ( Burr et al . , 1994 ) , although motion detection is still active ( Castet and Masson , 2000 ) .", "It is unclear why maintaining perceptual stability would require such a long , powerful , and selective suppression of sensory feedback , if it were purely related to perception , and independent of motor control .", "After all , there is a price to be paid to discard so much sensory information for some 100 ms . Thus , saccadic suppression is a complex phenomenon , for which a meaningful function has not been clearly identified .", "Here we phrase this problem in the framework of Bayesian estimation during closed-loop control of saccades .", "In this framework , we show theoretically that the timing of saccadic suppression is expected if the brain uses the same posterior beliefs about the state of the eye for perception and control .", "Indeed , our model shows that uncertainty about the instantaneous state of the eye should increase with motor commands as a result of signal-dependent noise and of sensorimotor delays , making delayed sensory information less reliable around the time of movement .", "In an optimal estimation framework , this gives lower weights to sensory inputs when we move .", "Our study thus shows how sensorimotor control can give rise to sensory suppression in an efficient brain , provided that the nervous system uses a common substrate for perception and for control .", "We discuss how this theoretical result may arise from shared neural resources supporting perceptual and motor systems ." ], [ "If we want to explore the relationship between saccadic suppression and control we need to model the underlying system .", "First , the nature of the representation matters: although saccades are often simplistically viewed as ballistic ( or open-loop ) movements , these movements are monitored online through the corollary discharge ( Van Gisbergen et al . , 1981; West et al . , 2009; Goossens and Van Opstal , 2000; Xu-Wilson et al . , 2011; Sommer and Wurtz , 2008; Optican , 2009 ) .", "Second , sensory feedback matters: we are not ‘blind’ during saccades .", "There is no peripheral interruption of sensory inflow , and information about specific spatiotemporal frequency or color is still good ( Burr et al . , 1994; Burr and Morrone , 1996 ) .", "Moreover , target jumps during long saccades can influence movement ( Gaveau et al . , 2003 ) .", "We should thus model saccades as driven by closed-loop control ( Figure 1a ) . 10 . 7554/eLife . 25073 . 003Figure 1 . Model architecture and simulations of eye movements .", "( a ) Schematic representation of the control and estimation architectures .", "We consider a closed loop controller based on optimal feedback control and state estimation .", "The dynamics of the eye plant corresponded to a second order system with time constants taken from the literature ( 13 ms and 224 ms ) .", "Bottom: Optimal state estimator based on usual Kalman filtering , and augmented with the extrapolation of sensory feedback to compensate for sensorimotor delays ( Sensory Extrapolation , red box ) .", "The symbolic representation of the signals in blue follows the same notations as in the Materials and methods: y ( t ) is the sensory feedback , x^ ( t|y ) is the extrapolation of sensory feedback , u ( . ) is the sequence of previous and current control commands , x^P ( . ) and x^ ( . ) are the prior and posterior estimates at the corresponding time steps .", "( b ) Top: Modeled saccadic eye movement from the first ( x1* ) to the second fixation target ( x2* ) .", "Bottom: Associated control function .", "Time zero corresponds to the end of the fixation period to the first target .", "( c ) Illustration of the sensory extrapolation performed in the state estimator .", "The simulated task is to track the target , which suddenly starts moving ( velocity jump ) with or without position jump in the opposite direction .", "The simulated eye trajectory shows how the extrapolation of target motion over the delay interval generates a catch up saccade ( black arrow ) .", "This compensatory movement is also illustrated in the velocity trace . DOI: http://dx . doi . org/10 . 7554/eLife . 25073 . 003 To describe saccades in the context of closed loop control , we model a controller that takes the sensory feedback and the corollary discharge as input , and outputs motor commands .", "We employ a Linear-Quadratic-Gaussian ( LQG ) controller , which can deal with noise both in sensory feedback and control signals .", "We use a second order model for the oculomotor plant ( see Materials and methods ) .", "This explicit model of saccadic control allows us to derive predictions of eye movement behaviors and gives us a control process that we can relate to saccadic suppression .", "The important feature of this control design in the context of this paper is its state estimator .", "The control of saccadic eye movements relies on the corollary discharges as well as on sensory feedback , which jointly allow state estimation .", "This state estimator has two main components .", "The first is a forward model that dynamically updates the current estimate based on the corollary discharge ( Figure 1a , bottom: Forward Model ) .", "The output is a prior estimate of the next state at the next step .", "The second component is the sensory extrapolation , which combines the delayed sensory feedback with the corollary discharge to estimate the current state ( Figure 1a: Sensory Extrapolation , red ) .", "This sensory extrapolation is critical for the behavior of the model .", "The presence of sensory extrapolation is supported by previous studies showing that error signals used to generate saccades depend on an estimate of the present state of the eye or of the target ( Bennett et al . , 2007; Ferrera and Barborica , 2010; Diaz et al . , 2013; Blohm et al . , 2005; de Brouwer et al . , 2002 ) , which clearly requires extrapolation of sensory feedback .", "Indeed , because the system only has access to the delayed feedback , this feedback must be extrapolated to compare it with the one step prediction , or prior .", "This operation does not appear explicitly in standard control models in which sensorimotor delays were considered ( Izawa and Shadmehr , 2008; Crevecoeur et al . , 2016; Todorov and Jordan , 2002; Crevecoeur and Scott , 2013 ) , because these previous studies used system augmentation , and the sensory extrapolation in this case falls out of the block-structure of the model .", "However , this component is necessary , and ignoring it can lead to instability ( Crevecoeur and Scott , 2013 ) .", "In the present model , the sensory extrapolation is performed explicitly ( Equation 5 ) , which also allows us to incorporate the impact of the signal-dependent noise that accumulates over the delay interval during the extrapolation ( see also Materials and methods ) .", "The model then corrects the one step prediction , weighting the difference between feedback and expected feedback optimally ( Figure 1a , K ( t ) is the Kalman gain ) .", "The first behavior that our model must describe is a saccade .", "The model reproduces stereotyped , step-like trajectories ( Figure 1b , top ) , like those found during real saccades .", "Moreover , the associated commands provide a typically wide agonist burst , followed by a short , sharp antagonistic inflection , which stabilizes the eye at the target ( Figure 1b , bottom ) .", "This pattern of control , shaped by the fast time constants of the oculomotor plant , is compatible with the pattern of burst neurons that generate saccades ( Van Gisbergen et al . , 1981 ) .", "Thus the model replicates both behavioral and physiological aspects of saccadic eye movements .", "A second behavior that our model can capture is smooth pursuit .", "We do not imply that these two behaviors are supported by the same neural hardware , and the model does not make any prediction about their neural implementation .", "Instead , we simply assume that optimal state estimation underlies both saccades and pursuit , which is in agreement with the hypothesis that these movements are distinct outputs of shared sensorimotor computations ( Orban de Xivry and Lefèvre , 2007; Krauzlis , 2004 ) .", "The model reproduces typical responses to changes in target velocity , occurring with or without initial target jump ( Figure 1c ) .", "When the target starts moving ( velocity jump ) , position error accumulates over the delay interval , which in turn requires a rapid compensatory movement to catch up with the target ( Figure 1b , light blue ) .", "Although the controller was not explicitly designed to model the interaction between saccades and pursuit , the catch-up saccade in Figure 1b naturally falls out of the simultaneous correction for errors both in position and velocity .", "In contrast , when the target jumps backwards at the onset of the velocity jump ( Figure 1c , dark blue ) , the eye starts moving smoothly and there is no catch-up saccade ( Rashbass , 1961 ) .", "The model also reproduces corrections following perturbations applied during movement through internal monitoring of the corollary discharge , as well as online corrections for target jumps occurring during long saccades ( simulations not shown ) .", "In all , the model generates typical trajectories and control commands associated with eye movements , and reproduces the dynamic estimation of the target resulting from the sensory extrapolation .", "Our muscles produce signal dependent noise; the stronger the muscles pull , the more noisy the state .", "The phasic activity associated with the agonist burst induces a peak in the variance of the control signal ( Figure 2a , solid ) .", "Thus motor commands produce instantaneous noise , and because of the delay , there is no way for the nervous system to directly subtract or filter out this noise .", "As a consequence , the extrapolation error computed over an interval that includes even a fraction of the control burst has higher variance .", "In other words , moving the eye effectively induces visual uncertainty , which can only go back to baseline after the end of muscle activation . 10 . 7554/eLife . 25073 . 004Figure 2 . Reduction of sensory weight .", "( a ) Variance of the control signal ( solid ) and of the extrapolation of sensory feedback ( dashed ) .", "Four times are represented to illustrate how signal-dependent noise impacts the extrapolation of sensory feedback is ( t1 = −100 ms , t2= −50 ms , t3= 0 ms , and t4= 50 ms ) .", "The dots with similar color represent the moment when the information at the corresponding time is available ( ti+100ms ) .", "Observe the increase in extrapolation variance associated with stimuli between t1 and t2 .", "( b ) Weight of the position feedback for correcting the estimate of the position .", "This weight is directly taken from the Kalman gain matrix .", "The reduction in Kalman gain at each selected time point is directly linked to the increase in variance .", "( c ) Representation of a modeled saccadic eye movement , with the gray area corresponding to the interval of time during which sensory input is given less weight as a result of the extrapolation variance .", "( d ) Control and extrapolation variance normalized to the maximum values obtained for saccades of 20 deg ( top traces ) .", "( e ) Weight of sensory feedback for the two simulations .", "Observe that although the catch-up saccade is very small ( ~2 deg ) , the transient increase in extrapolation variance gives rise to a reduction in weight .", "( f ) Illustration of the smooth pursuit task with ( dark blue ) or without ( light blue ) initial target jump occurring simultaneously with the velocity jump .", "The absence of target jump evokes a catch-up saccade , which is associated with ta reduction in sensory weight .", "There is no reduction with the initiation of smooth pursuit . DOI: http://dx . doi . org/10 . 7554/eLife . 25073 . 004 The time-varying variance induced by control-dependent noise has a direct impact on the weight of retinal signals , through the Kalman gains ( K ( t ) , Figure 2b ) .", "Recall that this matrix weights the difference between the current and expected estimates of both position and velocity , conditional upon the available visual information ( see Materials and methods , y ( t ) , Equation 4 and Figure 1 ) to correct the one-step prediction .", "We thus focus on the weight of position feedback , as it appears closely related to saccadic suppression .", "During saccades , the extrapolation variance increases as a result of signal dependent noise and of sensorimotor delays ( Vt , Equation 8 ) .", "As the Kalman gain is inversely proportional to this variance , the transient increase associated with the agonist burst generates a reduction in K ( t ) , and thus lowers the weight of position feedback about the eye position in the state estimator .", "And indeed , sensory suppression is seen before and during the time of simulated saccades ( Figure 2b ) .", "The period of suppression predicted by the model is long because the high variance period includes the movement time in addition to the delay ( gray rectangle in Figure 2c ) .", "The model predicts that the onset of saccadic suppression should precede movement onset by a time interval equal to the delay , which was fixed to 100 ms in the model ( see Materials and methods ) , although previous work suggested that it could be shorter ( Gaveau et al . , 2003 ) .", "Considering that processing times in the retina approach ~50 ms ( White et al . , 2009 ) , a conservative estimate for the onset of suppression according to the model ranges from 100 ms to 50 ms prior to movement onset .", "Our theoretical predictions thus indicate that feedback about retinal stimuli from this time window should be given less weight to optimally estimate the state of the eye .", "We can also see related effects in the simulated pursuit task .", "The presence of a catch up saccade , even a small one ( 4deg in Figure 2 ) , is sufficient to evoke a transient increase in extrapolation variance ( Figure 2d–f ) .", "This results in a reduction in the weight of sensory feedback with similar timing as for larger saccades .", "In contrast , when the eye starts moving smoothly ( Figure 2f ) , there is no catch-up saccade needed and the model predicts no visible change in the weight of sensory feedback .", "Because the model is linear , there is no transition between the simulated pursuit and saccade task , thus the apparent transition in the behavior results from the correction for the error in position that accumulates during the delay interval ( Figures 1 and 2 , light blue ) .", "The fact that suppression occurs for saccades specifically results from the high control signals required for these movements .", "In contrast , the pursuit task without a saccade uses smaller control signals that do not evoke any visible change in the weight of sensory feedback .", "The behavioral finding ( Schütz et al . , 2007 ) that saccades but not smooth pursuit elicit suppression of sensory feedback , and that the suppression scales with the amplitude of the catch-up saccade , directly results from this model .", "Behaviorally , we can analyze data from perception experiments .", "According to our hypothesis , suppression should occur prior to movement onset , reach a maximum close to movement onset ( Figure 3b ) , and scale with the movement amplitude with relatively invariant timing across amplitudes .", "Interestingly , this goes even down to the level of microsaccades , inducing partial suppression despite being very small in amplitude ( Hafed and Krauzlis , 2010 ) .", "These known properties of saccadic suppression are in line with the model prediction ( Figure 3b , black ) : contrast sensitivity is reduced and visual stimuli such as flashes , gratings , or small displacements are less likely to be accurately perceived ( Diamond et al . , 2000; Burr et al . , 1994; Watson and Krekelberg , 2011; Burr et al . , 1999; Bridgeman et al . , 1975; Beeler , 1967 ) .", "This even happens when the stimuli are chosen so that the eye movement does not change the retinal image , which is compatible with the model ( see the simulated contrast reduction of a white stripe , Figure 3a ) .", "Finally , although timing is preserved across amplitudes ( Ibbotson and Krekelberg , 2011 ) , the model predicts that the magnitude of suppression scales with the saccade amplitude as observed experimentally ( Ridder and Tomlinson , 1997 ) .", "This scaling is a direct consequence of signal-dependent noise . 10 . 7554/eLife . 25073 . 005Figure 3 . Representation of a simulated 20 deg saccade ad dynamic weight of sensory feedback , with the perisaccadic suppression highlighted in gray . These traces are similar as Figure 3a and c .", "The images represent the convolution of a horizontal stripe with a Gaussian kernel with variance proportional to the extrapolation variance to highlight that assigning higher variance may lead to reduced contrast , even when the movement is aligned with the stimulus orientation .", "Times correspond to the Figure 3 .", "The decrease in Kalman gain occurs from 0 to 150 ms relative to saccade onset ( solid trace: time locked ) , thus the window during which stimuli are suppressed corresponds to −100 to 50 ms ( dashed trace: stimulus-locked ) .", "( b ) Illustration of how the predicted saccadic suppression compares with previously reported suppression from behavioral and neural data .", "The duration of the perisaccadic suppression in the model is the sum of the temporal delay and of the movement time as represented above with the gray rectangle .", "Comparisons are approximate as movement time was not the same across all studies .", "The solid and dashed traces for saccadic suppression in SC indicates the range of onset and offset as given by Hafed and Krauzlis ( 2010 ) .", "Other intervals of saccadic suppression were drawn following the authors’ summary or based on visual inspection of the corresponding references . DOI: http://dx . doi . org/10 . 7554/eLife . 25073 . 005 A brief increase in the Kalman gain following the saccade can be observed in Figures 2 and 3 .", "This increase is due to the fact that the absolute value of the motor command is transiently lesser than the activity required to maintain the eye at the eccentric target .", "Thus , during this short interval , the Kalman gain becomes greater than during the simulated fixation .", "This feature resembles post-saccadic enhancement , which characterizes the enhanced motor response to a velocity jump in the target following a saccade ( Lisberger , 1998; Ibbotson et al . , 2007 ) .", "However , this transient increase in the model was not sufficient to generate a behavior similar to the one observed experimentally , despite the fact that larger differences in the Kalman gain can do so ( simulations not shown ) .", "This observation and the fact that motion detection is active during saccades ( Castet and Masson , 2000 ) suggest that the model needs further refinement to fully capture the processing of velocity signals during and after saccades .", "The model also predicts changes in neural activity relative to the timing of saccadic suppression .", "One way to implement the Kalman gains is to simply drive neurons less strongly when there is more uncertainty .", "This should predict reduced firing rates around the time of saccades .", "Indeed , a large number of experimental studies have found such a neural suppression across the hierarchy of visual areas .", "( Ibbotson et al . , 2008 ) .", "The pathways begin with the lateral geniculate nucleus ( LGN ) ( Reppas et al . , 2002 ) and the superior colliculus ( SC ) ( Hafed and Krauzlis , 2010 ) , and continue in the cortical areas V1 ( Kagan et al . , 2008 ) , MT , MST , MSTd ( Ibbotson et al . , 2008; Bremmer et al . , 2009 ) , and VIP ( Bremmer et al . , 2009 ) ( Figure 3b , colored bars ) .", "Interestingly , the timing is very similar across brain regions , which emerges naturally from the fact that the loop through the outside world with its delays is the dominating timescale .", "Thus there is suppression of visual signals across the entire visual hierarchy consistent with a sensorimotor origin of saccadic suppression ." ], [ "We have presented a feedback control model that assumes signal-dependent noise and delays , and uses state estimation to optimally control eye movements .", "It is built on the insight that motor noise is unavoidable and produces sensorimotor uncertainty .", "It is also based on the key assumption that saccades are supported by closed-loop control including retinal signals .", "This assumption is further developed below .", "This model allowed us to propose the hypothesis that saccadic suppression originates from efficient sensorimotor integration .", "Behaviorally , it describes the dynamics of both smooth pursuit and saccades .", "Perceptually , it describes the suppression of sensation around the time of saccades .", "Neurally , it captures the reduction of neural responses to visual stimuli presented before or during saccades .", "The important motivation behind this study was that cancelling the retinal shift induced by the saccade , as commonly assumed , does not explain the phenomenon of saccadic suppression .", "Indeed , suppression in this case should only occur when the eyes move , and should not be stronger than the moderate loss in performance associated with saccades simulated as a rapid displacement of the visual scene ( Diamond et al . , 2000 ) .", "All discarded information beyond movement-related effects would otherwise represent a net loss ( up to ~100 ms for some brain areas , Figure 3b ) .", "Thus it is clear that saccadic suppression is either very inefficient , or that maintaining a stable visual scene is just not its only purpose .", "We provide an alternative hypothesis that captures suppression qualitatively in the context of sensorimotor control .", "Rather than providing a definite answer to why suppression occurs , we highlight a plausible explanation and expect that it provide an insightful framework for interpreting data about visual processing .", "Although the model is not straightforward to test experimentally , our assumption about a common origin for saccadic suppression and movement control makes testable predictions for prospective experimental work .", "For instance , as we suggest that perception is impaired by sensorimotor control , it is conceivable that control might be impaired by a perceptual task .", "That is , if it were possible to train participants to pay attention to visual stimuli displayed during saccades , thereby increasing the weight of sensory feedback , then the theory predicts that movement trajectories should become more variable as a result of suboptimal state estimation .", "There is already clear evidence that the locus of attention and the goal of saccadic movements are linked ( Kowler et al . , 1995 ) ( and many references thereto ) .", "Here our specific prediction is that saccade trajectories should become more variable from trial to trial when participants are forced to use sensory information presented during the interval of saccadic suppression .", "As well , assuming that saccadic suppression is directly linked to the variance of sensory feedback through the Kalman filter , the model predicts that varying the reliability of sensory information may have an impact on the magnitude of saccadic suppression .", "Observe that these two predictions also assume that suppression can be flexibly modulated dependent on the behavioral context , which to our knowledge has not been documented .", "In addition to capturing the major aspects of behavioral and neural suppression , our model explains the previous findings of Watson and colleagues ( Watson and Krekelberg , 2011 ) , who investigated the detection of noisy gratings in humans , and found that the best explanation for saccadic suppression was a stimulus-independent reduction in the response gain .", "This result is a key aspect of saccadic suppression: the retinal images do not become intrinsically noisier; instead it is the visual system that responds less to a given stimulus .", "Our model also accounts for this observation: by reducing the sensory weight in the Kalman gain , the controller becomes less sensitive to sensory information .", "This is due to the uncertainty induced by the motor commands , which is clearly independent of the retinal image .", "The contribution of our model is to show that such stimulus-independent reduction in response gain may be rooted in efficient computations about the state of the eye .", "We propose this mechanism as a plausible origin of saccadic suppression , but cannot indicate how the visual system performs this operation at the level of neural circuits .", "We draw a qualitative link between Kalman filtering and the reduction in sensory weight or neural excitability , and thus this link remains speculative .", "However , the model does provide hints about what to look for .", "First , the increase in extrapolation variance clearly results from convolving the motor-dependent noise with the expected eye dynamics over the delay interval .", "Second , this increase is directly proportional to the integrated motor command .", "Thus , convolutional networks in the visual system receiving the corollary discharge as input may easily implement a reduction in the gain of neural responses that achieves statistically optimal sensory weighting .", "Any anatomical or functional similarity between these putative neural operations and neural data may thus provide insight into the circuitry underlying state estimation .", "A compelling aspect of our model is its simplicity , as the distinct behaviors and the dynamic estimation simply fell out of the simplest instance of linear stochastic optimal control ( LQG ) .", "Besides saccadic suppression , our model succeeded at the difficult task of controlling fast movements with comparatively long delays , without artificially interrupting the sensory inflow .", "While previous models of saccadic control tend to only consider open-loop controllers ( Harris and Wolpert , 1998 ) , or closed-loop control with internal feedback only ( Optican , 2009; Jürgens et al . , 1981; Chen-Harris et al . , 2008 ) , there is evidence that sensory information remains available and may influence online control .", "Indeed , motion detection during saccades is not suppressed ( Castet and Masson , 2000 ) , and peri-saccadic target jumps evoke adaptation ( Panouillères et al . , 2016 ) .", "In addition , a large retinal slip prior to saccade initiation can elicit curved movements ( Schreiber et al . , 2006 ) , indicating that retinal information prior to saccade onset can influence online control .", "Finally , Gaveau and colleagues reported partial corrections of eye trajectories following target jumps occurring during long saccades ( Gaveau et al . , 2003 ) .", "The fact that these corrections accounted for a small proportion of the target jump can be explained in the model , as a lower weight of sensory feedback leads to only partial correction of the target jump ( estimates take longer to converge to the true value ) .", "Thus , although evidence may not be definitive , these previous observations collectively suggest that sensory feedback must be considered in a model of neural control of saccades .", "Based on this assumption , our model predicts that sensory feedback must be strongly reduced , but not completely suppressed , as observed experimentally ( Castet and Masson , 2000 ) .", "This is because the Kalman filter achieves an optimal projection in the probabilistic sense , by making the estimation error orthogonal to ( or statistically uncorrelated with ) the estimated state .", "Thus , the decrease in the Kalman gain during movement indicates that state information prior to the saccade still carries some information about the current state , and thus can be exploited to derive optimal estimates .", "The resulting control law ( see Materials and methods , Equation 10 ) plays the role of a burst generator , and can be easily inserted as such in more complex models of gaze control .", "We have formulated the hypothesis that saccadic suppression originates from sensorimotor processing , although suppression has been characterized behaviorally as a perceptual phenomenon .", "Thus our theoretical developments imply that perception and control share a common neural substrate in the visual system .", "There are already strong pieces of evidence for shared resources .", "Indeed , previous work emphasized that perception and action share estimates of target speed ( Priebe and Lisberger , 2004 ) .", "Recently , a strong link between saccadic suppression and visual-motor neurons has been established in superior colliculus of macaque monkeys ( Chen and Hafed , 2017 ) .", "Furthermore , the motion on its own must not be suppressed to maintain perceptual stability , instead it must be equal to the commanded movement monitored online , thus perception is also conditional upon the ability to integrate extra-retinal signals accurately , both during saccades and pursuit ( Sommer and Wurtz , 2008; Blohm et al . , 2005; Hafed and Krauzlis , 2010; Bedell and Lott , 1996 ) .", "If perceptual and sensorimotor processes were completely decoupled , posterior beliefs about sensory information could be separated from movement-related effects , and perception around the time of saccades could be as good as during simulated saccades ( Figure 4 , H1 ) .", "Alternatively , a motor origin of saccadic suppression implies that the same posterior beliefs are shared for perception and control , which is suboptimal as it impacts perception of otherwise reliable sensory signals ( Figure 4 , H2 ) .", "Thus the hypothesis of shared resources requires a functional explanation .", "Although perception on its own is suboptimal during saccades ( we discard a lot of meaningful information ) , the shared resources model is clearly cheaper in terms of neural resources .", "It may thus be globally optimal to tolerate perceptual loss during saccades rather than commit to more neural resources for visual processing , considering perceptual and control systems together .", "Using the same posterior belief for perception and action also ensures self-consistency , in the sense that the same stimulus is not deemed more or less reliable dependent on how we use it .", "Self-consistency is known to characterize perceptual judgment tasks , where participants make continuous use of the hypothesis to which they previously committed ( Stocker and Simoncelli , 2007 ) .", "Our model suggests that similar principles may govern the use of posterior beliefs about the state of the eye for perception and control , indicating that these functions emerge from a shared neural substrate .", "We hope that future work will investigate whether this theory is generally applicable to other examples of active sensory suppression associated with voluntary actions such as force generation and reaching ( Chapman et al . , 1987; Blakemore et al . , 1999; Seki et al . , 2003 ) . 10 . 7554/eLife . 25073 . 006Figure 4 . Schematic illustration of separate or shared resources hypotehses . In the hypothesis of separated resources ( H1 ) , computations of the posterior belief are carried out independently for perception and control .", "In this scenario , the uncertainty induced by the control commands does not impact the perceptual estimate .", "This possible architecture is optimal in the sense that it would minimize loss of sensory information .", "In the hypothesis of shared resource ( H2 ) , the computation of the posterior belief about the state of a variable is shared for perception and control , thus both processes are similarly influenced by control-dependent noise .", "Although the first hypothesis is optimal , the second hypothesis is more efficient in terms of neural resources , and is also self-consistent ( see Discussion ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 25073 . 006" ], [ "We consider a second-order , low-pass filter as a biomechanical model of the oculomotor plant .", "Based on previous modeling work ( Robinson et al . , 1986 ) we set the time constants to τ1=224ms and τ2=13ms .", "In the sequel , scalars are represented with lower-case characters , vectors with bold lower-case and matrices with capitals .", "Thus the state-space representation of the continuous-time differential equation representing the eye dynamics was:[x . 1x . 2]= [01−1/ ( τ1τ2 ) − ( τ1+τ2 ) / ( τ1τ2 ) ][x1x2]+[01/ ( τ1τ2 ) ]u where x1 is the eye angle , x2 is the eye velocity , u is the command input and the dot operator is the time derivative .", "The explicit dependency on time was omitted for clarity .", "This representation takes the formx .", "=Ax+Bu with x:=[x1 x2]T representing the state of the system .", "The plant model was then augmented with the target position and target velocity , and transformed into discrete time model to include sensorimotor noise .", "The discrete-time stochastic dynamics governing the change of state over time , and the equation describing the visual signals available in the brain ( y", "( t ) ) are as follows: ( 3 ) x ( t+dt ) =Adx", "( t ) +Bdu", "( t ) +αεtBdu", "( t ) +ξt , ( 4 ) .", "y", "( t ) =x ( t−δt ) +σt The matrices Ad and Bd form the discrete-time state space representation of the continuous-time system defined in Equation 1 , which for a discretization step of dt corresponds to: Ad=edtA , and Bd= ( ∫0dtesAds ) B .", "The constant α>0 is a scaling parameter; εt , ξt and σt are Gaussian noise disturbances .", "The multiplicative noise ( εt ) is a scalar with zero mean and unit variance , whereas the additive sources of noise are 4-dimensional random disturbances with zero mean and variance set to Σξ , σ , which will be defined below .", "The measurement delay was δt=100ms in a agreement with measured and modeled latencies of rapid saccadic responses to visual stimuli ( Munoz and Everling , 2004; Stanford et al . , 2010 ) .", "The subscript t for the random noise disturbances was used to remind that these series do not have finite instantaneous variation; but they have finite variance over the discretization interval of dt .", "The variable y", "( t ) defined in Equation 4 represents the retinal information that is available to control the eye movement , which in the context of this paper is the state vector delayed by δt and corrupted by the sensory ( σt ) .", "Thus this definition captures the hypothesis that the available sensory information is a noisy and delayed measurement of the state .", "Recall that the augmented state vector includes position and velocity , as well as the target position and velocity .", "Optimal estimation and control of the stochastic system defined in Equations 3 and 4 can be derived in the framework of extended Linear-Quadratic-Gaussian control ( LQG ) , including the effect of control and state-dependent noise ( Todorov , 2005 ) .", "However this approach is not necessarily well suited for handling sensorimotor delays because it requires system augmentation ( Crevecoeur and Scott , 2013 ) , and as a consequence the estimator achieves optimal ( probabilistic ) projection of the prior estimate onto the delayed state measurement ( Anderson and Moore , 1979 ) .", "Since we know that the visual system extrapolates sensory information to compute the present state of the eye or of a moving target ( Bennett et al . , 2007; Ferrera and Barborica , 2010; Diaz et al . , 2013; Blohm et al . , 2005 ) , we were interested to derive an optimal estimator that explicitly extrapolates sensory signals , captured in y", "( t ) , over the interval δt ( see also Figure 2 ) .", "The key aspect of this estimator design is that , by taking into account the control function u", "( s ) , t−δt≤s≤t , the variance of the extrapolated sensory signal is dynamically adjusted as a function of the control-dependent noise ( 3rd term of Equation 3 ) .", "More precisely , we assume that neural processing of sensory signals consists in computing an estimate of the present state of the eye given the delayed sensory signals as follows: ( 5 ) x ( t|y ) =eδtAy", "( t ) +∫t−δtte ( t−s ) ABu", "( s ) ds .", "Using the notation M", "( t ) :=etA , it is easy to observe that the extrapolation error ( Δt ) follows a Gaussian distribution defined as follows: ( 6 ) x ( t|y ) =x", "( t ) +Δt , ( 7 ) Δt∼N ( 0 , Vt ) , ( 8 ) Vt=M ( δt ) ΣσM ( δt ) T+∫t−δttα′2M ( t−s ) Bu", "( s ) u", "( s ) TBTM ( t−s ) Tds , where α′:=α ( dt ) −1/2 was defined in agreement with the unit-variance Brownian noise disturbance considered for the stochastic differential equation .", "With these definitions , we can derive an adaptive estimator based on standard Kalman filtering using the extrapolated state ( Equation 8 ) instead of the available state measurement ( Equation 4 ) .", "The state estimate is computed in two steps as follows:x^P ( t+dt ) =Adx^", "( t ) +Bdu", "( t ) ( 10 ) x^ ( t+dt ) = x^P ( t+dt ) +K", "( t ) ( x ( t|y ) −x^", "( t ) ) , and the Kalman gain , K", "( t ) , as well as the covariance of the estimated state are updated iteratively following standard procedures ( Anderson and Moore , 1979 ) .", "Observe from Equation 10 that a reduction in the Kalman gain has a direct impact on the use of visual feedback through the relationship between this feedback ( y", "( t ) ) and the sensory extrapolation ( x ( t|y ) , Equation 5 ) .", "In other words , visual information conveyed in the sensory feedback participates less to the estimation when the Kalman gain is low .", "Observe that the separation principle does not hold because the variances of the one-step prediction and of Δt both depend on ut .", "Thus our approach is valid under the assumption that the control must not be jointly optimized with the state estimator .", "Instead of optimizing iteratively the controller and the state estimator as in the extended LQG framework ( Todorov , 2005; Phillis , 1985 ) , we computed the controller independently based on the heuristic assumption that the separation principle applied , and then optimized the state estimator defined in Equations 9–10 by taking into account the effect of control-dependent noise explicitly ( Equations 6–8 ) .", "The controller was thus obtained by solving the LQG control problem while ignoring the multiplicative noise in Equation 3 as follows: ( 11 ) .", "u", "( t ) = − ( R+BdTSt+1Bd ) −1BdTSt+1Ax^", "( t ) In Equation 11 , R represents the cost of motor commands , and the matrices St are computed offline following standard procedures ( Todorov , 2005; Astrom , 1970 ) .", "We developed this approach to include the extrapolation of sensory data while considering the control over the delay interval explicitly .", "Using feedback control based on a predicted state is known as finite spectrum assignment ( FSA ) , which is germane to a Smith predictor in that it aims at removing the delay from the feedback loop ( Zhong , 2010 ) .", "Here , FSA was chosen to reconstruct the predicted state ( instead of the system output as for the Smith predictor ) , allowing the use of position and velocity estimates in the control law ( Equation 11 ) .", "The only free parameters are α ( the scaling of the signal dependent noise ) , the covariance matrices of ξt and σt ( respectively Σξ and Σσ ) , and the cost-function used for control .", "We used the following values: the constant α was set to 0 . 08 , Σξ was 10−3×BdBdT and Σσ was 10−6 times the identity matrix of appropriate dimension .", "These parameters were manually adjusted so that when adding the signal-dependent term to the variance of the extrapolation error ( Equation 8 ) , the Kalman gains converged to steady-state values and the variances of the extrapolated state and of the motor noise were comparable during fixation .", "It is clear that changing the noise parameters may influence the results qualitatively .", "However the key feature of the adaptive estimator is that the extrapolation variance increases monotonically with the square of the motor command , which is why the extrapolated measurement is dynamically reduced during movement .", "This aspect does not depend on the different noise parameters .", "The cost parameters were adjusted to generate simulated saccades compatible with typical recordings of eye movements in humans , and these parameters do not impact the results qualitatively .", "For saccadic movements , we simulated two fixation periods at the initial ( x1* ) and final ( x2* ) targets during which the cost of position error was QFIXATION , i= ( x1−xi* ) 2 .", "The two fixation periods were separated by the movement time , which was a 50 ms window during which the eye was free to move without any penalty on the state vector .", "For the smooth movements in response to velocity jumps , we simulated a fixation to the target and changed the target state during a simulation run .", "The cost of motor commands in all cases was Ru", "( t ) 2 , with R∶=0 . 01 .", "Finally we used a discretization step of 5 ms . One difficulty is that the extrapolation requires that all state variables , including the target , be observed independently ( Equation 4 ) .", "This is not fully compatible with the visual system , because there is no measurement of the target state independent of the state of the eye .", "This limitation could be overcome by considering another observer that reconstructs the state vector prior to extrapolating the sensory feedback .", "Here , instead of considering such additional observer , we injected similar amounts of signal-dependent noise in the sensory feedback about the state of the eye as about the state of the target .", "This procedure was chosen for simplicity and captures the intuitive idea that if the eye position is very noisy , then information about the target location logically shares the same uncertainty ." ] ]

[ "Humans perform saccadic eye movements two to three times per second .", "When doing so , the nervous system strongly suppresses sensory feedback for extended periods of time in comparison to movement time .", "Why does the brain discard so much visual information ?", "Here we suggest that perceptual suppression may arise from efficient sensorimotor computations , assuming that perception and control are fundamentally linked .", "More precisely , we show theoretically that a Bayesian estimator should reduce the weight of sensory information around the time of saccades , as a result of signal dependent noise and of sensorimotor delays .", "Such reduction parallels the behavioral suppression occurring prior to and during saccades , and the reduction in neural responses to visual stimuli observed across the visual hierarchy .", "We suggest that saccadic suppression originates from efficient sensorimotor processing , indicating that the brain shares neural resources for perception and control ." ]

[ "Although we have the impression that our eyes move smoothly from place to place , we in fact perform rapid eye movements called saccades several times per second .", "Experiments have shown that our ability to perceive contrast and flashes decreases before and during each saccade .", "This phenomenon is known as saccadic suppression .", "A prevailing hypothesis to explain saccadic suppression suggests that by making vision temporarily less sharp for the rapid eye movement , the nervous system discards visual information about movement and helps us to perceive the world as stable .", "However , this does not explain the timing of saccadic suppression .", "Indeed , for saccades of about 50 milliseconds , the brain begins to reduce the sharpness of vision roughly 100 milliseconds before each eye movement begins .", "Why does the brain discard so much visual input ?", "To answer this question , Crevecoeur and Kording generated a computer model that took into account three properties that previous experiments have detected in animal nervous systems .", "First , transferring information between the retina and the neurons that control the movement of the eyes involves delays .", "Second , when neurons generate commands to move the eyes , they also show random fluctuations in activity that increase with the intensity of the commands .", "And third , visual information can still influence eye movement during a saccade .", "As a result of incorporating these three properties , the model predicted optimal timings for saccadic suppression that correspond to those that occur in real life .", "Visual perception and the control of eye movements have often been considered as separate functions of the brain .", "However , the model generated by Crevecoeur and Kording suggests that perception and the control of eye movement may in fact involve common brain regions .", "Further research is now needed to investigate predictions made by the model , which should provide new insights into how the brain supports vision ." ]

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

[ "neuroscience" ]

[ [ "How the brain processes sequences is a central question in cognitive science and neuroscience , and speech is a classic example of complex and rapid sequences that the human brain can effectively process .", "In general , speech utterances such as sentences are not memorized sequences , but instead are proposed to reflect compositional processes which allow us to understand and produce countless new sentences never heard before ( such as the one you are currently reading ) .", "Therefore , to derive the meaning of a speech sequence , the brain has to integrate information across words , the meaning of which are stored in long-term memory .", "Recent neurophysiological results have shown that when listening to speech , cortical activity is observed on multiple time scales that match the time scales of multiple levels of linguistic units , such as sentences , phrases , words , and syllables ( Brodbeck et al . , 2018; Broderick et al . , 2018; Ding et al . , 2017; Ding et al . , 2018; Keitel et al . , 2018; Makov et al . , 2017 ) .", "Critically , cortical responses on the time scales of phrases and sentences are observed even when the phrasal and sentential boundaries are not cued by prosodic features or by the transitional probability between words .", "The neural responses on the time scales of phrases and sentences have been taken as strong evidence that the brain applies grammatical rules to group words into chunks ( Ding et al . , 2017; Martin and Doumas , 2017 ) .", "Challenging this rule-based chunking interpretation , it has been argued that neural responses at the phrasal and sentential rates may not reflect neural construction of multi-word chunks based on rules and can instead be explained by neural tracking of properties of individual words alone ( Frank and Yang , 2018 ) .", "In the English materials used in Ding et al . ( 2017 ) , for example , all sentences have the same structure of adjective+noun+verb+noun , for example ‘new plans gave hope’ .", "Therefore , neural activity tracking lexical properties , such as part of speech information or lexical semantic information that distinguishes objects and actions , may appear to track sentences and phrases .", "For example , neural activity tracking verbs will occur at the sentence rate , since there is one verb per sentence .", "Consistent with this lexical property model , it has been shown that apparent neural tracking of sentences could be observed if the neural response independently represents each word using multidimensional features learned by statistical analysis of large corpora ( Frank and Yang , 2018 ) .", "In other words , neural populations that are tuned to lexical features of individual words may show activity that apparently tracks sentence structures .", "Furthermore , it is well established that the neural response to a word depends on the context and the response amplitude is smaller if the word is semantically related to previous words ( Kutas and Federmeier , 2011; Lau et al . , 2008 ) .", "In general , words within the same sentence are more related than words from neighboring sentences .", "Therefore , for a context-dependent neural response , its amplitude is expected to be stronger at the beginning of a sentence , leading to apparent neural tracking of sentences .", "This model considers semantic relatedness between consecutive words , but it does not consider the sentence structure: Semantic relatedness is evaluated the same way within and across sentence boundaries .", "Apparent sentence tracking behavior is generated since words within a sentence are more closely related .", "The lexical property model and semantic relatedness model do not assume chunk-level representations and therefore provide different explanations for sentential/phrasal-rate responses than the rule-based chunking model .", "The rule-based chunking model , however , has additional flexibility , allowing the same sequence of words to be grouped differently based on different sets of rules .", "This flexibility is most clearly demonstrated when processing structurally ambiguous sequences .", "For example , ‘sent her kids story books’ can be chunked as ‘sent [her kids] story books’ or ‘sent her [kids story books]’ ( Shultz and Pilon , 1973 ) .", "For such structurally ambiguous sentences , the rule-based chunking model , but not the lexical property model or the semantic relatedness model , would predict different phrase-tracking responses when the sentence is chunked differently .", "Notably , the three models introduced here are not exclusive , and neural encoding of lexical properties is a prerequisite to analyze the semantic relations between words or to build multi-word chunks .", "It is well established that the brain responses reflect encoding of lexical properties and semantic relations , but it remains debated whether the brain represents multi-word chunks .", "Therefore , the goal of the current study is to test whether neural activity on the time scales commensurate with multi-word chunks indeed indicates chunk-level neural representations or can be explained by the simpler word-level representations .", "Here , we distinguished the rule-based chunking model from the word-based models by asking the listeners to chunk a word sequence based on different rules in two experimental conditions .", "The word sequence was a sequence of nouns that describe either living ( L ) things or nonliving ( N ) things ( Figure 1A ) .", "The sequence had no syntactically defined chunks .", "Nevertheless , the participants explicitly learned artificial rules to chunk the sequence , and rules varied between two conditions .", "While participants performed the rule-based chunking task , their cortical responses were recorded using MEG .", "The word-level models , that is the lexical property model and the semantic relatedness model , predicted the same neural response when participants listened to the same word sequence regardless of how the sequence was chunked .", "The rule-based chunking model , however , predicted chunk-dependent neural responses ." ], [ "Participants were instructed to parse a sequence of words into chunks and each chunk consisted of two words .", "The words were drawn from two categories , that is living ( L ) and nonliving ( N ) things .", "The experiment contrasted two conditions in which the chunks were constructed based on different rules .", "In one condition , referred to as the same-category condition , the two words in a chunk belonged to the same semantic category ( Figure 1A , upper panel ) .", "In the other condition , referred to as the different-category condition , the two words in a chunk were drawn from different categories ( Figure 1A , lower panel ) .", "Based on these rules , there were two valid chunks in the same-category condition , that is LL and NN , and also two valid chunks in the different-category condition , that is NL and LN .", "The same- and different-category conditions were presented in separate blocks .", "In each condition , participants were asked to parse the sequences into chunks , that is pairs of words , and were instructed about the rules that valid chunks followed .", "They had to judge if any invalid chunk was presented in a sequence ( Figure 1B ) .", "The sequences in each condition ( N = 60 ) were further divided into two types , that is the alternating-order sequence ( N = 30 ) and the random-order sequence ( N = 30 ) .", "In the alternating-order sequence , the two valid chunks in each condition were interleaved while in the random-order sequence the two valid chunks were presented in random order .", "Equal numbers of alternating-order sequences and random-order sequences were intermixed and presented randomly in each block .", "The behavioral correct rate was 85 ± 2% and 86 ± 2% for the same- and different-category conditions respectively ( mean ± SEM across participants ) .", "The neural responses to these two types of sequences were separately analyzed .", "The alternating-order sequences allowed an intuitive comparison between the same- and different-category conditions , which would be detailed in the following .", "The random-order sequences were designed as fillers to avoid alternative strategies for the task ( see Materials and methods for details ) , but model simulations in the following showed that they could also distinguish the three models .", "Simulations of the neural responses to the alternating- and random-order sequences by the three models are shown in Figure 2 for both the same- and different-category conditions .", "The lexical property model considers two neural populations that idealize tuning to living and nonliving word meanings , respectively .", "Since the two neural populations are anti-correlated , only the neural population tuned to living things is shown ( Figure 2AB ) .", "The simulated neural response analyzed in the frequency domain demonstrates that neither the population shows a spectral peak at 1 Hz , that is the chunk rate , in the spectrum .", "In contrast to the lexical property model , the semantic relatedness model and the rule-based chunking model predict a 1 Hz response peak ( red and green curves respectively in Figure 2AB ) .", "A more fundamental difference between the semantic relatedness model and rule-based chunking model lies in their predictions about the 1 Hz response phase .", "The semantic relatedness model predicts a 180° phase difference between same- and different-category conditions , while the rule-based chunking model predicts a 0° phase difference between conditions ( Figure 2 ) .", "For the alternating-order sequence , these predictions are straightforward: These sequences are offset by one word between the same- and different-category conditions .", "Consequently , neural activity tracking semantic relatedness between words is offset by the duration of a word between conditions .", "For neural activity at 1 Hz , this time lag lead to a 180° phase difference .", "For the random-order sequence , although less straightforward , model simulation shows that the 1 Hz response has a 180° phase difference between conditions .", "For the rule-based model , however , the response is aligned with the chunk boundaries , which are not affected by the conditions and sequence types .", "Therefore , the rule-based model predicts the same response phase , that is a 0° phase difference , for the same- and different-category conditions .", "In summary , the three models considered in this study lead to different predictions about the neural responses ( Figure 2AB ) .", "Details about the model simulations are given in Figure 2—figure supplement 1A .", "The lexical property model and the semantic relatedness model assume ideal tuning to living/nonliving things .", "In the following , we turn to the actual neural responses obtained using MEG and evaluate their consistency with the simulations made for the three different models .", "The MEG responses were separately averaged for the same- and different-category conditions and the mean response was transformed to the frequency domain .", "We first analyzed the MEG responses to the alternating-sequences .", "The response spectrum averaged over all MEG gradiometers showed a clear peak at 1 Hz ( Figure 3A , left two plots ) .", "The 1 Hz response power was significant in both conditions ( F32 , 64 = 5 . 8 , p=2 . 0 × 10−9 and F32 , 64 = 6 . 5 , p=2 . 1 × 10−10 for the same- and different-category conditions respectively; F-test , FDR corrected ) .", "The 1 Hz spectral peak was consistent with the semantic relatedness model and the rule-based chunking model , but not with the lexical property model ( Figure 2A ) .", "On top of the 1 Hz response peak , a 2 Hz response peak was clearly observed and was significant ( F32 , 64 = 45 . 8 , p=9 . 7 × 10−33 and F32 , 64 = 35 . 9 , p=1 . 4 × 10−29 for the same- and different-category conditions respectively; F-test , FDR corrected ) .", "However , no significant peak was observed at 0 . 5 Hz ( F32 , 64 = 1 . 3 , p=0 . 17 and F32 , 64 = 1 . 0 , p=0 . 52 for the same- and different-category conditions respectively; F-test , FDR corrected ) .", "We then analyzed the phase difference between same- and different-category conditions at 1 Hz .", "In all the 306 MEG sensors , the phase difference averaged over participants was closer to 0° than 180° ( Figure 3E ) , and in 258 sensors the effect was significant ( p<0 . 01 , bootstrap , see Materials and methods , FDR corrected ) .", "These results were consistent with the rule-based chunking model ( Figure 2A ) .", "The phase difference averaged over all MEG sensors was significantly closer to 0° than 180° ( p=1 × 10−4 , bootstrap , see Materials and methods ) .", "The mean phase difference was 0 . 5° and the 99% confidence interval ranged from −22 . 9° to 18 . 7° .", "The main results on response spectrum and response phase difference could be reliably observed in individual participants ( Figure 3—figure supplement 1A ) .", "To further illustrate the response phase difference in the time domain , we analyzed the response waveforms .", "The Principal Component Analysis ( PCA ) was employed to extract major response patterns from all MEG sensors .", "The first PC captured the MEG response to the sound onset ( Figure 3—figure supplement 1B ) and the second PC captured the 1 Hz response , which oscillate in phase in the same- and different-category conditions ( Figure 3F ) .", "The MEG responses to random-order sequences were analyzed the same way as the responses to alternating-order sequences , and the results were similar: In the response spectrum , a clear peak was observed at 1 Hz ( Figure 3A , right two plots ) .", "The 1 Hz response power was significant in both conditions ( F32 , 64 = 4 . 5 , p=2 . 6 × 10−7 and F32 , 64 = 5 . 9 , p=1 . 5 × 10−9 for the same- and different-category conditions respectively; F-test , FDR corrected ) .", "The 1 Hz response power was not significantly different between random- and alternating-order sequences ( F32 , 32 = 1 . 0 , p=0 . 99 and F32 , 32 = 1 . 1 , p=0 . 90 for same- and different-category conditions respectively; F-test , FDR corrected; Figure 3B ) .", "The responses to random-order sequences also showed a significant 2 Hz response peak ( F32 , 64 = 48 . 5 , p=1 . 7 × 10−33 and F32 , 64 = 44 . 6 , p=2 . 0 × 10−32 for the same- and different-category conditions respectively; F-test , FDR corrected ) .", "The peak at 0 . 5 Hz was not significant ( F32 , 64 = 0 . 75 , p=0 . 81 and F32 , 64 = 0 . 91 , p=0 . 61 for the same- and different-category conditions respectively; F-test , FDR corrected ) .", "At 1 Hz , the phase difference between conditions was closer to 0° than 180° in all MEG sensors ( Figure 3E ) , and the effect was significant in 196 sensors ( p<0 . 01 , bootstrap , see Materials and methods , FDR corrected ) .", "The phase difference averaged over all MEG sensors was significantly closer to 0° than 180° ( p=4 × 10−4 , bootstrap , see Materials and methods ) .", "The mean phase difference was 20 . 3° and the 99% confidence interval over participants ranged from −0 . 1° to 56 . 6° .", "For both the neural responses to alternating-order and random-order sequences , in the response topography , the 1 Hz and 2 Hz responses showed bilateral activation ( Figure 3CD ) .", "Neural source localization results confirmed that both the 1 Hz and 2 Hz responses were mainly generated from bilateral temporal and frontal lobes ( Figure 3CD ) .", "An ROI analysis further revealed that the 1 Hz response peak was statistically significant in both temporal and frontal lobes ( F32 , 64 > 3 . 3 , p<1 . 9 × 10−5 for all conditions; F-test , FDR corrected ) , and the response is stronger in the left superior temporal gyrus than the left inferior frontal gyrus ( F128 , 128 = 2 . 0 , p=7 . 1 × 10−4; F-test , FDR corrected; Figure 3—figure supplement 1C ) ." ], [ "How chunks are represented during sequence processing is a prominent question in psychology and cognitive neuroscience .", "Here , we demonstrate that low-frequency neural activity can track multi-word chunks that are mentally constructed based on artificial chunking rules .", "Critically , the artificial rules here can dissociate the properties of individual words from the chunk structures and therefore provide strong evidence that low-frequency neural activity can encode chunks .", "Whether items in a sequence are mentally represented by hierarchically organized chunks is a heavily debated question in many cognitive domains , including language comprehension , action planning , and event perception ( Everaert et al . , 2015; Frank et al . , 2012; Lashley , 1951 ) .", "The multi-item chunks may be called schemas , scripts , or phrases in different fields .", "For example , for the routine behavior to prepare coffee , one view holds that it constitutes a schema with hierarchical structures: The schema of preparing coffee divides into sub-schemas such as adding sugar and adding milk , while each sub-schema further divides into finer-grained sub-schemas such as opening a package and pouring sugar into coffee ( Cooper and Shallice , 2006 ) .", "A contrasting view , however , is that hierarchical schemas are epiphenomenal and the internal cortical states generating basic actions are chained directly without any additional superordinate representation ( Botvinick and Plaut , 2004 ) .", "Similarly , for newly learned complex sequential processing tasks , some studies found that the underlying neural activity can be modeled by item-based state-transition models ( Mante et al . , 2013 ) while others provide evidence for chunk-level neural representations ( Geddes et al . , 2018; Jiang et al . , 2018 ) .", "Chunk-based models and item-based models favor different kinds of neural implementations .", "Representations of chunks could be implemented by sustained neural activation throughout the duration of a chunk ( Fuster , 2001 ) .", "In other words , the neural representation of a chunk is synchronized to the onset and offset of a chunk .", "Consistent with this idea , both computational models ( Cooper and Shallice , 2006; Martin and Doumas , 2017 ) and neural responses ( Ding et al . , 2017; Nozaradan et al . , 2011 ) show activity matching the time scales of hypothetical chunks during routine behavior and language/auditory perception .", "For item-based models , cortical state is updated item by item , which does not explicitly predict neural synchronization to chunks .", "Indeed , in some cognitive domains only item-rate neural activity is observed .", "For example , when a zebra finch sings a multi-syllable song , no slow neural activity matching the time scales of multi-syllabic chunks is observed .", "Instead , bursts of transient neural responses occur at syllable onsets , which strongly support the item-based state transition model ( Long et al . , 2010 ) .", "The zebra finch songs , however , are highly stereotyped .", "Future studies are needed to establish whether the chunk-rate responses observed here is limited to the processing of flexible complex sequences or limited to the primate brain .", "A potential way to link chunk-based and item-based processing mechanisms is that they both exist in the brain but are implemented in different brain areas and applied to different tasks ( Goucha et al . , 2017 ) .", "During language processing , for example , it has been proposed that item-based simple combination of words is implemented in the anterior temporal lobe ( Bemis and Pylkkanen , 2013; Brennan et al . , 2012 ) while rule-based chunk-level processing is implemented in the inferior frontal lobe ( Grodzinsky and Friederici , 2006; Grodzinsky and Santi , 2008 ) .", "Furthermore , experience may also affect the processing mechanism .", "For example , when participants have to perform a task based on rules that are learned explicitly , either rules in an executive control task or grammar in an unknown language , it is barely controversial that rule-based processing occurs in the brain , since there is not enough exposure to build up a state transition model .", "Nevertheless , for routine behavior or native language processing , extensive exposure allows the learning of statistical relations and it becomes difficult to distinguish rule-based models and item-based state transition models .", "In the current study , listeners explicitly apply artificial rules to group words into chunks , in contrast to previous studies in which the listeners can rely on implicit linguistic knowledge to group words into phrases .", "It remains elusive whether the brain relies on similar mechanisms for sequence chunking in different tasks .", "The primary motivation for a domain-general implementation of sequence-chunking tasks is that many sequence-chunking tasks share common computational principles .", "One such common computation is to find and encode the chunk boundaries .", "Consistent with a domain-general mechanism to encode chunk boundaries , studies have found similar EEG responses , that is the closure positive shift ( CPS ) , at the prosodic phrasal boundaries in speech ( Li and Yang , 2009; Steinhauer et al . , 1999 ) and music phrasal boundaries ( Zhang et al . , 2016 ) .", "Similarly , a transient increase in brain activity has been observed at event boundaries in movies ( Zacks et al . , 2001 ) and in non-speech sound sequences ( Chait et al . , 2007 ) .", "Another common computation in sequence-chunking tasks is to integrate information within a chunk .", "It has been suggested that low-frequency neural activity may also reflect sequential information integration within spoken phrases ( Ding et al . , 2017 ) and musical meters ( Nozaradan et al . , 2011 ) .", "During sequential decision making , it has been more quantitatively demonstrated that neural activity reflects accumulation of sensory evidence ( Barascud et al . , 2016; O'Connell et al . , 2012; Shadlen and Shohamy , 2016 ) .", "The task in the current experiment also engages sequential decision making but , unlike most previous sequential decision tasks , it does not allow the decision variable to be updated incrementally .", "Here , the first word in a chunk , whether an L or N , does not contribute to the decision , and the decision variable should only be updated when the second word is compared with the first word .", "Since the decision variable is updated every other words , that is every chunk , it could potentially contribute to the chunk-rate response .", "Nevertheless , the bilateral topography of chunk-rate response is not compatible with the previous finding that decision-related responses concentrate in central MEG channels ( de Lange et al . , 2010 ) .", "Furthermore , the decision-related centro-parietal positivity ( CPP ) in EEG is shown to be equivalent to the P300 ( O'Connell et al . , 2012; Twomey et al . , 2015 ) while the MEG counterpart of the P300 does not show a bilateral topography either ( Mecklinger et al . , 1998 ) .", "Therefore , the neural source of the chunk-rate response is not identical to the P300 , the dominant decision signal in previous EEG/MEG studies , but it may still reflect sequential decision making since such responses are widely distributed as shown by intracranial neural recordings ( Gold and Shadlen , 2007 ) .", "The idea that different sequence chunking tasks involve common computational principles does not necessarily imply that these principles are implemented in a common neural network .", "Nevertheless , there is indeed evidence for domain-general neural networks for sequence chunking .", "For example , functional MRI studies show that ventrolateral prefrontal cortex , including the Broca’s area , is not only a core area for language processing but also activated by rule-based nonlinguistic sequential processing tasks ( Koechlin and Jubault , 2006; Novick et al . , 2005; Thompson-Schill et al . , 2005 ) .", "There are also studies , however , arguing for domain-specific sequence processing mechanisms , especially for the processing of language ( Fedorenko et al . , 2011 ) .", "For example , it has also been shown that , for newly learned rules , rules of different complexity activate different parts of the frontal lobe , forming a posterior-to-anterior gradient ( Badre and Nee , 2018; Koechlin and Summerfield , 2007 ) .", "During language processing , however , syntactic rules of different complexity all activate Broca’s area ( Jeon and Friederici , 2013 ) .", "These findings lead to the hypothesis that automatic processes and more controlled processes rely on distinct neural circuits ( Jeon and Friederici , 2015 ) .", "Based on this hypothesis , the chunk-level task in the current study and syntactic analysis may engage different parts of the frontal lobe .", "With the spatial resolution of MEG , we cannot precisely localize the neural source of the chunk-rate response .", "However , it is found that the frontal lobe activation is bilateral with no clear lateralization between hemispheres ( Figure 3—figure supplement 1C ) , in contrast to the clearly left lateralized sentence-tracking response ( Ding et al . , 2016; Sheng et al . , 2019 ) .", "Similar to the sentence-tracking response , the chunk-rate response is also stronger in the temporal lobe than in the frontal lobe , suggesting that both sentences and chunks defined by temporary rules can drive large-scale chunk-rate responses in the temporal lobe .", "The results in the current study show that low-frequency neural activity primarily tracks rule-defined chunks instead of semantic relatedness .", "The semantic relatedness hypothesis , however , is based on solid evidence in the literature .", "It builds on the priming effect in the psychological literature ( Tulving and Schacter , 1990 ) and the neural adaptation effect in the neuroscience literature ( Grill-Spector et al . , 2006 ) , and is related to the predictive coding hypothesis ( Bar , 2007; Friston , 2005; Tian and Poeppel , 2013 ) .", "It is well established that if a word is preceded by a semantically related word , it is processed faster ( Collins and Loftus , 1988 ) and its neural response , especially the ERP N400 component and its MEG counterpart , is reduced ( Broderick et al . , 2018; Kutas and Federmeier , 2011; Lau et al . , 2009 ) .", "In this study , words from the same semantic category are more closely related than words drawn from different categories .", "Nevertheless , the categories used here are broad categories ( e . g . animals or plants ) .", "In general , words from a broad category , for example animals , are only weakly related compared with words from a narrower category , for example birds ( Quinn and Kinoshita , 2008; Vigliocco et al . , 2002 ) .", "A weak relationship between words predicts a weak priming effect on the neural response ( Federmeier and Kutas , 1999 ) , which may underlie why semantic relatedness between words does not drive a strong neural response .", "Furthermore , previous studies have identified two kinds of semantic priming , that is automatic and strategic priming ( Neely , 1977 ) .", "Automatic priming can be caused by , for example semantic relatedness between words in long-term memory .", "Strategic priming , however , can actively predict upcoming words based on temporally learned association rules .", "Behavioral experiments have demonstrated a cross-category priming effect if the prime word from one category , for example tools , is known to predict target words from a different category , for example animals ( Neely , 1977 ) .", "In other words , participants can make use of association rules learned during an experiment to actively predict words that have no long-term semantic relationship with the prime word .", "Different from automatic priming that can occur with very short stimulus onset asynchrony ( SOA ) between words , strategic priming occurs when the SOA between the prime and target words is relatively long , e . g . , >400 ms ( Hutchison , 2007 ) .", "In the current study , the SOA between words is 500 ms , allowing strategic priming to occur .", "Furthermore , since the chunking rule remains the same in each block , listeners can prepare in advance about how to parse the sequences , making strategic predictions to occur more easily .", "Based on the knowledge about valid chunks , the semantic category of the second word in each chunk is fully predictable in both the same-category condition and the different-category condition .", "The first word in each chunk is also predictable in the alternating-order sequences but not predictable in the random-order sequences .", "Since the alternating-order sequences and the random-order sequences are mixed , predictability is generally lower for the first word than for the second word in each chunk .", "Therefore , for strategic predictions , the predictability of words correlates with the chunk structure .", "This kind of strategic predictions , however , is based on rule-based chunking instead of semantic relatedness stored in long-term memory .", "Finally , the chunk-rate response in the current study is consistent with the rule-based chunking model .", "However , it may reflect the actual chunking process or downstream processes building on the multi-word chunks .", "After the chunk structure is parsed , the listener could synchronize their attention and predictions to the sequence .", "Previous studies have suggested that entrained neural oscillations may reflect both sequence parsing ( Ding et al . , 2017; Kösem et al . , 2016; Meyer and Gumbert , 2018; Meyer et al . , 2016; Wang et al . , 2017 ) and temporal attention/prediction ( Jin et al . , 2018; Morillon and Baillet , 2017; Rimmele et al . , 2018 ) , and could causally modulate speech perception ( Kösem et al . , 2018; Riecke et al . , 2018; Zoefel et al . , 2018 ) .", "The current results cannot distinguish which chunk-related process drives the chunk-rate response .", "What can , however , be concluded here is that the chunk-rate response cannot be fully accounted by neural tracking of individual words .", "Thus , the current study and previous studies ( Ding et al . , 2017 ) provide strong support to the notion that the brain can construct superordinate linguistic representations based on either long-term syntactic rules or temporary rules learned in an experiment ." ], [ "Sixteen participants took part in the study ( 19–27 years old; mean age 22 . 6; eight female ) .", "All participants were right-handed , with no self-reported hearing loss or neurological disorders .", "The experimental procedures were approved by the Research Ethics Committee of the College of Medicine , Zhejiang University ( 2019–047 ) and the Research Ethics Committee of Peking University ( 2019-02-05 ) .", "The participants provided written consent and were paid .", "All words were disyllabic words in mandarin Chinese and each syllable was a morpheme .", "For the noun sequences , each word was selected from a pool of 240 disyllabic concrete nouns .", "These concrete nouns equally divided into two categories , that is living ( L ) and nonliving ( N ) things .", "Living things further divided into 2 subcategories , that is animals ( N = 60; e . g . , monkey , panda ) and plants ( N = 60; e . g . , tulip , strawberry ) .", "Nonliving things also divided into two subcategories , that is small manipulatable objects ( N = 60; e . g . , teacup , toothbrush ) and large non-manipulatable objects ( N = 60; e . g . , playground , hotel ) .", "In each noun sequence , all living things were randomly drawn from a subcategory , that is animals or plants , and all nonliving things were also randomly drawn from a subcategory , that is manipulatable or non-manipulatable objects .", "Details about how the nouns constructed noun sequences are provided in the Sequence Structure section .", "Each disyllabic word was independently synthesized by the iFLYTEK synthesizer ( http://peiyin . xunfei . cn/; female voice , Xiaoying ) .", "All disyllabic words were adjusted to the same intensity and the same duration , that is 500 ms , following the procedure in Ding et al . ( 2017 ) .", "Within a word , no additional control was applied to the intensity and duration of individual syllables and coarticulation could exist between these syllables .", "Compared with speech materials in which each syllable was independently synthesized , the disyllabic words synthesized as a whole sounded more natural .", "When constructing sequences , the synthesized disyllabic words were directly concatenated , without any additional pause in between .", "Therefore , words were isochronously presented at 2 Hz .", "For speech stimuli generated according to this procedure , each disyllabic word was an acoustically independent unit and larger chunks consisting of multiple words had no acoustically defined boundaries .", "Pairs of nouns constructed chunks and chunks further constructed sequences .", "The experiment compared two conditions in which the chunks were constructed based on different rules .", "For the same-category condition , the two nouns in each chunk belonged to the same semantic category .", "For the different-category condition , however , the two nouns in each chunk were from different semantic categories .", "Since the study only considered two categories of words , there were two valid chunks in the same-category condition , that is LL and NN , and two valid chunks in the different-category , that is NL and LN .", "Each chunk was 1 s in duration .", "Each sequence consisted of 12 chunks and therefore was 12 s in duration .", "In each sequence , the two valid chunks were concatenated in either an alternating order or a random order ( Figure 1A ) .", "The alternating-order sequence in each condition had a fixed structure , repeating a four-words unit six times , that is NNLL for the same-category condition and NLLN for the different-category condition .", "The repeating four-words unit led to the 0 . 5 Hz rhythm in the lexical property model ( Figure 2 ) .", "In each random-order sequence , every chunk was randomly and independently chosen from the two valid chunks .", "After the category of each word was determined , the actual words were filled in .", "Each word was randomly drawn from a pool of 60 words ( see Speech Materials ) , with an additional constraint that no word repeated in a sequence .", "The alternating-order sequences had a highly regular structure , which led to a simple relationship between the alternating-order sequences in same- and different-category conditions: Any alternating-order sequence in the different-category condition could be converted to a same-category sequence by removing the first word in the sequence .", "Attributable to this property , the neural response phase could conveniently distinguish the word- and phrase-based models in Figure 2 .", "Nevertheless , this property also gave rise to an alternative strategy that could detect invalid chunks based on the same set of rules in both the same- and different-category conditions .", "For this strategy , the participants ignored the first word of each sequence in the different-category condition and treated the rest of the sequence as a same-category sequence .", "To eliminate this alternative strategy and to ensure that participants had to apply different rules in the same- and different-category conditions , the random-order sequences were designed as fillers to increase variability .", "Participants were familiarized with the synthesized words at the beginning of each experiment .", "In the familiarization session , after hearing a word , the participants pressed a key to see the word on a screen .", "Then , the participants could press one key to hear the word again or press another key to hear the next word .", "In the MEG experiment , the same-category condition and the different-category condition were presented in separate blocks and the order of the two blocks was counterbalanced across participants .", "In each condition , 30 alternating-order sequences and 30 random-order sequences were mixed and presented in a random order .", "In eight alternating-order sequences and eight random-order sequences , a living noun in one chunk was switched with a nonliving noun in another chunk so that the two chunks were no longer valid ( Figure 1B ) .", "These 16 sequences with invalid chunks were called outlier sequences .", "The outlier sequences ( N = 16 ) and normal sequences ( N = 44 ) were mixed and presented in a random order .", "The participants had a rest after listening to 30 sequences .", "Before MEG recording , participants received training .", "The same-category condition was trained first , followed by the different-category condition .", "For each condition , participants were explicitly instructed that the sequence was constructed by bi-word chunks and explicitly instructed about how valid chunks were constructed based on living/nonliving things .", "They were told that any chunk that violated the chunk construction rule was invalid chunks that they had to detect .", "After receiving instructions , participants were familiarized with two normal sequences , followed by two outlier sequences .", "When listening to the outlier sequences , they were asked to verbally report the invalid chunks as soon as they heard them .", "The sequence could be replayed upon request .", "The participants then went through a practice session , which was the same as the MEG experiment , except that it was carried outside the MEG scanner .", "During the practice session and during the MEG experiment , participants had to distinguish normal and outlier sequences and indicated their decisions by pressing different keys at the end of each sequence .", "After the key press , the next sequence was presented after a silent interval randomized between 1 s and 2 s ( uniform distribution ) .", "The practice session ended after the participants made four correct responses in five consecutive sequences .", "The MEG experiment started after participants finished the practice session for the different-category condition .", "Neuromagnetic responses were recorded using a 306-sensor whole-head MEG system ( Elekta-Neuromag , Helsinki , Finland ) at Peking University , sampled at 1 kHz .", "The system had 102 magnetometer and 204 planar gradiometers .", "Four MEG-compatible electrodes were used to record EOG at 1000 Hz .", "To remove ocular artifacts in MEG , the horizontal and vertical EOG were regressed out from the recordings using the least-squares method .", "Four head position indicator ( HPI ) coils were used to measure the head position inside MEG .", "The positions of three anatomical landmarks ( nasion , left , and right pre-auricular points ) , the four HPI coils , and at least 200 points on the scalp were also digitized before experiment .", "For MEG source localization purposes , structural Magnetic Resonance Imaging ( MRI ) data were collected from all participants using a Siemens Magnetom Prisma 3 T MRI system ( Siemens Medical Solutions , Erlangen , Germany ) at Peking University .", "A 3-D magnetization-prepared rapid gradient echo T1-weighted sequence was used to obtain 1 × 1 × 1 mm3 resolution anatomical images .", "In each condition , normal and outlier sequences were mixed and presented in a random order .", "However , only the neural responses to normal sequences were analyzed .", "Temporal Signal Space Separation ( tSSS ) was used to remove the external interference from MEG signals ( Taulu and Hari , 2009 ) .", "Since the current study only focused on responses at 0 . 5 Hz , 1 Hz , and 2 Hz , the MEG signals were bandpass filtered between 0 . 3 and 2 . 7 Hz using a linear-phase finite impulse response ( FIR ) filter ( −6 dB attenuation at the cut-off frequencies , 10 s Hamming window ) .", "The frequency response curve of the FIR filter was compensated in the response spectrum .", "The response during each sequence was extracted , downsampled to 20 Hz sampling rate , and was referred to as a trial .", "The MEG signals were further denoised using a semi-blind source separation technique , the Denoising Source Separation ( DSS ) .", "The DSS was a linear transform that decomposed multi-sensor MEG signals into components ( de Cheveigné and Parra , 2014 ) .", "The bias function of the DSS was chosen as the response averaged over trials within each condition .", "A common DSS for all conditions was derived based on the response covariance matrices averaged over conditions .", "The first six DSS components were retained and transformed back to the sensor space for further analysis .", "This DSS procedure was commonly used to extract cortical responses entrained to speech ( Ding et al . , 2017; Zhang and Ding , 2017 ) .", "To illustrate the response waveform , the PCA was employed to transform the 306-channel MEG data into components .", "Responses in the same- and different-category conditions and responses to the alternating- and random-order sequences were pooled in the PCA analysis .", "The first two PC were shown in Figure 3—figure supplement 1B and the 2nd PC , which captured the 1 Hz response , was filtered around 1 Hz and shown in Figure 3F ( FIR filter with 2 s Hamming window , cut-off frequency: 0 . 75 and 1 . 25 Hz ) .", "In the frequency-domain analysis , to avoid the response to the sound onset , the response during the first two seconds of each trial were removed .", "Consequently , the neural response was 10 s in duration for each trial .", "The average of all trials was transformed into the frequency domain using the Discrete Fourier Transform ( DFT ) without any additional smoothing window .", "The frequency resolution of the DFT analysis was 1/10 Hz .", "If the complex-valued DFT coefficient at frequency f was denoted as X ( f ) , the response power and phase were |X ( f ) |2 and ∠X ( f ) , respectively .", "The DFT was separately applied to each MEG sensor .", "For the MEG response power analysis , responses from the two collocated gradiometers were always averaged .", "When showing the spectrum , all MEG gradiometers were averaged .", "For the phase analysis , all magnetometers and gradiometers were separately analyzed .", "The circular mean was used to average the neural response phase over participants or sensors .", "The MEG responses averaged over trials were mapped into source space using cortex constrained minimum norm estimate ( MNE ) ( Hämäläinen and Ilmoniemi , 1994 ) , implemented in the Brainstorm software ( Tadel et al . , 2011 ) .", "The T1-weighted MRI images were used to extract the brain volume , cortex surface , and innermost skull surface using the Freesurfer software ( http://surfer . nmr . mgh . harvard . edu/ ) .", "In the MRI images , the three anatomical landmarks ( nasion , left , and right pre-auricular points ) were marked manually .", "Both three anatomical landmarks and digitized head points were used to align the MRI images with MEG sensor array .", "The forward MEG model was derived based on the overlapping sphere model ( Huang et al . , 1999 ) .", "The identity matrix was used as noise covariance .", "Source-space activation was measured by the dynamic statistical parametric map ( dSPM ) ( Dale et al . , 2000 ) and the value was in arbitrary unit ( a . u . ) .", "Individual source-space responses , consisting of 15 , 002 elementary dipoles over the cortex , was rescaled to the ICBM 152 brain template ( Fonov et al . , 2011 ) for further analyses .", "Two ROIs were defined in each hemisphere .", "A frontal-lobe ROI included the pars opercularis and pars triangularis , and a temporal-lobe ROI included the superior temporal area .", "Anatomical areas are defined according to an automated landmark-based registration algorithm ( Desikan et al . , 2006 ) .", "In source space , the response of all dipoles were transformed to the frequency domain , and the 90th percentile of response power was calculated for each ROI , at each frequency .", "When comparing the response between ROIs , the results were averaged over the alternating-order and random-order sequences and over the same-category and different-category conditions .", "Pulse sequence: In all three models , the smallest unit being considered was the word , and the model output was updated word by word .", "Therefore , in the simulations , each model was first simulated using a pulse sequence ( Figure 2—figure supplement 1A ) , in which a pulse was placed at the onset of each word and the pulse amplitude was described in the following .", "The lexical property model and the semantic relatedness model were simulated based on lexical features .", "For the model illustrated in Figure 2 , only two features were considered , that is living and nonliving things .", "Each feature took a binary value , that is one when the feature was present and 0 otherwise , and the pulse amplitude for each word equaled this binary value .", "The semantic relatedness model built on the lexical property model: The semantic relatedness between the current word and the previous word was characterized by the correlation coefficient between lexical representations ( Broderick et al . , 2018 ) .", "The correlation coefficient was a scalar .", "Additionally , since the neural response to a stimulus is usually weaker instead of stronger if the stimulus is preceded by a similar stimulus , we used one minus the correlation coefficient to modulate a pulse sequence .", "In the rule-based chunking model , pulses of unit amplitude were placed at the chunk onset .", "The neural responses were smooth waveforms rather than sharp pulses .", "Therefore , neural response waveforms were further simulated by convolving the pulse sequence with a response function , which was a 500 ms duration Gaussian window .", "Here , the rule-based chunking model was simulated by a response time locked to the chunk onset .", "In general , however , the model only assumed a consistent response within the duration of a chunk .", "Results in Figure 2—figure supplement 1B confirmed that the key predictions of the model were not affected by the waveforms ." ] ]

[ "Chunking is a key mechanism for sequence processing .", "Studies on speech sequences have suggested low-frequency cortical activity tracks spoken phrases , that is , chunks of words defined by tacit linguistic knowledge .", "Here , we investigate whether low-frequency cortical activity reflects a general mechanism for sequence chunking and can track chunks defined by temporarily learned artificial rules .", "The experiment records magnetoencephalographic ( MEG ) responses to a sequence of spoken words .", "To dissociate word properties from the chunk structures , two tasks separately require listeners to group pairs of semantically similar or semantically dissimilar words into chunks .", "In the MEG spectrum , a clear response is observed at the chunk rate .", "More importantly , the chunk-rate response is task-dependent .", "It is phase locked to chunk boundaries , instead of the semantic relatedness between words .", "The results strongly suggest that cortical activity can track chunks constructed based on task-related rules and potentially reflects a general mechanism for chunk-level representations ." ]

[ "From digital personal assistants like Siri and Alexa to customer service chatbots , computers are slowly learning to talk to us .", "But as anyone who has interacted with them will appreciate , the results are often imperfect .", "Each time we speak or write , we use grammatical rules to combine words in a specific order .", "These rules enable us to produce new sentences that we have never seen or heard before , and to understand the sentences of others .", "But computer scientists adopt a different strategy when training computers to use language .", "Instead of grammar , they provide the computers with vast numbers of example sentences and phrases .", "The computers then use this input to calculate how likely for one word to follow another in a given context .", "\"The sky is blue\" is more common than \"the sky is green\" , for example .", "But is it possible that the human brain also uses this approach ?", "When we listen to speech , the brain shows patterns of activity that correspond to units such as sentences .", "But previous research has been unable to tell whether the brain is using grammatical rules to recognise sentences , or whether it relies on a probability-based approach like a computer .", "Using a simple artificial language , Jin et al . have now managed to tease apart these alternatives .", "Healthy volunteers listened to lists of words while lying inside a brain scanner .", "The volunteers had to group the words into pairs , otherwise known as chunks , by following various rules that simulated the grammatical rules present in natural languages .", "Crucially , the volunteers’ brain activity tracked the chunks – which differed depending on which rule had been applied – rather than the individual words .", "This suggests that the brain processes speech using abstract rules instead of word probabilities .", "While computers are now much better at processing language , they still perform worse than people .", "Understanding how the human brain solves this task could ultimately help to improve the performance of personal digital assistants ." ]

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

[ "structural biology and molecular biophysics" ]

[ [ "Transient receptor potential ( TRP ) channels belong to a large and diverse superfamily of tetrameric cation channels that contain six transmembrane helices per subunit ( Liao et al . , 2013; Hellmich and Gaudet , 2014 ) , similar to voltage-activated ion channels ( Long et al . , 2007 ) , ryanodine receptors ( Yan et al . , 2015 ) and IP3 receptors ( Fan et al . , 2015 ) ( Figure 1A ) .", "Several TRP channels are exquisitely temperature-dependent , enabling them to serve as biological thermosensors throughout the animal kingdom ( Vay et al . , 2012 ) .", "The vanilloid-sensitive TRPV1 channel is a weakly voltage-dependent and cation-selective channel that is expressed in nociceptive sensory neurons , where it acts as a sensor for noxious stimuli ( Caterina and Julius , 2001 ) including heat , extracellular acidic pH , vanilloid compounds ( e . g capsaicin ) , and venom toxins ( e . g . double-knot toxin or DkTx ) ( see [Hilton et al . , 2015] for review ) .", "The sensitivity of TRPV1 to diverse modulators is fascinating because many stimuli are capable of influencing the responses of TRPV1 to other signals .", "For example , protons can sensitize the channel to activation by heat ( Jordt et al . , 2000 ) and capsaicin ( Jordt et al . , 2000; Ryu et al . , 2003 ) , and both capsaicin ( Voets et al . , 2004; Matta and Ahern , 2007; Gregorio-Teruel et al . , 2014 ) and protons ( Lee and Zheng , 2015 ) can sensitize the channel to activation by voltage .", "Conceptually , these observations can be explained by allosteric models wherein distinct stimulus-sensing domains are coupled to each other ( Latorre et al . , 2007 ) , and to an internal gate within the pore that opens and closes the ion permeation pathway ( Salazar et al . , 2009; Cao et al . , 2013 ) ( Figure 1A ) . 10 . 7554/eLife . 13356 . 003Figure 1 . Substitution of extracellular Na+ with NMDG+ increases TRPV1-mediated currents .", "( A ) Side view in ribbon representation of the transmembrane domains of two opposing TRPV1 subunits ( as indicated by the black arrow , extracellular face on the top , intracellular face on the bottom ) in the apo state ( refined TRPV1 structural model [Bae et al . , 2016] ) .", "The dashed boxes denote the location of the two constrictions proposed to serve as gates .", "Side chains of residues forming the binding site for capsaicin ( purple ) or determining activation of TRPV1 by protons ( blue ) are shown as sticks .", "( B ) Representative time-course of whole-cell TRPV1-mediated currents elicited by 100-ms voltage pulses from -90 mV ( triangles ) to +90 mV ( circles ) at 300 ms intervals and at room temperature .", "The colored horizontal lines signal the onset of rapid-solution exchange as indicated by the labels .", "The dotted red line indicates the zero-current level .", "( C ) Normalized TRPV1 current-voltage ( I-V ) relations obtained from 1s-duration voltage-ramps , following the same solution-exchange sequence as in ( B ) .", "The darker curves are the mean and lighter-colored envelopes the standard error ( n = 8 ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 13356 . 00310 . 7554/eLife . 13356 . 004Figure 1—figure supplement 1 . Substitution of external Na+ with NMDG+ induces channel rundown at room temperature with high cell-to-cell variability .", "( A ) Two representative TRPV1 current time courses obtained from a train of voltage ramps in the whole-cell configuration , constructed by plotting the mean currents at -120 ( triangles ) and +120 mV ( circles ) for each ramp within the train as a function of time .", "Rapid switching between extracellular solutions containing either 130 mM Na+ ( Nao , gray ) or 130 mM NMDG+ ( 130 NMDGo , yellow ) is indicated by the color of the symbols .", "The rate of channel rundown in the absence of external Na+ exhibited large cell-to-cell variability , as observed when comparing the experiment on the left ( prominent inactivation ) to the one on the right ( modest and slow inactivation ) .", "Rundown could not be prevented by adding ATP and/or diC8-PIP2 to the intracellular solution or by maintaining the intracellular milieu intact in perforated patch recordings ( data not shown ) .", "The dotted red lines indicate the zero-current level .", "( B ) Rundown could be slowed down if cells were kept for longer periods of time in the presence of 130 mM external Na+ .", "DOI: http://dx . doi . org/10 . 7554/eLife . 13356 . 00410 . 7554/eLife . 13356 . 005Figure 1—figure supplement 2 . Activation of rat TRPV1 channel orthologues by substituting external Na+ with NMDG+ .", "Representative current families recorded from outside-out patches containing TRPV1 channels from different species ( mouse , human and chicken ) at room temperature .", "Currents were elicited by voltage steps of 100 ms duration going from -120 to +140 mV in 10-mV increments , and different solutions were applied using the fast solution-exchange system .", "The red-dotted lines denote the zero-current level . DOI: http://dx . doi . org/10 . 7554/eLife . 13356 . 00510 . 7554/eLife . 13356 . 006Figure 1—figure supplement 3 . Comparison of TRPV1 channel I-V relations measured using voltage steps and voltage ramps .", "( A ) Representative whole-cell TRPV1 current families obtained at room temperature in response to 100-ms voltage pulses from -120 to +140 mV in 10-mV increments and recorded in the absence and presence of external Na+ or NMDG+ , with and without saturating capsaicin .", "The dotted red lines indicate the zero-current level .", "( B ) Superposition of the normalized I-V relations obtained from voltage ramps ( continuous curves , Figure 1C ) or from families of voltage pulses ( crossed circles ) as in ( A ) .", "For the ramps , the dark gray curves are the mean and the colored envelopes the standard error ( n = 8 ) .", "For the pulses , data are shown as mean ± SEM ( n = 7 ) .", "For both ramps and pulses , normalization was done as indicated on the y-axis label . DOI: http://dx . doi . org/10 . 7554/eLife . 13356 . 00610 . 7554/eLife . 13356 . 007Figure 1—figure supplement 4 . Theoretical I-V relations in the presence and absence of external Na+ obtained with the Goldman-Hodgkin-Katz current equation . Superposition of the I-V relations obtained from voltage ramps ( Figure 1C ) and theoretical I-V curves calculated using the Goldman-Hodgkin-Katz current equation ( red curves ) with a permeability of TRPV1 for Na+ ions that is 20-fold larger than that for NMDG+ .", "Theoretical I-V relations were calculated with the following equation: I ( V ) =N ( Po , min+Po , max−Po , min1+exp ( −zF ( V−V12 ) RT ) ) [PX1zX12VF2RT[X1]i1+exp ( −zX1FVRT ) −fPX1zX22VF2RT[X2]o1+exp ( +zX2FVRT ) ] , where I ( V ) is the current as a function of voltage ( V ) , N is the number of channels , Po , minis the minimal open probability at V << 0 , Po , max is the maximal open probability at V >> 0 , z is the gating charge of the channel , V1/2 is the voltage of half-maximal channel activation , F is Faraday’s constant , R is the gas constant , T is the temperature , PX1 is the permeability of the intracellular cation ( i . e . Na+ ) , zX1 and zX2 are the charges of the intracellular and extracellular cations , respectively , [X1]iand [X2]o are the molar concentrations of the intracellular ( Na+ ) and extracellular ( Na+ or NMDG+ ) cations , respectively , and f is the permeability ratio for cations 1 and 2 ( PX2/PX1 ) .", "At saturating capsaicin , the parameters used were: Po , min = 0 . 05; Po , max = 0 . 9; z = 0 . 31 e0; V1/2 = 71 mV and f = 1 for 130 Nao or 0 . 05 for 130 NMDGo .", "For 130 NMDGo the parameters were: Po , min = 0; Po , max = 0 . 30; z = 0 . 72 e0; V1/2 = 99 mV and f = 0 . 05 .", "A permeability for Na+ of 2 . 04721 x 10–19 m/s was used . DOI: http://dx . doi . org/10 . 7554/eLife . 13356 . 007 The regions of TRPV1 targeted by some activating stimuli have been identified , including the vanilloid binding site within internal regions of the S1-S4 domain ( Jordt and Julius , 2002; Cao et al . , 2013 ) , and both proton- ( Jordt et al . , 2000 ) and DkTx-binding sites within the external pore ( Bohlen et al . , 2010; Cao et al . , 2013; Bae et al . , 2016 ) ( Figure 1A ) .", "However , we currently do not understand the structural basis by which the binding of protons , capsaicin or DkTx promotes opening of the internal pore .", "Moreover , there is no consensus about the site and mechanism of temperature sensitivity .", "Mutations throughout TRPV1 alter the temperature sensitivity of the channel ( Feng , 2014 ) , and distinct models have been proposed to explain temperature sensitivity in these channels ( Voets , 2012 ) .", "For example , in the conventional allosteric model , a specialized temperature-sensitive transition is coupled to a temperature- and voltage-insensitive opening transition ( Brauchi et al . , 2004; Matta and Ahern , 2007 ) .", "In contrast , others have suggested that temperature sensitivity arises from a weakly voltage-sensitive opening transition ( Voets et al . , 2004 ) .", "Indeed , one recent study on voltage-activated potassium ( Kv ) channels suggests that the opening transition in these related channels can be made steeply temperature-dependent when the coupling between the voltage-sensing and pore domains is weakened ( Yang et al . , 2014 ) .", "It has also been suggested that TRP channels may not contain localized temperature-sensors , but that their steep temperature-dependence results from changes in solvation of hydrophobic residues throughout the protein ( Clapham and Miller , 2011 ) .", "Recent landmark near-atomic resolution structures of the TRPV1 channel confirm the presence of an internal gate related to that present in Kv channels ( Liu et al . , 1997; del Camino and Yellen , 2001 ) .", "In addition , the external pore and selectivity filter change conformation in the combined presence of DkTx and the vanilloid resiniferatoxin ( RTx ) ( Cao et al . , 2013; Liao et al . , 2013 ) .", "Given our limited understanding of the temperature-sensitivity of TRPV1 , it is unclear whether these structures provide insight into the mechanisms of heat activation .", "However , the structural rearrangements within the external pore are intriguing because mutations in this region have been reported to alter activation of the channel by protons ( Jordt et al . , 2000 ) and heat ( Grandl et al . , 2010; Cui et al . , 2012; Yang et al . , 2014 ) .", "In addition , external Na+ ions have been reported to inhibit the TRPV1 channel at room temperature ( Ohta et al . , 2008 ) , raising the possibility that an external cation binding site may regulate these channels .", "In the present study , we demonstrate the presence of a critical external Na+ ion-binding site that must be occupied for the channel to remain closed at room temperature , and to be available for opening by noxious heat .", "Our results also show that multiple steeply temperature-dependent transitions are allosterically coupled to an opening transition , and that binding of Na+ or DkTx to the external pore strongly influences the activity of the temperature-sensor and opening of the pore ." ], [ "To investigate the extent to which the large outward currents in external NMDG+ result from a change in driving force when exchanging external solutions , we obtained current-voltage ( I-V ) relations using rapid voltage ramps ( Figure 1C ) or voltage steps in cells exhibiting minimal rundown at room temperature ( Figure 1—figure supplement 3 ) .", "I-V relations for fully activated channels ( 10 µM capsaicin ) in the presence of external Na+ or NMDG+ superimpose at positive voltages ( Figure 1C and Figure 1—figure supplement 3 ) , suggesting that the unitary conductance of TRPV1 channels is similar under these conditions .", "In addition , theoretical I-V relations calculated from the Goldman-Hodgkin-Katz equation predict little change in unitary conductance at positive voltages ( Figure 1—figure supplement 4 ) , and we confirmed that exchanging external solutions does not affect the single-channel current amplitude ( i ) at positive voltages in outside-out patches containing a few channels ( Figure 2A ) .", "From these results , we conclude that exchanging external Na+ with NMDG+ leads to spontaneous opening of the TRPV1 channel at room temperature . 10 . 7554/eLife . 13356 . 008Figure 2 . Extracellular sodium ions are allosteric inhibitors of the TRPV1 channel .", "( A , left )", "Representative recordings at +90 mV performed on outside-out patches containing a few TRPV1 channels in the presence of 130 mM external Na+ ( top ) or NMDG+ ( bottom ) at room temperature .", "The continuous horizontal line represents the zero-current level ( C , all channels closed ) and the dotted red lines indicate current value levels corresponding to one ( O1 ) , two ( O2 ) or three ( O3 ) simultaneously open channels .", "( A , right )", "All-points current amplitude histograms constructed from the recordings on the left .", "The red curves are fits to a sum of Gaussian functions .", "( B ) Normalized I-V relations constructed from voltage-ramps measured in the whole-cell configuration with 260 mM internal Na+ ( Nai ) and the extracellular solutions indicated in the figure .", "The dark curves are the mean and the lighter-colored envelopes the standard error ( n = 4 ) .", "( C ) I-V relations obtained from families of 100 ms voltage-pulses in either the inside-out ( left ) or outside-out ( right ) configuration ( mean ± SEM , n = 4–5 ) .", "( D ) Sodium dose-response relations ( mean ± SEM , n = 7 ) obtained from voltage-ramps in the whole-cell configuration using solutions with different Na+/NMDG+ ratios , all adding up to a total cation concentration of 130 mM .", "The continuous curves are fits to the Hill equation with a Hill coefficient of 1 . 2 ± 0 . 1 .", "( E ) K1/2-V relation from fits as in ( D ) .", "The red curve is a fit to K1/2 ( V ) = K1/2 ( 0 ) x exp ( -zδV/kBT ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 13356 . 00810 . 7554/eLife . 13356 . 009Figure 2—figure supplement 1 . Monovalent cation selectivity of the external Na+-binding site in TRPV1 . ( A ) Voltage-ramps ( darker thin curves are the mean , lighter-colored envelopes the SEM , n = 5 ) obtained in the whole-cell configuration in the presence of different extracellular cations ( and 130 mM internal Na+ ) , and normalized to the current measured at +140 mV in the presence of 130 mM external NMDG+ .", "( B ) Fractional current at +120 mV ( mean ± SEM , n = 5 ) measured from ramps as shown on the left in the presence of each extracellular monovalent cation tested , relative to the current measured in the presence of external NMDG+ at +120 mV . DOI: http://dx . doi . org/10 . 7554/eLife . 13356 . 009 To distinguish whether channel activation results from the removal of Na+ or the addition of NMDG+ , we obtained voltage ramps in 130 mM NMDGo in the additional presence of 130 mM Nao ( with 260 mM internal Na+ ) and observed only small outward currents at positive voltages that superimposed with those obtained in the presence 260 mM Nao ( Figure 2B ) .", "This result indicates that NMDG+ does not activate the channel in the presence of Na+ , but rather removal of external Na+ leads to channel activation .", "Removal of internal Na+ in recordings using inside-out patches produced no channel activation ( Figure 2C , left panel ) , whereas removing external Na+ using outside-out patches led to robust activation ( Figure 2C , right panel ) , demonstrating that the inhibitory influence of Na+ is limited to the external side of the TRPV1 channel .", "We also measured the concentration-dependence for external Na+ inhibition over a range of voltages ( Figure 2D ) and found that inhibition was only weakly voltage-dependent , with an extrapolated apparent affinity ( K1/2 ) at 0 mV of 2 mM ( Figure 2E ) .", "These results suggest that external Na+ stabilizes a closed state of the TRPV1 channel by binding to a site on the extracellular side of the protein .", "This ion-binding site is not highly selective for Na+ , as external K+ can stabilize the closed state almost as effectively as Na+ , followed by Rb+ , Tris+ , Cs+ and Li+ ( Figure 2—figure supplement 1 ) .", "An external Na+ binding site that stabilizes TRPV1 in a closed state is interesting because titratable acidic residues are commonly found in such sites ( Lev et al . , 2013 ) and acid is a physiological stimulus for this channel , with mildly acidic conditions sensitizing the channel to either capsaicin or heat , and strongly acidic conditions causing channel activation ( Jordt et al . , 2000;Ryu et al . , 2003 ) .", "To explore a possible mechanistic link between the external Na+ site and acid regulation , we changed external pH from 7 . 4 to 6 and observed a pronounced shift in the concentration-dependence for Na+ inhibition to higher Na+ concentrations ( Figure 3A ) .", "Moreover , activation of the channel by pH 6 could be completely overcome by raising the Na+ concentration to 300 mM .", "These results are consistent with the expectation that protonation of acidic residues forming the Na+ binding site would lower the affinity of the metal ion , suggesting that the inhibitory Na+ binding site is intimately involved in the regulation of the TRPV1 channel by acid .", "However , acidic solutions further stimulated channel activation in the absence of external Na+ ( Figure 3A ) , indicating that protons also activate TRPV1 channels through a Na+-independent mechanism .", "These findings support the idea that proton-mediated channel activation and proton-dependent potentiation of responses to other stimuli have distinct mechanisms ( Jordt et al . , 2000; Ryu et al . , 2007 ) . 10 . 7554/eLife . 13356 . 010Figure 3 . External Na+ , H+ and DkTx modulate the TRPV1 channel through overlapping mechanisms involving E600 in the extracellular pore .", "( A ) Extracellular Na+ dose-response relations measured at different extracellular pH values at +120 mV in the whole-cell configuration in response to voltage ramps ( mean ± SEM , n = 3–7 ) .", "Experiments at different pH values were recorded from independent cells at room temperature .", "The data on the left of the axis break reflect activation of TRPV1 channels by protons in the absence of external Na+ .", "The continuous curves are fits to the Hill equation .", "The obtained parameters were: s = 1 . 2 ± 0 . 08 and K1/2 = 7 ± 0 . 15 mM at pH 7 . 4 ( n = 7 ) ; s = 1 . 5 ± 0 . 08 and K1/2 = 51 . 2 ± 11 . 5 mM at pH 6 . 0 ( n = 3 ) .", "The Hill equation was not fit to the data at pH 5 . 5 ( n = 5 ) , but the Hill function shown ( green curve ) has s = 2 . 0 and K1/2 = 2 . 0 M . Mean K1/2 values from the fits to data at pH 7 . 4 and 6 . 0 are shown on the right panel insert .", "For all cells , data were normalized to the currents measured in the absence of external Na+ at pH 7 . 4 ( 130 mM NMDG+ for pH 7 . 4 and 5 . 5; 300 mM NMDG+ for pH 6 . 0 ) .", "( B ) Representative whole-cell WT TRPV1 current time-course at -90 and +90 mV constructed from a train of voltage-ramps at room temperature .", "Horizontal thick lines denote the removal of external Na+ ( yellow ) or the application of DkTx ( blue ) .", "The dotted line denotes the zero-current level .", "( C ) Mean normalized I-V relations ( mean – thin darker curves – ± SEM – lighter-colored envelopes , n = 4 ) from WT TRPV1 channels obtained from whole-cell recordings at room temperature in response to voltage ramps with 130 mM intracellular Na+ and the extracellular solutions indicated by the labels at the bottom .", "The order of the labels reflects the order in which the different solutions were tested in each experiment .", "( D ) Mean normalized I-V relations ( mean ± SEM , n = 4 ) from TRPV1 channels with the E600Q mutation , obtained as in ( C ) .", "In these experiments , the 0 Nao-solution was tested before application of DkTx .", "( E ) Normalized I-V relations for TRPV1 Δ604–626 obtained in the whole-cell configuration at room temperature ( continuous curves are the mean , lighter envelopes the SEM , n = 6 ) in response to voltage ramps .", "( F ) External Na+ dose-response relations for TRPV1 Δ604–626 ( open circles , mean ± SEM , n = 4 ) measured at +120 mV from voltage-ramps in the whole-cell configuration .", "The dotted curve is a fit to the Hill equation with parameters: s = 2 . 2 ± 0 . 7 , K1/2 = 0 . 7 ± 0 . 1 mM .", "Closed circles are data for WT TRPV1 at +120 mV ( Figure 2D and 3A - pH 7 . 4 ) .", "The continuous curve is a fit to the Hill equation with parameters indicated in ( A ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 13356 . 01010 . 7554/eLife . 13356 . 011Figure 3—figure supplement 1 . The location of E600 , the extracellular pore turret and the binding site for DkTx within the outer pore of TRPV1 , and the role of external Mg2+ ions in TRPV1 modulation .", "( A ) Side view of a ribbon representation of the transmembrane domain of the TRPV1 channel bound to DkTx/RTx ( refined structural model for TRPV1 with the docked solution structure of DkTx ) ( Bae et al . , 2016 ) .", "The pore domains of two adjacent subunits in contact with the K1 lobe of DkTx ( shown in green ) are colored in teal and yellow , and their respective S1-S4 domains are colored in lighter blue and yellow , respectively .", "All other subunits ( including that in contact with the K2 lobe of DkTx , shown in cyan ) are colored in white .", "The DkTx molecule bound to the two subunits in the back was omitted for clarity .", "Residue E600 is shown in stick representation and colored in dark blue .", "The red coloring near E600 indicates the position from which the extracellular pore turret was deleted in the construct used for structure determination .", "( B ) Amino acid sequence alignment corresponding to the pore region of several TRPV1 channel orthologues together with rat TRPV2 , highlighting the location and sequence conservation of the extracellular pore turret denoted by the thick orange line .", "The green thick lines delimit the location of the S5 and S6 transmembrane regions as based on the structure of the rat TRPV1 channel ( Cao et al . , 2013; Liao et al . , 2013 ) .", "The purple line denotes the pore helix and the blue line the location of the selectivity filter .", "The intensity of the blue text background denotes sequence conservation between the aligned proteins , with darker coloring representing higher conservation .", "The vertical arrow denotes the position of E600 within the sequence .", "( C ) Normalized I-V relations constructed from voltage-ramps measured in the whole-cell configuration with 260 mM internal Na+ ( Nai ) and the extracellular solutions indicated in the figure .", "The dark curves are the mean and the lighter-colored envelopes are the standard error ( n = 4 ) .", "The data without magnesium are the same as in Figure 2B , as activation by NMDG+ and Mg2+ was tested in the same experiment . DOI: http://dx . doi . org/10 . 7554/eLife . 13356 . 011 We next set out to determine whether other stimuli acting on the extracellular pore activate TRPV1 through a mechanism associated with the binding of external Na+ .", "The double-knot tarantula toxin ( DkTx ) is an interesting candidate as it activates the TRPV1 channel with high avidity by binding to the periphery of outer pore of the channel ( Figure 3—figure supplement 1A ) ( Bohlen et al . , 2010; Cao et al . , 2013; Bae et al . , 2016 ) .", "We recently solved the solution structure of DkTx and performed a detailed analysis of its interactions with the TRPV1 channel , which suggested that the toxin activates TRPV1 by disrupting a cluster of hydrophobic residues at the extracellular half of the S5 and S6 segments ( Bae et al . , 2016 ) .", "Interestingly , removal of external Na+ before application of the toxin activates TRPV1 to a similar extent when compared with a saturating concentration of DkTx ( Figure 3B and C ) , and removal of external Na+ did not produce further activation of DkTx-bound channels .", "In contrast , application of a saturating concentration of capsaicin produced further activation ( Figure 3C ) .", "The lack of additivity for the activation of TRPV1 by DkTx and the removal of external Na+ contrasts with our results with acidic extracellular pH in the absence of external Na+ ( Figure 3A ) , and suggests that DkTx and external Na+ modulate the TRPV1 through convergent mechanisms .", "The unambiguous identification of the Na+-binding site would require a high resolution structure of TRPV1 .", "However , a glutamate at position 600 near the top of S5 ( Figure 3—figure supplement 1A ) has been previously associated with the inhibition of TRPV1 by external Na+ ( Ohta et al . , 2008 ) , and has also been shown to be involved in the proton-dependent potentiation of TRPV1 responses to heat and capsaicin ( Jordt et al . , 2000 ) .", "To confirm a key role of this residue in modulation of TRPV1 by external Na+ , we investigated the effect of removing external Na+ on E600Q mutant channels , and found that the removal of external Na+ no longer produced constitutive activation even though the mutant remained responsive to capsaicin ( Figure 3D ) .", "Consistent with DkTx and external Na+ having convergent mechanisms for modulating TRPV1 , E600Q channels were not responsive to a concentration of DkTx that is saturating in WT channels ( Figure 3D ) .", "These results demonstrate an important role for E600 in mediating channel inhibition by external Na+ and activation by DkTx , and suggest that this residue may be a part of the Na+ binding site .", "Another interesting region within the outer pore of TRPV1 is the pore turret ( Δ604–626 , Figure 3—figure supplement 1B ) , a stretch of 22 residues that was removed in the construct of TRPV1 recently used for structure determination ( Liao et al . , 2013 ) and that is located near to E600 and to the binding site for DkTx ( Figure 3—figure supplement 1A ) .", "We therefore explored whether the deletion of the pore turret retains modulation by external Na+ .", "Initially , we found that the removal of external Na+ produced robust channel activation at depolarized potentials in channels lacking the pore turret ( Figure 3E ) , and that the addition of 10 µM capsaicin in the presence of external Na+ further activated the channels , similar to WT TRPV1 .", "Interestingly , the removal of the pore turret increased the apparent affinity of the channel for Na+ ( Figure 3F ) , indicating that the pore turret does not form the Na+ binding site , but influences the cation-binding site through an allosteric mechanism .", "However , we also made the surprising discovery that the removal of external Na+ in the presence of 10 µM capsaicin resulted in enhanced channel activation at positive voltages relative to 10 µM capsaicin in the presence of 130 mM external Na+ ( Figure 3E ) , in stark contrast with WT channels where I-V relations under these two conditions superimpose at depolarized potentials due to maximal channel activation ( Figure 1C and Figure 3C ) .", "Increasing the concentration of capsaicin from 10 to 40 µM did not further activate Δ604–626 TRPV1 channels in the presence of external Na+ ( Figure 3E ) , indicating that 10 µM is a saturating concentration and that capsaicin is a partial agonist for this construct .", "This reveals that the pore turret is mechanistically associated with the modulation of the channel not only by stimuli acting on the extracellular pore ( e . g . external Na+ ) , but also by capsaicin that binds near the intracellular part of the protein ( Figure 1A ) .", "Finally , we were interested in determining whether external Mg2+ ions activate the TRPV1 channel directly , as was suggested in two previous studies that analyzed the effect of substituting external Na+ with Mg2+ without considering the inhibitory effects of external Na+ ( Cao et al . , 2014; Yang et al . , 2014 ) .", "We tested the ability of 130 mM external MgCl2 to activate the TRPV1 channel in the presence of 130 mM external NaCl ( and 260 mM intracellular NaCl ) , as we did previously in Figure 2B to determine whether NMDG+ was an activator of TRPV1 .", "Contrary to what we observed for NMDG+ , Mg2+ ions produced robust activation of the TRPV1 channel in the presence of 130 mM external Na+ ( Figure 3—figure supplement 1C ) , indicating that Mg2+ can activate the TRPV1 channel directly .", "Collectively , our results thus far highlight a central role of the outer pore in the allosteric control of TRPV1 channel gating .", "Furthermore , they suggest that the binding of DkTx , protons and external Na+ to the extracellular pore of TRPV1 modulate channel function through at least partially overlapping mechanisms .", "We next set out to determine whether the external Na+ site has an influence in the mechanism of temperature-sensitivity of the TRPV1 channel , as protons have been shown to potentiate responses of the channel to heat ( Jordt et al . , 2000 ) , and the outer pore has been associated with temperature-sensitivity ( Myers et al . , 2008; Grandl et al . , 2010; Cui et al . , 2012; Cao et al . , 2014; Yang et al . , 2014 ) .", "TRPV1 is widely agreed to be activated by noxious heat above 35°C in the absence of other stimuli ( Liu et al . , 2003; Yao et al . , 2010 ) , but whether the channel responds to lower temperatures has not been carefully examined because the open probability ( Po ) in this temperature range is very low ( Oseguera et al . , 2007 ) .", "If TRPV1 remains sensitive to changes in temperature below 35°C , it is possible that the spontaneous activation that we observed upon removing external Na+ at room temperature might be caused by an alteration in the temperature-dependence for channel activation .", "We therefore began by investigating TRPV1 channel activity over a broad temperature range in the presence of external Na+ using cells with widely varying expression levels of the channel .", "We measured macroscopic current-temperature ( I-T ) relations between ~8°C and 45°C using slow temperature ramps and observed steep temperature-dependent activation over the entire range of temperatures explored ( Figure 4A and B ) .", "Although these I-T relations largely reflect temperature-dependent changes in Po , the rate of ion conduction through open channels is also weakly temperature-dependent and thus will contribute to the slope of I-T relations .", "To directly evaluate temperature-dependent changes in Po , we estimated the temperature-dependence of ion conduction ( Figure 4—figure supplement 1B–E ) and then calculated Po-T relations from the macroscopic I-T relations ( Figure 4C and Figure 4—figure supplement 1F , see Materials and methods ) .", "To qualitatively compare the slopes of Po-T relations between cells and conditions , we fit a single exponential function ( Equation 1 , see Materials and methods ) to the data over defined temperature ranges to obtain apparent enthalpy ( ΔHapp ) values ( see Figure 4—figure supplement 2C–E ) .", "At temperatures > 35°C , we observed steep temperature-dependence to Po at positive membrane voltages ( Figure 4C and D ) , which became even steeper at negative membrane voltages ( Figure 4D , Figure 4—figure supplement 2B and E ) , consistent with previous studies ( Voets et al . , 2004; Yao et al . , 2010 ) .", "At temperatures between 8 and 25°C , a range in which the temperature-sensitivity of TRPV1 has not been previously investigated , we also observed steep temperature-dependence to Po ( Figure 4C and D , Figure 4—figure supplement 2C–E ) .", "However , the entire Po-T relationship could not be described by a single temperature-dependent transition due to the presence of an apparent plateau near 22°C , raising the possibility that multiple temperature-dependent transitions are involved in the gating mechanism of TRPV1 . 10 . 7554/eLife . 13356 . 012Figure 4 . Temperature-dependent gating of TRPV1 in the presence of external Na+ .", "( A ) Representative whole-cell current family in the presence of external Na+ elicited by a train of pulses from -90 to +90 mV while increasing temperature ( temperature vs time plot is shown on the right panel ) using the temperature-controlled recording chamber ( Figure 4—figure supplement 1A ) .", "The traces obtained at 15–30°C are shown at higher magnification in the middle panel .", "Dotted lines denote the zero-current level .", "( B ) Mean current-temperature ( I-T ) relations obtained from experiments as in ( A ) by plotting the steady-state mean current values at -90 ( triangles ) and +90 mV ( circles ) for each voltage-pulse within a train as a function of temperature ( mean ± SEM , n = 14 , I-T relations for individual cells are shown in Figure 4—figure supplement 2A and B ) .", "The dotted line denotes the zero-current level .", "( C ) Normalized Po-T relations ( mean ± SEM , grey circles; individual cells are shown as colored curves , n = 14 ) obtained from I-T relations as in ( B ) at +90 mV as described in Methods and illustrated in Figure 4—figure supplement", "1 . ( D ) ΔHapp from fits of Equation 1 to Po-T relations ( see Figure 4—figure supplement 2 for individual cells ( circles ) and their mean ± SEM ( squares ) ) .", "The mean ΔHapp for the fits to data with external Na+ at T > 25°C ( Po-T relations in ( C ) colored in green , yellow , orange and red ) is shown as an open square ( mean ± SEM , n = 10 ) .", "The mean ΔHapp from fits to data in external Na+ at T < 25°C ( Po-T relations in ( C ) colored in blue ) is shown as a closed square ( mean ± SEM , n = 4 ) .", "ΔHapp values for data at -90 mV were obtained from fits of Equation 1 to I-T relations ( Figure 4—figure supplement 2B ) , followed by subtracting the enthalpy associated with ion conduction ( 9 kcal/mol ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 13356 . 01210 . 7554/eLife . 13356 . 013Figure 4—figure supplement 1 . Estimation of the temperature-dependence of ion conduction through an open channel for obtaining Po-T relations .", "( A ) Scheme of the custom-modified temperature-controlled microincubator stage ( HCMIS and PTC-10 controller , Ala Scientific , Farmingdale , NY ) used to obtain I-T relations .", "A round glass coverslip was attached to the bottom of the stage and an additional stainless steel 25 mm chamber ( MS-502S , Ala Scientific ) was added to the stage to reduce the volume of recording solution ( 500 µL ) .", "Thermal grease was applied between the stainless-steel chamber ( outer chamber ) and the incubator stage .", "The metal surface of the outer chamber was covered with a thin layer of silicon or teflon spray for electrical insulation .", "Finally , a copper block of ~1 cm3 with an embedded thermistor probe and an axial slit ( 0 . 7 mm diameter ) in one face was positioned to make contact with the outer chamber .", "Once the whole-cell configuration was obtained , the cells were lifted and the pipette tip introduced into the slit in the copper block , which served as a 'buffer' to ensure that the temperature at the pipette tip closely matched that of the embedded thermistor .", "The addition of the copper block significantly increased the accuracy of temperature measurements , as judged from experiments in which the resistances of open pipettes were recorded during temperature ramps ( data not shown ) .", "A fan-based heat sink ( Koolance , Warner Instruments , Hamden , CT ) for the Peltier elements in the temperature controlled-stage was used during heating when performing temperature-ramps , whereas a refrigerated water circulator ( NESLAB RTE7 , Thermo Scientific , Waltham , NA ) at 6 . 3°C was used for cooling .", "( B ) Normalized I-T ( filled circles ) and Po-T relations ( empty symbols , mean ± SEM , n = 11 ) at +90 mV constructed from whole-cell TRPV1 current recordings in the presence of 130 mM external Na+ and saturating capsaicin ( 10 µM ) .", "I-T ( and Po-T ) relations from individual cells were normalized relative to the current at 22°C before averaging ( as denoted by the vertical and horizontal dotted lines and the y-axis labels ) .", "The red curve is a fit to the mean I-T relation using Equation 1 ( see Materials and methods ) with an enthalpy ( ΔH≠ ) of 9 kcal/mol , which reflects the temperature-dependence of ion conduction through an open channel .", "Po-T relations were obtained from the quotient between individual I-T relations and Equation 1 with an enthalpy of 9 kcal/mol ( see Materials and methods for further details ) .", "The enthalpies resulting from fits of Equation 1 to I-T relations from individual cells ( open circles ) are plotted together with their mean ± SEM ( black square , 9 . 0 ± 0 . 5 kcal/mol ) on the insert to the right .", "( C and D )", "Representative recordings at two temperatures obtained from two outside-out patches containing a few channels , one in the presence of 130 mM external Na+ ( C ) and other in the absence of external Na+ ( D ) .", "Currents were elicited by trains of 500 ms pulses from -90 to +90 mV during a temperature ramp using the temperature-controlled chamber described in ( A ) .", "The continuous horizontal red line represents the zero-current level ( all channels closed ) and the dotted lines denote the current levels for one ( O1 ) , two ( O2 ) or three simultaneously open channels .", "( E ) Single channel current amplitudes obtained from recordings as in ( C ) and ( D ) at several temperatures with 130 Nao ( grey squares , n = 3 ) or 0 Nao ( yellow squares , n = 7 ) .", "The red curves are fits of Equation 1 to the data with ΔH≠ = 9 . 0 kcal/mol .", "Estimates of i from different patches at similar temperatures were averaged and are plotted as mean ± SEM .", "The standard errors for temperature ( i . e . x-axis SEM ) are also included but are negligible .", "( F ) Normalized ( mean ± SEM , n = 14 ) I-T ( closed circles ) and Po-T ( open circles ) relations obtained in the presence of 130 mM extracellular Na+ .", "The dotted lines denote that both relations were normalized to their corresponding values at 22°C .", "Po-T relations were calculated as the quotient of individual I-T relations and Equation 1 with ΔH≠ = 9 kcal/mol . DOI: http://dx . doi . org/10 . 7554/eLife . 13356 . 01310 . 7554/eLife . 13356 . 014Figure 4—figure supplement 2 . Individual Po-T relations in the presence of external Na+ measured over a wide range of temperatures uncover the presence of multiple temperature-dependent components in the gating mechanism of the TRPV1 channel .", "( A ) Normalized I-T relation ( mean ± SEM , grey circles ) obtained from data in the presence of 130 mM external Na+ ( Figure 4B ) at +90 mV with superimposed I-T relations from individual cells ( colored continuous curves , n = 14 ) .", "( B ) The inward currents at -90 mV measured in the same experiments as the data at +90 mV shown in ( A ) were used to construct I-T relations at -90 mV .", "Each individual I-T relation at -90 mV was normalized to the current at 22°C from the I-T relation at +90 mV obtained from the same cell .", "Of the 14 cells included in ( A ) , only those that exhibited a substantial mono-exponential increase in the inward currents ( at -90 mV ) at higher temperatures were analyzed and are included in the figure .", "Those cells that were not included had inward currents that were still too small as compared to the leak in the measured range of temperatures .", "The resulting normalized mean I-T relation ( mean ± SEM , n = 5 ) is shown as grey triangles .", "The individual I-T relations are shown as colored continuous curves with data from each cell colored the same as in ( A ) .", "The dotted line denotes the zero-current level .", "( C ) Mean Po-T relation ( grey circles ) obtained from the data in ( A ) with superimposed Po-T relations from individual cells ( colored continuous curves , same coloring for each cell as in ( A ) ) .", "The continuous black curve is the prediction from model i ( Figure 7A ) calculated with the parameters in Figure 7—source data 2A .", "( D ) Mean Po-T relation shown in ( C ) with superimposed fits of Equation 1 ( see Materials and methods ) to each individual Po-T relation , with each fit extending over the temperature-range in which it was constrained during the fitting procedure .", "The color of the fits matches the color of their corresponding Po-T curves in ( C ) .", "( E ) Individual apparent enthalpy values for data at +90 mV corresponding to the fits in ( D ) as indicated by matching colors .", "The mean enthalpy for the fits at higher temperatures ( mean ± SEM , n = 10 , fits colored in green , yellow and red ) is shown as an open square .", "The mean enthalpy from fits at low temperatures ( mean ± SEM , n = 4 , fits colored in blue ) is shown as a closed square .", "The apparent enthalpy values for data at -90 mV were obtained from fits of Equation 1 to the I-T relations in ( B ) , followed by subtracting the enthalpy associated with ion conduction ( 9 kcal/mol , Figure 4—figure supplement 1B–E ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 13356 . 014 Having found that TRPV1 remains temperature-sensitive at room temperature and below in the presence of external Na+ , we measured macroscopic current-temperature ( I-T ) relations in the absence of external Na+ ( Figure 5A and B ) and constructed Po-T relations ( Figure 5C ) .", "Remarkably , both I-T and Po-T relations displayed greatly diminished slopes and readily detectable channel activity at the lowest experimentally accessible temperatures ( Figure 5A–C ) .", "In addition , removal of external Na+ produced marked temperature-dependent inactivation of TRPV1 channels between 25 and 38°C ( Figure 5B , see yellow arrow ) , a process that typically occurs after repetitive cycles of heating and cooling in the presence of extracellular Na+ ( Joseph et al . , 2013 ) and at temperatures above ~50°C in single heat-activation experiments ( Figure 5B , see purple arrow ) .", "Notably , temperature-inactivated channels could not be subsequently activated by heating , but were partially responsive to saturating capsaicin ( Figure 5—figure supplement 1A ) ( Cao et al . , 2014 ) .", "This facilitation of temperature-dependent inactivation in the absence of external Na+ can explain why removing Na+ also leads to progressive rundown of the TRPV1 channel at room temperature ( Figure 1—figure supplement 1 ) .", "We also observed that capsaicin diminishes rundown at room temperature when external Na+ was removed ( Figure 5—figure supplement 1B ) , consistent with a mechanistic link between temperature-dependent inactivation and channel rundown in the absence of external Na+ at room temperature . 10 . 7554/eLife . 13356 . 015Figure 5 . External Na+ has a strong influence on temperature-dependent gating of TRPV1 . ( A ) Representative whole-cell current family obtained as in Figure 4A in the absence of external Na+ .", "The temperature vs time plot is shown on the right panel .", "Dotted lines denote the zero-current level .", "( B ) Mean I-T relations in the absence ( obtained from data as in ( A ) , mean ± SEM , n = 7 ) and presence ( same data as in Figure 4B ) of external Na+ at +90 mV ( circles ) and -90 mV ( triangles ) .", "The I-T relation in purple is from an experiment in the presence of 130 mM external Na+ with pronounced temperature-dependent inactivation at T < 50°C .", "The purple and yellow arrows denote the approximate onset of inactivation for data with and without external Na+ , respectively .", "The dotted red line denotes the zero-current level .", "All relations are normalized to peak-current values at +90 mV .", "( C , left )", "Mean Po-T relations ( +90 mV ) in the presence ( grey ) and absence ( yellow ) of external Na+ .", "Po values at room temperature for scaling Po-T relations on an absolute Po-scale were estimated from macroscopic I-V relations and noise analysis as described in Methods and Figure 5—figure supplement", "2 . The dotted vertical line delimits the lower range of experimentally accessible temperatures .", "Po-T relations from individual cells in the absence of external Na+ are shown in Figure 6—figure supplement 1A .", "( C , right ) ΔHapp from fits of Equation 1 ( see Materials and methods ) to Po-T relations at +90 mV from individual cells ( circles ) and their mean ± SEM ( squares ) .", "The mean ΔHapp for the fits to data with external Na+ at T > 25°C is shown as an open square ( mean ± SEM , n = 10 ) .", "The mean ΔHapp from fits to data in external Na+ at T < 25°C is shown as a closed square ( mean ± SEM , n = 4 ) .", "( D ) Normalized I-T relations ( +90 mV ) from ( B ) plotted on a log-scale ( small circles ) with superimposed I-T relations obtained from rapid temperature-jumps from 8°C to higher temperatures ( large circles , mean ± SEM , n = 3–8 , see Materials and methods and Figure 5—figure supplement 3 ) .", "The blue bars denote the increased inactivation observed in the I-T relation in the absence of external Na+ obtained from slow temperature ramps relative to that obtained with rapid temperature-jumps .", "( E ) Schematic representation of the essential features on a log-scale of Po-T relations at +90 mV in the presence ( grey ) or absence ( yellow ) of external Na+ .", "The dashed lines denote plateaus in which the Po does not visibly change with temperature .", "Arrows denote portions of the relations in which Po steeply increases with temperature .", "Monoexponential fits of Equation 1 correspond on a log-scale to straight lines with slope ~ΔHapp . DOI: http://dx . doi . org/10 . 7554/eLife . 13356 . 01510 . 7554/eLife . 13356 . 016Figure 5—figure supplement 1 . Temperature-dependent inactivation of TRPV1 channels in the absence of external Na+ can be partially reversed by capsaicin .", "( A ) Representative whole-cell current time course ( lower panel ) elicited by voltage steps from -90 to +90 mV while increasing temperature ( time course for temperature is shown on the top panel ) using the temperature-controlled chamber ( Figure 4—figure supplement 1A ) .", "The continuous horizontal colored lines indicate the composition of the external solution in the recording chamber .", "The vertical dotted blue line denotes the onset of inactivation in the absence of external Na+ while the temperature is still increasing .", "The dotted red line denotes the zero-current level .", "Note that no detectable TRPV1-mediated current remains after inactivation at high temperatures , but substantial TRPV1 channel activation could still be attained by the addition of saturating capsaicin at room temperature .", "( B ) The addition of a subsaturating concentration of capsaicin prevents rundown at room temperature in the absence of external Na+ .", "Representative whole-cell TRPV1 channel current time-course at room temperature obtained from voltage ramps .", "Only current values at –90 ( triangles ) and +90 ( circles ) mV are shown .", "The dotted red line indicates the zero-current level .", "The teal open squares are the mean current time course ( ±SEM , n = 6 ) in the absence of external Na+ + 100 nM capsaicin calculated from identical experiments by normalizing each individual time-course to its initial current value ( i . e . the first open square ) in the solution of interest for subsequent averaging .", "Only data points at 1-min intervals are shown . DOI: http://dx . doi . org/10 . 7554/eLife . 13356 . 01610 . 7554/eLife . 13356 . 017Figure 5—figure supplement 2 . Scaling of Po-T relations based on estimates of absolute Po at room temperature from macroscopic I-V relations and noise-analysis .", "( A ) Mean normalized I-V relations at room temperature obtained in the whole-cell configuration using voltage ramps in the presence of extracellular solutions containing different Na+ concentrations or capsaicin .", "The darker thin curves are the mean , the lighter envelopes the SEM ( n = 7–9 ) .", "( B ) Mean normalized I-V relations obtained as in ( A ) from cells with very high levels of expression ( mean ± SEM , n = 6 ) .", "( C ) I-V relations from ( A ) and ( B ) ( same color codes ) scaled and plotted together on a log scale .", "The dotted line denotes that all curves were scaled relative to the current value at saturating capsaicin at +90 mV .", "The numbers on the right are the corresponding Po for each condition at room temperature if the Po for saturating capsaicin at +90 mV is 0 . 9 ( see Materials and methods ) .", "( D ) Representative WT TRPV1 variance ( σ2I ) vs mean current ( Imean ) plot obtained from noise analysis at ~3°C and +90 mV in the whole-cell configuration using the temperature-controlled chamber ( Figure 4—figure supplement 1A ) .", "Data points on the left of the plot ( yellow dots ) were calculated from recordings in the absence of external Na+ , whereas data on the right ( black dots ) were calculated from recordings in the same cell in the presence of 130 Nao + 10 µM capsaicin .", "Colored curves are fits to Equation 2 ( σI2=Imeani−Imean2N , N is the number of channels in the cell , i is the single-channel current at +90 mV ) with parameters: green fit ( unconstrained fitting ) , N = 1064 , i = 1 . 08 pA; red fit ( i was constrained based on single-channel recordings , see Figure 4—figure supplement 1C–E ) , N = 439 , i = 2 . 15 pA .", "The Po values shown at the top of the graph were calculated from the steady-state mean current at saturating capsaicin ( Imean , ss ) and the parameters from the fits of Equation 2 ( constrained in red or unconstrained in green ) with Equation 3: Po=Imean , ssiN .", "( E ) Representative Imean from the experiment in ( D ) in the absence of external Na+ ( yellow ) or in the presence of 130 mM external Na+ and 10 µM capsaicin ( black ) at 3°C and +90 mV .", "The dotted red line denotes the zero-current level .", "( F ) WT TRPV1 single-channel current amplitudes ( +90 mV ) at different temperatures estimated from recordings from outside-out patches expressing a few channels ( yellow and grey circles with red envelopes , data from Figure 4—figure supplement 1E shown as mean ± SEM ) or from noise analysis ( green circles , individual symbols correspond to estimates from independent cells ) from free-parameter fits of Equation 2 to σ2I vs Imean relations as in ( E ) .", "Estimates of i for TRPV1 channels lacking the extracellular pore turret ( TRPV1 Δ604–626 , see Figure 9—figure supplement 1 ) also obtained from noise analysis are shown as green triangles .", "Noise analysis systematically underestimated i by a factor of ~2 , as illustrated by i-values obtained from noise analysis after multiplication by 2 ( blue symbols ) .", "The red and green curves are fits of Equation 1 with ΔH≠ = 9 kcal/mol , corresponding to the temperature-dependence of ion conduction through an open channel estimated from macroscopic I-T relations in saturating capsaicin ( Figure 4—figure supplement 1B ) .", "( G ) Po-T relations for 0 Nao and 130 Nao + 10 µM capsaicin obtained from noise analysis at different temperatures using two different methods and Equation", "3 . Po values calculated from unconstrained fits of Equation 2 to σ2I vs Imean relations ( e . g . green fit in ( D ) ) are shown as circles with green envelopes .", "Po values for the same cells calculated by constraining i in Equation 2 to the values obtained from direct single-channel recordings ( e . g . red fit in ( D ) ) are shown as circles with a red envelope .", "Yellow circles correspond to Po-values obtained from Equation 3 and Imean , ss in the absence of external Na+ ( e . g . yellow trace in ( E ) ) , whereas black circles were calculated from Imean , ss from data in the presence of external Na+ and saturating capsaicin .", "Black and yellow dotted lines denote Po values of 0 . 9 and 0 . 2 , respectively . DOI: http://dx . doi . org/10 . 7554/eLife . 13356 . 01710 . 7554/eLife . 13356 . 018Figure 5—figure supplement 3 . Perfusion-mediated temperature control .", "( A ) Schematic illustration of the perfusion-based temperature-control system used for rapid temperature jumps and I-V relations at low temperatures .", "Solutions kept in elevated reservoirs ( for gravity-driven flow ) were passed through glass capillary spirals immersed in water baths at different temperatures , and recordings were performed in a small-volume ( 200–500 µL ) chamber during constant perfusion .", "Temperature was measured with a thermistor located very close to the pipette tip .", "Separate perfusion lines were used for each solution ( e . g . one for 130 mM external Na+ , shown in grey in the figure , and one for external NMDG+ , shown in yellow ) .", "( B ) Representative current time courses obtained during rapid perfusion-induced temperature changes showing both temperature ( red traces , top panels ) and current ( open symbols , bottom panel ) recorded in the whole-cell configuration at -90 mV ( triangles ) and +90 mV ( circles ) .", "Horizontal thick lines denote changes in extracellular solution composition , and the red dotted lines indicate the zero-current level . DOI: http://dx . doi . org/10 . 7554/eLife . 13356 . 018 We next explored why the TRPV1 channel lacks steep temperature-dependent activation in the absence of external Na+ .", "One possibility is that the mechanism of heat activation remains intact , but that increases in Po upon heating cannot be seen because channels run down after opening during temperature ramps ( see Figure 7—figure supplement 5 and Materials and methods for an illustration using an allosteric model for channel gating ) .", "If this is the case , increasing the rate at which temperature is changed in the absence of external Na+ should diminish the effects of rundown and reveal the presence of heat activation .", "To examine this possibility , we measured I-T relations in either the presence ( Figure 5D , large grey circles ) or absence ( Figure 5D , large orange circles ) of external Na+ by rapidly jumping between solutions at different temperatures ( Figure 5—figure supplement 3 ) , and observed that I-T relations in the absence of external Na+ obtained from temperature-ramps and from jumps agreed up to ~25°C , after which they deviated markedly ( Figure 5D , note deviation between orange circles and small yellow circles as highlighted by the blue lines ) .", "Additionally , the rate of temperature-dependent inactivation in the absence of external Na+ at T > 45°C became so fast that the speed of our temperature-jumps was not sufficient to visualize the increase in Po before significant inactivation occurred ( note the last orange circle in Figure 5D ) .", "These results suggest that temperature-dependent activation has been greatly perturbed in the absence of external Na+ at temperatures below 25°C , but that activation at higher temperatures appears largely intact ( Figure 5D , compare orange and grey large circles ) .", "Taken together , these findings suggest that the Na+ regulatory site is strongly coupled to a temperature-dependent transition that governs TRPV1 activation at low temperature , and confirm the presence of multiple temperature-dependent transitions .", "The results obtained from measurements of Po-T relations are depicted schematically in Figure 5E , where dotted horizontal lines represent plateau regions where steep temperature-dependence cannot be readily observed ( ΔHApp~0 ) , and arrows represent regions where Po increases steeply with temperature ( ΔHApp>> 0 ) .", "The vertical dotted grey line indicates the lowest temperature that we can achieve experimentally , with the yellow arrow crossing that temperature representing a hypothetical temperature-sensitive transition in the absence of external Na+ .", "We include this transition because the Po-T relation in the absence of Na+ shows some temperature-dependence to Po at T < 10°C ( Figure 5C and 6A ) .", "To verify the existence of this transition in the absence of external Na+ , we tested two manipulations that should shift the temperature range over which that transition operates .", "Capsazepine has been proposed to shift heat activation of TRPV1 to higher temperatures ( Matta and Ahern , 2007 ) , and we therefore tested whether this allosteric inhibitor might produce a rightward shift in the Po-T relation , making the temperature- sensitive transition more readily detectable in the absence of external Na+ .", "Indeed , the addition of capsazepine resulted in clear temperature-dependence of Po at low temperatures in the absence of external Na+ ( Figure 6A and C ) .", "Next , we tested intermediate external Na+ concentrations to see if temperature-dependent changes in Po could be detected at lower temperatures .", "We obtained Po-T relations at 10 , 30 and 65 mM external Na+ , and observed clear temperature-dependence at low temperatures ( Figure 6B ) , with enthalpies close to those measured in the presence of 130 mM external Na+ in the same temperature range ( Figure 6C ) .", "Taken together , these findings suggest that the TRPV1 channel undergoes the same conformational transitions in response to heat regardless of whether external Na+ is present , and thus that the enthalpy ( ΔHo ) associated with Na+ binding is not itself responsible for temperature-dependent gating ( for a more detailed discussion see Materials and methods ) .", "Rather , it seems that external Na+ modifies the midpoint ( or entropy , △S ) of the temperature-sensitive transition that operates at low temperatures .", "Additionally , external Na+ appears to stabilize a closed state of the channel independently of its effect on temperature-sensitivity , as the Po values corresponding to the inferred plateau regions ( denoted by dotted lines in Figure 6B - insert ) decrease as the concentration of external Na+ is increased . 10 . 7554/eLife . 13356 . 019Figure 6 . The response of the TRPV1 channel to heating is dominated by distinct conformational transitions over different temperature ranges .", "( A ) Normalized Po-T relations ( mean ± SEM , n = 3 ) obtained in the presence of 0 Nao and capsazepine ( Cpz ) .", "Data in 0 Nao without capsazepine are shown for comparison .", "The essential features of the Po-T relations are schematized in the insert on the right as done in Figure 5E , showing that capsazepine causes a shift in the Po-T relations to higher temperatures in the absence of external Na+ , uncovering temperature-dependent gating at low temperatures in the absence of Na+ .", "The dotted vertical line delimits the lower range of experimentally accessible temperatures .", "( B ) Po-T relations obtained in the presence of different concentrations of external Na+ at +90 mV ( mean ± SEM , n = 4–14 , see Figure 6—figure supplement 1 for individual cell data ) .", "Po-T relations are schematized as in Figure 5E on the right panel insert , showing that increasing the concentration of external Na+ shifts TRPV1 channel Po-T relations to higher temperatures .", "The dotted vertical line delimits the lower range of experimentally accessible temperatures .", "( C ) ΔHapp obtained from fits of Equation 1 to Po-T relations from individual cells ( circles ) and their mean ± SEM ( open squares ) .", "The filled square is the mean ΔHapp from fits to data in 130 Nao at T < 25°C ( see Figure 4—figure supplement 2C–E ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 13356 . 01910 . 7554/eLife . 13356 . 020Figure 6—figure supplement 1 . Po-T relations from individual cells obtained with different concentrations of external Na+ .", "( A ) Mean Po-T relation at +90 mV in the absence of external Na+ ( 0 Nao , large yellow circles , Figure 5C , no-T relation predicted by model i ( Figure 7A ) in the absence of external Na+ using the parameters in Figure 7—source data 2A .", "( B–D )", "Po-T relations from individual cells obtained in the presence of different concentrations of external Na+ from slow temperature ramps as in Figure 4A .", "Theoretical Po-T relations calculated for model i ( Figure 7A ) with parameters shown in Figure 7—source data 2A are also included as continuous colored curves . DOI: http://dx . doi . org/10 . 7554/eLife . 13356 . 020 These features of our results can be depicted in a simple allosteric model wherein two temperature-sensitive transitions are allosterically coupled to a temperature-insensitive opening transition , with Na+ regulating both the midpoint ( △S ) of the temperature-sensitive transition operating at low temperatures and the open/closed equilibrium ( Figure 7A , see Materials and methods for an in-depth discussion on the choice of model and the inherent assumptions ) .", "In the presence of Na+ , the two temperature-sensitive transitions have similar midpoints , such that the plateau between the two is barely noticeable in the Po-T relation ( Figure 6B insert , grey arrows ) .", "Upon Na+ removal , the midpoint of the first temperature-sensitive transition shifts to lower temperatures , effectively extending the middle plateau where Po is temperature-independent ( Figure 6B insert , yellow lines and arrows ) and the channel mostly occupies the open/closed equilibrium highlighted with orange squares in Figure 7A .", "Additionally , external Na+ also negatively impacts the opening transition through allosteric coupling , which results in the intermediate plateau between the two temperature-dependent transitions in Po-T relations having a lower Po in the presence of external Na+ .", "This simple model can qualitatively reproduce our results at intermediate Na+ concentrations ( Figure 7B and Figure 6—figure supplement 1 ) .", "One important feature of the model is that the opening transition is relatively temperature-insensitive , and that the channel can open even when the temperature sensor is in the deactivated state ( Figure 7A , C – O transitions with blue squares ) .", "Although we would not rule out measurable changes in ΔHo during opening , as shown by an alternate model that also satisfactorily reproduced our experimental results ( Figure 7—figure supplement 1A ) , such a mechanism would require divergent temperature-sensitive transitions depending on whether external Na+ is bound to the channel , which we consider to be more conceptually complex than the allosteric model proposed here ( see Materials and methods for a more detailed discussion ) .", "Additionally , in the alternate model ii , external Na+ influences the temperature-dependent transition that occurs at lower temperatures in the same way as in Model i ( Figure 7—figure supplement 1A ) .", "We also cannot rule out the possibility that the temperature sensors have to activate for the channel to open ( i . e . , occupancy of the open states with blue squares in Figure 7A is negligible ) , as the gating of TRPV1 channels remains temperature-sensitive even at the lowest temperatures examined .", "However , in the absence of constraining experimental data , we have opted for the more general mechanism depicted in Figure 7A . 10 . 7554/eLife . 13356 . 021Figure 7 . An allosteric framework for TRPV1 channel gating .", "( A ) Scheme for Model i with two temperature-dependent transitions ( horizontal arrows ) given by equilibrium constants J1 and J2 of the form J ( T ) = exp ( - ( ΔHo-TΔSo ) /RT ) ) and a temperature-independent opening transition ( vertical arrows ) with equilibrium constant L . The first and second temperature-dependent transitions promote the open state by increasing L by a factor D or E , respectively .", "Binding of external Na+ ( denoted by the subscript 'Na' , diagonal arrows ) , which is given by equilibrium constant K1 , decreases J1 by a factor G , J2 by a factor H and L by a factor F . The color of the letters indicates a positive allosteric effect ( green , allosteric factor > 1 ) or a negative allosteric effect ( red , factor < 1 ) on the associated equilibrium constants .", "The analytical expression for Po is given in Figure 7—source data 1A .", "( B ) Mean experimental Po-T relations from data obtained in different concentrations of external Na+ ( colored curves , data from Figure 6B ) with the predictions of model i superimposed ( black curves ) .", "Model parameters are provided in Figure 7—source data 2A . DOI: http://dx . doi . org/10 . 7554/eLife . 13356 . 02110 . 7554/eLife . 13356 . 022Figure 7—source data 1 . Analytical expressions for Po in different models .", "DOI: http://dx . doi . org/10 . 7554/eLife . 13356 . 02210 . 7554/eLife . 13356 . 023Figure 7—source data 2 . Parameters for the mathematical gating models .", "DOI: http://dx . doi . org/10 . 7554/eLife . 13356 . 02310 . 7554/eLife . 13356 . 024Figure 7—figure supplement 1 . Alternative models for describing temperature-dependent gating of TRPV1 and its modulation by external Na+ .", "( A ) Scheme for Model ii with individual conformational states represented as squares .", "‘C’ labels denote closed states , ‘O’ open states and the subscript ‘Na’ denotes Na+-bound states .", "In the absence of external Na+ ( upper row in the scheme ) , one temperature-dependent transition between closed states ( given by temperature-dependent equilibrium constant J1 ) precedes a temperature-independent opening transition ( given by the equilibrium constant L ) .", "A second temperature-dependent transition ( given by J2 ) can occur between open states .", "The binding of external Na+ ( lower row in the scheme ) increases the midpoint temperature for the first temperature-dependent transition ( given in the presence of Na+ by G x J1 ) , and induces a temperature-dependent opening transition ( given by J3 ) that is not observed in the absence of external Na+ .", "The analytical expression for the Po for model ii can be seen in Figure 7—source data 1B .", "( B ) Superimposed predictions of model i ( continuous black curves , scheme in Figure 7A ) and ii ( dashed black curves , parameters in Figure 7—source data 2B ) together with experimental data from Figure 6B .", "( C ) Scheme for Model iii showing the four coupled equilibria considered and their allosteric couplings .", "Model iii is equivalent to model i ( Figure 7A ) , but with a single temperature-dependent transition ( horizontal transitions ) .", "Activation of the temperature sensor is controlled by equilibrium constant J1 ( T ) .", "The opening transition is controlled by equilibrium constant L ( vertical transitions ) and the binding of Na+ ( diagonal transitions ) by constant K1 .", "The strength of the allosteric coupling is given by the allosteric factors D ( between temperature-sensor and the opening transition ) , F ( between the binding of external Na+ and the opening transition ) and G ( between the temperature-sensor and the binding of external Na+ ) .", "The color of the letters indicates a positive allosteric effect ( green , allosteric factor > 1 ) or a negative allosteric effect ( red , factor < 1 ) on the associated equilibrium constants .", "The analytical expression for Po corresponding to this model is provided in Figure 7—source data 1C .", "( D ) Theoretical Po-T relations ( black curves ) calculated using model iii and parameters in Figure 7—source data 2C for different external Na+ concentrations .", "The experimental data shown ( mean ± SEM , colored curves ) is that from Figure 6B for different concentrations of external Na+ .", "DOI: http://dx . doi . org/10 . 7554/eLife . 13356 . 02410 . 7554/eLife . 13356 . 025Figure 7—figure supplement 2 . High concentrations of external Na+ do not fully prevent TRPV1 channel activation by positive voltages , capsaicin or heat .", "( A ) Normalized I-V relations obtained from voltage ramps measured in the whole-cell configuration with an intracellular solution containing 600 mM NaCl and the following extracellular solutions ( in mM ) : 600 NaCl ( grey ) , 0 NaCl ( 600 NMDGCl , yellow ) , 600 NaCl + 10 µM capsaicin ( black ) and 0 NaCl ( 600 NMDGCl ) + 10 µM capsaicin ( green ) .", "Dark thin curves are the mean , and the colored envelopes the SEM ( n = 4 ) .", "The colored squares are the normalized current values ( mean ± SEM ) at +90 mV from the I-V relations in Figure 1C , which were obtained with solutions containing a concentration of permeant cations of 130 mM . ( B ) Representative whole-cell I-T relations obtained in the presence of 600 mM intra- and extracellular Na+ .", "Data wwere obtained using the same procedure as for I-T relations shown in Figure 4A and B . Data for three different cells ( yellow , purple and blue ) are shown .", "Each curve was normalized to the peak current value at +90 mV .", "The red-dotted line indicates the zero-current level . DOI: http://dx . doi . org/10 . 7554/eLife . 13356 . 02510 . 7554/eLife . 13356 . 026Figure 7—figure supplement 3 . Assessing the influence of voltage on temperature-dependent gating of TRPV1 channels in the absence of external Na+ .", "( A ) Representative whole-cell TRPV1 current families obtained at 8°C in response to 400 ms voltage pulses from -120 to +140 mV in 10 mV increments ( only traces for steps to even voltages are shown for clarity ) and recorded in the absence and presence of external Na+ or NMDG+ , with and without saturating capsaicin .", "Solutions were delivered though a temperature-controlled perfusion system as illustrated in Figure 5—figure supplement 3A .", "The dotted red lines indicate the zero-current level .", "( B ) Normalized conductance-voltage ( G-V ) relations obtained in 130 Nao + 10 µM capsaicin from current families as in ( A ) ( ~8°C , n = 5 ) or as in Figure 1—figure supplement 3A ( ~22°C , n = 7 ) .", "Conductance was calculated from the tail currents at -90 mV right after each test pulse and normalized to the tail current amplitude after the voltage pulse to +140 mV .", "( C ) Fraction of current activated in the absence of external Na+ relative to 130 Nao + 10 µM capsaicin as a function of voltage at 8 and 22°C , obtained from current families as in ( A ) ( for 8°C data ) or as in Figure 1—figure supplement 3A ( 22°C ) .", "Data are shown as mean ± SEM . DOI: http://dx . doi . org/10 . 7554/eLife . 13356 . 02610 . 7554/eLife . 13356 . 027Figure 7—figure supplement 4 . The effect of a change in heat capacity associated with the operation of the temperature-sensor on the predictions of the allosteric gating model .", "( A ) Graph showing how J1 ( T ) ( blue ) and J2 ( T ) ( red ) ( see model i in Figure 7A ) change as a function of temperature when they are associated with a change in heat capacity ( dashed curves , parameters in Figure 7—source data 2D ) or not ( continuous curves , parameters in Figure 7—source data 2A ) .", "The vertical dotted black lines denote the range of temperatures in which the experimental data was obtained .", "( B ) Theoretical Po-T relations ( dashed black curves ) calculated using model i ( Figure 6A ) with a heat capacity difference associated with the two temperature-dependent transitions governed by J1 ( T ) and J2 ( T ) ( see Materials and methods and model parameters in Figure 7—source data 2D ) .", "Predictions of model i with temperature-independent changes in enthalpy and entropy ( i . e . , no change in heat capacity ) associated with temperature-sensor function are shown as continuous black curves .", "Experimental Po-T relations ( colored circles , data from Figure 6B ) for different external Na+ concentrations are also included . DOI: http://dx . doi . org/10 . 7554/eLife . 13356 . 02710 . 7554/eLife . 13356 . 028Figure 7—figure supplement 5 . Incorporating temperature-dependent inactivation into the allosteric model .", "( A ) Theoretical plot of temperature vs time used for introducing temperature-dependent inactivation into the predictions of model i ( see Materials and methods ) .", "( B ) Theoretical Po-T relations ( model i with the parameters in Figure 7—source data 2A , see Materials and methods for details ) in which temperature-dependent inactivation was taken into account ( continuous colored curves ) .", "The dotted black lines are the predictions of the model without inactivation , and experimental data are shown as colored circles ( data from Figure 6B ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 13356 . 028 Voltage is also known to influence temperature-sensitivity of TRPV1 ( Figure 4B and D ) ( Voets et al . , 2004; Matta and Ahern , 2007; Yao et al . , 2010 ) .", "However , the low permeability of NMDG+ precluded examination of the influence of external Na+ on temperature-sensitivity of TRPV1 at negative voltages .", "Nonetheless , our data show that at the highly depolarized potentials where we performed our experimental studies and modeling , voltage does not have a predominant influence on temperature-sensitivity of this channel ( see Figure 7—figure supplement 3 and Materials and methods for a more detailed discussion ) , and therefore we did not include voltage in our modeling .", "Capsaicin is arguably the best characterized TRPV1 agonist , activating the TRPV1 channel to a maximal Po close to 1 ( Premkumar et al . , 2002; Hui et al . , 2003 ) , and has also been shown to influence temperature-sensitivity of this receptor ( Voets et al . , 2004; Matta and Ahern , 2007; Yao et al . , 2010 ) .", "We were therefore interested in determining whether it exerts a similar influence on the temperature-sensitivity of TRPV1 as external Na+ .", "To explore this possibility , we measured Po-T relations using slow temperature ramps in the presence of 130 mM external Na+ and different concentrations of capsaicin ( Figure 8A and Figure 8A—figure supplement 1A for Po-T relations from individual cells ) .", "Although we observed detectable temperature-dependent changes in Po at low temperatures for subsaturating concentrations of capsaicin , the Po values varied by more than 100-fold over the range of capsaicin concentrations , suggesting that capsaicin has a larger effect on the closed/open transition compared to the temperature-dependent transition ( Figure 8A - insert ) .", "This contrasts with the effects of removing external Na+ , which produces much larger shifts in the Po-T relation and smaller changes in Po ( Figure 6B ) .", "Notably , incorporating these features in our model ( Figure 8B ) can qualitatively describe our experimental findings with capsaicin ( Figure 8A and Figure 8—figure supplement 1A ) .", "The Po-T relation in saturating capsaicin exhibited marginal temperature-dependent changes , consistent with a large influence of capsaicin on the open/closed equilibrium of the channel leading to full receptor activation at a saturating concentration of this agonist ( Figure 8A ) . 10 . 7554/eLife . 13356 . 029Figure 8 . The effect of capsaicin on temperature-dependent gating of TRPV1 . ( A ) Po-T relations ( mean ± SEM , n = 4–14 ) obtained in the presence of 130 mM external Na+ and different concentrations of capsaicin ( see Figure 8—figure supplement 1A for Po-T relations from individual cells ) .", "The numbers in parenthesis indicate the mean Po at 22°C and +90 mV estimated from IV relations ( see Materials and methods ) .", "Continuous curves are the predictions from Model i including capsaicin shown in ( B ) with Po given in Figure 7—source data 1A and parameters in Figure 7—source data 2A ( see Figure 8—figure supplement 1B and C for theoretical Po-T relations over a larger temperature range ) .", "The insert on the right shows schematized Po-T relations on a log scale as in Figure 5E .", "( B ) Schematic depiction of the allosteric Model i ( Figure 7A ) including capsaicin with four coupled equilibria denoted by the numbers in parenthesis: ( 1 ) opening and closing of the pore , determined by the temperature-independent equilibrium constant L; ( 2 ) two-step activation/deactivation of the temperature sensor ( s ) , given by temperature-dependent equilibrium constants J1 and J2 of the form J ( T ) =e−∆Ho−T∆SoRT; binding/unbinding of sodium ( 3 ) or capsaicin ( 4 ) , determined by temperature-independent equilibrium constants K1 and K2 , respectively .", "Arrows indicate allosteric coupling between equilibria , with red denoting allosteric inhibition ( allosteric factor < 1 ) and green stimulation ( allosteric factor > 1 ) .", "The magnitude of the coupling is determined by the multiplicative temperature-independent allosteric factors next to the arrows . DOI: http://dx . doi . org/10 . 7554/eLife . 13356 . 02910 . 7554/eLife . 13356 . 030Figure 8—figure supplement 1 . Po-T relations from individual cells for different concentrations of capsaicin and predictions of the allosteric model over an extended range of temperatures .", "( A ) Theoretical Po-T relations ( thick continuous curves , model i with capsaicin – Figure 8B , Po-equation in Figure 7—source data 1A , parameters in Figure 7—source data 2A ) calculated for three concentrations of capsaicin and 130 mM extracellular Na+ with superimposed Po-T data for individual cells ( thin colored lines ) .", "( B and C )", "Same graphs as in Figure 7B and 8A with an extended temperature-axis to illustrate predictions of the model over extremes of temperature .", "Theoretical Po-T relations are shown in black . DOI: http://dx . doi . org/10 . 7554/eLife . 13356 . 030 If capsaicin has a relatively modest influence on the activation of the temperature-sensor ( as compared to its larger effects on the open/closed equilibrium ) , then it should be possible to observe temperature-dependent changes in saturating capsaicin in a construct where capsaicin-bound channels are not fully activated , such as the turret deletion ( Figure 3E ) .", "To test this possibility , we first determined whether TRPV1 channels lacking the pore turret are temperature-sensitive , as this is a controversial issue ( Yang et al . , 2010; Yao et al . , 2010; Cui et al . , 2012; Liao et al . , 2013 ) .", "Interestingly , we found that the turret deletion can still be robustly activated by heat , albeit at somewhat higher temperatures compared to the WT channel ( Figure 9A ) , confirming that the turret is not the temperature sensor . 10 . 7554/eLife . 13356 . 031Figure 9 . Deletion of the outer pore turret uncovers temperature-sensitive gating of TRPV1 in saturating capsaicin .", "( A ) Three representative TRPV1 Δ604–626 whole-cell I-T relations measured in 130 mM external Na+ in response to pulses from -90 to +90 mV as in Figure 4A and B . The dotted line is the zero-current level .", "( B ) Normalized I-V relations for TRPV1 Δ604–626 obtained in the whole-cell configuration at 22°C ( continuous curves are the mean , lighter envelopes the SEM , n = 6 , same data as Figure 3E ) or 8°C ( circles , mean ± SEM , n = 9 ) in response to voltage ramps or families of voltage pulses , respectively .", "The arrows indicate the fractional activation by capsaicin in the presence of external Na+ at both 8 and 22°C .", "( C ) Po-T relation ( mean ± SEM , n = 12 ) for TRPV1 Δ604–626 ( open circles ) at +90 mV scaled based on the I-V relations in ( B ) at 22°C ( assuming maximal Po of 0 . 9 at room temperature in the presence of 10 µM capsaicin without external Na+ ) .", "The mean Po-T relation for the WT channel in the presence of 130 mM external Na+ and 10 µM capsaicin is shown as closed black circles ( data from Figure 4—figure supplement 1B , mean ± SEM , n = 11 ) .", "The insert on the right schematizes as in Figure 5E the Po-T relations for both WT ( long-dash line ) and Δ604–626 ( short-dash line and arrows ) channels in the presence of saturating capsaicin and external Na+ .", "See Figure 9—figure supplement 1B for Po-T relations from individual cells . DOI: http://dx . doi . org/10 . 7554/eLife . 13356 . 03110 . 7554/eLife . 13356 . 032Figure 9—figure supplement 1 . Influence of the pore turret of TRPV1 on temperature-dependent gating and modulation by sodium .", "( A ) External Na+ dose-response relations for TRPV1 Δ604–626 ( open circles , mean ± SEM , n = 4 ) measured at +90 mV from voltage-ramps in the whole-cell configuration .", "Closed circles are data for WT TRPV1 at +90 mV .", "The grey and black continuous curves are the predicted normalized dose-response relations generated with model i ( Figure 7A and 8B with capsaicin ) for TRPV1 Δ604–626 ( parameters in Figure 7—source data 2E ) or WT TRPV1 ( parameters in Figure 7—source data 2A ) , respectively .", "( B ) Mean Po-T relation ( large open circles , mean ± SEM , n = 12 , data from Figure 9C ) for TRPV1 Δ604–626 at +90 mV .", "The Po-T relations for all individual cells are shown as continuous colored curves ( each cell has a different color ) .", "The open squares are the mean Po ± SEM ( n = 4–5 for each temperature-range ) obtained from noise analysis at different temperatures ( see Materials and methods and Figure 5—figure supplement 2D–G ) for TRPV1 Δ604–626 , after averaging Po-data obtained at similar temperatures in the presence of 130 Nao + 10 µM caps .", "( grey open squares ) or 0 Nao +10 µM caps .", "( green open squares ) .", "The continuous black and light-green curves are predictions of model i for 130 Nao + 10 µM caps .", "( black ) and 0 Nao +10 µM caps .", "( light green ) using parameters in Figure 7—source data 2E . DOI: http://dx . doi . org/10 . 7554/eLife . 13356 . 032 To look for temperature-dependent changes in Po for the turret-deletion construct at saturating capsaicin , we initially examined the effects of lowering temperature from 22 to 8°C on capsaicin activation in the presence of external Na+ compared to maximal activation in the presence of saturating capsaicin with no external Na+ ( Figure 9B ) .", "At 8°C , the fractional activation by saturating capsaicin in the presence of external Na+ was substantially lower than what we observed at 22°C , revealing that lowering temperature further decreased the efficacy of capsaicin ( Figure 9B; 8°C colored circles; 22°C colored ramps; see black arrows ) .", "We then obtained the full Po-T relationship in the presence of external Na+ and capsaicin , as before for the WT channel , but for the turret deletion we observed large temperature-dependent changes in Po , with two components in the relation separated by a clear plateau at intermediate temperatures ( Figure 9C and Figure 9—figure supplement 1B for Po-T relations from individual cells ) .", "These findings with the turret deletion further support the allosteric framework for conceptualizing the mechanism by which Na+ and capsaicin influence temperature-dependent gating .", "Notably , we could accurately describe the Po-T relations in saturating capsaicin and the dose-response for external Na+ for the turret deletion using model i ( Figure 9—figure supplement 1A and B ) , simply by assuming that the deletion of the pore turret increased the affinity of the channel for external Na+ and reduced the strength of the coupling between the binding of capsaicin and the opening of the pore ( Figure 7—source data 2E ) .", "Together , these results qualitatively show that capsaicin influences both the activity of the temperature-sensor and the open/closed equilibrium , but appears to have a larger influence on the latter .", "Even though the values of the coupling parameters in our modeling are not well-determined , we believe that the larger influence of capsaicin on the open/closed equilibrium is robust enough to support this conclusion .", "The results thus far demonstrate that the external pore of TRPV1 contains Na+ ion binding sites that exert strong control over temperature-dependent activation and inactivation , as well as the closed/open equilibrium .", "Our previous results with DkTx suggest external Na+ and DkTx modulate the TRPV1 through a common mechanism ( Figure 3B–D ) .", "In addition , our recent analysis of the interaction between DkTx and TRPV1 suggests that the toxin activates the channel by inducing a displacement of the S5-S6 segments relative to the S1-S4 domain ( Figure 10—figure supplement 1A ) , which results in the disruption of a cluster of buried hydrophobic residues at the extracellular ends of the S5 and S6 segments ( Bae et al . , 2016 ) .", "This is interesting in light of a recent demonstration in the Shaker Kv channel that movement of hydrophobic residues between buried and solvent-exposed environments imparts temperature-dependence ( Chowdhury et al . , 2014 ) .", "We therefore measured Po-T relations in the presence of external Na+ and DkTx to test whether the binding of the toxin has an effect on temperature-sensitivity , and observed that activation of the channel by heat was effectively ablated between 5 and 35°C ( Figure 10 , and Figure 10—figure supplement 1B for Po-T relations from individual cells ) .", "This finding is particularly striking when considering that the Po is situated well below the theoretical upper limit of 1 . 0 .", "These results demonstrate that DkTx also exerts strong control over temperature-sensor activation , which in our model can be achieved simply by trapping the channel in the intermediate plateau and preventing temperature-sensor deactivation or full activation ( Figure 10 , insert ) . 10 . 7554/eLife . 13356 . 033Figure 10 . The binding of DkTx to the outer pore of TRPV1 effectively ablates temperature-dependent gating over a wide range of temperatures . Po-T relation ( mean ± SEM , n = 9 ) for WT TRPV1 obtained in the presence of external Na+ and DkTx .", "The dotted line denotes the mean Po at 22°C and +90 mV as estimated from the I-V relations in DkTx relative to saturating capsaicin shown in Figure 3C .", "The insert to the upper right represents experimental Po-T relations on a log scale measured in the presence of external Na+ and saturating capsaicin ( dashed black line denoting the absence of temperature-dependent gating ) , external Na+ and DkTx ( dashed blue line , no temperature-dependent gating ) and external Na+ alone ( two temperature-dependent transitions ) .", "See Figure 10—figure supplement 1B for Po-T relations from individual cells . DOI: http://dx . doi . org/10 . 7554/eLife . 13356 . 03310 . 7554/eLife . 13356 . 034Figure 10—figure supplement 1 . DkTx binding to TRPV1 . ( A ) Overlay of the side views of the S3-S6 segments of one TRPV1 subunit and the S5-S6 segments of an adjacent subunit in the apo ( subunit 1 , S3-S4 in light pink , S5-S6 in magenta; subunit 2 , S5-S6 in light grey ) and the DkTx/RTx bound state ( subunit 1 , S3-S4 in light blue , S5-S6 in teal; subunit 2 , S5-S6 in light orange ) .", "The structures shown are the refined structural models of TRPV1 from ( Bae et al . , 2016 ) , with the docked solution structure of DkTx ( K1 in green and K2 in cyan ) .", "E600 is shown in stick representation and colored in dark blue , and the red highlight denotes the location from which the pore turret was deleted in the structure used for structure determination ( Cao et al . , 2013; Liao et al . , 2013 ) .", "( B ) Mean Po-T relation ( mean ± SEM , n = 9 , data from Figure 10 , ) for WT TRPV1 obtained in the presence of external Na+ and DkTx .", "The Po-T relations for individual cells are shown as colored continuous curves . DOI: http://dx . doi . org/10 . 7554/eLife . 13356 . 034" ], [ "The present results examining the regulation of temperature-dependent gating by external Na+ and capsaicin have four important implications for understanding the gating mechanisms of the TRPV1 channel .", "First , our results demonstrate that the TRPV1 channel has evolved external Na+ binding sites that must be occupied for the channel to remain closed at physiological body temperatures in mammals .", "The steeply temperature-sensitive transition that normally opens TRPV1 in response to noxious heat still operates in the absence of Na+ , yet without external Na+ the channel would be spontaneously open at physiological temperatures , many channels would be inactivated , and responses to noxious heat would therefore be diminished .", "Our results also show that the Na+ sites will play physiologically important roles in activation of the channel by acid , as protonation of acidic residues involved in binding Na+ leads to diminished Na+ occupancy and activation of the channel .", "This connection between Na+ and pH regulation provides a mechanistic explanation for previous observations where mildly acidic solutions potentiate activation of TRPV1 by heat ( Jordt et al . , 2000 ) .", "Thus , this Na+ regulatory mechanism plays a fundamental role in tuning the properties of TRPV1 to serve as a detector of noxious heat and acid , and possibly other stimuli that have yet to be examined .", "Second , our results reveal the existence of multiple temperature-sensitive transitions that are coupled to the opening transition ( see model in Figure 7A ) .", "Although we see only hints of the presence of distinct temperature-dependent transitions in the presence of external Na+ ( Figure 4C ) , they become unambiguous when Na+ is removed ( Figure 5D ) and when studied in the presence of capsaicin with the turret deletion construct ( Figure 9C ) .", "Our results suggest that the role of the first temperature-sensitive transition is to maintain the channel in a closed state at physiological temperatures , whereas the second transition functions to activate the channel in response to noxious heat .", "It is interesting that both Na+ and capsaicin do not seem to have a substantial impact on the second transition , at least to the extent that we could study this component in the presence of inactivation .", "In contrast , external Na+ exerts strong control over the first transition .", "Capsaicin also modifies the shape of macroscopic Po-T relations ( Figure 8A ) , but we were able to detect temperature-dependent transitions at low temperatures even in the presence of near-saturating capsaicin concentrations ( e . g . 100 nM ) that increase the Po at room temperature to values higher than those measured in the absence of external Na+ or in the presence of DkTx ( Figure 3C ) .", "In contrast , we observed marginal to no temperature-dependence in the Po-T relations at low temperatures in the absence of external Na+ or in the presence of DkTx ( Figure 5D and Figure 10 ) .", "These observations suggest that the vanilloid has a larger influence on the open/closed equilibrium than on the temperature-sensing machinery .", "The existence of multiple temperature-sensitive transitions also provides a possible explanation for why Po-T relations at negative voltages are at least two-fold steeper than at positive voltages ( Figure 4B ) ( Voets et al . , 2004; Yao et al . , 2010 ) .", "If hyperpolarization shifts the temperature-sensitive transition operating at lower temperatures so as to coincide with the second temperature-sensitive transition , the △Ho of the two transitions would add to make the Po-T steeper .", "Overall , the present results on the influence of Na+ and capsaicin on temperature-dependent gating in TRPV1 provide a set of essential constraints for understanding the gating mechanism of the channel using an allosteric framework .", "Third , our results provide clues about where these important Na+ binding sites are located .", "Because Na+ inhibition has only very weak voltage-dependence , these Na+ binding sites are likely to be located outside the ion permeation pathway within each subunit .", "Indeed , the large outward Na+ currents we observe on removing external Na+ at positive voltages indicate that the permeation pathway must have a high Na+ occupancy even in the absence of external Na+ .", "Mutating acidic residues forming the external Na+ site would weaken Na+ binding affinity , possibly leading to lower Na+ occupancy in the presence of 130 mM external Na+ .", "However , such an effect would not necessarily cause spontaneous TRPV1 activity because the lower Na+ occupancy would be expected to promote the form of inactivation that we observed on removing external Na+ .", "Additionally , removing external Na+ would be expected to cause less activation of the channel compared to the WT protein because ion occupancy would be diminished by the mutant .", "E600 in the extracellular loop just after the S5 helix ( Figure 3—figure supplement 1A ) has been proposed to be responsible for proton-mediated potentiation of heat- and capsaicin-dependent activation of the channel ( Jordt et al . , 2000 ) , making it an excellent candidate for forming the Na+ binding site .", "The E600Q mutant displayed modest spontaneous activity when recording at room temperature ( Ohta et al . , 2008 ) , and only weak activation when removing external Na+ or applying DkTx when compared with the WT channel , consistent with a role in forming the Na+ binding site .", "Interestingly , E600 is located near the pore turret ( Liao et al . , 2013 ) ( Figure 3—figure supplement 1A ) , which when deleted from TRPV1 causes a dramatic increase in the apparent affinity of Na+ ( Figure 3F ) , and this residue is also located very close to where DkTx binds ( Cao et al . , 2013 ) ( Figure 3—figure supplement 1A and Figure 10—figure supplement 1A ) .", "Finally , our results have important implications for interpreting the recent cryo-EM structures of TRPV1 and in localizing the region of the protein involved in temperature sensing .", "In the cryo-EM studies on the turret deletion construct of TRPV1 , capsaicin appears to produce a partial opening of the internal pore , and the combined influence of RTx and DkTx to produce further expansion of the internal pore together with a conformational change within the selectivity filter ( Cao et al . , 2013 ) ( Figure 10—figure supplement 1A ) .", "These differences between the two structures might indicate that there are two gates that can be differentially regulated by activating stimuli ( Cao et al . , 2013 ) ( Figure 1A ) .", "However , we discovered that capsaicin is a partial agonist of the turret deletion construct , suggesting that the partially open features of the capsaicin-bound channel may not represent a unique conformation of the protein , but an average of closed and open states .", "The cryo-EM structure of the apo state of TRPV1 ( Liao et al . , 2013 ) , however , reveals that the internal pore must expand for the pore to support ion permeation , consistent with accessibility studies ( Salazar et al . , 2009 ) .", "The observation of temperature-independent plateaus in Po-T relations under diverse conditions that produce submaximal open probabilities suggests that the opening of the pore does not require the two temperature-dependent transitions to occur , and that the open/closed equilibrium itself is not the source of the temperature-sensitivity of TRPV1 .", "If opening of the internal pore is indeed temperature-insensitive , what is the significance of the structural changes in the external pore observed in the structure of the RTx/DkTx-bound channel ?", "The present findings with external Na+ and DkTx suggest that these structural changes in the external pore are tightly associated with the temperature-sensing machinery , either by participating in allosteric coupling or in the actual mechanism of temperature-sensing ." ], [ "The WT rat TRPV1 channel in pcDNA3 . 1 vector was kindly provided by Dr . David Julius ( UCSF ) , and subcloned into the low-expression pcDNA1 vector .", "The E600Q point mutation and Δ604–626 deletion were introduced into rTRPV1 using a two-step PCR mutagenesis technique and the resulting constructs were verified by sequencing .", "HEK293 cells were cultured following standard protocols ( Li et al . , 2015 ) and transiently transfected with plasmids for the expression of TRPV1 and GFP ( pGreen-Lantern , Invitrogen , Carlsbad , CA ) using FuGENE6 ( Promega , Madison , WI ) .", "Standard whole-cell patch clamp recordings from transiently transfected HEK293 cells at room temperature ( 22–24°C ) were performed unless stated otherwise .", "The data were acquired with an Axopatch 200B amplifier ( Molecular Devices , Sunnyvale , CA ) , filtered with an 8-pole low-pass Bessel filter ( model 900 , Frequency Devices , Ottawa , IL ) and digitized with a Digidata1322A interface and pClamp10 software ( Molecular Devices ) .", "All data were analyzed using Igor Pro 6 . 34A ( Wavemetrics , Portland , OR ) .", "Pipettes were pulled from borosilicate glass and heat-polished to final resistances between 1 and 5 MΩ .", "80–95% series resistance ( Rs ) compensation was used in all whole-cell recordings except those involving changes in temperature .", "The intracellular recording solution consisted of ( in mM ) : 130 NaCl , 10 HEPES , 10 EGTA , pH 7 . 4 ( NaOH/HCl ) .", "For experiments involving changes in temperature , 10 mM MgCl2 was added to the intracellular solution to block endogenous inward-rectifying and low-threshold temperature-sensitive currents that were observed in some recordings .", "The addition of the divalent did not cause any appreciable effect on TRPV1 channel currents , consistent with a previous report ( Yang et al . , 2014 ) .", "Moreover , we did not observe significant contaminating currents at room temperature when magnesium was omitted from the pipette solution , except in a small subset of cells that exhibited noticeable inward currents that were easily recognizable due to the outward-rectifying character of TRPV1 , and thereby could be readily discarded .", "The control extracellular solution ( 130 Nao ) consisted of ( mM ) : 130 NaCl , 10 HEPES , 10 EGTA , pH 7 . 4 ( NaOH/HCl ) .", "The zero-sodium extracellular solution ( 0 Nao ) consisted of ( mM ) : 130 NMDGCl , 10 HEPES , 10 EGTA , pH 7 . 4 ( NMDG/HCl ) .", "For solutions with different extracellular Na+/NMDG+ concentrations , NaCl was replaced with NMDGCl .", "Capsaicin and capsazepine ( 100 mM ) stock solutions were prepared in ethanol or 1:1 ethanol/DMSO , respectively .", "All chemicals and reagents were from Sigma-Aldrich ( San Luis , MO ) .", "DkTx was produced recombinantly , folded and purified as described previously ( Bae et al . , 2012 ) .", "A holding potential of -90 mV was used in all experiments .", "The data were acquired at 5–10 kHz and low-pass filtered at 2 kHz .", "For voltage-ramps , voltage was initially stepped down to -120 mV for 50 ms , then ramped up to +140 mV over 1 s before returning to -90 mV .", "Ramps were applied every 5 s .", "For I-V relations obtained with steps , voltage was stepped from -90 mV to the test potential for 100 ( room temperature recordings ) or 400 ms ( low temperature recordings , due to slow kinetics ) , and returned to -90 mV after each pulse .", "Test pulses went from -120 to +140 mV in 10-mV increments .", "Current time courses at a fixed voltage were obtained by applying trains of pulses from -90 to +90 mV of either 100 ( experiments at room temperature ) or 300 ms duration ( experiments starting at lower temperatures ) .", "The pulse interval was varied , using 3–5 s for I-T relations and 100 ms for time courses at room temperature .", "In all experiments at room temperature , a gravity-fed rapid solution exchange system ( RSC-200 , BioLogic , France ) was used .", "The cells were lifted from the coverslip and placed in front of glass capillaries perfused with different solutions .", "For I-V relations at a low temperature and fast temperature-jumps , a custom-made temperature-controlled perfusion system was used , as described below and in Figure 5—figure supplement 3A .", "I-T relations in response to slow temperature ramps were obtained in a temperature-controlled microincubator stage ( see below and Figure 4—figure supplement 1A ) .", "Po-T relations were constructed by factoring out the temperature-dependence of conduction through an open channel from I-T relations ( see below ) , and then scaled based on the relative current magnitudes at each condition measured at +90 mV and at room temperature from voltage-ramps ( see below for details ) .", "The scaling of Po-T relations was also verified using noise analysis ( see below ) .", "To obtain a qualitative assessment of the temperature-dependence of channel function , the steepest portions of I-T relations were fit to Equation 1: ( 1 ) I ( T ) =exp ( −∆Happ−T∆SappRT ) where I ( T ) is the current as a function of temperature , ΔHapp and ΔSapp are the apparent enthalpy and entropy , respectively , T is the temperature ( in Kelvins ) and R is the gas constant .", "Approximate Q10 values were calculated from Q10≈e∆Happ20 .", "All dose-response curves were obtained with voltage-ramps and fit to the Hill equation: IImax=Imin+Imax−Imin1+ ( K1/2[X] ) s , where Imin and Imax are the currents at saturating concentrations of Na+ and 0 Na+ , respectively , K1/2 is the concentration of half-maximal inhibition , s is the Hill coefficient and [X] is the molar concentration of Na+ .", "Dose-response relations for extracellular Na+ at pH 7 . 4 and 5 . 5 were obtained using 130 mM internal Na+ and , for all test solutions , a total concentration of external monovalent cations ( Na+ + NMDG+ ) of 130 mM .", "For the dose-response relation at pH 6 . 0 , 300 mM intracellular Na+ was used with a total concentration of external cations of 300 mM .", "For experiments at pH 6 and 5 . 5 , methanethiosulfonic ( MTS ) acid was used as a counterion instead of chloride to ablate endogenous proton-activated chloride currents ( Lambert and Oberwinkler , 2005 ) .", "Solutions at pH 6 . 0 or 5 . 5 were buffered with 10 mM Bis/Tris or 10 mM MES , respectively ." ] ]

[ "TRPV1 channels in sensory neurons are integrators of painful stimuli and heat , yet how they integrate diverse stimuli and sense temperature remains elusive .", "Here , we show that external sodium ions stabilize the TRPV1 channel in a closed state , such that removing the external ion leads to channel activation .", "In studying the underlying mechanism , we find that the temperature sensors in TRPV1 activate in two steps to favor opening , and that the binding of sodium to an extracellular site exerts allosteric control over temperature-sensor activation and opening of the pore .", "The binding of a tarantula toxin to the external pore also exerts control over temperature-sensor activation , whereas binding of vanilloids influences temperature-sensitivity by largely affecting the open/closed equilibrium .", "Our results reveal a fundamental role of the external pore in the allosteric control of TRPV1 channel gating and provide essential constraints for understanding how these channels can be tuned by diverse stimuli ." ]

[ "Humans and other mammals sense elevated heat and other painful stimuli via a sensory ion channel protein called TRPV1 .", "Ion channels create pores in the outer membrane of cells and act as gates that open and close to regulate the flow of ions into and out of cells .", "This flow of ions generates electrical signals that sensory neurons use to communicate information about the environment to the brain .", "The TRPV1 channel is opened by heat , but also by venom toxins , acid and the active ingredient in hot chilli peppers .", "The fluid that surrounds animal cells often contains high levels of sodium ions , and these ions flow through TRPV1 channels when they are open .", "Also , when sodium ions are removed from the fluid surrounding a cell , TRPV1 channels will spontaneously open .", "It is not known why this happens , but it suggests that sodium ions help to regulate the activity of the TRPV1 channel , and despite having been extensively studied , it remains unclear how this channel senses temperature and responds to other harmful stimuli .", "Jara-Oseguera et al . have now investigated the role of sodium ions in activating TRPV1 in cells grown in the laboratory .", "The experiments involved a technique that records the movement of ions through TRPV1 channels in the cell surface membrane while exposing the cell to various stimuli that open or close the channels .", "The results showed that TRPV1 contains an important site on its outer surface that binds sodium ions and fine-tunes the channel’s properties .", "This enables the channel to be activated by potentially damaging temperatures .", "Further analysis revealed that TRPV1 undergoes at least two distinct physical changes in response to heat and that the binding of sodium ions to the channel’s outer structure regulates one of these changes .", "Jara-Oseguera et al . went on to discover that the binding of a tarantula toxin to the outer surface of TRPV1 renders the channel insensitive to changes in temperature .", "These findings suggest that structural changes to the external part of this channel protein are closely associated with its ability to sense temperature , possibly because this region of the protein is directly responsible for detecting changes in temperature .", "These findings shed light on how this channel detects and responds to dangerously high temperatures , as well to other harmful stimuli .", "An aim for future studies is to pinpoint the specific regions of the protein that form the sodium-binding sites and to test the hypothesis that temperature sensors are located on the outer surface of the protein ." ]

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

[ "neuroscience" ]

[ [ "In our daily lives , we are constantly exposed to complex acoustic environments composed of multiple sound sources , for instance , while shopping in crowded markets or listening to an orchestra .", "Although we do it effortlessly , the separation of such mixtures of sounds into perceptually distinct sound sources is a highly complex task .", "In spite of being a topic of intense investigation for several decades , the neural bases of auditory object formation and segregation still remain to be fully explained ( Cherry , 1953; McDermott , 2009; Griffiths et al . , 2012 ) .", "The most commonly used signal for probing auditory perceptual organization is a sequence of two pure tones alternating in time that , under certain conditions , can ‘stream’ or segregate into two sources ( van Noorden , 1975; Bregman , 1990 ) .", "Much work using these streaming signals has been carried out to elucidate the neural substrates and computations that underlie auditory segregation ( Moore and Gockel , 2012; Snyder et al . , 2012 ) .", "In a series of seminal experiments , Fishman and colleagues recorded multi-unit activity from the auditory cortex of macaques in response to a simple streaming sequence ( Fishman et al . , 2001; 2004 ) .", "For large frequency differences and fast presentation rates , which promote two distinct perceptual streams , they observed spatially segregated responses to the two tones .", "This pattern of segregated cortical activation , proposed to underlie the streaming percept , has since been widely replicated ( e . g . , Bee and Klump , 2004; 2005; Micheyl et al . , 2007a; Bidet-Caulet and Bertrand , 2009 ) and attributed to basic physiological principles of frequency selectivity , forward masking and neural adaptation ( Fishman and Steinschneider , 2010a ) .", "These properties are considered to contribute to stream segregation by promoting the activation of distinct neuronal populations in the primary auditory cortex ( A1 ) that are well separated along the tonotopic axis ( McCabe and Denham , 1997; Carlyon , 2004; Micheyl et al . , 2007a; Moore and Gockel , 2012 ) .", "Human imaging studies that directly correlated the perceptual representation of streaming sequences with brain responses also support the correspondence between the streaming percept and the underlying neural activity in A1 ( Gutschalk et al . , 2005; Snyder et al . , 2006; Wilson et al . , 2007; Cusack , 2005 ) .", "However , similar effects have also been demonstrated in the auditory nerve , suggesting that processes contributing to segregation might occur earlier in the ascending auditory pathway rather than be mediated exclusively by the auditory cortex ( Beauvois and Meddis , 1991; Pressnitzer et al . , 2008 ) .", "A major drawback of the streaming paradigm is that it uses relatively simple , temporally regular narrowband signals which do not capture the rich spectrotemporal complexity of natural acoustic scenes .", "Moving beyond streaming , Kidd and colleagues developed a spectrally rich signal referred to as the ‘informational masking’ ( IM ) stimulus ( Kidd et al . , 1994 , 1995 , 2011; Kidd and Mason , 2003 ) .", "IM refers to a type of non-energetic or central masking that is associated with an increase in detection thresholds due to stimulus uncertainty and target-masker similarity that is distinct from peripheral energetic masking ( Pollack , 1975; Durlach et al . , 2003 ) .", "These multi-tone masking experiments required listeners to detect tonal target signals in the presence of simultaneous multi-tone maskers , often separated by a ‘spectral protection region’ ( a certain frequency region around the target with little masker energy ) that promoted the perceptual segregation of the target from the masker tones .", "Results demonstrate that target detection is critically dependent on the width of the spectral protection region , and the ‘density’ of the maskers ( Micheyl et al . , 2007b; Gutschalk et al . , 2008; Elhilali et al . , 2009b ) , and has been hypothesized to rely on the same adaptation-based mechanisms as proposed in the context of simple streaming signals ( Micheyl et al . , 2007b ) .", "In contrast , the sounds we are required to segregate in everyday life are distinct from the narrowband targets used in streaming and IM paradigms; they are often broadband with multiple frequency components that are temporally correlated and overlap with other signals in the environment ( McDermott and Simoncelli , 2011 ) .", "Indeed , the ability of models inspired by such paradigms to explain segregation is currently under debate .", "Recently , Elhilali et al . ( 2009a ) demonstrated that when the two tones in a streaming signal are presented synchronously , listeners perceive the sequence as one stream irrespective of the frequency separation between the two tones , a result that is inconsistent with predictions based on adaptation-based models .", "Instead , the authors argued that in addition to separation in feature space , temporal coherence between different elements in the scene is essential for segregation such that temporally incoherent patterns tend to result in a segregated percept while temporal coherence promotes integration ( Shamma et al . , 2011; Fishman and Steinschneider , 2010b; Micheyl et al . , 2013a , b ) .", "To investigate systematically the emergence of an auditory object from a random stochastic background , we developed a new stimulus ( Stochastic figure-ground; SFG ) consisting of coherent ( ‘figure’ ) and randomly varying ( ‘background’ ) components that overlap in spectrotemporal space and vary only in their statistics of fluctuation ( Figure 1A; Teki et al . , 2011 ) .", "The components comprising the figure vary from trial to trial so that it can be extracted only by integrating across both frequency and time .", "The appearance of a brief figure embedded in background components thus simulated perception of a coherent object in noisy listening environments .", "Two spectrotemporal dimensions of the figure were manipulated in each experiment—the ‘coherence’ , or the number of repeating components , and the ‘duration’ , or the number of chords that comprised the figure . 10 . 7554/eLife . 00699 . 003Figure 1 . Examples of Stochastic Figure-Ground stimuli . All stimuli in this example contain four identical frequency components ( only for illustrative purposes: these were selected randomly in the experiments ) with Fcoh = 1016 . 7 Hz , 2033 . 4 Hz , 3046 . 7 Hz , and 4066 . 8 Hz repeated over 6 chords and indicated by the black arrows .", "The figure is bound by a black rectangle in each stimulus .", "( A ) Chord duration of 50 ms: stimulus comprises of 40 consecutive chords each of duration 50 ms with a total duration of 2000 ms . ( B ) Chord duration of 25 ms: stimulus comprises of 40 consecutive chords each of duration 25 ms with a total duration of 1000 ms . ( C ) Ramped figures: stimulus comprises of 40 consecutive chords each of duration 50 ms each ( like A ) but the frequency components comprising the figure increase in frequency in steps of 2*I or 5*I , where I = 1/24th of an octave , represents the resolution of the frequency pool .", "( D ) Isolated figures: stimulus comprises only of the ‘figure present’ portion without any chords preceding or following the figure .", "The duration of the stimulus is given by the number of chords .", "( E ) Chords interrupted by noise: stimulus comprises of 40 consecutive chords alternating with 40 chords comprising of loud , masking broadband white noise , each 50 ms in duration .", "In experiment 6b , the duration of the noise was varied from 100 ms to 500 ms ( see ‘Materials and methods’ ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 00699 . 003 We used psychophysics to examine listeners’ ability to extract complex figures and tested segregation behavior in the context of various spectral and temporal perturbations .", "Our results demonstrate that listeners are remarkably sensitive to the emergence of such figures ( Figure", "2 ) and can withstand a variety of stimulus manipulations designed to potentially disturb spectrotemporal integration ( Figures 1B–E and 4 ) .", "We also show that a model based on the detection of temporal coherence across frequency channels ( Shamma et al . , 2011 , 2013 ) accounts for the psychophysical data ( Figure 3 and Figure 3—figure supplement 1 ) .", "The work demonstrates an automatic , highly robust segregation mechanism that is sensitive to temporal correlations across frequency channels ." ], [ "In experiment 1 , the basic SFG stimulus sequence was used to probe figure-detection performance ( Figure 1A; see ‘Materials and methods’ ) .", "Listeners’ responses were evaluated to obtain d′ for each combination of coherence and duration of the figure .", "The results ( Figure 2A ) show a clear effect of increasing coherence and duration .", "Hit rates ( not shown ) mirror d′ with listeners achieving mean hit rates of 93 ± 2% for the most salient coherence/duration combination .", "It is notable that the patterns were very brief ( longest figure duration was 7 chords or 350 ms ) , yet very high levels of performance were observed ( and without extensive practice ) .", "This is consistent with the idea that this task based on the SFG stimulus taps low-level , finely tuned segregation mechanisms .", "What underlies this sensitivity ?", "Since ‘figure-absent’ and ‘figure-present’ signals were controlled for overall number of components ( see ‘Materials and methods’ ) , a global power increase per se associated with the emergence of the figure , can be discounted as a potential cue .", "However , it is possible that the decisions of the listeners are based on other changes within the stimulus , for example , the emergence of a figure might be associated with a change in the temporal modulation rate of a few frequency channels .", "The purpose of experiment 2 was to investigate whether the detection of figures involves a specific figure-ground decomposition , namely whether the figure components are grouped together as a detectable ‘perceptual object’ distinct from the background components , or whether listeners were rather just detecting some low-level changes within the ongoing stimulus .", "To address this issue , we created stimulus triplets with different background patterns in which each stimulus contained a figure but where figure components were identical in two out of the three signals .", "Listeners were required to identify the ‘odd’ signal that contained a different figure from the other two signals with identical figures in this AXB psychophysical paradigm ( see ‘Materials and methods’ for details ) .", "Results ( Figure 2B ) indicate that for the very short figure duration ( 4 chords , or 200 ms ) listeners had difficulty with this discrimination task ( d′ = 0 . 31 ± 0 . 18; not significantly different from 0: p=0 . 12 , t = 1 . 72 ) , but that performance increased significantly for a figure duration of 8 chords ( 400 ms; d′ = 1 . 75 ± 0 . 34 ) and reached ceiling for a figure duration of 12 chords ( 600 ms; d′ = 2 . 93 ± 0 . 26 ) .", "This pattern of results indicates that figure detection in these stimuli is associated with a segregation mechanism whereby coherent components are grouped together as a distinct perceptual object . 10 . 7554/eLife . 00699 . 004Figure 2 . Behavioral performance in the basic and figure identification task . The d′ for experiments 1 ( A; ‘chord duration of 50 ms’; n = 9 ) and 2 ( B; ‘figure identification’; n = 9 ) are plotted on the ordinate and the duration of the figure ( in terms of number of 50 ms long chords ) is shown along the abscissa .", "The coherence of the different stimuli in experiment 1 is color coded according to the legend ( inset ) while the coherence in experiment 2 was fixed and equal to six .", "The AXB figure identification task was different from the single interval alternative forced choice experiments: listeners were required to discriminate a stimulus with an ‘odd’ figure from two other stimuli with identical figure components .", "Error bars signify one standard error of the mean ( SEM ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 00699 . 004 In experiment 3 , the length of each chord in the SFG stimulus was halved to 25 ms , thereby reducing the corresponding durations of the figure and the stimulus ( Figure 1B ) .", "Here , we aimed to test whether figure-detection performance would be affected by such temporal scaling , that is , whether performance would vary as a function of the total duration of the figure ( twice as long in experiment 1 vs experiment 2 ) or the number of repeating chords that comprised the figure ( same in experiments 1 and 2 ) .", "Behavioral results ( Figure 4A ) reveal good performance , as in experiment 1 .", "Listeners achieved hit rates of 92 ± 3% for the highest coherence/duration combination used .", "An ANOVA with coherence and duration as within-subject factors and experimental condition ( 50 ms vs 25 ms chords ) as a between-subject factor revealed no significant effect of condition ( F1 , 15 = 2; p=0 . 174 ) , suggesting that performance largely depends on the number of repeating chords irrespective of the time scale .", "Finally , as expected , model predictions were consistent with the experimental findings .", "Thus , correlations across the spectrogram channels remained significant , but now occurred at higher rates than in experiment 1 ( 40 Hz vs 20 Hz ) , reflecting the faster rate of tone presentations in the figure ( Figure 3—figure supplement 1A ) .", "In the preceding experiments , figure components were identical across several chords .", "In experiment 4 , we manipulated the figure components such that they were not identical across chords but rather ramped , that is , increasing in frequency from one chord to the next ( Figure 1C ) .", "The components in the frequency pool used to generate the SFG signals are separated equally by 1/24th of an octave; and in the following two experiments we increased the frequency steps from one chord to the next by two times ( experiment 4A; Figure 4B—thick lines ) or five times ( experiment 4B; Figure 4B—thin lines ) the frequency resolution ( i . e . , 2/24th octave and 5/24th octave respectively ) .", "Performance in these experiments was robust ( maximum hit-rates of 0 . 97 and 0 . 83 were obtained for figures with coherence equal to 8 and duration equal to 7 for the two ramp levels of 2 and 5 respectively ) and a comparison with experiment 1 using an ANOVA with coherence and duration as within-subject factors and experimental condition ( repeating vs ramp size 2 vs ramp size", "5 ) as a between-subject factor revealed a significant effect of condition: F2 , 25 = 19; p<0 . 001 .", "Performance was significantly worse for the ramp = 5 vs ramp = 2 condition ( F1 , 18 = 21 , p<0 . 001 ) , but , remarkably , listeners exhibited above-chance performance even for the steeper slope .", "This suggests that the segregation mechanisms in question are more susceptible to spectral than temporal perturbations ( as in experiments 3 , and 6 below ) but can still integrate over dynamically changing , rather than fixed , figure components .", "Finally , as with previous experiments , there were significant correlations among the channels predicting the saliency of the figure .", "However , the optimal rate at which the correlations occurred here was slightly lower ( at 10 Hz; Figure 3—figure supplement 1B , C ) than that of experiment 1 ( 20 Hz ) , perhaps because two 50 ms chords are integrated as a single unit to define the ramp .", "The stimuli in previous experiments consisted of a sequence of ‘background-only’ chords , prior to the onset of the figure , and another sequence of ‘background-only’ chords after figure offset .", "From first principles , segregation could be considered to be mediated by adaptation to the ongoing background statistics and detection of the figure as a deviation from this established pattern .", "In order to test this hypothesis , in experiment 5 , we removed the ‘background’ context which preceded the occurrence of the figure ( Figure 1D; see ‘Materials and methods’ section ) .", "The stimulus consisted simply of the chords which contained a figure ( between 3 and 7 chords ) .", "Similar to previous experiments , the results ( Figure 4C ) show a marked effect of coherence and duration , and performance improved with increasing salience of the figures with listeners reaching hit rates of 89 ± 5% for the most salient condition .", "To evaluate behavior with respect to experiment 1 , an ANOVA with coherence and duration as within-subject factors and experimental condition ( with background vs no background ) as a between-subject factor was used which yielded no significant effect of condition: F1 , 16 = 0 . 033; p=0 . 859 , suggesting that the ‘background-only’ chords which preceded the figure did not affect performance .", "Modeling for this experiment replicated the results of experiment 1 in that the correlations increased with the coherence and duration of the figure and showed maximum response at 20 Hz ( Figure 3—figure supplement 1D ) , corresponding to the rate of presentation of the chords comprising the figure .", "In experiment 6 , we incorporated 50 ms of loud , broadband masking noise between successive 50 ms long SFG chords ( Figure 1E ) , in an attempt to disrupt binding of temporally successive components .", "If figure detection is accomplished by low level mechanisms which are sensitive to a power increase within certain frequency bands , the addition of the noise bursts would disrupt performance by introducing large power fluctuations across the entire spectrum , thus reducing the overall power differences between channels .", "The results ( Figure 4D ) show good behavioral performance ( maximum hit rate of 0 . 93 was obtained for the most salient condition ) which varied parametrically with the coherence and duration of the figure .", "An ANOVA with coherence and duration as within-subject factors and experimental condition ( 50 ms repeating chords vs 50 ms chords alternating with white noise ) as a between-subject factor revealed no significant effect of condition ( F1 , 17 = 0 . 004; p=0 . 953 ) .", "Interleaving the noise bursts between successive chords does not therefore affect performance .", "Model predictions in this experiment ( Figure 3—figure supplement 1E , F ) are broadly consistent with the findings in that detection became easier with more coherent tones , and with longer figure intervals .", "The reason is simply because the noise weakens but does not eliminate the correlation among the tones , at least when computed at slower rates .", "A natural question that arises from the preceding experiment is—what are the temporal limits or the range over which such a higher-order mechanism operates ?", "In order to test this question , we gradually varied the duration of the intervening noise bursts between stimulus chords in a set of three related experiments with different durations of noise for a particular combination of coherence ( 6 ) and duration ( 6; see ‘Materials and methods’ ) .", "Results ( Figure 4E ) indicate robust performance for all durations of noise up to 300 ms and surprisingly , supra-threshold performance ( d′ = 1 . 00 ± 0 . 30; significantly different from 0: p=0 . 01; t = 3 . 29 ) for a noise duration of 500 ms . This remarkable ability of listeners to integrate coherent patterns over 3 s long ( in the case of 500 ms noise bursts ) suggests that the underlying higher-order mechanisms are very robust over such long time windows .", "Model predictions of these findings are still possible if correlations are measured over longer windows ( or slower rates—e . g . , 3 . 33 Hz as in Figure 3—figure supplement 1F ) .", "Temporal windows of integration , as long as 500 ms , have rarely been reported in the context of auditory object formation in complex scenes such as those examined here .", "The results suggest the existence of a central mechanism that is not affected by interfering broadband noise that integrates repeating pure tone components as belonging to a distinct object over multiple time scales .", "The long temporal windows here implicate cortical mechanisms at or beyond primary auditory cortex ( see e . g . , Overath et al . , 2008 demonstrating a range of ‘cortical windows’ between 20 ms and 300 ms ) ." ], [ "The temporal coherence model proposes that segregation is determined not only based on separation in feature-space but rather by the temporal relationship between different elements in the scene , such that temporally coherent elements are grouped together , while temporally incoherent channels with independent fluctuation profiles are perceived as belonging to separate sources ( Shamma et al . , 2011 ) .", "Specifically , the model incorporates two stages: firstly , a feature analysis stage that performs multidimensional feature analysis by distinct populations of neurons in the auditory cortex that are tuned to a range of temporal modulation rates and spectral resolution scales .", "Auditory features such as pitch , timbre and loudness are computed by different neuronal groups at this initial stage , the output of which is fed to a second stage that involves analysis of temporal coherence .", "Elhilali et al . carried out work implicating a critical role for temporal coherence in the assignment of common elements within a stream: they showed that a pair of synchronous repeating tones produces the same coherent pattern of modeled central activity as a single stream irrespective of the frequency separation between them , suggesting that temporal coherence is an important factor governing segregation ( Elhilali et al . , 2009a ) .", "This was substantiated by direct neurophysiological recordings from ferret auditory cortex which showed that synchronous and alternating cortical responses were equally segregated despite their perceptual differences , and hence that the temporal factors are more important in inducing the one and two streams percept ( Shamma and Micheyl , 2010; Pressnitzer et al . , 2011; Shamma et al . , 2011 , 2013 ) .", "In the case of the more complex SFG stimuli , the modeling results suggest that temporal coherence is modulated as a function of the coherence and the duration of the figure in a manner similar to the modulation of figure detection performance .", "Although this is not causal evidence in favor of the model , it behaves similar to human listeners in complex acoustic conditions as used here .", "The data suggest temporal coherence as a correlate of stimulus salience by which the brain picks out the most important sounds in busy auditory scenes: a process that may not be computed by dedicated structures but could be achieved by binding across distributed feature channels without significant changes in ensemble activity .", "Similar accounts of binding in vision based on coherence of the temporal structure have been put forward previously ( e . g . , Sporns et al . , 1991; Alais et al . , 1998; Blake and Lee , 2005 ) .", "It is still not known how temporal coherence may be computed and which brain areas perform these computations .", "Temporal coherence may be implemented by neurons that show strong sensitivity to temporal coherence across distant frequency channels , or by neurons that act as multiplexers and are more selective to particular combinations of inputs ( Elhilali et al . , 2009a; Shamma et al . , 2011 ) .", "Elhilali et al . ( 2009a ) sought such cells in the primary auditory cortex of the ferret but were unable to demonstrate any in passively listening animals but have preliminary evidence in behaving ferrets ( Shamma et al . , 2013 ) .", "Although previous brain imaging studies have identified activity in A1 that was correlated with the streaming percept ( e . g . , Gutschalk et al . , 2005; Snyder et al . , 2006; Wilson et al . , 2007 ) , we found no evidence of modulation of BOLD signal in A1 as a function of figure emergence in a passive listening paradigm ( Teki et al . , 2011 ) .", "However , we found activity in the intraparietal sulcus ( IPS ) to be strongly modulated by the salience of the figure , similar to the modulation of temporal coherence observed here .", "The IPS activation likely reflect bottom-up stimulus-driven processing of figures and is consistent with accumulating literature which suggests that areas outside the conventional auditory system , such as the parietal cortex may have a role in auditory segregation ( Cusack , 2005; Dykstra et al . , 2011 ) .", "Although not relevant to the passive fMRI experiment ( Teki et al . , 2011 ) , attention also influences segregation .", "In this regard , the parietal cortex is in an ideal position to integrate both bottom-up auditory input as it receives auditory input from the temporoparietal cortex ( Pandya and Kuypers , 1969; Divac et al . , 1977; Hyvärinen , 1982; Cohen , 2009 ) as well as top-down attentional input from the prefrontal cortex ( Andersen et al . , 1985; Barbas and Mesulam , 1981; Petrides and Pandya , 1984; Stanton et al . , 1995 ) .", "IPS is associated with both bottom-up and top-down attention and is a key structure implicated in saliency map models of visual search ( Koch and Ullman , 1985; Itti and Koch , 2001; Walther and Koch , 2007 ) where low-level feature maps may combine with top-down cognitive biases to represent a global saliency map ( Gottlieb et al . , 1998; Geng and Mangun , 2009; Bisley and Goldberg , 2010 ) .", "IPS ( and its monkey homologue ) has been implicated in mediating object representations , binding of sensory features within and across different modalities , as well as attentional selection .", "We hypothesize that IPS may represent a neural correlate of the figure percept where the representation will depend on the salience of auditory figures .", "In our model , this perceptual representation depends on the computation of temporal coherence across multiple frequencies that are initially represented in the auditory cortex .", "Neurophysiological recordings from parietal neurons might in future determine whether such sensory analysis ( before perceptual representation ) involves parietal neurons or is established in auditory cortex first ." ], [ "We developed a new stimulus ( Stochastic figure-ground [SFG] stimulus; Teki et al . , 2011 ) to model naturally complex situations characterized by a figure and background that overlap in feature space that are only distinguishable by their fluctuation statistics .", "Contrary to previously used signals , the spectrotemporal properties of the figure vary from trial to trial and the figure can only be extracted by binding the spectral components that comprise the figure across frequency and time .", "Figure 1 presents examples of the SFG stimulus which consists of a sequence of random chords , each 50 ms in duration with 0 ms inter-chord-interval , presented for a total duration of 2000 ms ( 40 consecutive chords ) .", "Each chord contains a random number ( average: 10 and varying between 5 and 15 ) of pure tone components .", "The spectral components are randomly selected from a set of 129 frequencies equally spaced on a logarithmic scale between 179 Hz and 7246 Hz such that the separation between successive components is 1/24th of an octave .", "The onset and offset of each chord are shaped by a 10 ms raised-cosine ramp .", "In half of these stimuli , a random number of tones are repeated across a certain number of consecutive chords ( e . g . , in Figure 1 , four components marked by arrows are repeated across 6 chords ) which results in the percept of a ‘figure’ that readily pops out of the random tonal background .", "To eliminate correlation between the number of figure and background components , the figure was realized by first generating the random background and then adding additional , repeating components to the relevant chords .", "To avoid the problem that the interval containing the figure might , on average , also contain more frequency components , and to prevent listeners from relying on this feature in performing the figure detection task , the remaining 50% of the stimuli ( those containing no figure ) also included additional tonal components , which were added over a variable number ( 2–7 ) of consecutive chords at the same time as when a figure would have appeared .", "But these additional components changed from chord to chord and did not form a coherent figure .", "In the present study , we parametrically varied the number of consecutive chords over which the tones were repeated ( ‘duration’ ) and the number of repeated frequency components ( ‘coherence’ ) .", "The onset of the figure was jittered between 15 and 20 chords ( 750–1000 ms ) post stimulus onset .", "All participants tested in this set of experiments reported normal hearing and had no history of audiological or neurological disorders .", "Experimental procedures were approved by the research ethics committee of University College London ( Project ID number: 1490/002 ) , and written informed consent was obtained from each participant .", "For each experiment we report the number of listeners whose data is included in the final analysis .", "In each experiment , a few listeners ( 2–3 ) were excluded from analysis because of their inability to perform the task .", "9 listeners ( 2 females; aged between 20 and 47 years; mean age: 26 . 9 years ) took part in experiment 1 .", "9 listeners ( 6 females; aged between 22 and 28 years; mean age: 23 . 8 years ) participated in experiment 2 based on the AXB design .", "10 listeners ( 5 females; aged between 20 and 36 years; mean age: 25 . 7 years ) took part in experiment 3 .", "10 listeners ( 5 females; aged between 23 and 31 years; mean age: 26 . 8 years ) participated in experiment 6a .", "27 listeners ( Group 1: 9 listeners; 5 females , aged between 19 and 27 years; mean age: 21 . 1 years; Group 2: 10 listeners; 3 females; aged between 19 and 25 years; mean age: 21 . 3 years; Group 3: 8 listeners; 3 females; aged between 19 and 29 years; mean age: 22 . 4 years ) participated in experiment 6b .", "10 listeners ( 6 females; aged between 21 and 34 years , and mean age of 24 . 7 years ) participated in experiment 4a with ramp step equal to 2 and another group of 10 listeners ( 3 females; aged between 20 and 30 years and mean age of 24 . 5 years ) took part in experiment 4b with ramp step of", "5 . 10 listeners ( 5 females; aged between 22 and 31 years , mean age: 24 . 8 years ) participated in experiment", "5 . SFG stimuli in experiment 1 consisted of a sequence of 50 ms chords with 0 ms inter-chord interval and 2 s duration ( 40 consecutive chords ) .", "The coherence of the figure varied between 1 , 2 , 4 , 6 or 8 and the duration of the figure ranged from 2 to 7 chords .", "Stimuli for all combinations of coherence and duration were presented in a separate block ( total of 30 blocks ) where 50% of the trials ( 50 trials per block ) contained a figure .", "The stimuli in experiment 2 consisted of 50 ms chords and a figure coherence value of", "6 . Figure duration varied between 4 , 8 and 12 ( in separate blocks ) .", "Stimuli , all containing a figure , were presented in triplets as in an AXB design ( e . g . , Goldinger , 1998 ) .", "The background patterns were different in all three signals but two of them ( either A and X or B and X ) contained identical figure components .", "Listeners were required to indicate the ‘odd’ figure ( A or B ) by pressing a button .", "Three blocks of 60 trials each were presented for each duration condition .", "Stimuli in experiment 3 were identical to those in experiment 1 except that chord duration was reduced to 25 ms . The coherence of the figure varied between 2 , 4 , 6 or 8 and the duration of the figure ranged from 2–7 chords resulting in a total of 24 blocks .", "In experiment 4a and 4b , stimuli were similar to those in experiment 1 except that in this condition , the successive frequencies comprising the figure were not identical from one chord to the next but increased across chords in steps of 2*I or 5*I , where I = 1/24th of an octave is the resolution of the frequency pool used to create the SFG stimulus .", "The coherence of the figure was 4 , 6 , or 8 and duration was 5 , 7 or 9 chords resulting in a total of 9 blocks for each condition .", "Note that in this experiment , the maximum duration of the figure ( 9 chords ) is longer than the maximum duration of the figure in the remaining experiments ( 7 chords ) .", "The stimuli for experiment 5 were same as in experiment 1 except that they comprised of the figure only ( 3–7 chords or 150–350 ms ) without any chords that preceded or succeeded the figure as in previous experiments .", "The coherence of the figure was 2 , 4 , 6 , or 8 chords and this resulted in a total of 20 blocks .", "For experiment 6a , we modified the SFG stimulus so that successive chords were separated by 50 ms broadband noise burst .", "The loudness of the noise was set to a level 12 dB above the level of the stimulus chords .", "The coherence of the figure was 2 , 4 , 6 or 8 and the duration of the figure ranged from 3 to 7 chords resulting in a total of 20 blocks .", "The stimuli in experiment 6b were identical to the previous experiment save for the following differences:", "( a ) coherence and duration were fixed at a value of 6;", "( b ) the duration of the noise was varied in three different experiments in increasing order: group 1: 50 , 100 , 150 ms; group 2: 100 , 200 , 250 ms; group 3: 100 , 300 , 500 ms respectively .", "The 100 ms condition was chosen as an anchor and only those participants who performed above a threshold of d′ = 1 . 5 in this condition were selected for the whole experiment .", "Prior to the study , training was provided which consisted of listening to trials with no figures , easy-to-detect figures , difficult-to-detect figures and one practice block of fifty mixed trials .", "For the main experiment , the value of coherence and duration was displayed before the start of each block and participants were instructed to press a button as soon as they heard a figure pop out of the random background ( for the brief figures used here , these sounded like a ‘warble’ in the on-going random pattern ) .", "Feedback was provided .", "Blocks with different values of coherence and duration were presented in a pseudorandom order .", "The participants self-paced the experiment and the study lasted approximately an hour and a half .", "The procedure was identical across all experiments .", "Participants’ responses were measured in terms of sensitivity ( d prime , or d′ ) .", "We also report hit rates for certain conditions as mean ± one standard error .", "All stimuli were created online using MATLAB 7 . 5 software ( The Mathworks Inc . , Natick , MA ) at a sampling rate of 44 . 1 kHz and 16 bit resolution .", "Sounds were delivered diotically through Sennheiser HD555 headphones ( Sennheiser , Germany ) and presented at a comfortable listening level of 60–70 dB SPL ( self adjusted by each listener ) .", "Presentation of the stimuli was controlled using Cogent ( http://www . vislab . ucl . ac . uk/cogent . php ) .", "Listeners were tested individually in an acoustically shielded sound booth .", "The apparatus was identical for all experiments .", "The temporal coherence model was run for a range of temporal modulation rates: 2 . 5 , 5 , 10 and 20 Hz for experiments 1 , 4 , 5 , and 6a and 5 , 10 , 20 and 40 Hz for experiment 3 respectively .", "Additionally , we used a rate of 3 . 33 Hz corresponding to the rate of presentation of 300 ms white noise segments in experiment 6b .", "These rates cover the range of physiological temporal modulation rates observed in the auditory cortex .", "A single spectral resolution scale of 8 cycles per octave ( corresponding to the bandwidth of streaming; 4 cycles per octave for experiment 4b where larger frequency steps are required to extract a ramped figure ) was used .", "The analysis was conducted by entering the SFG stimulus for each experimental condition to the input stage of the model .", "For experiments 1 and 3 , the entire stimulus duration was fed to the model input and for the remaining experiments a stimulus without the pre- and post-figure chords was entered .", "This was based on the prediction that the background chords before and after the figure onset contribute little to the cross-correlation matrix unlike the chords comprising the figure .", "The simulations were performed separately for the stimuli containing a figure and without a figure and repeated across 500 iterations .", "To establish differences between the resultant coherence matrices , we computed the maximum value of the cross-correlation across all time points .", "This spectral decomposition helps us to examine which channels are correlated with each other ( hence , the channels with repeating figure components could possibly be bound together as one object , or the ‘figure’ ) , and not significantly correlated with each other ( hence , the channels with random correlation between channels may not be perceived as a single object ) .", "The difference in the average values of the maxima between the figure and the ground stimuli was calculated as the model response and plotted like the psychophysical curves to obtain model responses ( see Figure 3 and Figure 3—figure supplement 1 ) ." ] ]

[ "In contrast to the complex acoustic environments we encounter everyday , most studies of auditory segregation have used relatively simple signals .", "Here , we synthesized a new stimulus to examine the detection of coherent patterns ( ‘figures’ ) from overlapping ‘background’ signals .", "In a series of experiments , we demonstrate that human listeners are remarkably sensitive to the emergence of such figures and can tolerate a variety of spectral and temporal perturbations .", "This robust behavior is consistent with the existence of automatic auditory segregation mechanisms that are highly sensitive to correlations across frequency and time .", "The observed behavior cannot be explained purely on the basis of adaptation-based models used to explain the segregation of deterministic narrowband signals .", "We show that the present results are consistent with the predictions of a model of auditory perceptual organization based on temporal coherence .", "Our data thus support a role for temporal coherence as an organizational principle underlying auditory segregation ." ]

[ "Even when seated in the middle of a crowded restaurant , we are still able to distinguish the speech of the person sitting opposite us from the conversations of fellow diners and a host of other background noise .", "While we generally perform this task almost effortlessly , it is unclear how the brain solves what is in reality a complex information processing problem .", "In the 1970s , researchers began to address this question using stimuli consisting of simple tones .", "When subjects are played a sequence of alternating high and low frequency tones , they perceive them as two independent streams of sound .", "Similar experiments in macaque monkeys reveal that each stream activates a different area of auditory cortex , suggesting that the brain may distinguish acoustic stimuli on the basis of their frequency .", "However , the simple tones that are used in laboratory experiments bear little resemblance to the complex sounds we encounter in everyday life .", "These are often made up of multiple frequencies , and overlap—both in frequency and in time—with other sounds in the environment .", "Moreover , recent experiments have shown that if a subject hears two tones simultaneously , he or she perceives them as belonging to a single stream of sound even if they have different frequencies: models that assume that we distinguish stimuli from noise on the basis of frequency alone struggle to explain this observation .", "Now , Teki , Chait , et al . have used more complex sounds , in which frequency components of the target stimuli overlap with those of background signals , to obtain new insights into how the brain solves this problem .", "Subjects were extremely good at discriminating these complex target stimuli from background noise , and computational modelling confirmed that they did so via integration of both frequency and temporal information .", "The work of Teki , Chait , et al . thus offers the first explanation for our ability to home in on speech and other pertinent sounds , even amidst a sea of background noise ." ]

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

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

[ [ "Bacteria can have profound effects on the normal development of diverse animal taxa ( McFall-Ngai et al . , 2013 ) .", "One of the most pervasive examples of bacteria stimulating development is the induction of animal metamorphosis by bacteria ( Hadfield , 2011 ) .", "During these interactions in marine environments , surface-bound bacteria often serve as environmental triggers that induce mobile animal larvae to settle on a surface and undergo metamorphosis .", "Although the stimulation of metamorphosis by bacteria is critical for diverse animal-mediated processes such as coral reef formation ( Webster et al . , 2004; Whalan and Webster , 2014 ) , the recruitment of stocks for marine fisheries ( Dworjanyn and Pirozzi , 2008; Yu et al . , 2010 ) and the fouling of submerged surfaces like the hulls of ships ( i . e . biofouling ) ( Khandeparker et al . , 2006; Nedved and Hadfield , 2008 ) , we know little about the mechanisms that govern this microbe–animal interaction .", "Despite the fact that the link between bacteria and animal metamorphosis was first discovered in the 1930s ( Zobell and Allen , 1935 ) , few bacterial products have been described that stimulate this developmental transition .", "To date , identified bacterial cues can all be classified as small molecules .", "Two examples are the small bacterial metabolite tetrabromopyrrole , which induces partial or complete metamorphosis of corals ( Sneed et al . , 2014; Tebben et al . , 2011 ) and the polar molecule histamine from algae or associated microbes , which induces urchin metamorphosis ( Swanson et al . , 2007 ) .", "To our knowledge , however , no proteinaceous bacterial cues have yet been identified that stimulate animal metamorphosis .", "To investigate how bacteria induce animal metamorphosis , we have previously studied the interaction between the tubeworm Hydroides elegans ( hereafter Hydroides ) and the bacterium Pseudoalteromonas luteoviolacea ( Hadfield et al . , 1994; Huang and Hadfield , 2003; Nedved and Hadfield , 2008; Shikuma et al . , 2016 ) .", "We found that P . luteoviolacea produces arrays of Metamorphosis Associated Contractile structures ( MACs ) that induce the metamorphosis of Hydroides larvae ( Huang et al . , 2012; Shikuma et al . , 2014 ) .", "MACs are an example of a Contractile Injection System ( CIS ) ; macromolecular machines that are specialized to puncture membranes and often deliver proteinaceous effectors into target cells ( Brackmann et al . , 2017; Taylor et al . , 2018 ) .", "Like other CISs , MACs are evolutionarily related to the contractile tails of bacteriophages ( bacterial viruses ) and are composed of an inner tube protein ( homologous to gp19 from phage T4 and Hcp from type six secretion systems ) surrounded by a contractile sheath , a tail-spike , and a baseplate complex ( Shikuma et al . , 2014 ) .", "The conserved mechanism is driven by contraction of the sheath , which propels the inner tube/spike into the target cell .", "Different ways of loading effectors onto a CIS have been suggested .", "Translocation mechanisms of effectors via the spike complex of a CIS have been well characterized ( Quentin et al . , 2018; Shneider et al . , 2013 ) .", "The presence of an alternative pathway of loading effectors into the inner tube lumen has been speculated but is only poorly understood .", "For example , an amorphous density inside the inner tube of the Antifeeding prophage ( Afp ) was attributed to either the toxin payload or a tape-measuring protein ( Heymann et al . , 2013 ) .", "Other classes of effectors were found to interact with the inner tube protein ( Hcp ) and are likely released post-firing by tube dissociation in the target cytoplasm ( Sana et al . , 2016; Silverman et al . , 2013 ) .", "In this study , we set out to identify a potential metamorphosis-inducing effector that MACs inject into Hydroides larvae , as well as the loading of such an effector into MACs .", "We show that a previously identified genomic region in P . luteoviolacea ( Shikuma et al . , 2016 ) encodes a bacterial protein that localizes to the inner tube lumen of the MAC structure and is necessary for inducing the metamorphosis of Hydroides larvae .", "Our results identify a proteinaceous effector stimulating animal metamorphosis and provide a direct visualization of an effector in the tube lumen of an assembled CIS ." ], [ "We previously identified a genomic locus in P . luteoviolacea encoding six genes ( gene numbers JF50_12590 , JF50_12595 , JF50_12600 , JF50_12605 , JF50_12610 and JF50_12615 ) that was essential for inducing the larvae of Hydroides to undergo metamorphosis ( Shikuma et al . , 2016 ) .", "Here we analyzed biofilms of strains with in-frame deletions of each of the six genes and tested their ability to induce Hydroides metamorphosis .", "The ΔJF50_12605 and ΔJF50_12615 mutants exhibited a reduced ability to induce metamorphosis ( less than 20% , Figure 1A ) , while mutation of the other four genes had no observable effect .", "When JF50_12605 and JF50_12615 were replaced back into their native chromosomal loci , metamorphosis induction was restored ( Figure 1B ) .", "We confirmed the effect of MACs on Hydroides metamorphosis by producing cell-free MAC array preparations .", "While larvae exposed to MACs from a ∆JF50_12615 mutant did not induce metamorphosis ( even at high concentrations ) , MACs from a ∆JF50_12605 mutant induced metamorphosis when added at higher doses ( Figure 1—figure supplement 1A/B ) .", "Our results suggest that JF50_12615 was essential for the induction of metamorphosis , while JF50_12605 contributed but was dispensable .", "Based on our results here and below , we name the protein encoded by JF50_12615 as ‘Mif1’ for Metamorphosis-Inducing Factor 1 .", "To search for structural differences between MACs from wildtype P . luteoviolacea and the specific gene deletion mutants , we employed cryo-electron tomography ( cryoET ) imaging .", "Deletion of the full JF50_12590–JF50_12615 locus ( Figure 1—figure supplement 2 ) , or each of the six genes individually ( Figure 1—figure supplement 3 ) , did not impair the formation of ordered arrays of MACs , featuring both extended and contracted conformations .", "Upon detailed analyses , we observed that extended MACs from both ΔJF50_12605 and Δmif1 strains exhibited a central lumen with very low density .", "By contrast , MACs from wildtype and the other deletion mutants possessed a density distribution that was homogeneous and a lumen was not discernable ( Figure 1C–G and Figure 1—figure supplement 3 ) .", "We refer to these structural phenotypes as ‘empty’ and ‘filled’ respectively .", "Strikingly , quantitative analyses showed that the empty phenotype in ΔJF50_12605 and Δmif1 MACs correlated with the inability to induce metamorphosis ( Figure 1A/H ) .", "The replacement of mif1 and JF50_12605 back into their native chromosomal loci reverted the empty phenotype back to filled ( Figure 1—figure supplement 4 ) .", "To investigate whether the structural differences between wildtype and Δmif1/ΔJF50_12605 MACs represented potential cargo , we performed sub-tomogram averaging of the extended sheath-tube complex ( resolution estimation in Figure 2—figure supplement 1 ) .", "The resulting MAC structures for both wildtype and Δmif1 revealed densities corresponding to the sheath and the inner tube ( Figure 2A–F ) , similar to the structures of homologous CISs ( Jiang et al . , 2019 ) .", "While the Δmif1 structure lacked any discernible density inside the ~4 nm-wide tube lumen ( Figure 2E , F ) , the wildtype structure exhibited repeating packets of density inside the tube ( Figure 2A–D ) , suggesting the presence of a potential cargo .", "The densities in the tube lumen reinforced less strongly compared to the sheath-tube complex , which could be caused by one or a combination of the following factors:", "1 ) during averaging , the alignment of the cargo was affected by the strong densities from the sheath-tube ,", "2 ) the cargo was not structured or flexible , and/or", "3 ) the cargo was present at a sub-stoichiometric amount compared to tube subunits or tube-rings .", "In any case , it is likely that the packet-like shape of the cargo density was caused by alignment artifacts .", "This is supported by a difference map between the wildtype and ∆mif1 structure , which shows a continuous density filling the inner tube lumen ( Figure 2G , H ) .", "It is important to note , however , that the cargo and tube densities were separated by a low-density region ( arrowheads in Figure 2B ) .", "Furthermore , any expelled tubes from triggered MACs always showed an ‘empty’ phenotype in cryotomography images ( Figure 2—figure supplement 2 ) .", "These results together could indicate weak or entirely absent interactions between cargo and tube , possibly facilitating rapid release of the cargo from the tube upon contraction .", "To test whether JF50_12605 and/or Mif1 were present within the MAC complex and represented the cargo within the tube lumen , we performed protein identification by mass spectrometry of purified MACs .", "In two independent experiments , we detected Mif1 but not JF50_12605 in wildtype MAC samples ( Figure 3A ) .", "MACs from the ΔJF50_12605 mutant exhibited considerably fewer spectral counts for the Mif1 protein .", "These results are consistent with the ‘empty’ phenotype observed by cryoET imaging of the ΔJF50_12605 strain ( Figure 1D and Figure 1—figure supplement 3E ) .", "To further corroborate the association of Mif1 with MAC arrays , we tagged Mif1 with a FLAG-tag in its native chromosomal locus in three different locations .", "After purifying MACs , we detected Mif1 strongly associated with MACs from two Mif1-FLAG-tagged strains and one at a reduced level by dot-blot and an anti-FLAG antibody ( Figure 3B ) .", "Because JF50_12605 is important for localizing Mif1 within the MAC complex , we analyzed protein–protein interactions between JF50_12605 and Mif1 .", "To this end , we performed a reciprocal pull down of S-tagged JF50_12605 and 6xHis-tagged Mif1 .", "We detected JF50_12605 when pulling down Mif1 by nickel chromatography , and we detected Mif1 when pulling down JF50_12605 with S-tag antibodies ( Figure 3C ) .", "To determine whether Mif1 or JF50_12605 associated with other components of the MAC complex , we utilized a bacterial two-hybrid system based on the interaction-mediated reconstruction of a cyclic AMP ( cAMP ) signaling cascade ( Karimova et al . , 2000 ) .", "When JF50_12605 , Mif1 and MacT1 ( JF50_12680 , tube ) were screened for interactions , we found a significant interaction between JF50_12605 and Mif1 as well as JF50_12605 with itself ( Figure 3D–F ) .", "However , neither JF50_12605 , nor Mif1 interacted with MacT1 ( JF50_12680 , tube ) .", "Together , these data indicate that Mif1 is present within the MAC structure and represents the densities seen in the tube lumen , while JF50_12605 might act as a chaperone that helps to localize Mif1 inside the MAC tube .", "Mif1 , however , could also associate with MACs independently of JF50_12605 in an inefficient manner .", "This is shown by", "1 ) the residual presence of Mif1 in MACs from a ∆JF50_12605 mutant as detected by mass spectrometry ( Figure 3A ) , and", "2 ) the fact that high concentrations of a cell-free ∆JF50_12605 MAC extract can induce metamorphosis ( Figure 1—figure supplement 1 ) .", "Because our results suggested that Mif1 was loaded into the MAC tube lumen , we next tested whether Mif1 was sufficient for stimulating metamorphosis when delivered to Hydroides larvae .", "We therefore purified N-terminally His-tagged Mif1 by nickel chromatography ( Figure 4A ) and verified its identity by western blot with a Mif1-specific antibody ( Figure 4B ) .", "As controls , we purified JF50_12605 and GFP under the same conditions .", "Mif1 protein provided exogenously to competent larvae of Hydroides at concentrations of up to 250 ng/µl did not stimulate metamorphosis ( Figure 4—figure supplement 1 ) .", "We reasoned , however , that the Mif1 protein might require intracellular delivery into host cells to initiate metamorphosis of Hydroides .", "To this end , we utilized a custom electroporator that was previously successfully used for other marine invertebrates ( Zeller , 2018; Zeller et al . , 2006 ) .", "Successful translocation of protein was confirmed by anti-GFP western blotting of larval lysate after electroporation ( Figure 4—figure supplement 2 ) .", "When we delivered Mif1 into competent Hydroides larvae , a significant percentage of the larvae underwent metamorphosis ( Figure 4C ) .", "In contrast , neither JF50_12605 nor GFP stimulated metamorphosis when electroporated under the same conditions .", "Our results suggest that Mif1 was sufficient to stimulate Hydroides metamorphosis when delivered by electroporation ." ], [ "In conclusion , our data indicate that the bacterium P . luteoviolacea induces metamorphosis of animal larvae by delivering the effector protein Mif1 via the tube lumen of an extracellular contractile injection system ( MACs ) .", "These insights are significant for different fields of research as discussed below .", "First , previously reported bacterial cues for animal metamorphosis are all classified as small molecules ( Sneed et al . , 2014; Swanson et al . , 2007; Tebben et al . , 2011 ) .", "The identification of a protein ( Mif1 ) that stimulates tubeworm metamorphosis requires us to expand the scope of possible biomolecules and mechanisms by which bacteria stimulate animal development .", "Our findings suggest that rather than MACs stimulating metamorphosis solely by physical puncturing and depolarization of larval membranes , the delivered Mif1 protein could have an enzymatic activity .", "This challenges previous hypotheses on metamorphosis induction ( Carpizo-Ituarte and Hadfield , 1998; Yool et al . , 1986 ) , however , it would be in line with many studies on other eukaryote-targeting CIS effectors with enzymatic activities ( Jiang et al . , 2016; Ma and Mekalanos , 2010; Vlisidou et al . , 2019 ) .", "Future investigations will shed light on the molecular mechanism by which Mif1 triggers animal metamorphosis , which is a challenging task , given that Mif1 does not feature any recognizable conserved protein domains .", "Second , our results directly showed the previously hypothesized possibility of effector delivery via the tube lumen of a CIS ( Heymann et al . , 2013; Sana et al . , 2016; Shneider et al . , 2013; Silverman et al . , 2013 ) .", "Interestingly , the comparison of MACs with a different class of CIS , namely the Type Six Secretion System ( T6SS ) , reveals significant differences .", "The T6SS effectors that are thought to be delivered by the T6SS tube lumen show protein–protein interactions between the T6SS effector and the T6SS tube protein ( Hcp ) ( Sana et al . , 2016; Silverman et al . , 2013 ) .", "By contrast , we did not detect such interactions between Mif1 and MAC tube protein .", "One possible explanation could be that the biophysical characteristics of the T6SS tube and the MAC tube are different .", "While the T6SS tube is inherently unstable and disassembles soon after contraction ( Szwedziak and Pilhofer , 2019 ) , inner tubes of MACs and other extracellular CISs ( and contractile phages ) can be readily detected by electron microscopy and therefore seem to be much more stable .", "Given our observation that expelled MAC tubes were always empty ( e . g . Figure 2—figure supplement 2 ) , this poses the question of how the effectors exit such a stable tube after contraction .", "We hypothesize that this could be the very reason for weak or entirely absent interactions between Mif1 and MAC tube , as well as for the low-density region that was seen in subtomogram averages separating Mif1 and MAC tube ( Figure 2B ) .", "Another mechanistic consequence of low affinity between Mif1 and tube could be the requirement of an assembly factor , that is JF50_12605 , that allows for efficient targeting of Mif1 to the tube .", "Third , our insights into MAC function could be significant to re-engineer the system for future medical and biotechnological applications .", "As micron-scale , syringe-like structures , MACs have potential for being developed as delivery systems targeting eukaryotic cells .", "Extracellular CISs such as MACs are of particular interest , because they are released from the producing bacterial cell and autonomously bind to the target cell’s surface .", "Extracellular CISs that target bacterial pathogens are already under development as narrow host-range antimicrobial agents ( Scholl , 2017 ) .", "The identification of effectors carried by MACs provides the basis for loading MACs with a cargo of choice .", "We recently reported a second effector that MACs deliver to eukaryotic cells ( Rocchi et al . , 2019 ) , expanding the number of MAC effectors that could be engineered .", "Intriguingly , a cryotomogram of the ∆pne1 ( JF50_12610 ) mutant shows that the tube has a filled phenotype ( Figure 1—figure supplement 3 ) .", "The filled phenotype suggests that Pne1 is not found within the inner tube , but instead could be loaded in a different location ( e . g . spike ) within the MACs complex .", "Understanding tube lumen-delivered effectors could be particularly helpful , based on the potential of a higher payload per CIS , as compared to spike-bound effectors .", "Fourth , the identification of Mif1 and its delivery mechanism will facilitate the investigation of how bacterial factors trigger animal signaling systems , leading to metamorphosis .", "This could have potential practical applications for preventing biofouling , improving aquaculture husbandry , restoring degraded ecosystems like coral reefs , and as a biotechnology platform .", "The fact that bacteria are known to stimulate metamorphosis in every major group of animals alive today ( Hadfield , 2011 ) , combined with the detection of MAC-like gene clusters in microbes from diverse environments including the ocean , terrestrial environments and even the human gut ( Böck et al . , 2017; Jiang et al . , 2019 ) , underscores the huge diversity of bacterium–animal interactions that remains to be explored ." ], [ "Bioassays were conducted with specimens of Hydroides elegans obtained from Quivira Basin , San Diego , California .", "Embryos were obtained and maintained as previously described ( Nedved and Hadfield , 2008; Shikuma et al . , 2016 ) .", "Competent larvae were exposed to biofilms of P . luteoviolacea wild type , as a positive control , to P . luteoviolacea mutants , and to P . luteoviolacea strains unable to produce MAC structures ( ∆macB ) , as well as to artificial seawater ( - ) .", "The percent of larvae that underwent metamorphosis was scored 24 hr after the induction of metamorphosis .", "Metamorphosis was scored visually by observing the number of individuals that formed branchial radioles , and a primary and secondary tube .", "Four biological replicates of approximately 30 larvae each were performed for each treatment on three separate occasions with larvae spawned from different adults .", "All bacterial strains , plasmids and primer sequences used are listed in the supplemental Tables 1 and 2 .", "All deletion and fusion strains were created according to previously published protocols ( Rocchi et al . , 2019; Shikuma et al . , 2016; Shikuma et al . , 2014 ) .", "Plasmid insert sequences were verified by DNA sequencing .", "Deletion and insert strains were confirmed by PCR .", "All E . coli strains were grown in Lysogeny-Broth ( LB ) media at 37°C shaking at 200 revolutions per minute ( RPM ) .", "All P . luteoviolacea cultures were grown in seawater tryptone ( SWT ) media ( 35 . 9 g/l Instant Ocean , 2 . 5 g/l tryptone , 1 . 5 g/l yeast extract , 1 . 5 ml/l glycerol ) at 25°C shaking at 200 RPM .", "Media that contained antibiotics were at a concentration of 100 mg/ml unless otherwise stated .", "P . luteoviolacea was grown in 50 ml SWT media in 250 ml flasks at 30 °C for 6 hr or overnight ( 12–14 h ) .", "Cells were centrifuged for 30 min at 4000 g and 4 °C and resuspended in 5 ml cold extraction buffer ( 20 mM Tris , pH 7 . 5 , 1M NaCl ) .", "Cultures were centrifuged for 30 min at 4000 g and 4 °C and the supernatant was isolated and centrifuged for 30 min at 7000 g and 4 °C .", "The pellet was resuspended in 20–100 µl cold extraction buffer and stored at 4°C for further use .", "Plunge freezing was performed as implemented in Weiss et al . ( 2017 ) .", "In essence , gentle MAC extractions were seeded with 10 nm BSA-coated colloidal gold particles ( 1:4 v/v , Sigma ) and 4 μl of the mixture was applied to a glow-discharged holey-carbon copper EM grid ( R2/1 , Quantifoil ) .", "The grid was backside blotted in a Vitrobot ( FEI Company ) by using a Teflon sheet on the front pad , and plunge-frozen in a liquid ethane-propane mixture ( 37%/63% ) cooled by a liquid nitrogen bath .", "Frozen grids were stored in liquid nitrogen .", "The gentle MAC extractions were imaged by cryo-electron tomography ( cryoET ) ( Weiss et al . , 2017 ) .", "Images were recorded on a Titan Krios TEM ( FEI ) equipped with a Quantum LS imaging filter operated at a 20 eV slit width and K2 Summit ( Gatan ) .", "Pixel sizes at specimen level ranged from 2 . 14 Å to 2 . 72 Å .", "Tilt series were collected using a bidirectional tilt-scheme from −30° to +60° and −32° to −60° in 2° increments .", "Total dose was ~90 e-/Å2 and defocus was kept at −5 to −6 µm .", "Some tilt series were recorded in focus using a Volta phase plate ( Danev et al . , 2014 ) .", "Tilt series were acquired using SerialEM ( Mastronarde , 2005 ) and reconstructed and segmented using the IMOD program suite ( Kremer et al . , 1996 ) .", "Density plots to determine filled and empty phenotypes were done using Fiji ( Schindelin et al . , 2012 ) .", "Contrast enhancement of some tomograms was done using the tom_deconv deconvolution filter ( https://github . com/dtegunov/tom_deconv ) .", "Tomograms used for structure identification and picking were binned by a factor of 4 .", "Defocus was estimated using Gctf ( Zhang , 2016 ) and CTF correction , exposure filtering and backprojection was done using IMOD .", "SR data were binned by a factor of 2 resulting in a pixel size of 4 . 29 Å/px .", "The discrete extended MAC structures were identified visually in individual tomograms and their longitudinal axes were modeled with open contours in 3dmod ( Mastronarde , 2008 ) .", "Individual model points were added at defined intervals of about 12 nm along the contours using the addModPts program from the PEET package ( Heumann et al . , 2011 ) resulting in 24’721 initial particles for filled tubes and 37’024 initial particles for empty tubes .", "Models were imported into Dynamo ( Castaño-Díez et al . , 2012 ) , particles were extracted , the azimuth angle was randomized and all particles were averaged to obtain an initial reference .", "Four times binned subtomograms ( 17 . 14 Å/px ) were used for four iterations of initial alignment with the reference low-pass filtered to about 50 Å .", "No symmetry was applied , but rotational search was limited to +/- 30° .", "Before unbinning , subtomograms were cleaned by distance and cross correlation coefficient leaving 20’358 filled and 20’782 empty tube particles .", "Two times binned subtomograms ( 8 . 57 Å/px ) were extracted using the refined coordinates and the dataset was split in two half sets .", "Half sets were aligned independently for five iterations .", "Unbinned subvolumes ( 4 . 29 Å/px ) were extracted using the refined coordinates and aligned for eight more iterations .", "Subvolumes were cleaned by cross correlation coefficient and final averages were generated using 13’039 particles and 13’425 particles for filled and empty tubes , respectively .", "UCSF Chimera ( Pettersen et al . , 2004 ) was used for visualization of the 3D models and to generate the difference map between wildtype and ∆mif1 structure .", "Bacterial two-hybrid Analysis was performed following the protocols detailed previously ( Karimova et al . , 2000 ) .", "Briefly , proteins of interest were cloned into one of four Bacterial Two Hybrid ( BTH ) plasmids pUT18 , pUT18C , pKT25 , and pKNT25 .", "These produced individual N- or C-terminal fusions between the proteins of interest and the T18 and T25 subunits on of the adenylate cyclase ( CyaA ) protein .", "All plasmid sequences were confirmed by PCR .", "Plasmid combinations containing the genes of interest were then electroporated into BTH101 electrocompetent cells that lacked a native CyaA gene .", "The BTH101 cells were grown on LB agar containing ampicillin ( 100 mg/ml ) , kanamycin ( 100 mg/ml ) and 1% glucose .", "Glucose was used to suppress the expression of proteins before performing the assay .", "Protein–protein interactions were quantified by performing a β-galactosidase assay with cells being grown overnight at 37°C and shaking at 200 RPM .", "Protein expression was induced with 1 . 0 mM IPTG .", "The cultures were incubated at 25°C shaking at 200 RPM for 6 hr before being mixed with a one-step ‘β-gal’ mix ( Schaefer et al . , 2016 ) .", "A plate reader was then used to measure the absorbance at 420 nm and 600 nm .", "The optical densities were used to calculate Miller Units as previously described ( Miller , 1972 ) .", "To purify JF50_12615 , JF50_12605 and GFP proteins , genes of interest were cloned into the pET15b plasmid and grown in E . coli BL21 pLysE .", "Bacteria were struck out on LB agar plates with ampicillin ( 100 µg/µl ) and grown at 37°C for 24 hr .", "A single colony was inoculated into 5 ml LB with ampicillin ( 100 µg/µl ) and grown at 37°C shaking at 200 RPM for 14–16 hr .", "The overnight culture was diluted 1:500 into 500 ml LB with ampicillin ( 100 µg/µl ) , grown at 37°C shaking at 200 RPM until the culture reached an OD600 of 0 . 95 .", "Protein expression was induced with 0 . 1 mM IPTG and grown for 25°C for 16 hr .", "The culture was centrifuged at 4000 g for 20 min and the supernatant was removed .", "The pellet was then resuspended in lysis buffer ( 20 mM imidazole , 25 mM tris-HCl , 500 mM NaCl , pH 8 ) with a protease inhibitor cocktail ( 100 µM leupeptin , 1 µM pepstatin and 5 µM bestatin ) .", "The culture was French pressed twice ( 1000 psi ) and sonicated 3 times for 10–30 s each time .", "The lysed culture was then spun down at 12 , 000 g for 20 min and the supernatant was discarded .", "Inclusion bodies were purified from the pellet by first washing the pellet twice with 20 mM tris pH 8 , 2 M urea , 2% triton X-100 , 500 mM NaCl .", "The remaining pellet was resuspended using 5 ml 6M guanidinium HCl , 5 mM imidazole , 20 mM tris pH 8 , 500 mM NaCl .", "The 6XHIS tagged proteins were then bound to Ni-agarose beads which had been pre-equilibrated to the resuspension buffer .", "The proteins were refolded by adding 1 ml/min of 5 mM imidazole , 20 mM tris pH 8 , 500 mM NaCl up to a total of 50 ml .", "After refolding , the beads were loaded onto a vacuum column and washed twice with 10 ml of refolding buffer .", "The protein was then eluted using 250 mM imidazole , 20 mM tris pH 8 , 500 mM NaCl .", "Fractions containing the protein were buffer exchanged into a storage buffer ( 25 mM tris , 250 mM NaCl , pH 7 . 6 ) and stored at −80°C .", "A Bradford protein assay ( BioRad ) was done in order to quantify the amount of protein present .", "An antibody produced against the Mif1-specific peptide sequence CERSKGEFTEGKPKP ( Genscript ) was used to confirm expression and purification .", "Protein samples and lysates were first normalized using Bradford protein assay to quantify protein concentrations .", "Equal concentrations of protein were loaded onto BioRad stain-Free SDS-PAGE gels 4–20% ( catalog no . 4568093 ) and imaged prior to transfer to confirm equal loading using BioRad gel doc ez system and stain free tray .", "The protein gel was then used to transfer protein to a PVDF membrane via semi-dry transfer system .", "The membranes were blocked in 5% milk-TBST ( 50 mM Tris-Cl , pH 7 . 6; 150 mM NaCl , 0 . 1% tween-20 ) for 30 min .", "The primary antibody was added at 1:1000 dilution ( unless otherwise stated ) to 5% milk-TBST and rocked overnight at 4°C .", "The membrane was washed three times for 10 min each in TBST .", "Secondary antibody was added at 1:20 , 000 to TBST and rocked for 1 hr at room temperature .", "The membrane was washed three more times for 15 min each before chemiluminescent substrate was added and visualized using BioRad XRS imaging cabinet .", "Antibodies include a custom Mif1 antibody ( Genscript ) , S-Tag antibody ( GenScript catalog no . A00625 , RRID:AB_915085 ) , DYKDDDDK antibody ( Thermo Fisher Scientific catalog no . 701629 , RRID:AB_2532497 ) , GFP antibody ( Thermo Fisher Scientific catalog no . G10362 , RRID:AB_2536526 ) and Goat anti-Rabbit IgG ( H+L ) secondary antibody , HRP ( Thermo Fisher Scientific Catalog no . 31460 , RRID:AB_228341 ) .", "The method for electroporation of Hydroides larvae was adapted from those established for ascidian embryos ( Zeller , 2018; Zeller et al . , 2006 ) .", "Specifically , 50 µl of 0 . 77 M mannitol , 20 µl of concentrated larvae ( approximately 30 larvae ) , and 10 µl of purified protein ( 1 . 25–12 . 5 µg , 15 . 6–156 ng/µl final concentration based on protein recovery from inclusion bodies ) were mixed and added to a 2 mm electroporation cuvette .", "The mixture was then electroporated with 30 V ( 150 V/cm ) at 10 ohms and 3000 µF using a custom electroporation apparatus as previously described ( Zeller et al . , 2006 ) .", "After electroporation , the mixture was immediately removed from the cuvette and mixed with 1 ml filtered artificial sea water and transferred into a 24-well plate .", "The larvae were then observed for metamorphosis 24 to 72 hr later ( dependent on WT MACs positive control ) .", "Purification of proteins was performed on three separate occasions and each purification was electroporated twice , for a total of six independent biological replicates , each yielding similar outcomes .", "To test whether purified protein is transferred to tubeworm larvae by electroporation , larvae were concentrated to 30 larvae/µl .", "Final GFP concentration used was 0 . 625 µg/µl ( 50 µg total per electroporation ) .", "20 µl of larvae were either electroporated by adding them to 50 µl 0 . 77 M mannitol and 10 µl of 5 µg/µl purified GFP , then electroporated at 30 V ( 150 V/cm ) at 10 ohms and 3000 µF or not electroporated .", "Larvae were recovered from the electroporation cuvette by adding 1 ml instant ocean and moved to microcentrifuge tubes .", "Larvae were then washed 5 times with 1 ml instant ocean by first spinning down at 4000 g for 30 s and removing all but 50 µl of liquid .", "After the five washes larvae were then spun down at 4000 for 2 min and the sea water was removed .", "On ice , the larvae were then lysed in 100 µl 50 mM tris pH 8 , 150 mM NaCl , 1% Triton X-100 , and vortexed three times for 30 s each .", "Cell debris was pelleted by centrifuging at 21 , 000 g for 10 min and the pellet was discarded .", "The lysate was then quantified by first diluting a small aliquot of lysate ( 5 µl ) 1:10 and using a Bradford protein assay .", "The GFP standard curve was created by performing 2-fold serial dilutions of the original purified GFP and using densitometry of the western blot .", "8 . 76 µg of larvae lysate was loaded onto the same gel , with both the mock zap negative control and the electroporated larvae .", "Recovered protein was calculated using densitometry and the GFP standard curve .", "This was repeated for four biological replicates each with separate western blots .", "E . coli containing a dual expression plasmid with 12605 , Mif1 , or both 12605 and Mif1 was grown in 50 ml of LB supplemented with chloramphenicol ( 100 µg/ml ) until an OD600 0 . 6 at 37°C .", "The E . coli plasmid was induced with 1 mM IPTG and the temperature was lowered to 20°C , then expression was allowed to proceed overnight ( 16 hr ) .", "Cells were recovered by pelleting at 4000 g for 10 min and media was discarded .", "Cells were resuspended in 25 mM tris pH 7 . 6 , 150 mM NaCl and protease inhibitor cocktail ( 100 µM leupeptin , 1 µM pepstatin and 5 µM bestatin ) .", "Cells were lysed twice by French press 1000 Psi and then centrifuged at 10 , 000 g to remove cellular debris where the supernatant was then transferred to a new tube and the pellet was discarded .", "The conjugated agarose beads ( Ni-NTA agarose and S-Tag binding agarose , EMD Millipore 69704–3 ) were equilibrated with the lysis buffer and 1 ml of agarose was added in batch to the recovered supernatant .", "Agarose was then recovered on a column and washed an additional two times with the lysis buffer to remove non-specific bound proteins .", "Proteins were eluted using either , 500 µl 3 M MgCl2 , 20 mM tris pH 7 . 6 ( S-TAG beads ) , or 500 µl 250 mM imidazole , 500 mM NaCl , 20 mM Tris pH 7 . 6 ( Ni-NTA beads ) .", "20 µg protein was loaded onto SDS-PAGE gel and then transferred onto PVDF membrane for western blot using either the endogenous Mif1 antibody or S-Tag antibody ( GenScript Cat# A00625 , RRID:AB_915085 ) .", "P . luteoviolacea was grown in 50 ml Marine Broth ( MB ) media in 250 ml flasks at 30°C for 6 hr or overnight ( 12–14 hr ) .", "Cells were centrifuged for 30 min at 7000 g and 4°C and resuspended in 5 ml cold extraction buffer ( 20 mM Tris , pH 7 . 5 , 1 M NaCl ) .", "The resuspensions were centrifuged for 30 min at 4000 g and 4°C and the supernatant was isolated and centrifuged for 30 min at 7000 g and 4°C .", "The pellet was resuspended in 20–100 µl cold extraction buffer and stored at 4°C for further use .", "All mass spectrometry was done by the Functional Genomics Center Zurich ( FGCZ ) .", "To prep the MAC extracts for mass spectrometry , the extracts were precipitated by mixing 30 µl of sample with 70 µl water and 100 µl 20% TCA .", "The samples were then washed twice with cold acetone .", "The dry pellets were dissolved in 45 µl buffer ( 10 mM Tris/2 mM CaCl2 , pH 8 . 2 ) and 5 µl trypsin ( 100 ng/µl in 10 mM HCl ) .", "They were then microwaved for 30 min at 60°C .", "The samples were dried , then dissolved in 20 µl 0 . 1% formic acid and transferred to autosampler vials for LC/MS/MS .", "1 µl was injected ." ] ]

[ "The swimming larvae of many marine animals identify a location on the sea floor to undergo metamorphosis based on the presence of specific bacteria .", "Although this microbe–animal interaction is critical for the life cycles of diverse marine animals , what types of biochemical cues from bacteria that induce metamorphosis has been a mystery .", "Metamorphosis of larvae of the tubeworm Hydroides elegans is induced by arrays of phage tail-like contractile injection systems , which are released by the bacterium Pseudoalteromonas luteoviolacea .", "Here we identify the novel effector protein Mif1 .", "By cryo-electron tomography imaging and functional assays , we observe Mif1 as cargo inside the tube lumen of the contractile injection system and show that the mif1 gene is required for inducing metamorphosis .", "Purified Mif1 is sufficient for triggering metamorphosis when electroporated into tubeworm larvae .", "Our results indicate that the delivery of protein effectors by contractile injection systems may orchestrate microbe–animal interactions in diverse contexts ." ]

[ "Many marine animals , including corals and tubeworms , begin life as larvae swimming in open water before transforming into adults that anchor themselves to the seabed .", "These transformations , known as metamorphoses , are often triggered by certain types of bacteria that form friendly relationships ( or “symbioses” ) with the animals .", "One such symbiosis forms between a bacterium called Pseudoalteromonas luteoviolacea and a tubeworm known as Hydroides elegans .", "Previous studies have shown that P . luteoviolacea produces syringe-like structures known as Metamorphosis Associated Contractile structures ( or MACs for short ) that are responsible for stimulating metamorphosis in the tubeworm larvae .", "Some viruses that infect bacteria use similar structures to inject molecules into their host cells .", "However , it was not clear whether MACs were also able to inject molecules into cells .", "Here , Ericson , Eisenstein et al . used a technique called cryo-electron tomography combined with genetic and biochemical approaches to study how the MACs of P . luteoviolacea trigger metamorphosis in tubeworms .", "The experiments identified a protein in the bacteria named Mif1 that was required for the tubeworms to transform .", "The bacteria loaded Mif1 into the tube of the MAC structure and then injected it into the tubeworms .", "Further experiments showed that inserting Mif1 alone into tubeworms was sufficient to activate metamorphosis .", "Mif1 is the first protein from bacteria to be shown to activate metamorphosis , but it is likely that many more remain to be discovered .", "Since other marine animals also form symbioses with bacteria , understanding how Mif1 and other similar proteins work may inform efforts to restore coral reefs and other fragile ecosystems , and increase the production of oysters and other shellfish .", "Furthermore , MACs and related structures may have the potential to be developed into biotechnology tools that deliver drugs and other molecules directly into animal cells ." ]

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

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

[ [ "Anatomical atlases have played a crucial role in research into organogenesis , anatomy , physiology , and pathology ( Marquart et al . , 2017; Chon et al . , 2019; Liu et al . , 2020 ) and have been used for a wide range of applications , including investigating developmental biology ( Kunst et al . , 2019 ) , defining stereotactic coordinates ( Franklin and Paxinos , 2013 ) , identifying patterns of neural activity associated with behavior ( Renier et al . , 2016; Kim et al . , 2017 ) , and integrating morphological , transcriptomic , and neural activity data across species ( Marquart et al . , 2017 ) .", "Ongoing initiatives continue to develop these resources , including the Human Cell Atlas ( Regev et al . , 2019 ) , the BRAIN Initiative ( Ecker et al . , 2017; Koroshetz et al . , 2018 ) , the Human Brain Project ( Amunts et al . , 2016; Bjerke et al . , 2018 ) , and centralized repositories of data , including the Allen Brain Atlas ( Lein et al . , 2007; Jones et al . , 2009; Ng et al . , 2007 ) .", "Macroscale imaging , such as magnetic resonance imaging ( MRI ) , routinely generates data from intact whole organs ( Huang and Luo , 2015; Thompson et al . , 2014a; Jahanshad et al . , 2013; Sudlow et al . , 2015; Miller et al . , 2016; Alfaro-Almagro et al . , 2018; Walhovd et al . , 2018 ) in contrast to microscale imaging , such as light microscopy , which has typically relied on physically cutting thin sections leading to misalignment artifacts .", "Technological advances in microscopy have facilitated a dramatic increase in imaging of high-resolution , intact whole organs using serial two-photon tomography ( STPT ) ( Ragan et al . , 2012 ) or tissue clearing techniques ( e . g . CLARITY [Chung et al . , 2013] , 3DISCO [Ertürk et al . , 2012] , and CUBIC [Susaki et al . , 2014] ) and lightsheet microscopy ( Chhetri et al . , 2015; Meijering et al . , 2016; Mano et al . , 2018; Hillman et al . , 2019; Cai et al . , 2019; Ueda et al . , 2020 ) .", "These 3D microscopy methods have been applied to map cytoarchitecture and cellular connectivity in complex tissues , including the brain ( Cai et al . , 2019; Kim et al . , 2015; Murakami et al . , 2018 ) , heart ( Gilbert et al . , 2019; Bai et al . , 2015 ) , and lung ( Li et al . , 2012; Ardini-Poleske et al . , 2017 ) .", "Accurate analysis of imaging data from whole organs requires atlases of the corresponding organ , species , developmental age , resolution , and dimensions ( Allen Institute for Brain Science , 2017; Susaki and Ueda , 2016; Niedworok et al . , 2016; Watson and Watkins , 2019 ) .", "The progression to 3D microscopy data necessitates 3D anatomical atlases to reference data to existing anatomically-defined labels ( Puelles and Rubenstein , 2003; Watson et al . , 2012 ) .", "Although numerous atlases based on 2D physical sections exist ( Franklin and Paxinos , 2013; Ardini-Poleske et al . , 2017; Savolainen et al . , 2009; de Boer et al . , 2012; Thompson et al . , 2014b ) , most suffer from label misalignments or insufficient resolution in the third dimension , limiting their utility for 3D imaging ( Wang et al . , 2020 ) .", "The most recent version of the Allen Common Coordinate Framework atlas ( CCFv3 ) of the adult ( P56 ) C57BL/6J mouse brain represents one of the most complete 3D atlases at microscopic resolution to date ( Wang et al . , 2020 ) .", "A team of neuroanatomists and illustrators redrew structures natively in 3D , sometimes using the original annotations as seeds to guide the redrawing process , producing 658 structural annotations ( Wang et al . , 2020 ) .", "Even with this monumental effort , 23% of labels from the original Allen Reference Atlas were not included in the 3D version , largely due to the time and labor constraints of further manual curation ( Wang et al . , 2020 ) .", "To date , this titanic effort to generate a 3D atlas at microscopic resolution has not been repeated for most combinations of organ , species , and developmental stage , despite the numerous existing 2D-based atlases .", "Automated methods to convert existing 2D-derived atlases into fully 3D atlases could leverage existing , established atlases without the time-consuming effort required for manual 3D curation , or provide the substrate for more rapid manual fine-tuning .", "This conversion needs to address multiple limitations in atlases based on 2D physical sections , as demonstrated by the challenges of using the existing 3D reconstruction of eight mouse brains , each at a different developmental time point , in the Allen Developing Mouse Brain Atlas ( ADMBA ) ( Thompson et al . , 2014b; Figure 1A ) .", "First , all atlases in the ADMBA are missing labels across large regions , typically the lateral planes of one hemisphere and the entire opposite hemisphere .", "Second , warping and asymmetrical placement of samples in the histological images prevents simply mirroring labels from the annotated to the non-annotated hemisphere .", "Third , as noted in the original Allen Reference Atlas ( Ng et al . , 2007; Niedworok et al . , 2016; Wang et al . , 2020 ) , the assembled 3D volume is smooth in the sagittal planes , in which the atlas annotations were drawn , but suffers from substantial edge artifacts in the coronal and axial planes ( Figure 1A ) .", "Several automated approaches have partially addressed some of these issues .", "The Waxholm Space rat brain atlas used two standard reference atlases as templates to yield an atlas with relatively smooth 3D structures through manual and semi-automated segmentation , although some label edge artifacts remain ( Papp et al . , 2014 ) .", "Erö et al . , 2018 performed non-rigid alignments between histological slice planes of the Common Coordinate Framework ( CCFv2 , 2011 ) , applying the same deformations to the label images .", "This registration improved alignment; however , the labels often appear to extend beyond the underlying histological planes .", "Rather than registering planes , Niedworok et al . , 2016 applied a smoothing filter on the annotations themselves to reduce label artifacts , though many label edge irregularities persisted .", "Here , we present a pipeline to automatically generate fully 3D atlases from existing 2D-derived partial atlases and apply it to the full ADMBA .", "We performed three steps ( Figure 1A ) : ( 1 ) Extension and mirroring of 2D data to approximate missing labels , ( 2 ) Smoothing with 3D watershed and morphological methods to align jagged edges between 2D sections , and ( 3 ) Edge detection from microscopy images to guide the 3D watershed to align boundaries with anatomical divisions , which we call ‘edge-aware’ refinement ( example atlas shown in Figure 1B ) .", "We demonstrate qualitative and quantitative improvements over the existing 3D ADMBA .", "To assess the resulting 3D atlas , we generated 3D imaging data from 15 C57BL/6J wild-type mouse brains at postnatal day 0 ( P0 ) using tissue clearing and lightsheet microscopy .", "The 3D labels in our refined atlas match the brain structures better than the initial atlas , as demonstrated by closer alignment between labels and anatomical edges and decreased variability within labels at the cellular level .", "We provide the pipeline as open source software called MagellanMapper to apply the algorithms to equivalent atlases and provide the resulting ADMBA 3D atlases as a resource for immediate application to automated whole-brain 3D microscopy analysis ." ], [ "The ADMBA contains data for eight mouse brains , each at a different development stage: embryonic day ( E ) 11 . 5 , E13 . 5 , E15 . 5 , E18 . 5 , postnatal day ( P ) 4 , P14 , P28 , and P56 .", "The data are generated from 158 to 456 serial 2D sagittal sections of 20–25 µm stained with Nissl or Feulgen-HP yellow and imaged with a light microscope at a lateral resolution of 0 . 99 to 1 . 049 µm .", "For the three earliest stages of development , E11 . 5 , E13 . 5 , E15 . 5 , the entire embryo was imaged , rather than just the brain .", "Each of the eight atlases are annotated with expertly curated labels of brain structures ( Thompson et al . , 2014b; Allen Institute for Brain Science , 2013b ) in a hierarchy starting with the largest structures ( e . g . neural plate ) extending through 13 sublevels to the smallest annotated structures ( e . g . parvicellular part of Lat ) .", "Viewed sagittally , these labels cover the majority of tissue in each brain with smooth , anatomically aligned edges ( Figure 2A ) ; however , only sections on the left side of each brain are annotated and , for six atlases , labels are not present for the most lateral 14–24% sagittal planes of the brain ( 4–5% by volume; Figure 2—figure supplement 1; Figure 2B ) .", "Labels extend slightly beyond the midline for several atlases , helping to annotate brains with a midline skew , yielding a median label to atlas volume ratio of 51% .", "To focus our analysis on the brain tissue , we excluded non-CNS tissue for the three embryos for which the brain was not dissected prior to imaging .", "Across all eight atlases the similarity between the microscopy images and the original label annotations was estimated by taking the threshold of each sagittal section on the left hemisphere of the brain as an approximation of ground truth and comparing the label coverage using the Dice Similarity Coefficient ( DSC ) ( Dice , 1945 ) , calculated by the Insight Segmentation and Registration Toolkit ( ITK ) ( Tustison and Gee , 2009 ) .", "Higher DSCs reflect greater similarity between the images and labels , with a maximum possible value of 1 .", "The observed DSCs for the original labels in the annotated hemisphere ranged from 0 . 85 to 0 . 97 ( median 0 . 91; Figure 2 ) .", "To generate labels across the entire brain , we extended the existing labels to the lateral edges by following histological boundaries , before mirroring the labels on the opposite hemisphere ( Figure 2 ) .", "The most lateral labeled sagittal section provided the initial seed from which to grow labels laterally .", "First , we resized this plane to match the extent of corresponding microscopy signal in the next , unlabeled lateral sagittal section ( Figure 2—figure supplement 2A , B ) .", "Next , we refined label boundaries by eroding each label and regrowing it along histological boundaries , which we call ‘edge-aware’ refinement .", "To model these boundaries , we generated gross anatomical maps in 3D using edge-detection methods on the volumetric histology images .", "Taking the Laplacian of Gaussian ( Marr and Hildreth , 1980 ) of each histological volumetric image highlighted broad regions of similar intensities , using a relatively large Gaussian sigma of 5 to capture only well-demarcated boundaries .", "A zero-crossing detector converted this regional map into a binary anatomical edge map ( Figure 2—figure supplement 2B ) .", "After eroding each label , we next used this anatomical map to guide the regrowth of labels by a compact watershed algorithm ( Neubert and Protzel , 2014 ) step , which adds a size constraint to the classic watershed algorithm .", "Distances of each pixel to its nearest edge map pixel formed anatomically based watershed catchment areas , while each eroded label served as a large seed from which filling of the nearest catchment areas began and grew until meeting neighboring labels .", "Thus , we extended a labeled sagittal plane to an unlabeled one , refined by the histology .", "This process was repeated iteratively across all remaining lateral sections ( Figure 2—figure supplement 2B , C ) .", "By using a 3D anatomical map for the watershed and seeding it with the prior plane’s labels , we ensured continuity between planes .", "To model the tapering of labels laterally , we preferentially eroded central labels by weighting each label’s filter size based on the label’s median distance from the tissue section’s outer boundaries .", "Central labels thus eroded away faster , while the next most central labels grew preferentially to fill these vacated spaces .", "In the E18 . 5 atlas , for example , this approach allowed the basal ganglia to taper off and give way to the dorsal and medial pallium ( Figure 2B ) .", "Although edges between planes remained somewhat jagged , this extension step creates a substrate for further refinement .", "After completing label coverage for the left hemisphere , both the labels and underlying microscopy images were reflected across the sagittal midline to cover the remaining hemisphere .", "Care was taken to ensure that the sagittal midline was identified correctly by inspecting and rotating the 3D image and midline plane from multiple angles ( Figure 2C ) .", "Recalculating the DSC between the microscopy images and labels for the left hemisphere showed greater similarity across all eight atlases with a median DSC improvement of 0 . 02 ( p=0 . 02 , WSRT ) and a resulting DSC range of 0 . 87 to 0 . 97 ( median 0 . 93 , Figure 2D ) .", "Equivalent analysis of DSC for the whole brain would show substantial improvement due to the absence of labels on the right side in the original .", "The ADMBA atlases have been provided as 3D volumetric images , combined computationally from the original 2D sagittal reference plates; 2D sections can be generated from these 3D images in orthogonal dimensions to the original sagittal view .", "Visual inspection of labels in the axial and coronal planes reveals high-frequency artifacts along most edge borders , likely from the difficulty of drawing contiguous borders in dimensions orthogonal to the drawing plane ( Figure 1 , Figure 3 ) .", "To quantify the degree of label smoothness , we used the unit-less compactness metric ( Ballard and Brown , 1982 ) .", "The compactness measure applied in 3D incorporates both surface area and volume , allowing for quantification of smoothness to measure shape irregularity .", "Of note , compactness is independent of scale or orientation , facilitating comparison across all labels , despite size differences , and its sensitivity to noise allows finer detection of label irregularity ( Bribiesca , 2008 ) .", "Measuring the compactness of each label and taking the weighted mean based on label volume for each atlas gave a median compactness of 13 , 343 ( mean: 34 , 099 , standard deviation ( SD ) : 44 , 988 ) .", "For context , across all eight atlases the thresholded 3D whole-brain microscopy images were more compact ( median compactness: 1895 , mean: 2615 , SD: 2305; p=0 . 02 , WSRT , Bonferroni corrected for three comparisons; Figure 3—figure supplement 1A ) , consistent with the observed irregularity in the label images compared to anatomical images ( Figure 2B and Figure 3 ) .", "To reduce this irregularity , we applied a smoothing filter iteratively to the 3D image of each label .", "Prior approaches to this problem used a Gaussian filter ( kernel SD of 0 . 5 , applied in two passes ) ( Niedworok et al . , 2016 ) .", "While this visually improved the smoothness of label edges , we observed sharp angles along the edges , presumably from the limitation of rounding blurred pixel values to the integers required for label identities rather than allowing the subtler floating-point gradients from the Gaussian kernel ( Figure 3—figure supplement 2A ) .", "In addition , the Gaussian filter expanded the volume of each label , leading to label loss as larger labels enveloped smaller ones ( Figure 3—figure supplement 2B ) .", "Optimal smoothing would maximize the smoothness of each label ( i . e . compactness; Bribiesca , 2008 ) whilst minimizing changes in shape and location .", "It would also not lead to label loss .", "To calculate the improvement in compactness , we defined ‘compaction’ as the difference in compactness between the original and smoothed label over the compactness of the original label with a range from 0 or 1 , with 1 being optimal .", "For changes in shape and location , we defined ‘displacement’ as the fraction of the smoothed label that was outside of the original label with a range from 0 or 1 , with 0 being optimal .", "We defined a ‘smoothing quality’ metric to reflect the balance of compaction and displacement , calculated as the difference between these two measures with a range from −1 or 1 , with 1 being optimal .", "To estimate atlas-wide smoothing quality , we took the weighted sum by volume of smoothing quality for all labels in the atlas .", "Assessing the quality of smoothing using Gaussian blur with increasing Gaussian sigmas , we observed label loss in all atlases in the ADMBA , even with a small sigma where labels remained visibly jagged .", "At this sigma value , the median atlas-wide smoothing quality across all eight atlases was 0 . 54 ( mean 0 . 53 ) , rising to a peak of 0 . 57 ( mean 0 . 56 ) at sigma 0 . 5 and 0 . 49 ( mean 0 . 53 ) at a sigma of 1 . 0 , but with substantial loss of labels ( median 14% lost at sigma 0 . 25 , rising to 42% lost at sigma 1; mean 11% and 47% , respectively Figure 3—figure supplement 2B ) .", "To refine smoothing while minimizing label loss , we changed the filter from a Gaussian to a morphological opening filter ( Serra , 1983 ) .", "This filter first erodes each label to remove artifacts , followed by dilation to restore its original volume .", "To avoid label loss caused by excessive erosion of small labels , we halved the size of its structuring element for labels with ≤5000 pixels .", "A few labels were split into numerous tiny fragments that would disappear with an opening filter .", "For these small labels , the opening filter was replaced by a closing filter , reversing the process by dilating before eroding to reconnect these components .", "With this adaptive opening filter approach , labels became more compact with smoother edges , while retaining their overall shape as seen in both 2D and 3D visualizations ( Figure 3A ) .", "Quantifying the improvement with the adaptive opening filter approach , using only filter sizes that completely eliminated label loss , we obtained a median atlas-wide smoothing quality of 0 . 61 ( mean 0 . 62 ) across all eight atlases , and improvement over the Gaussian filter approach ( sigma 0 . 25; p=0 . 008; Figure 3—figure supplement 2C ) .", "The optimal filter size varied between atlases , ranging from 2 to 7 ( E15 . 5 and P14 shown in Figure 3C , D; all ADMBA shown in Figure 3—figure supplement 3 ) .", "The median overall compactness improved significantly ( 13 , 343 ( SD = 44 , 988 ) for unsmoothed labels vs . 2527 ( SD = 2634 ) for smoothed labels , p=0 . 02 , WSRT , Bonferroni corrected for the three comparisons; mean 34 , 099 vs . 3172 ) to a level that did not differ from that observed for the microscopy images of whole brains ( p=1 . 00 , WSRT , Bonferroni corrected for the three comparisons; Figure 3—figure supplement 1B , C ) .", "Because morphological filters such as erosion classically operate globally , a drawback to these filters is the potential loss of thin structures , such as loss of the thin portion of the alar plate of the evaginated telencephalic vesicle ( Figure 3A ) .", "Smoothing in-place also does not address gross anatomical misalignment .", "We address these issues in subsequent steps .", "The extending , mirroring , and smoothing steps lead to a more complete set of labels for the ADMBA and correct the irregular borders in orthogonal planes to which the original labels were drawn; however , in several locations the labels do not align closely to the anatomical edges seen in the underlying histology images , for example the basal ganglia do not follow the curve of the lateral septal nuclei ( Figure 2C ) .", "To better map the anatomical fidelity of annotations in all dimensions , without manual relabeling , we leveraged our method for extending labels laterally based on gross anatomical edges to further refine all labels ( Figure 4 ) .", "Using the same gross anatomical map in 3D ( shown for an example plane in Figure 4A , second from left ) , we first quantified the distances from 3D label edges to the expected anatomical position .", "Assuming that the nearest gross anatomical edge was the correct one , we measured the distance from each label edge to the nearest gross anatomical edge .", "We can visualize this distance as a color gradient , in which higher intensity of color represents a greater distance to each anatomical edge ( Figure 4A , right columns ) .", "To modify the labels in light of the gross anatomical edge map , we again incorporated the edge-aware algorithm , this time in 3D .", "We made the assumption that the core of each label is annotated accurately , whereas label edges are more prone to inaccuracies from plane-to-plane misalignments or the difficulty of assessing histological edges .", "To preserve the core while reannotating the periphery , we first eroded each label to remove edges .", "These eroded labels became the seed for the watershed step , which re-grew labels back toward their original size but now guided by the gross anatomical edge map .", "Normally , the erosion step would lead to loss of thin structures within labels because erosion operates globally on the entire label .", "To preserve these thin structures , we skeletonized each label in 3D , which thins the label to its core structure ( Lee et al . , 1994 ) , and added the skeleton back to the eroded label .", "We used a much more lightly eroded version of the label for the skeletonization to avoid retaining label edges in the skeleton .", "By combining erosion with skeletonization when generating seeds for the watershed , we retained thin structures such as the alar plate of the evaginated telencephalic vesicle located anterior to the basal ganglia .", "After performing this edge-aware step , we ran the adaptive morphological opening filter smoothing step ( Figure 4B ) .", "Because the edge-aware step partially smooths structures , we could use smaller filter sizes for smoothing to avoid loss of thin structures .", "The resulting labels show considerable improvement , for example the basal ganglia now curve around the lateral septal nuclei ( Figure 4C ) .", "By adapting the morphological filter sizes during both the edge-aware and final smoothing steps , we avoid label loss and minimize volume changes relative to label size as seen in the smaller labels ( Figure 4—figure supplement 2 ) .", "Visualization of the color gradient of distances to anatomical edges also confirms substantial improvement in label alignment compared with the original labels or smoothing or edge-aware steps alone ( Figure 4—figure supplement 1B ) .", "To quantify this improvement brain-wide , we calculated the sum of edge distances for each pixel at label surfaces across the ADMBA .", "We observed a significant reduction from a median of 187 million to 86 million µm ( p=0 . 008 , WSRT; Figure 4B , C , F ) , with a median Dice Similarity Coefficient between original ( mirrored ) and smoothed ( edge-aware ) labels of 0 . 76 ( mean 0 . 80 ) and 9% median ( mean 12% ) volume reassignment .", "Example planes from all atlases in the ADMBA before and after refinement are depicted in Figure 5 , and movies across all planes are shown in Figure 5—videos 1–16 .", "Anatomical edges in microscopy images reflect differences in intensity between regions .", "Therefore , we would expect accurate labels to have smaller variation in intensity in the underlying microscopy images than inaccurate labels , although such a difference would need to be apparent even though the majority of the label is unchanged .", "We used the coefficient of variation of intensity values within each label to quantify this expectation and demonstrated significantly lower variation with edge-aware labeling ( median from all labels weighted by size decreased from 0 . 290 to 0 . 282 , p=0 . 008 , for all eight atlases , WSRT; Figure 4D , E ) .", "Furthermore , edge-aware labeling decreased the absolute coefficient of variation for 92 of the 100 individual labels represented by all atlases .", "The few labels that showed increased variation were frequently in regions of relatively subtle intensity changes , such as the hindbrain , where histological edges were less well-defined ( Figure 4D ) .", "As an internal control , we also registered the atlases to their original , asymmetric microscopy images to compare the impact of atlas reconstruction in the originally labeled hemispheres and the contralateral , asymmetric hemispheres on which the atlases were not derived and found similar improvement in anatomical fidelity ( Figure 4—figure supplement 3 ) .", "We also compared the computationally generated labels and partially labeled lateral regions not included in the 3D reconstruction with manually annotated regions in the P28 atlas , which showed an increase in the Dice Similarity Coefficient in the reconstructed labels ( Figure 4—figure supplement 4 ) .", "To explore how the edge-aware approach handles delicate sub-regions , which may not have clearly demarcated anatomical boundaries , we focused in greater depth on two complicated sub-regions in the P28 atlas ( Figure 4—figure supplement 5 ) and two regions described in the original ADMBA paper ( Thompson et al . , 2014b ) as demarcated by in-situ hybridization markers in the E13 . 5 and E15 . 5 atlases ( Figure 4—figure supplement 6 ) , showing that if anatomical data does not support moving the boundary , our approach smooths the boundary in the axial and coronal planes without moving the location in any of the planes .", "A major goal of 3D atlas refinement is for automated label propagation to optically sectioned , volumetric microscopy images generated from intact tissue using recently developed tissue clearing techniques .", "As the accuracy of atlas registration is ultimately dependent on the fidelity of the underlying atlas in all dimensions , we sought to test and quantify improvement from our atlas refinements in cleared mouse whole brains .", "Among the many available methods to clear whole organs , we chose CUBIC ( Susaki et al . , 2014; Susaki et al . , 2015 ) given its balance of overall transparency , morphological retention with minimal tissue expansion , and ease of handling as a passive aqueous technique ( Kolesová et al . , 2016; Xu et al . , 2019a; Bossolani et al . , 2019 ) .", "After clearing C57BL/6J WT mouse pup brains ( age P0 ) with simultaneous incubation in SYTO 16 nuclear dye for 2 weeks , we imaged intact whole brains by lightsheet microscopy at 5x to obtain volumetric images at cellular resolution ( Figure 6—figure supplement 1 ) , taking approximately 3 hr to image and generating approximately 500 GB of data per brain ( n = 15; 10 male , 5 female ) .", "To detect nuclei throughout cleared whole brains automatically , we implemented a 3D blob detection algorithm using the Laplacian of Gaussian filter , which has been shown to work well in equivalent image data ( Schmid et al . , 2010; Figure 6 ) .", "To make the nuclei approximately spherical for blob detection , we interpolated the images axially to match the lateral resolution .", "Due to the large quantity of data , processing was performed in parallel on small chunks .", "Preprocessing and detection settings were optimized using hyperparameter tuning against a ‘truth set’ of 1118 nuclei selected from multiple brain regions that had been verified by manual visualization ( Figure 6—figure supplement 2A , B ) .", "The resulting model achieved a recall ( sensitivity ) of 90% and precision ( positive predictive value ) of 92% .", "Furthermore , the model showed high correlation with total lightsheet microscopy intensity levels brain-wide ( r > 0 . 99 , p≤1 × 10−16 , Spearman’s rank correlation coefficient ( SRCC ) for both original ( mirrored ) and smoothed ( edge-aware ) atlases ) and nuclei vs . intensity densities ( original: r = 0 . 89; smoothed: r = 0 . 93; p≤1 × 10−16 for both , SRCC ) , suggesting the performance was accurate outside the narrow target of the truth set ( Figure 6—figure supplement 2C–E ) .", "Using the E18 . 5 brain as the closest atlas to our P0 C57BL/6J wild-type mouse brains ( birth typically at E19 Murray et al . , 2010 ) , we then registered the atlas volumetric histology images to each sample brain .", "We chose to register brains using the Elastix toolkit ( Klein et al . , 2010; Shamonin et al . , 2014 ) , which has been validated on CUBIC cleared brains and balances computational efficiency and accuracy ( Nazib et al . , 2018 ) .", "After downsampling the volumetric images to the same dimensions as the atlas for efficiency , we applied rigid , followed by non-rigid , deformable registration based on a multi-resolution scheme , optimizing the cross-correlation similarity metric as implemented by the SimpleElastix ( Nazib et al . , 2018; Marstal et al . , 2016 ) programmable interface to the Elastix toolkit ( Klein et al . , 2010; Hammelrath et al . , 2016 ) .", "After registration , the median DSC between the registered atlas volumetric histology images and the lightsheet microscopy images of each sample brain was 0 . 91 ( mean: 0 . 91; 95% confidence interval: 0 . 90–0 . 92; Figure 6—figure supplement 3 ) , although inspection of registered brains also revealed slight misalignments with the atlas , mostly in caudal regions including the cerebellum , a structure known to be challenging for registration ( Nazib et al . , 2018 ) .", "Variations in sample preparation , including the completeness of the dissected hindbrain and expansion of the third ventricle during tissue clearing may also have contributed to these misalignments .", "To evaluate whether our updated , smoothed 3D labels improved the analysis of true volumetric data , we registered both the original and refined labels to these 15 tissue-cleared , nuclei-labeled whole wild-type mouse brains ( 10 male , five female ) .", "We would expect improved 3D labels to correspond more closely to the underlying anatomy .", "To test this expectation , we generated gross anatomical edge maps for each cleared brain and measured distances from label borders to anatomical edges .", "Edge distances significantly improved for almost all labels ( overall decrease from a median of 153 million µm to 96 million µm , p=0 . 007 , WSRT , Bonferroni corrected for all 120 labels across all hierarchical levels , mean 152 million to 99 million µm; Figure 7A , C–D; Figure 7—figure supplement 1A ) .", "We would also expect improved 3D labels to have lower variation in image intensity within each label , as we observed in assessing the refined labels with the original brain images from which they were derived ( Figure 4E ) .", "We observed a small , but significant decrease in the intensity coefficient of variation at the whole-brain level ( 0 . 309 to 0 . 301 , p=0 . 007 , WSRT , Bonferroni corrected for the 120 comparisons , mean 0 . 311 to 0 . 304 ) and most sub-labels ( Figure 7—figure supplement 1B ) .", "For 22 labels ( 18% of all labels ) the variability worsened , however this was only significant ( p=0 . 04 ) for a single label .", "The majority of these 22 labels describe small structures ( combined 7% of total volume ) and therefore sensitive to slight perturbations in border location , and located ventrally in the brain , where signal was prone to being distorted by glue artifacts from the mount for lightsheet imaging .", "For context , variability improved for 98 labels ( 82% of all labels ) with 27 labels showing significant improvement .", "These two analyses support an overall improved performance of the refined 3D labels with the volumetric images , however we can also test nuclei density - a key use case for whole-brain imaging methods ( Murakami et al . , 2018 ) .", "After detecting nuclei throughout each brain ( Figure 6 ) , we assigned each nucleus to an atlas label based on the 3D location of the nucleus .", "Using the numbers of nuclei per label and the volume per registered label , we calculated nuclei densities using the original and refined labels ( Figure 7—figure supplement 2 ) .", "As with the volumetric image data , we would expect accurate labels to encapsulate regions with more constant nuclei density .", "We therefore assessed the coefficient of variation for nuclei density and observed a small , but significant improvement with the refined labels overall ( median 0 . 629–0 . 625 , p=0 . 007 , WSRT , Bonferroni corrected for the 120 hierarchical labels , mean 0 . 629–0 . 625 ) and across the majority of labels in the hierarchy ( Figure 7—figure supplement 1C ) .", "A median of 13 . 5% ( 4 . 7 million ) nuclei ( mean 13 . 2% [4 . 6 million] nuclei ) were reassigned from original to refined labels .", "Examples of these nuclei reassignments in a wild-type mouse forebrain are depicted in Figure 7B .", "As an independent assessment of label alignment based on nuclei density alone , we measured nuclei clustering within each label under the expectation that well-aligned labels would group nuclei of similar densities , whereas poorly aligned labels would form isolated pockets of nuclei along borders between regions of contrasting nuclei densities .", "We employed Density-Based Spatial Clustering of Applications with Noise ( DBSCAN ) ( Ester et al . , 1996 ) , an algorithm useful for both clustering and noise or anomaly detection ( Ali et al . , 2010 ) , and indeed found that label refinement reduced isolated , unclustered nuclei by 30% ( median 4 . 4 × 105 to 3 . 1 × 105 nuclei; p=0 . 007 , WSRT , Bonferroni corrected for the 120 labels; mean 4 . 4 × 105 to 3 . 1 × 105; Figure 7E–F; Figure 7—figure supplement 3 ) , suggesting greater nuclei assignment to labels with nuclei clusters of similar density .", "To facilitate both automated atlas refinement and visual inspection of large 3D microscopy images , we developed the open-source MagellanMapper software suite .", "This Python-based ( Oliphant , 2007; Millman and Aivazis , 2011 ) image processing software consists of both a graphical user interface ( GUI ) for visualization and a command-line interface for automation ( Figure 7—figure supplement 4 ) .", "A major goal of the suite’s GUI is to enable unfiltered , raw visualization and annotation of original 2D images in a 3D context .", "The GUI consists of three main components: ( 1 ) A region of interest ( ROI ) selector and 3D visualizer with 3D rendering , ( 2 ) A serial 2D ROI visualizer and annotator , tailored for building truth sets of 3D nuclei positions , and ( 3 ) A simultaneous orthogonal plane viewer with atlas label editing tools , including painting designed for use with a pen and edge interpolation to smoothly fill in changes between two edited , non-adjacent planes .", "The command-line interface provides automated pipelines for processing of large ( terabyte-scale ) volumetric images , including 3D nuclei detection at full image resolution .", "The suite makes extensive use of established computer vision libraries such as scikit-image ( van der Walt et al . , 2014 ) , ITK ( Yoo et al . , 2002 ) ( via SimpleITK Lowekamp et al . , 2013 and SimpleElastix Klein et al . , 2010; Marstal et al . , 2016 ) , visualization toolkits such as Matplotlib ( Hunter , 2007 ) and Mayavi ( Ramachandran and Varoquaux , 2011 ) , and statistical and machine learning libraries such as Numpy ( van der Walt et al . , 2011 ) , SciPy ( Virtanen et al . , 2020 ) , Pandas ( McKinney , 2010 ) , and scikit-learn ( Pedregosa et al . , 2018 ) .", "The cross-platform capabilities of Python allow the suite to be available in Windows , MacOS , and Linux .", "We also leverage additional open-source imaging ecosystems such as the ImageJ/Fiji ( Schindelin et al . , 2012; Rueden et al . , 2017 ) suite for image stitching through the BigStitcher plugin ( Hörl et al . , 2019 ) , integrated into our automated pipelines through Bash scripts ." ], [ "Mouse whole-brain imaging has been used to understand models of human traits , including sexual dimorphism ( Kim et al . , 2017 ) and models of human disorders , including Alzheimer’s disease ( Liebmann et al . , 2016; Delafontaine-Martel et al . , 2018; Whitesell et al . , 2019 ) , serotonin dysregulation ( Ellegood et al . , 2018 ) , epilepsy ( Intson et al . , 2019 ) , and autism spectrum disorder ( ASD ) ( Suetterlin et al . , 2018 ) .", "Such analyses would be augmented by accurate 3D reference atlases , allowing the detection of subtle quantitative changes not readily appreciable in individual slices .", "As we have described and demonstrated here , anatomically guided completion and refinement of reference atlases more fully leverages the many existing and established atlases by expanding them to full coverage and greater accuracy at cellular resolution .", "The completed and refined 3D ADMBA serves as a resource to help identify biological differences in the multiple models of human disorders and traits across brain development ." ], [ "The ADMBA series provides atlases for multiple developmental time points from embryonic through adult stages ( Thompson et al . , 2014b ) .", "Each atlas consists of two main images given in a volumetric format ( . mhd and its associated . raw file ) , a microscopy image ( ‘atlasVolume’ ) and a labels image ( ‘annotation’ ) , which are each composed of multiple 2D sagittal planes .", "As outlined in the ADMBA technical white paper ( Allen Institute for Brain Science , 2013a ) and online application programming interface ( API ) documentation ( Allen Institute for Brain Science , 2018 ) , each microscopy plane is from imaging of a C57BL/6J mouse brain specimen cryosectioned into 20 µm ( E11 . 5-P4 ) or 25 µm ( P14-P56 ) thick sagittal sections stained with Feulgen-HP yellow nuclear stain ( E11 . 5-E18 . 5 ) or Nissl staining ( P4-P56 ) , and imaging planes were assembled into a 3D volume .", "To create the atlas labeling , an expert anatomist used Adobe Illustrator CS to annotate 2D planes , which were interpolated to create annotations in 3D ( Thompson et al . , 2014b; Allen Institute for Brain Science , 2013b; Allen Institute for Brain Science , 2018 ) .", "The annotation planes correspond to each microscopy plane , with integer values at each pixel denoting the label at the finest structural level annotated for the corresponding microscopy voxel .", "While the ADMBA covers a large proportion of unique areas within each brain , the atlas labels leave the lateral edges of one hemisphere and the entire other hemisphere unlabeled .", "To fill in missing areas without requiring further manual annotation , we made use of the existing labels to sequentially extend them into the lateral unlabeled histology sections .", "This process involves ( 1 ) resizing the last labeled plane to the next , unlabeled plane , ( 2 ) refitting the labels to the underlying histology , and ( 3 ) recursively labeling the next plane until all unlabeled planes are complete ( Figure 2—figure supplement 2 ) .", "To extend lateral edges of the labeled hemisphere , we first identified the lateral-most labeled plane of the atlas microscopy images .", "We started from the first sagittal plane on the labeled side of the image and moved toward and into the brain , checking each plane for the presence of any label .", "Once we identified a contiguous stretch of planes with labels , we used the most lateral labeled plane as the template for subsequent labels to be extended out laterally , in the opposite direction .", "In a few atlases ( e . g . P28 ) , the lateral-most labeled planes are only partially complete , in which case the most lateral completely labeled plane was manually specified instead .", "This last labeled lateral plane contained one or more discrete structures to extend label coverage , typically the cortex and sometimes the cerebellum .", "To find each structure and its associated labels , we first generated a mask of the histology by taking a slightly dilated version of the labels ( morphology . binary_dilation method in scikit-image , typically with a disk shaped structuring element of size 5 ) to capture nearby unlabeled histological structures , thresholded this histology plane by a value of 10 , removed small objects of size less than 200 connected pixels ( default connectivity of 1 in morphology . remove_small_objects ) , and identified bounding boxes of connected histology components ( measure . regionprops ) and their matching labels .", "These bounding boxes contain the discrete structures that we will follow laterally , using the corresponding labels for each structure as templates to extend into the rest of each structure .", "After identifying each structure and its labels , we fit the labels to the next lateral plane and recursively generated a new template for the subsequent plane .", "To fit the labels to the next plane , we found the histology bounding box for each structure and resized its labels to this box .", "We assumed for simplification that each structure is the same or smaller size in each subsequent plane , such as the tapering profile of the cortex laterally , and took only the single largest object found within the structure’s bounding box .", "Spline interpolation , anti-aliasing , and range rescaling were turned off during the resize operation to avoid introducing new label values .", "Some atlas labels contain empty space such as ventricles .", "To ensure that the ventricles close as they progress laterally , we employed an in-painting approach .", "Using the Euclidean distance transform method from the Scipy ( Virtanen et al . , 2020 ) library ( ndimage . distance_transform_edt ) , we identified the indices of the nearest neighboring pixel for any unlabeled pixel whose corresponding histology intensity value was above threshold and filled this missing pixel label with the value of this neighbor .", "While the labels from one plane could serve as an approximate template for the next plane , we curated this template to fit the underlying anatomy .", "To map this anatomy , we generated gross anatomical edge maps of the histology images through 3D edge detection .", "First , we smoothed the volumetric microscopy image using a Gaussian filter with a sigma of 5 followed by an edge detection with a Laplacian filter using a default operator size of 3 , using both filters implemented in scikit-image ( filters . gaussian and filters . laplace , respectively ) .", "This relatively large Gaussian sigma allowed for capture of broad anatomical edges such as the cortex and basal ganglia while minimizing detection of artifactual boundaries .", "To enhance edge detection of the outermost boundaries , we identified and removed background by thresholding the original microscopy image with an Otsu threshold ( filters . threshold_otsu ) and combining it with a mask of all the original labels to fill in any missing holes in the thresholded image .", "Finally , we reduced the edge-detected image to a binary image of edges alone by applying a zero-crossing detector .", "This detector separately erodes and dilates ( morphology . erosion and morphology . dilation , respectively , with a ball-shaped structuring element of size", "1 ) the edge-detected image to find borders , taking all pixels where this image changed signs as gross anatomical edges ( Figure 4A ) .", "This edge map allowed us to conform labels to local anatomy .", "To refit labels , we eroded and regrew them in a watershed transformation guided by the anatomical edge map .", "First we eroded labels individually ( morphology . binary_erosion in scikit-image with a ball-shaped structuring element of manually determined radii for each atlas ) .", "These eroded labels served as seeds for a compact watershed ( morphology . watershed implemented in scikit-image with a compactness parameter of 0 . 005 ) ( Neubert and Protzel , 2014; Kornilov and Safonov , 2018 ) , guided by the anatomical edge map .", "We used the Euclidean distance transform of the anatomical edge map to define the catchment basins for the watershed transformation , where voxels farther from anatomical edges are at the bottoms of basins and fill faster , guiding the regrowth of eroded labels .", "Labels typically crossed several anatomical edges but tended to meet neighboring labels at common edges ( Figure 4B ) .", "To limit the watershed to atlas foreground , which prevents label spillover across empty spaces , we set the watershed mask parameter to the original total labels foreground smoothed by an opening filter ( morphology . binary_opening with a ball structuring element of radius 0 [off] to two determined for each atlas to avoid label loss and minimize volume changes ) to remove artifacts around small ventricular spaces that might otherwise be crossed .", "The eroded labels thus regrew to fit anatomical guides and became the new , anatomically refined template to extend labels into the next plane .", "To model the tapering and disappearance of labels laterally , we allowed labels to erode completely .", "We weighted erosion toward central labels within each structure by multiplying the erosion filter size for each label by the label’s median distance to the structure perimeter divided by the maximum distance .", "Instead of simply eroding away the smallest labels , this approach preferentially eroded labels farthest from the perimeter , typically central labels .", "In atlases such as the ADMBA E18 . 5 atlas , a few spurious labeled pixels from other regions regrew to create artifacts .", "To filter these spurious pixels , we applied smoothing to the initial labels template using the smoothing approach described below .", "Also , we applied skeletonization as outlined below to avoid loss of thin structures during erosion .", "Each plane of labels thus conformed to its underlying anatomy and became the template for the next plane , keeping labels inherently connected from one plane to the next .", "While this approach is in serial 2D rather than fully 3D because the starting labeled plane is 2D , a subsequent step will further refine labels in 3D .", "To fill the missing hemisphere in the labels image , we initially simply mirrored the present labels and underlying histology planes across the first unlabeled sagittal plane on the opposite side .", "We noticed , however , that this mirroring frequently duplicated midline structures because the labels extend slightly past the true sagittal midline .", "When we shifted the mirroring to the sagittal plane closest to midline , we found that many labels were lost in the final image .", "Many of the developing atlases contain at least a few labels positioned solely across the midline in the otherwise unlabeled hemisphere .", "While it is possible that these labels represent structures unique to one hemisphere , at least some of these labels are on the other side of the midline in other atlases of the ADMBA and thus more likely represent artifact .", "In some cases ( e . g . P4 and P14 ) , mirroring just slightly past the sagittal midline preserved these labels , whereas other atlases ( e . g . P28 ) contained over 100 labels past the midline .", "One approach to preserve these labels would be to compress the near-midline labels to bring all labels into a single hemisphere at the expense of potentially misaligning otherwise appropriately positioned labels .", "To avoid this side effect , we elected to cut off a few labels to preserve the placement of the majority of labels .", "Upon closer inspection , many of the brains are slightly rotated in two or three dimensions , which contributed to loss of midline labels during mirroring .", "To rotate images volumetrically in 3D , we applied the scikit-image rotate function to all 2D planes along any given axis for each rotation .", "For each atlas , we applied this volumetric rotation along all necessary axes until the sagittal midline was parallel to an image edge , manually inspecting each brain in our 2D/3D orthogonal viewer to ensure symmetry .", "In some cases , such as the E18 . 5 atlas , the brain skewed laterally along its rostrocaudal axis .", "To avoid introducing a gap between midline labels and the corrected midline after rotation , we filled all planes on the unlabeled hemisphere side with the last labeled plane before rotation .", "Mirroring would then overwrite all of these repeated labels except those that filled in potential gaps left by rotation .", "After rotation , we noticed that mirroring reduced label loss in at least some atlases ( e . g . E13 . 5 ) .", "The distal spinal cord of the E11 . 5 atlas is strongly skewed and would be duplicated during mirroring .", "The skew is complicated by the cord’s coiling back on itself and progressive skew along its length .", "To bend the distal cord to the sagittal midline , we developed a method for piecewise 3D affine transformation .", "This transformation allows a cuboid ROI within a volumetric image to be sheared while maintaining a specified attachment point along another axis , reducing discontinuity with neighboring areas .", "First , we specify an axis along which to shear and the degree of shearing for a given ROI .", "Each full plane along this axis is shifted in the indicated direction by a progressively larger amount , overwriting pixels into which the plane is shifted and filling vacated pixels with background to shear the stack smoothly in 3D .", "If an axis of attachment is also specified , each plane is sheared line-by-line along this axis so that the resulting parallelogram remains fully connected at one end of each axis .", "The resulting ROI is thus sheared in 3D while remaining smoothly connected to its surrounding space along two orthogonal faces of the original cuboid ROI to minimize disruption .", "Applied to the skewed spinal cord , this approach allowed us to shear the cord one section at a time as it curved back along itself ( Figure 5—figure supplement 1 , left column ) , with the connected faces ensuring that each piece remains attached to one another .", "First we sheared the entire distal cord from the start of the skew ( Figure 5—figure supplement 1 , middle left column ) .", "This shift brought the proximal section toward midline , but the more distal cord remained skewed where it curved back on itself .", "To correct this skew , we sheared again but starting from a more distal point and along an orthogonal angle to follow the cord’s curve ( Figure 5—figure supplement 1 , middle right column ) .", "Now most of the cord lined up with the sagittal midline , but the shear exacerbated the skew of the most distal cord .", "Finally , we applied a third affine , this time on only the most distal section and in the opposite direction as the prior affine ( Figure 5—figure supplement 1 , right column ) .", "After mirroring the resulting brain and cord , no duplication could be seen .", "This piecewise , overlapping affine of targeted regions allowed straightening of the cord without breakages or alteration of surrounding areas .", "Although surrounding non-CNS tissue suffered noticeable breakage , they were stripped out as described below .", "The extended labels allowed us to mask and crop out non-CNS tissue , including the rest of the embryo present in several of the embryonic stage atlases .", "While distinguishing this tissue based on intensity characteristics alone would be challenging , especially for areas where the spinal cord extends the length of the embryo , the extended , mirrored labels provide a map of relevant CNS tissue .", "For each of the atlases with non-CNS tissue ( E11 . 5-E15 . 5 ) , we first cropped the atlas to the bounding box of these labels along with a small padding ( 5px ) to remove much of the non-CNS tissue , such as the entire unlabeled body in the E15 . 5 atlas .", "We removed non-CNS tissue remaining within the cropped areas by using the labels as a mask to remove all histology pixels outside of the labels mask , including the body surrounding the labeled spinal cord in E11 . 5 .", "To avoid missing tissue that may have been unlabeled , we dilated the labels mask slightly ( morphology . binary_dilation with a ball-shaped structuring element of size", "2 ) so that the mask encompasses both the labels and its immediate surroundings .", "The resulting histology thus contains all pixels in the near vicinity of labels , including pixels that should be labeled but are not .", "While labels appear generally smooth when viewed from the sagittal plane , label edges are noticeably jagged when seen from the orthogonal directions .", "A previous smoothing solution proposed by Niedworok et al . , 2016 utilized a Gaussian blur with a sigma of 0 . 5 in two iterations to minimize ragged edges in an atlas derived from the Allen Reference Atlas P56 mouse brain atlas .", "We applied this approach by iteratively applying the Gaussian filter implemented in scikit-image ( filters . gaussian ) with a range of sigmas ( 0 . 25–1 . 25 ) to each label in 3D ( Figure 3—figure supplement 2 ) .", "To extract each label in 3D , we found the bounding box of the label using the scikit-image measure . regionprops method and added additional empty padding space around the box .", "The Gaussian filter was applied to the label , and the original label in the bounding box was replaced with the smoothed label .", "Each label smoothing left small gaps vacated by the previously ragged borders .", "To fill in these gaps , we employed the in-painting approach described above .", "Finally , we replaced the original bounding box with that of the smoothed label in the volumetric labels image .", "We repeated the process for all labels from largest to smallest to complete the smoothing .", "To enhance smoothing while retaining the original underlying contour of each label , we devised an adaptive opening morphological filter approach .", "The morphological opening filter first erodes the label to remove artifacts such as ragged edges , followed by dilation of the smoothed label to bring it back toward its original size .", "In place of the Gaussian filter , we applied this opening filter ( morphology . binary_opening in scikit-image ) .", "For smaller labels , the filter occasionally caused the label the disappear , particularly for sparsely populated labels with disconnected pixels .", "To avoid label loss , we employed an adaptive filter approach by halving the size of the filter’s structuring element for small labels ( ≤5000 pixels ) .", "For any label lost in spite of this filter size reduction , we switched the filter to a closing morphological filter ( morphology . binary_closing ) , which dilates first before eroding and tends to preserve these sparse labels at the expense of potentially amplifying artifact .", "To evaluate the quality of smoothing and to optimize morphological filter structuring element sizes , we developed a smoothing quality metric with the goal of balancing smoothness while maintaining the overall original shape and placement .", "Since the major effect of label smoothing is to minimize high frequency aberrations at label borders and thus make each label more compact , we measured this amount of smoothing by the 3D compactness metric .", "We termed the difference in compactness before and after smoothing as ‘compaction . ’ As the goal of smoothing is to remove these artifacts while preserving the overall shape of the label , we introduced a penalty term of ‘displacement , ’ measured by the volume shifted outside of the label’s original bounds .", "We used the classical measure of volumetric compactness , where lower values are more compact ( Ballard and Brown , 1982 ) : ( 1 ) Compactness=SA3/Vol2 To measure the surface area of each label in 3D , we employed the marching cubes algorithm ( Lorensen and Cline , 1987 ) as implemented in scikit-image ( measure . marching_cubes_lewiner ) , which also accounts for anisotropy .", "The algorithm extracts surfaces in 3D by dividing the volume into cubes and marching through these cubes to find where isosurfaces intersect with each cube , forming triangular surfaces within each cube wherever an isosurface passes through the cube .", "Using a mask of each label , we obtained the edge surface as a mesh from which we measured the surface area ( measure . mesh_surface_area ) .", "We took the volume as the total number of mask pixels and multiplied by the product of the voxel spacing to account for anisotropy .", "With the surface area and volume , we calculated the labels compactness ( 1 ) .", "To quantify the fractional change in compactness before and after smoothing , we took the original compactness minus the smoothed compactness and normalized the difference to the original compactness to give the unitless value that we termed ‘compaction’: ( 2 ) Compaction=Corig−CsmoothCorigwhere C is the 3D compactness given above .", "To measure ‘displacement , ’ we measured the volume shifted outside of the label’s original bounds .", "Taking the mask of the smoothed label , we combined it with the inverse of the mask of the original label through a boolean ‘and’ before totaling the number of pixels .", "Similarly to the compactness measure , we normalized the displacement volume to generate a unitless value constrained between 0 and 1 , dividing this displaced smoothed volume by the total smoothed volume: ( 3 ) Displacement=Volsmooth∉VolorigVolsmoothwhere Vol is the volume of the given label .", "As a measure of smoothness quality , we took the difference of the compaction and displacement: ( 4 ) Smoothingquality=Compaction−Displacement To quantify the smoothing quality for the entire atlas , we took the weighted arithmetic mean of all the labels’ smoothing qualities , weighting by each label’s volume .", "The atlas-wide smoothing quality metric can be summarized in the following equation: ( 5 ) Atlas−WideSmoothingQuality=∑i=1NSmoothingQualityiVoli , orig∑i=1NVoli , origwhere N is the total number of original labels .", "While maximizing compaction would reduce surface area the most by transforming the label into a perfect sphere , the displacement from the label’s original space would penalize this over-compaction , allowing us to target the balance of compaction and displacement to find the optimal smoothing quality .", "As an automated method of quantifying the correspondence between labels and anatomical edges , we used the anatomical edge maps generated earlier to measure the distance between anatomical and label edges .", "We reduced each label to its edges in 3D by eroding the very outer surface of each label ( morphology . binary_erosion with a cross-shaped structuring element with connectivity of one ) and subtracting this eroded label from the original label .", "To measure distances between label and anatomical borders , we performed the Euclidean distance transform ( ndimage . distance_transform_edt in Scipy ) on the anatomical edge map to measure distances from any given voxel to the anatomical edges , using the sampling parameter to specify the microscopy image spacing in µm .", "Using the labels edge map , we next took only the voxels in the distance map corresponding to these label borders as a map of distances from each label voxel to its nearest anatomical edge .", "As overall measures of distance from label borders to the nearest anatomical border , we summed the edge distances for each label to compare before and after label refinement .", "Generating edge images provided a map of the boundaries between anatomically distinct regions not only to measure distances between borders , but also to curate the labels themselves with these anatomical edges .", "To reannotate the existing unsmoothed labels , we eroded and regrew them through an anatomically guided watershed transformation similar to the approach described above but now in 3D .", "We tested seeds with multiple erosion filter sizes and found that a structuring element size of 8 reduced the seed size sufficiently to correct label bounds around several grossly abnormal labels , including the lateral septal nuclei and basal ganglia .", "As a global operator , erosion typically leads to loss of thin structures , especially with larger structuring elements .", "To avoid this loss , we first extracted the core structure of each label by finding its 3D skeleton ( morphology . skeletonize_3d in scikit-image ) .", "We added the skeletonized image back to the eroded label to recover the location of thin structures , allowing the watershed to regrow these labeled areas in addition to the eroded label .", "To limit branches in the skeleton , which could counter the effect of the erosion , we input a lightly eroded version of the labels ( structuring element half the size of that for the main erosion ) to the skeletonization .", "The resulting watershed segmentation also served as preliminary smoothing but introduced its own label edge artifacts , though of lower frequency than in the original labels .", "We thus deferred the smoothing algorithm until after this watershed step and could use smaller filter sizes to generate a final smooth image .", "Each atlas in the ADMBA required a different set of refinement features and settings , including specialized adjustments such as the 3D piecewise affine only for a specific atlas ( E11 . 5 ) .", "To allow for customized settings , we defined separate profiles of parameters within our software suite for each atlas .", "The microscopy images depict a specimen in embryonic stage with the caudal end including the spinal cord wrapping around itself and deviated laterally from the rest of the body .", "As with most other atlases in this series , one half of the labels were missing .", "Making the image symmetric would allow us to mirror labels from the existing side onto this missing side and minimize bias when using the atlas for automated registration tasks .", "We started by rotating the image by 5° in the axial planes toward the left of the brain and 1° in the coronal planes to raise the right side of the brain , bringing the sagittal midline parallel to an image edge .", "To shift the distal end of the spinal cord back toward the sagittal midline , we applied the piecewise 3D affine transformation described above ( Figure 5—figure supplement 1 ) .", "Prior to the affine , we applied an additional rotation of 30° in the sagittal planes to position the skewed distal cord within a single cuboid ROI parallel to the image .", "With the embryo now symmetric , we mirrored the labels and microscopy signal along the embyro’s midline , measured as 52% across the sample along the z-axis .", "To highlight the central nervous system and remove breakages introduced by the affine transformations in non-CNS tissue , we stripped out non-CNS tissue as described earlier .", "The E11 . 5 atlas uniquely contains labeled ventricles .", "To ensure that the Laplacian of Gaussian edge-detection algorithm appropriately found ventricular edges , we used only the thresholded atlas rather than incorporating the labels to find the background .", "We also included the ventricular space as foreground for purposes of the DSC calculations between microscopy and labels to account for the ventricular labeling .", "This atlas did not require lateral extension .", "This atlas also contains the full embryo , but the spinal cord is symmetric along the sagittal midline of the brain and did not require the affine transformations as in the E11 . 5 atlas .", "After extending the lateral edges , we rotated the atlas by 4° in the axial planes toward the left of the brain and 2° in the coronal planes to lift the right side of the brain , making the atlas symmetric before mirroring the atlas at 48% along the sagittal planes .", "We again stripped non-CNS tissue the same way as for the E11 . 5 atlas .", "This atlas is the final one to include the complete embryo , although only the very rostral end of the spinal cord includes labels .", "We extended the lateral edges , rotated the images by 4° in the axial planes toward the left side of the brain , mirrored the atlas at 49% along the sagittal planes , and stripped away the entire embryo outside of the brain .", "A stepwise shift in sagittal planes is apparent at several planes ( e . g . 104 and 129 ) in both the histology and label images in the original atlas , which we smoothed slightly in the labels during the smoothing step .", "A small subset of labels from sagittal planes 103–107 were compressed along the dorsoventral axis .", "To match them with their neighboring label planes and the underlying atlas , we resized them similarly to the lateral edge extension .", "Starting with the first plane in this subset , we thresholded the microscopy image , removed small objects , and obtained the largest bounding box of connected histology components .", "Taking only this largest connected structure allowed us to avoid including extraneous tissue visible on the ventral aspect of the brain , which was unlabeled .", "We repeated the process on the labels to obtain its compressed bounding box and resized it to the size of the microscopy bounding box .", "Finally , we repeated the entire process on the rest of the planes in this subset ( Figure 2—figure supplement 3 ) .", "For the lateral edge extension , we noted that the basal ganglia in the most lateral planes are slightly larger than in more medial planes .", "Under the assumption that the basal ganglia would be tapering laterally , we skipped these planes and started the extension at 13 . 7% along the sagittal planes to take the plane with smallest basal ganglia label .", "After rotating the atlas by 1 . 5° in the axial planes toward the right of the brain and 2° in the coronal planes to lift the left side of the brain , we mirrored microscopy and labels at 52 . 5% along the sagittal planes for symmetry .", "This atlas is reminiscent of E18 . 5 but without the necessity of setting the lateral edge extension starting plane explicitly or expanding any compressed labels .", "After lateral edge extension and rotating the atlas by 0 . 22° in the axial planes toward the right of the brain , we mirrored microscopy and labels at 48 . 7% along the sagittal planes for symmetry .", "The most laterally labeled plane is discontinuous with the rest of the labeled planes in this atlas , so we skipped this plane during lateral extension and started extending only from the first contiguous set of planes .", "After rotating the atlas by 0 . 4° in the axial planes toward the left of the brain , we mirrored the microscopy and labels images at the 50% mark along the sagittal planes .", "The most lateral planes had incomplete labels , requiring use of a more medial plane with complete labels at 11% along the sagittal planes for the lateral edge extension .", "After rotating the atlas by 1° in the axial planes toward the right of the brain , we mirrored the microscopy and labels images at the 48% mark along the sagittal planes .", "The ADMBA contains a P56 mouse similar to the adult P56 but following the same ontological labeling scheme as in the rest of the ADMBA .", "This atlas uniquely contains bilateral labels , although the far lateral section is still missing .", "We again extended the lateral edges and mirrored the microscopy and labels images , starting extension at 13 . 8% and mirroring at 50% along the sagittal planes , respectively .", "The most lateral labeled plane contains two distinct labeled structures , the cortex and cerebellum , requiring separate extension for each distinct structure as outlined in our method above .", "All procedures and animal care were approved and performed in accordance with institutional guidelines from the University of California , San Francisco Laboratory Animal Research Center ( LARC ) .", "All strains were maintained on a C57BL/6J background .", "Animals were housed as one breeding pair per cage in a vivarium with a 12 hr light , 12 hr dark cycle .", "For timed pregnancies , noon on the day of the vaginal plug was counted as embryonic day 0 . 5 .", "Pups were harvested at P0 ( postnatal day 0 ) .", "For assessing the eight atlases in the ADMBA , sample size was determined by the number of atlases available in the original 2D resource ( n = 8 ) .", "The size of our validation cohort ( P0 wild-type mouse brains , n = 15 ) was chosen to detect an effect size of 1 . 5 after correction for multiple comparisons using a power calculation based on a paired t-test .", "At the time of experiment , P0 pups were anesthetized on ice and perfused transcardially with ice-cold 1X PBS supplemented with 10 U/mL heparin and then with 4% PFA in 1X PBS , followed by brain isolation .", "P0 brains were post-fixed overnight at 4°C in 4% PFA in 1X PBS .", "The next day the excess fixative was removed by washing the brains with 1X PBS supplemented with 0 . 01% ( wt/vol ) sodium azide ( Sigma-Aldrich ) for at least 2 hr at room temperature ( RT ) .", "Samples were cleared using the advanced CUBIC clearing protocol for whole-brain and whole-body clearing ( Susaki et al . , 2015 ) .", "In short , samples were immersed in 1/2-water-diluted Reagent-1 containing 1 µM SYTO 16 ( Thermo Fisher ) and incubated at 37°C for 6 hr ( Reagent-1: 25 weight% ( w% ) Urea , 25 wt% Quadrol , 15 wt% Triton X-100 , and dH2O ) .", "1/2 diluted Reagent-1 was replaced with Reagent-1 containing 1 µM SYTO 16 and incubated overnight at 37°C on a rotator .", "The next day , solution was replaced with fresh Reagent-1 containing 1 µM SYTO 16 and was incubated overnight at 37°C .", "Reagent-1 was replaced with fresh Reagent-1 containing 1 µM SYTO 16 every 48 hr for a total of 8 days .", "Tissue clearing was stopped by washing the sample with 1X PBS supplemented with 0 . 01% ( wt/vol ) sodium azide at RT once for 2 hr , once overnight and again once for 2 hr .", "After the wash step , samples were immersed in 10 mL of 1/2-PBS-diluted Reagent-2 ( Reagent-2: 25 wt% Urea , 50 wt% sucrose , 10 wt% Triethanolamine , and dH2O ) .", "Vials containing brains in 1/2-PBS-diluted Reagent-2 were placed in a vacuum desiccator with gentle shaking overnight at RT .", "The following day , the solution was replaced with Reagent-2 and incubated at 37°C overnight on a rotator .", "The next day , Reagent-2 solution was replaced with fresh Reagent-2 and incubated at 37°C overnight on a rotator .", "This step was repeated four times .", "We imaged and processed a total of n = 15 from 5 female and 10 male mice .", "Samples were imaged within 7 days of completing the clearing protocol at the Gladstone Institutes Histology and Light Microscopy Core on a Zeiss Lightsheet Z . 1 microscope .", "Lightsheet imaging of a mouse P0 brain required approximately 50 tiles ( 5 × 10 ) per brain at 5x ( Zeiss EC Plan-Neofluar 5x , NA 0 . 16 , RI 1 . 45 , WD 5 . 6 mm ) , which afforded a level of resolution that allowed for nuclei detection ( 0 . 913 µm/px lateral resolution ) .", "The microscope requires specimen to be suspended between fixed illuminator and detector objectives , typically using capillaries to embed small specimens .", "To suspend the relatively larger whole brain , we designed a custom 3D-printed rod with a platform to maximize surface area for gluing the ventral surface of the brain to the mount ( Figure 7—figure supplement 1 ) .", "The rod also contains a neck to position the brain directly under the mount holder , allowing full movement of the brain throughout the chamber to capture the brain it its entirety .", "The mount oriented the brain axially toward the detector to minimize the path of the illuminators on opposite sides laterally through the tissue as well as the emission path superiorly to the detector .", "We designed the mount in Onshape and printed it using a Stratasys uPrint 3D printer .", "To glue the brain to the mount , we placed the brain , ventral surface facing up , on a custom sieve to drain reagent and rolled cotton bud applicators on the ventral surface of the brain to dry it .", "We applied glue ( Scotch Super Glue Liquid ) with a brush applicator to the mount platform surface and attached it the brain .", "After flipping the brain to dorsal side up , we placed the mount in a custom holder to allow the glue to dry over 3 min and dribbled Reagent-2 media on the dorsal surface to ensure that it did not dry out .", "After the glue dried , we suspended the mount from the microscope manipulator before immersing the mounted brain into the microscope chamber filled with Reagent-2 .", "With the lasers initiated , we aligned the two opposite illuminators along the z-axis by visual inspection at a central region of the brain .", "We ranged the z-stack from just beyond the first and last z-planes with visible nuclei , typically 800–1000 planes using a slice interval of 4 . 935 µm with a lightsheet thickness of 10 . 44 µm , and set the image tiling to include the farthest lateral and anterior-posterior nuclei with a 10% overlap per tile .", "We illuminated the brain with 488 nm excitation and 30 ms dwell time through the Z . 1", "LSFM 5x/0 . 1 illuminators , LBF 405/488/561/640 laser blocking filter , SBS LP 560 secondary beam splitter , and BP 505–545 band pass filter .", "The microscope paused for 20 s between each tile to allow tissue settling after repositioning for the next tile .", "We controlled the microscope through the Zeiss Zen microscopy software suite and saved images in a CZI multi-tile format with all tiles stored in a single archive .", "To stitch the tiled microscopy images in an automated fashion , we used the Fiji/ImageJ ( Schindelin et al . , 2012; Rueden et al . , 2017 ) BigStitcher plugin ( Hörl et al . , 2019 ) , a successor to the Stitching plugin ( Preibisch et al . , 2009 ) that allows for better memory management and multiprocessing as well as a graphical interface to verify alignments .", "As we needed to stitch multiple brains , we accessed this plugin headlessly through its scriptable interface , manually intervening only to visually verify alignments before proceeding with the fusion step .", "After importing the CZI file into the BigStitcher HDF5-based format , the plugin auto-detected the brightest illuminator for each tile , discarding planes from the other illuminator .", "We chose to simply select planes from the optimal illuminator rather than fusing planes from both illuminators after our inspection revealed that illuminators rarely if ever aligned perfectly throughout the tile , leading to artifacts such as apparent elongation of nuclei from imperfect overlap of the same nuclei from different illuminators .", "The plugin calculated tile shifts using a phase correlation method , and we filtered out links below a correlation threshold of r = 0 . 8 before applying shifts through the two-round iterative global optimization strategy .", "To account for occasional tile misalignments , we manually inspected every pre-stitched brain to reposition any misaligned tiles , which occurred in approximately 10% of brains .", "Once tiles aligned , the plugin fused them into a single large TIFF image per channel .", "We imported this fused file via Python-Bioformats and Javabridge , libraries that allow access to life science formats via Bio-Formats , to a Numpy array format for image processing in our Python-based software as outlined below .", "We detected nuclei in cleared mouse brains using a 3D Laplacian of Gaussian blob detection technique throughout each whole brain .", "To perform detections in large images several hundred gigabytes ( GB ) to over a terabyte ( TB ) in size , we subdivided the image into many smaller chunks to reduce RAM requirements and maximize parallel processing .", "We loaded images through the Numpy library’s memory mapped method ( load with the mmap_mode option ) to load only the necessary parts of the image on-the-fly , allowing us to load small images a chunk at a time without reading the entire volumetric image into memory .", "We divided the image shape into overlapping chunks to ensure that nuclei at borders would not be missed , with overlap size of approximately the nucleus diameter .", "After determining the offset and shape of each chunk , we set the image array as a class attribute , initiated multiprocessing ( the multiprocessing . Pool in the standard Python library ) , and accessed each chunk as a view in a separate process via class methods to avoid duplicating arrays in memory .", "Thus , we could control total memory usage by the size of chunks and the number of CPU ( central processing unit ) cores available for separate processes .", "3D cell detection poses a number of challenges including adapting to local variation such as staining inhomogeneity , background variation , and autofluorescence , in addition to overlapping cells in dense tissue ( Shuvaev et al . , 2017 ) .", "To address these issues , we analyzed images in a local manner by further subdividing each chunk for preprocessing based on its immediate surroundings .", "We split each chunk into sub-chunks using the same approach as above but ran each sub-chunk serially within each CPU process .", "In each sub-chunk , we first clipped intensity values at the 5th and 98 . 5th percentiles ( percentile in Numpy ) to remove extreme outliers , rescaled the intensities from 0 to 1 , and further saturated signal by clipping the rescaled intensities at the 50th percentile .", "We next enhanced edges by using unsharp masking with a Gaussian sigma of 8 ( filters . gaussian in scikit-image ) to identify sharp details as the difference between an image and its blurred version ( which we amplified by a factor of 0 . 3 ) and adding back those details to the original image .", "We mildly eroded the resulting signal with an erosion filter using an octahedron structuring element of size 1 ( morphology . erosion with morphology . octahedron in scikit-image ) to separate out blobs .", "To detect blobs , we implemented the 3D Laplacian of Gaussian blob detector from the scikit-image library ( feature . blob_log ) as a multi-scale interest point operator ( Lindeberg , 1993 ) .", "We set the minimum and maximum sigma based on the microscopy resolution , with 10 intermediate values , detection threshold of 0 . 1 , and overlap fraction threshold of 0 . 55 below which duplicated blobs are eliminated .", "Initially we missed many nuclei positioned above one another in the z-direction , likely because the anisotropy necessitated by the relatively thick lightsheet at 5x in our setup limited resolution in the z-direction .", "To improve detection along the z-axis , we interpolated the images in this direction to near isotropy before detection ( transform . resize in scikit-image ) .", "The blob detector had a tendency to cluster detections in the bottom and topmost z-planes in each ROI from nuclei visible within the ROI but whose centroids are outside .", "To avoid this clustering and minimize duplication with adjacent ROIs , we cropped nuclei from these planes on the assumption that they would be captured better in the adjacent , overlapping ROIs .", "Overlapping chunks minimized missing nuclei at edges but also necessitated pruning blobs duplicately detected in adjacent chunks .", "Pruning involves checking for duplicates within all potentially overlapping regions .", "Since the overlapping portions of the regularly spaced chunks collectively form a grid pattern throughout the full volumetric image , we could limit our search to these grid planes along each axis .", "After completing detection on the whole image , we first pooled all detected blobs into a single array .", "Along a given axis of the full image , we determined the boundaries for each overlapping region and all of its blobs .", "Within each overlapping region , we found all blobs close to another blob by taking the absolute value of the difference between all blobs with one another and finding blobs within a given tolerance in all dimensions .", "The tolerance was titrated so that the ratio of final blobs in overlapping regions to the next adjacent regions of same volume was about 1:1 .", "For each close pair of blobs found , we replaced both blobs with a new blob that took the mean of their coordinates .", "To minimize memory usage , we checked smaller groups of blobs against one another until completing all comparisons .", "We checked overlapping regions simultaneously in multiprocessing along a given axis for efficiency , re-pooled all blobs , and pruned along the next axis to account for blobs that may have been duplicately detected in overlapping chunks along multiple axes , at grid intersections .", "Parameters for preprocessing , detections , and pruning steps were optimized through a Grid Search approach , a type of hyperparameter tuning , to check combinations of parameters systematically .", "To evaluate the accuracy of each set of parameters , two students in our lab generated truth sets of nuclei locations and radii using our serial 2D nuclei annotation tool , taking ROIs of size 42×42×32 pixels ( x , y , z ) from representative ROIs of all major brain structures ( n = 15 forebrain , eight midbrain , and seven hindbrain; n = 2766 nuclei ) .", "They separately generated additional truth sets at a slightly lower magnification ( 4x ) , size 60×60×14 pixels ( n = 40 ROIs , 1116 nuclei ) to increase representation .", "After detecting nuclei on these images with a given set of parameters , matches between detections and ground truth was determined using the Hungarian algorithm , a combinatorial optimization method to determine optimal assignments between two sets ( Kuhn , 1955 ) , as implemented in optimize . linear_sum_assignment in Scipy .", "After scaling nuclei coordinates for isotropy , we found the Euclidean distances between detected and truth nuclei points through distance . cdist , which serves as the cost matrix input to optimize . linear_sum_assignment to find optimal pairings between points based on closest distance .", "We took correctly identified detections , or true positives ( TP ) , as pairings within a given tolerance distance .", "Unpaired detections or those in pairs exceeding this threshold were considered false positives ( FP ) , and the same for ground truth were false negatives ( FN ) .", "Since a match for a given nucleus within the ROI may lie outside of it and thus go unseen , we first searched for pairings only within an inner sub-ROI , followed by a secondary search for pairings between only unmatched inner sub-ROI objects and the rest of the ROI ( Ho et al . , 2017 ) .", "This approach reduced the total number of nuclei available ( n = 1118 nuclei ) but avoided missed border matches .", "As measures of performance of our detection compared with ground truth , we used the following standard equations: ( 6 ) Sensitivity ( Recall ) =TPTP+FN ( 7 ) PositivePredictiveValue ( PPV , orPrecision ) =TPTP+FP To assign nuclei to the proper brain label , we employed automated label propagation by registering the E18 . 5 atlas to each of our imaged mouse brains using SimpleElastix ( Marstal et al . , 2016 ) , a toolkit that combines the programmatic access of SimpleITK ( Lowekamp et al . , 2013 ) to the Insight Segmentation and Registration Toolkit ( ITK ) ( Yoo et al . , 2002 ) with the Elastix ( Klein et al . , 2010; Shamonin et al . , 2014 ) image registration framework .", "Elastix has been recently validated as a computationally efficient and accurate tool for registration of mouse brains cleared by CUBIC ( Nazib et al . , 2018 ) .", "As stitched images are typically several hundreds of GBs per file , image downsampling was necessary to reduce memory utilization during image registration .", "To reduce file size efficiently in both time and memory usage , we employed the same chunking strategy as used during nuclei detection except with larger , non-overlapping units to resize multiple sections of the image simultaneously .", "We also reduced memory required for the output array by saving directly to disk with a memory-mapped array ( lib . format . open_memmap in Numpy ) .", "We matched the target final size to the E18 . 5 atlas , which would be registered to each downsampled image .", "Registration involved rigid followed by non-rigid alignment .", "For rigid registration , we employed a translation ( translation parameter map in SimpleElastix ) with default settings except increasing to 2048 iterations ( MaximumNumberOfIterations setting ) followed by an affine ( affine parameter map ) with 1024 iterations , applied with an ElastixImageFilter , to shift , resize , and shear the atlas microscopy image to the same space as that of the sample brain .", "For non-rigid alignment , we employed a b-spline strategy ( bspline parameter map ) guided by the AdvancedNormalizedCorrelation similarity metric ( Nazib et al . , 2018; Hammelrath et al . , 2016 ) with grid spacing of size 60 , measured in voxels rather than physical units ( FinalGridSpacingInVoxels setting in place of FinalGridSpacingInPhysicalUnits ) , over 512 iterations .", "The TransformixImageFilter in SimpleElastix allowed us to apply the identical registration transformation to the atlas labels image , except that we set the final b-spline interpolation order ( FinalBSplineInterpolationOrder ) to 0 to avoid interpolating any new values , preserving the labels’ specific set of integer values .", "We applied this identical transformation to both the mirrored and edge-refined atlas labels .", "To evaluate the level of alignment from registration , we measured the similarity between each registered atlas histology and its corresponding sample image using a Dice Similarity Coefficient ( DSC ) ( Dice , 1945 ) as implemented by the GetDiceCoefficient function in SimpleElastix/SimpleITK , given by the equation ( Tustison and Gee , 2009 ) : ( 8 ) DiceSimilarityCoefficient ( DSC ) =2|S∩T||S|+|T|where S and T are two different sets of voxels .", "We took the foreground of each atlas and sample microscopy image to be its mean threshold ( filters . threshold_mean function in scikit-image ) and input them to a LabelOverlapMeasuresImageFilter to take the DSC .", "Registration of the atlas to our volumetric nuclear-stained brain microscopy images allowed us to quantify nuclei per label for comparison with the original ( mirrored ) and smoothed ( edge-aware ) atlases .", "We first measured volumes per label by taking a mask of each registered label and summing the foreground pixels within each mask before multiplying this volume by the microscopy pixel resolution ( scaled for downsampling ) to obtain volumes in physical units ( mm3 ) .", "For nuclei densities , we first constructed a nuclei heat map by converting the nuclei coordinates to nuclei per voxel within the downsampled image .", "We scaled the coordinates to the scaling of the downsampled image , rounding to the nearest integer , and found the counts of nuclei at each coordinate ( unique with return_counts option in Numpy ) .", "We next indexed these coordinates directly into an empty Numpy array of the same shape as that of the downsampled image to assign them to the corresponding nuclei counts at each voxel .", "We used the same label mask previously obtained to find the number of nuclei within each given label .", "Dividing the number of nuclei by the volume within each label gave the label nuclei density .", "To measure the variability of nuclei within each label before and after label reannotation , we measured the coefficient of variation within each label given by the standard equation: ( 9 ) Coefficientofvariation ( CV ) =σμwhere σ is the standard deviation , and µ is the mean .", "A lower coefficient of variation indicates lower variability and thus tighter capture of a more homogeneous label .", "As a raw proxy for nuclei variation , we first measured the variation of intensities within the nuclear-stained lightsheet images .", "Using the same label masks , we took the standard deviation of voxel intensities and divided it by the mean of intensities within the label ( std and mean , respectively , in Numpy ) to obtain the intensity coefficient of variation .", "Similarly , we measured the nuclei coefficient of variation by measuring the standard deviation and mean values of nuclei counts per label within the nuclei heat map .", "We also stratified nuclei ROIs into low/medium and high-density regions by taking the Otsu threshold of their density , which corresponded with their density histogram , and separately measured recall and precision in each group ( Figure 6—figure supplement 2D ) .", "As another measure of label alignment at the nuclei level , we measured nuclei clustering using Density-Based Spatial Clustering of Applications with Noise ( DBSCAN ) ( Ester et al . , 1996 ) as implemented by the scikit-learn library ( Pedregosa et al . , 2018 ) .", "DBSCAN clusters tightly packed points within a neighbor distance defined by the parameter E and a minimum number of points given as another parameter , with isolated points in lower density regions that cannot be clustered considered ‘outliers’ or ‘noise . ’ The minimum number of samples is typically taken as 2 · ndim , where ndim is the number of dimensions ( Schubert et al . , 2017 ) , thus giving 6 for our 3D nuclei point cloud .", "To find E , the nearest-neighbor distance of the 2 · ndim − 1 neighbor for each point is sorted and plotted to find the distance at the ‘elbow’ point , the point of maximum curvature ( Schubert et al . , 2017 ) , which we found to be at least 20 µm ( Figure 7—figure supplement 3A ) .", "For each label in the original ( mirrored ) atlas registered to each wild-type brain , we extracted the nuclei coordinates within the label and clustered them by DBSCAN to find the number of clusters , nuclei per cluster , and ‘noise , ’ or number of isolated nuclei that remained unclustered .", "We repeated the same process using the same nuclei coordinates for each brain but with the smoothed ( edge-aware ) atlas registered identically to the brain .", "As the ‘elbow’ distance of maximum curvature can be difficult to define , and E can strongly influence the clustering , we repeated this process for a range of E values through the elbow region ( Figure 7—figure supplement 3B ) and highlighted a conservative distance of 20 µm ( Figure 7—figure supplement 3C–E ) .", "Statistical tests used for comparisons are described in the relevant Results sections and figure legends .", "Bonferroni correction for p-values is applied when multiple statistical tests are performed for each figure sub-panel as indicated for the number of tests .", "We provide the MagellanMapper image software suite as a tool to assist with visualization , annotation , and automated processing of volumetric images ( Figure 7—figure supplement 4 ) .", "The suite consists of a graphical user interface ( GUI ) to aid visualization of 2D images in a 3D context and command-line interface ( CLI ) for non-interactive processing in workstation and cloud environments .", "The main GUI integrates ROI selection with 3D point and surface rendering through the Mayavi toolkit ( Ramachandran and Varoquaux , 2011 ) .", "Users can load volumetric image files and specify ROI boundaries through sliders and text boxes or load a previously saved ROI .", "3D point rendering provides a voxel-based visualization of the ROI with minimal filtering , whereas the 3D surface rendering utilizes VTK ( The Visualization Toolkit ) ( Schroeder et al . , 1998 ) for cleaner images .", "The interface is implemented in TraitsUI for integration with Mayavi .", "To inspect raw images , the user can launch two types of mixed 2D/3D interfaces from the main GUI to display and annotate the original 2D images for each plane .", "The first 2D interface is a serial 2D ROI viewer that shows each successive 2D plane within the ROI side-by-side , allowing the user to follow objects such as nuclei that come and go from plane to plane .", "Larger overview images at different magnifications show context and synchronize with the smaller views to effectively zoom in on a given plane .", "These overview images are also scrollable along the z-axis to visualize subtle object shifts in place .", "The interface also provides annotation tools geared toward blob detection such as nuclei .", "Detected blobs appear as circles along with optional segmentations , and the user can drag , resize , or cut/copy/paste circles to improve placement and flag their correctness .", "We have used this simplified blob annotator to generate truth sets for blob detection verification and optimization .", "In addition to ROI viewers , we provide a simultaneous orthogonal viewer to visualize and annotate atlases in all three dimensions .", "It displays orthogonal planes of the full volumetric image in three separate panels , with crosshairs in each panel denoting the corresponding planes in other panels .", "Clicking on or scrolling within any panel updates crosshairs and synchronizes the other displayed planes .", "The user can also load label maps to overlay directly on atlas microscopy images with an adjustable labels opacity to allow close inspection of annotation alignment with anatomical structures .", "To distinguish an arbitrary number of labels from one another , we use a custom discrete colormap with randomly generated colors , each assigned to a single label .", "We display all images as views of one another to minimize memory requirement and update all orthogonal planes in real-time .", "As a method for simple , rapid editing of these labels , the user can enter an editing mode to simply click and drag on individual labels to paint them into other spaces .", "We have used the interface with a tablet and electronic pen to edit labels by drawing .", "As hand-editing any given plane likely introduces edge artifacts seen in other orthogonal directions , we also designed a method to interpolate contours between two distant planes .", "After the user edits the same label ID at start and ending planes and initiates the interpolation , it takes the signed distance transforms of the label masks ( ndimage . distance_transform_edt in Scipy combined with a mask of the original label to identify distances inside versus outside the label ) and interpolates those distances for each intervening plane ( interpolate . interpn applied across a mesh-grid of the planes ) .", "The interpolation provides label border extensions that are generally smooth in all dimensions after manually editing only the first and last plane .", "The annotation interfaces are implemented in Matplotlib ( Hunter , 2007 ) .", "Blobs are stored in an SQLite ( Hipp et al . , 2021 ) database , while atlas edits are saved directly to their underlying 3D image file .", "In addition to a GUI for interactive visualization and verification , MagellanMapper provides automated pipelines for non-interactive image processing such as whole brain nuclei detection in cloud-based work environments .", "Users can access the suite via its CLI , and the suite provides Bash scripts to connect MagellanMapper with other tools such as Fiji/ImageJ for image stitching and Amazon Web Services ( AWS ) in a platform-independent manner .", "For input/output ( I/O ) , the suite utilizes standard 3D image formats for portability with other software libraries .", "Microscopy images stored in proprietary formats such as Zeiss CZI format can be imported to a standard Numpy array archive using the Bio-Formats library ( Linkert et al . , 2010 ) ( via the Javabridge and Python- Bioformats libraries developed for CellProfiler [Lamprecht et al . , 2007] libraries ) along with a separate Numpy archive containing image metadata extracted from the original file .", "Loading the imported Numpy array as a memory-mapped file ( load function with mmap_mode option ) allows users to access small parts of large files to minimize load time and memory usage by only loading the requested ROI rather than the potentially TB-sized full volumetric image .", "In addition to Numpy array archives , other 3D image formats such as MetaImage , NIfTI , and DICOM are supported through the SimpleITK/SimpleElastix library .", "Annotated images are saved to their respective formats .", "As an example of an automated pipeline for image import , a user can launch the script from a cloud-based server instance to first retrieve and decompress an archived microscopy file previously saved in a cloud storage location .", "After extracting the microscopy image , the pipeline script launches Fiji/ImageJ to run a custom headless script for the BigStitcher plugin , which stitches the tiled microscopy image as described above .", "After allowing the user to verify and adjust tile placements through the BigStitcher interface , the script continues the BigStitcher tile fusion operation , resulting in TIFF formatted images for each channel .", "The pipeline script next calls MegellanMapper to import these TIFF images into a single Numpy array archive .", "Subsequent pipelines can be run to process the imported image for automated nuclei detections , downsample or transpose the image , register images to an atlas , or automatically refine new atlases in 3D .", "We provide MagellanMapper as open-source software in the hope of facilitating both interactive and headless processing of large volume microscopy images and the atlases to which they will be registered .", "The suite API includes 3D image processing functions designed to be useful as library methods for other applications .", "Library functions for the Python and R plots depicted here are also provided to reproduce similar graphs .", "As an open-source , Python-based tool , our vision for the suite is that it will work alongside , integrate with , and itself become refined by the many other excellent image processing software suites and libraries available to the scientific community .", "The MagellanMapper suite is written in Python with the following versions and Python libraries: Python 3 . 6 , Numpy 1 . 15 , Scipy 1 . 1 , scikit-image 0 . 14 , scikit-learn 0 . 21 , Matplotlib 3 . 0 , Matplotlib ScaleBar 0 . 6 , Mayavi 4 . 6 , TraitsUI 6 . 0 , PyQt 5 . 11 , Python-Bioformats 1 . 1 , Javabridge 1 . 0 , SimpleElastix 1 . 1 , and Pandas 0 . 23 .", "We used Conda 4 . 7 for Python library management .", "We stitched images with Fiji/ImageJ 1 . 52 using BigStitcher 0 . 2 . 10 .", "We computed additional statistics in R 3 . 5 with RStudio 1 . 1 .", "To interact with AWS , we used AWS CLI 1 . 16 and Boto3 1 . 9 .", "For image acquisition , we used Zeiss Zen 2014 .", "We conducted most software development on a Mid-2014 MacBook Pro ( Intel Core i7-4980HQ , 16 GB RAM , 1TB SSD ) with MacOS 10 . 13 , image stitching and nuclei detections on AWS EC2 c5 . 9xlarge instances ( 36 vCPUs , 72 GB RAM , typically configured with 1TB SSD ) running RHEL 7 . 5 and Ubuntu 18 . 04 , and volume measurements and aggregation on a Dell Precision T7500 workstation ( 64 GB RAM , 256 GB SSD and 2 . 2TB HDD ) with Ubuntu 18 . 04 .", "We conducted additional cross-platform compatibility testing on a Microsoft Surface Pro ( 2017 ) laptop ( 8 GB RAM , 256 GB SSD ) with Microsoft windows 10 ( build 1803 ) , Windows Subsystem for Linux running Ubuntu 18 . 04 , and Windows 10 virtual machines in VirtualBox 6 . 0 ." ] ]

[ "3D imaging data necessitate 3D reference atlases for accurate quantitative interpretation .", "Existing computational methods to generate 3D atlases from 2D-derived atlases result in extensive artifacts , while manual curation approaches are labor-intensive .", "We present a computational approach for 3D atlas construction that substantially reduces artifacts by identifying anatomical boundaries in the underlying imaging data and using these to guide 3D transformation .", "Anatomical boundaries also allow extension of atlases to complete edge regions .", "Applying these methods to the eight developmental stages in the Allen Developing Mouse Brain Atlas ( ADMBA ) led to more comprehensive and accurate atlases .", "We generated imaging data from 15 whole mouse brains to validate atlas performance and observed qualitative and quantitative improvement ( 37% greater alignment between atlas and anatomical boundaries ) .", "We provide the pipeline as the MagellanMapper software and the eight 3D reconstructed ADMBA atlases .", "These resources facilitate whole-organ quantitative analysis between samples and across development ." ]

[ "The research community needs precise , reliable 3D atlases of organs to pinpoint where biological structures and processes are located .", "For instance , these maps are essential to understand where specific genes are turned on or off , or the spatial organization of various groups of cells over time .", "For centuries , atlases have been built by thinly ‘slicing up’ an organ , and then precisely representing each 2D layer .", "Yet this approach is imperfect: each layer may be accurate on its own , but inevitable mismatches appear between the slices when viewed in 3D or from another angle .", "Advances in microscopy now allow entire organs to be imaged in 3D .", "Comparing these images with atlases could help to detect subtle differences that indicate or underlie disease .", "However , this is only possible if 3D maps are accurate and do not feature mismatches between layers .", "To create an atlas without such artifacts , one approach consists in starting from scratch and manually redrawing the maps in 3D , a labor-intensive method that discards a large body of well-established atlases .", "Instead , Young et al . set out to create an automated method which could help to refine existing ‘layer-based’ atlases , releasing software that anyone can use to improve current maps .", "The package was created by harnessing eight atlases in the Allen Developing Mouse Brain Atlas , and then using the underlying anatomical images to resolve discrepancies between layers or fill out any missing areas .", "Known as MagellanMapper , the software was extensively tested to demonstrate the accuracy of the maps it creates , including comparison to whole-brain imaging data from 15 mouse brains .", "Armed with this new software , researchers can improve the accuracy of their atlases , helping them to understand the structure of organs at the level of the cell and giving them insight into a broad range of human disorders ." ]

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

[ "evolutionary biology" ]

[ [ "Crown-group animals ( metazoans ) originated around 850 million years ago ( Ma ) ( Dohrmann and Wörheide , 2017 ) , when atmospheric oxygen ( O2 ) was likely <10% of its present level ( Lenton and Daines , 2017 ) .", "Less atmospheric oxygen would had led to extensive deep-ocean anoxia and high redox variability in marine surface waters ( Reinhard et al . , 2016 ) , thereby placing energetic constraints on the diversity , abundance , and physiology of heterotrophic eukaryotes , particularly animals ( Fenchel and Finlay , 1995 ) .", "In all previously examined animal lineages , decreasing cellular oxygen concentrations , whether environmentally or metabolically driven , alter gene expression via the hypoxia-inducible factor ( HIF ) , a key pathway for maintaining physiological oxygen homeostasis ( Loenarz et al . , 2011 ) .", "The HIF pathway consists of two bHLH-PAS-domain transcription factors ( HIFa and HIFb/ARNT ) that form a heterodimer to initiate transcription when oxygen concentrations at the cellular level are low ( Semenza , 2007 ) .", "At higher oxygen levels , HIFa is hydroxylated by a member of the 2-oxoglutarate-dependent oxygenase family , HPH ( Bruick and McKnight , 2001 ) , also called PHD or EGL9 , and is then signaled for destruction by the von Hippel-Lindau ( VHL ) protein , thereby avoiding its dimerization with HIFb and subsequent transcriptional activation ( Min et al . , 2002 ) .", "Proteins in the HIF pathway have been characterized in many metazoan groups ( Kaelin and Ratcliffe , 2008 ) , including placozoans ( Loenarz et al . , 2011 ) and cnidarians ( Wang et al . , 2014 ) , but have not been evaluated in sponges ( Phylum Porifera ) and comb-jellies ( Phylum Ctenophora ) -- the two most likely candidates that have been discussed as the sister-lineage to all remaining animals ( e . g . King and Rokas , 2017 ) , but see Simion et al . ( 2017 ) ; Feuda et al . , 2017 ) .", "While previous experiments on the demosponge Halichondria panicea demonstrated survival under 1 . 6–12 . 7 µM O2 ( Mills et al . , 2014 ) , no transcriptional data from sponges kept under low-oxygen have been reported ( Mentel et al . , 2014 ) .", "Therefore , it remains unclear whether the HIF pathway extends to all living animals , which would parsimoniously suggest that HIF is an ancestral feature of animal life ." ], [ "Using comparative genomics ( see data sources in Supplementary file 1 ) , we found that all animal groups -- as well as the single-celled eukaryotic outgroup , Capsaspora owczarzaki ( Suga et al . , 2013 ) -- have bHLH-PAS family proteins ( Figure 1a , b ) .", "No members of this protein family were found in the genomes or transcriptomes of choanoflagellates , the sister lineage to animals ( King et al . , 2008; Fairclough et al . , 2013; Carr et al . , 2017 ) .", "While one-to-one orthologs of HIFa are found in cnidarians and placozoans , sponges and ctenophores lack true one-to-one HIFa orthologs ( Figure 1a , b ) .", "The phylogeny obtained for the bHLH-PAS family ( Figure", "2 ) reveals that vertebrate HIF1a arose from a series of duplications that gave rise to HIFa , SIM1 , SIM2 , NPAS1 and NPAS3 , and that the function of the ancestral protein is unclear ( Figure 2 ) .", "The position of both sponge and ctenophore sequences in the tree suggests that sponge and ctenophore orthologs are sister to all HIF-SIM-NPAS ( HSN ) proteins , and that the lack of one-to-one HIFa orthologs reflects the primary absence of HIFa in these phyla , rather than a secondary loss .", "Despite the apparent absence of one-to-one orthologs in sponges , we examined whether key features of HIFa may be found in other sponge bHLH-PAS proteins .", "In the presence of oxygen , HIFa is hydroxylated at a key proline in the motif L[D/E]x[L/R]AP[F/Y]I that signals the protein for degradation .", "The motif varies between clades , where the LAPY motif in vertebrate HIFa is instead RAPY in most protostomes , RAPY or RAPF in cnidarians , and LAPF in placozoans ( Figure 3 ) .", "None of these motifs were found in any of the sequences from the 26 sponge genomes and transcriptomes in our set .", "However , a similar motif ( LAMRAPYI ) was found in the C-terminal domain of a bHLH-PAS protein from the ctenophore Euplokamis dunlapae , but not in any of the other eight ctenophores .", "The residues surrounding this motif do not align well with the ODD domains from HIFa in other animals , thus the role of this protein cannot be determined by this study .", "However , these results must be interpreted cautiously , particularly as much of the data for our study species derive from transcriptomes .", "The C-terminal domains of nearly all bHLH-PAS proteins are enriched for repeats of proline , serine , and glutamine , which makes them difficult to align and may cause downstream problems in tree generation .", "As prolines are highly abundant ( Figure 3b ) , LAP or RAP motifs can be identified in many sponge sequences by chance , but also proteins that are not known to have oxygen-dependent degradation , including human NPAS1 and NPAS3 , suggesting that this short motif may occur without functional relevance .", "Additionally , prolines have a unique structural role in proteins as they are mostly inflexible , meaning they rarely can be exchanged for other amino acids .", "For this reason , alignment programs may cluster sequences around prolines , resulting in apparent alignments of non-homologous domains ( Figure 3c , d ) .", "Since we do not find the full hydroxylation motif in any sponge bHLH-PAS protein , none of these proteins are expected to function like HIFa .", "VHL orthologs were not found in any ctenophore or sponges outside of a few demosponges ( Figure 1b , Figure 2—figure supplement 1 ) .", "We were also unable to find EGL9/PHD orthologs in any sponge or ctenophore , although we found related genes in microbial eukaryotes , including choanoflagellates ( Figure 1b , Figure 2—figure supplement 2 ) .", "We were , however , able to identify homologs of the Factor Inhibiting HIF ( FIH/HIF1AN ) in sponges ( Figure 1b , Figure 2—figure supplement 3 ) , a protein involved in regulation of HIF by hydroxylating C-terminal asparagines ( Zhang et al . , 2010 ) .", "However , this protein has other targets ( Wilkins et al . , 2012 ) and could serve a more general function in sponges , unrelated to oxygen metabolism .", "Overall , the absence of essential components of the HIF pathway in sponges and ctenophores further suggests that these taxa are unable to modulate their transcriptional state in response to low oxygen levels , at least not in the same way as the remaining animal phyla .", "To test whether sponges regulate their transcription in response to low-oxygen availability , we examined transcriptomes from clones of the demosponge Tethya wilhelma ( Sarà et al . , 2001 ) kept at different oxygen levels over the course of 4 days .", "Between our low-oxygen treatments , which involved progressively lowering the oxygen concentration until it reached 0 . 25–2% of modern atmospheric saturation ( AS ) ( 0 . 5–4 µM O2 at 26°C and a salinity of 32 ) , and our high-oxygen controls ( 94–100% AS or 198–211 µM O2 ) , we found 128 differentially expressed genes ( Benjamini-Hochberg corrected p-value<0 . 01 ) out of the 37 , 000 total genes predicted in the genome ( Francis et al . , 2017 ) ( Supplementary file 2 ) .", "None of these differentially expressed genes have predicted functions related to metabolism or stress ( based on similarity to any SwissProt protein or Amphimedon queenslandica proteins ) , while 46 have no matches to any known protein in our set .", "This response contrasts sharply with HIF-mediated changes in other animals and supports the assessment that sponges lack the HIF pathway , and have not independently evolved a comparable oxygen-sensing transcription factor .", "For example , in the placozoan Trichoplax adhaerens , exposure to 10 µM O2 resulted in the upregulation of glycolytic enzymes , and death within 5 hr ( Loenarz et al . , 2011 ) .", "In cnidarians , polyps and medusae of Aurelia sp . 1 exhibited higher expression of HIFa after exposure to 16 µM O2 for 18 hr , relative to polyps and medusae kept under 250 µM O2 for the same length of time ( Wang et al . , 2014 ) .", "Therefore , the transcriptional response of T . wilhelma to low oxygen is clearly distinct from the responses seen in HIF-bearing non-bilaterian animals ( i . e . placozoans and the cnidarians ) .", "After exposing T . wilhelma to complete anoxia in a closed , non-circulating system for 1 hr , 5981 genes were differentially expressed ( Benjamini-Hochberg corrected p-value<0 . 01 ) relative to the 4 day , high-oxygen control experiments .", "Of this set , 569 genes were differentially overexpressed ( log fold change ≥ 2 ) and 142 genes were differentially underexpressed ( log fold change ≤ −2 ) in treated vs . control sponges ( Supplementary file 3 ) , and include some metabolic genes ( glycogen debranching enzyme , three heme binding proteins , 10 ubiquitin ligases ) , implying that the sponges may be stressed by these conditions .", "As with the low-oxygen treatment , half of the differentially expressed genes lack a reliable BLAST hit to an annotated protein , making it difficult to define which biological processes are most affected .", "Curiously , one of the most strongly upregulated genes is a cryptochrome , a light-dependent regulator of bHLH-PAS proteins involved in circadian rhythms ( Rivera et al . , 2012 ) , suggesting potential for cross-talk between circadian rhythms and redox state , as seen in some vertebrates ( Rutter et al . , 2001; Hirayama et al . , 2007 ) .", "All six bHLH-PAS proteins were upregulated to some degree , whereby the log fold change was greater than one for only the HSN-like group one proteins ( see Figure", "2 ) and the ARNT homolog , although it is unclear what controls their expression , whether any of these proteins regulate themselves , or what the downstream targets may be .", "We did not explore how long T . wilhelma can survive complete anoxia .", "Overall , our data suggests that metabolic function in T . wilhelma is unaffected by oxygen levels as low as 0 . 25% AS , the lowest levels observed , and that complete anoxia in still water induces stress -- at least when assayed at the mRNA level .", "T . wilhelma performs periodic full-body contractions under air-saturated oxygen levels ( Nickel , 2004 ) .", "Accepting this as normal behavior , we tested whether contractile dynamics were influenced by reduced oxygen concentrations .", "During our 4-day experiments , we observed no significant difference in contraction rate under oxygen concentrations between 4 and 100% AS ( 8 . 45–211 μM O2 ) ( Figure 4a , Video 1 ) .", "Full-body contractions did cease , however , at ≤1 . 86% AS ( 3 . 9 µM O2 ) ( Figure 4a ) .", "We are uncertain as to why contractions ceased under these low-oxygen levels , but in separate oxygen drawdown experiments , we observed elevated rates of oxygen uptake during the contraction period ( Figure 4d ) , potentially signifying that the sponges were energetically limited under ≤1 . 86% AS .", "However , the transcriptomes of the treatment and control sponges did not significantly differ ( Figure 5a; Adonis Pseudo-F = 1 . 6138 , p=0 . 06 ) , showing that the cessation of contraction was not associated with any shifts in gene regulation .", "Furthermore , local sub-contractions across the sponge surface resumed under oxygen levels as low as 0 . 25% AS ( 0 . 53 µM O2 ) .", "In summary , independently of whether these sponges were unable to contract , or no longer needed to -- perhaps to regulate their internal redox balance , as seen in the demosponge Geodia barretti ( Hoffmann et al . , 2005 ) -- exposure to 0 . 25–1 . 86% AS ( 0 . 53–3 . 9 µM O2 ) was not associated with any visible or transcriptional indicators of stress or death , suggesting that T . wilhelma remains viable under these levels ." ], [ "As shown here , the HIF pathway evolved only once in animals , after the last common ancestor ( LCA ) of Bilateria + Cnidaria + Placozoa split from sponges and ctenophores .", "Therefore , the HIF pathway is not a universal metazoan trait , as previously thought ( Loenarz et al . , 2011 ) .", "While sponges and ctenophores lack the capacity to sense oxygen via transcriptional regulators like HIF , they likely detect and respond to oxygen availability at the cellular level via other mechanisms , such as the allosteric regulation of heme proteins , or changes in the conductance of O2-sensitive ion channels ( Chandel and Schumacker , 2000 ) .", "At the mitochondrial level , electron transport and oxidative phosphorylation cease under anoxia , while hypoxia reversibly lowers the maximum reaction rate ( Vmax ) of the cytochrome C oxidase , thereby lowering the mitochondrial redox state and promoting the production of reactive oxygen species ( Chandel and Schumacker , 2000 ) .", "Cytochrome C is also inhibited by hydrogen sulfide ( Powell and Somero , 1986; Cooper and Brown , 2008 ) , which may serve as an indirect mechanism of oxygen detection , as environmental or metabolic sulfide can accumulate under anoxia and is removed in the mitochondria by a series of O2-dependent reactions to produce sulfate .", "Most of the enzymes involved in this sulfide-removal pathway are found in all non-bilaterian groups ( Figure 1—figure supplement 1 ) , implying that these mechanisms are conserved , and that the absence of oxygen can be more universally detected among animals at the mitochondrial level through the presence of sulfide .", "Many metazoan transcription factors contain bHLH domains with diverse roles ( Simionato et al . , 2007 ) .", "PAS domains are found universally , including in the oxygen-sensing FixL in bacteria ( Green and Paget , 2004 ) , and the circadian rhythm protein WC1 in the fungus Neurospora crassa ( Tauber et al . , 2004 ) .", "However , the combination of the bHLH and PAS domains was found only in metazoans and the single-celled eukaryote C . owczarzaki , showing that this domain combination pre-dates the origin of animals ( Sebé-Pedrós et al . , 2012 ) .", "PAS domains have binding pockets capable of holding small molecules , such as heme in the case of fixL , and flavin for WC1 .", "Recent crystal structures of several mouse bHLH-PAS proteins suggest potential binding pockets in both PAS domains , as well as the dimerization interface ( Wu et al . , 2016 ) .", "Thus , even bHLH-PAS proteins uninvolved in the HIF pathway could be indirectly affected by the redox state of ligands , such as flavins .", "Conceivably , the presence of oxidized or reduced factors , such as FAD or NADH , could affect dimerization , which may affect downstream transcription .", "We identified a bHLH-PAS protein with a similar ODD motif ( Figure 3d ) in the ctenophore E . dunlapae , presenting a conundrum of whether HIFa was ancestrally present in ctenophores and then lost .", "We consider this possibility unlikely based on the bHLH-PAS tree ( Figure 2 ) , where the E . dunlapae protein groups with other ctenophore proteins and not with HIFa ( or with SIM/NPAS ) .", "Since we were unable to find EGL9 or VHL in any available ctenophore transcriptome or genome , the function of the E . dunlapae bHLH-PAS protein is unlikely be the same as HIFa , or at least cannot involve the same binding partners .", "Interestingly , E . dunlapae belongs to the sister group to all other ctenophores ( Simion et al . , 2015 ) , yet the homologs of the E . dunlapae protein in other ctenophores lack the ODD motif , raising the question of how and why the ODD motif was lost in the rest of the phylum .", "Overall , additional experimental investigation and genome sequencing -- particular on E . dunlapae and closely related species -- are still needed to resolve the question of oxygen regulation in ctenophores .", "The phylogenetic position of ctenophores has been a topic of recent controversy ( King and Rokas , 2017 , e . g . Ryan et al . , 2013; Pisani et al . , 2015 ) .", "The majority of analyses recovered either sponges or ctenophores as sister to all other animals , but agreed on the position of Placozoa as the sister to a Cnidaria + Bilateria clade .", "Most components of the HIF pathway are absent in both sponges and ctenophores , with all the components present in the clade of Bilateria + Cnidaria + Placozoa .", "Therefore , the acquisition of the HIF pathway is independent of the phylogenetic position of ctenophores , as it occurred after the divergence of both ctenophores and sponges from all other animals ( Figure 6 ) .", "Furthermore , recent molecular clock estimates suggest that all non-bilaterian phyla emerged in rapid succession between 850 and 820 Ma , and that this timing was independent of the phylogenetic position of ctenophores ( Dohrmann and Wörheide , 2017 ) .", "Despite lacking the HIF pathway , both sponges and ctenophores live under low oxygen in nature .", "Sponges are found at oxygen concentrations as low as ~3–8 µM O2 along the lower boundary of the Peruvian oxygen-minimum zone ( OMZ ) ( Mosch et al . , 2012 ) , and at ~4 µM O2 on the lower summit of the Volcano 7 Seamount , Eastern Tropical Pacific ( Levin , 2003 ) .", "Therefore , natural sponge populations live at oxygen concentrations approaching those under which we observed normal transcription in T . wilhelma ( <4 µM O2 ) .", "Some sponges can also withstand seasonal anoxia .", "For example , marine sponges in Lake Lough Hyne , Ireland can apparently endure anoxia during summer thermal stratification ( Bell and Barnes , 2000 ) , and freshwater sponge gemmules can survive anoxia for months ( Reiswig and Miller , 1998 ) .", "Therefore , at least some sponge species can establish themselves under low-oxygen levels in nature , despite lacking the HIF pathway .", "Ctenophores , the only other animal phylum predicted to lack the HIF pathway , are well known for inhabiting low-oxygen environments ( Purcell et al . , 2001 ) .", "For example , in the OMZ of the North Chilean coast , the highest abundance of ctenophores ( as determined through rRNA abundance ) was found at oxygen levels of 6 . 1 µM O2 , defining the upper oxic-anoxic interface ( Parris et al . , 2014 ) .", "Ctenophores were also found , although at lower abundance , in the anoxic core of the OMZ ( Parris et al . , 2014 ) .", "Similarly , in the Eastern Tropical North Pacific , plankton nets collected the highest abundance of ctenophores in OMZ waters with oxygen concentrations as low as 3–30 μM ( Beatteay , 2012 ) .", "Overall , ctenophores have low metabolic oxygen demands , and feature most metabolically active cells in direct contact with the environment ( Thuesen et al . , 2005 ) , thereby facilitating life under low-oxygen ( Purcell et al . , 2001 ) .", "Cnidarians , which unlike ctenophores possess the HIF pathway ( Loenarz et al . , 2011; Wang et al . , 2014 ) , have similar distributions along modern oxygen gradients ( Purcell et al . , 2001; Parris et al . , 2014 ) , demonstrating that adaptation to life in low-oxygen environments is not predicated on the possession of HIF .", "Despite their morphological simplicity , placozoans appear to be relatively sensitive to low-oxygen conditions , as seen in T . adhaerens , which dies after 5 hr of exposure to 10 µM O2 ( Loenarz et al . , 2011 ) .", "In nature , placozoans are found in shallow coastal waters <20 m deep ( Eitel et al . , 2013 ) and have not , at least to our knowledge , been reported from any low-oxygen systems .", "Placozoans phagocytose microbial prey via their upper epithelia , and feed through external digestion and osmotrophy via their lower epithelia ( Eitel et al . , 2013 ) , and therefore occupy trophic levels predicted to be supported in low-oxygen environments ( Fenchel and Finlay , 1995 ) .", "Indeed , most filter-feeding sponges ( in contrast to carnivorous sponges ) also subsist on small microbial prey and dissolved organics , and therefore occupy the same trophic levels as placozoans ( Mills and Canfield , 2017 ) .", "The sensitivity of placozoans to low-oxygen conditions is therefore difficult to explain in terms of their body plan and trophic style .", "All metazoans outside of ctenophores and sponges are predicted to have the HIF pathway ( Loenarz et al . , 2011 ) .", "Outside of animals , similar mechanisms of transcriptional regulation by oxygen-dependent degradation of transcription factors have been identified in fungi ( Lee et al . , 2009 ) , the social amoeba Dictyostelium ( van der Wel et al . , 2005 ) , and plants ( Licausi et al . , 2011 ) -- although these systems all use different protein components .", "Overall , it appears that oxygen-dependent transcriptional control has evolved convergently in multiple multicellular eukaryotic lineages .", "The ancestral absence of the HIF pathway in metazoans suggests that stem-group metazoans and the metazoan LCA did not regulate their transcription in response to oxygen availability .", "While it is unclear how much oxygen crown-group animals ancestrally required ( Mills and Canfield , 2014 ) , animals most likely originated prior to the establishment of modern atmospheric oxygen levels and the permanent oxygenation of the deep ocean ( Lenton and Daines , 2017 ) .", "In this case , the ancestral absence of HIF in animals suggests that the earliest metazoans could have functioned and respired under environmental oxygen concentrations as low as 0 . 25% of modern atmospheric saturation , and perhaps even lower , and did not need to respond to low-oxygen availability at the transcriptional level ." ], [ "Our T . wilhelma specimens developed from buds grown in the laboratory , and were ultimately clones of specimens collected from the type locality in the aquarium of the Wilhelma Zoological and Botanical Garden ( Stuttgart , Germany ) .", "Sponges were maintained in aquaria of artificial seawater kept at 26°C and salinity of 32 .", "For our long-term experiments , individual sponges were placed in a flow-through system that continuously subjected the sponges to circulating aquarium water sparged with a controlled air-N2 mixture .", "For a given experimental run , an individual sponge was placed inside a 100-mL blue cap bottle , where the sponge rested upon black polypropylene mesh netting , supported by a ring of black polyurethane foam , overlying a magnetic stir bar .", "For photographic documentation , the outside of the blue cap bottle was covered with black felt to enhance the contrast between the sponge and its background , with an opening in the felt for visibly exposing the sponge .", "The lid of the blue cap bottle was modified to accommodate two hollow glass tubes for the introduction and removal of artificial seawater .", "The O2 content of the bottle was measured using a two-channel Firesting oxygen meter ( Pyroscience , Germany ) , with a sensor spot affixed to the inside of the bottle , and an optical fiber mounted correspondingly on the outside .", "Artificial seawater circulated throughout our system within a single , self-contained loop via a peristaltic pump set at 3 . 0 mL min-1 .", "First , water was drawn out of the main aquarium through silicone tubing that entered and coiled within a 2-L glass reservoir filled with filter-sterilized ( 0 . 22 µm pore size ) artificial seawater .", "This reservoir water was sparged with an air-N2 mixture set by a gas mixer , and well mixed with a magnetic stir bar to control its O2 content .", "After the circulating aquarium water equilibrated with the sparged reservoir water ( thereby reaching the desired O2 concentration ) , it exited the reservoir and was transported ( via Iso-Versinic and glass tubing ) into the 100-mL blue cap bottle containing the single T . wilhelma specimen .", "Water exited the 100-mL blue cap bottle through the lid via overpressure and was redirected back to the aquarium system , thereby completing the circulation loop .", "The 2-L glass sparging reservoir and the 100-mL blue cap bottle housing the sponge both sat in a water bath maintained at ~26°C using a circulating temperature controller , monitored with the Firesting through its external temperature function and thermometer .", "Firesting O2 and temperature measurements were taken every 30 s using the associated Profix software ( Pyroscience , Germany ) .", "The sponge , while inside the 100-mL blue cap bottle , was photographed every 30 s with a Panasonic DMC-GX7 digital camera .", "Photos were subsequently uploaded , and analyzed in MATLAB using an edge-detection algorithm that identified the borders of the sponge against the background of each frame , and calculated the sponge’s projected area in pixels .", "Absolute projected area was then calculated after calibrating the scale ( a transparent metric ruler placed in front of the blue cap bottle , with the sponge positioned as close to the glass side as possible ) present in certain frames .", "Each experimental run ( 4 in total ) lasted 93 . 6 hr ( SD ± 5 . 5 hr ) and began with the calibration of the oxygen sensor and the introduction of the sponge to the blue cap bottle , initially filled with air-saturated water ( [O2] = 90–100% AS = 190–211 µM O2 given T = 26°C and S = 32 ) .", "The gas mixture was immediately set to 90% N2 and 10% air , ultimately bringing the reservoir water , and then the circulating aquarium water , to an O2 concentration of ~10% AS .", "This concentration was met relatively quickly ( 15–20 min ) in the sparged reservoir water , but took several hours to level out in the blue cap bottle .", "The set O2 level ( 10% AS ) was then maintained for an entire 24 hr after leveling out .", "O2 was then lowered again , leveling out ≤5% AS , where it was maintained for another 15–20 hr .", "Targeting the same O2 levels was difficult , even with identical gas mixture and peristaltic pump settings , so while two experimental runs leveled out 4–5% AS after transitioning from 10% AS , the two other runs leveled out closer to 2% AS ( perhaps indicating higher levels of O2 uptake in the experimental system ) .", "After this second stable O2 level was reached and maintained , O2 was lowered a final time to the lowest levels investigated , 0 . 43–1 . 85% AS .", "These levels again were maintained for another 15–20 hr before the experiments were terminated .", "To account for any drift in the oxygen signal , each experimental run ended with a second calibration of the oxygen sensor .", "We also conducted three negative controls in the same setup , where the gas mixture was set to 0% N2 and 100% air , thereby subjecting the sponges to oxygen levels > 90% AS for the duration of the experiment ( 91 . 3 hr , ±SD 4 . 8 hr ) .", "At the end of every low-oxygen experiment and high-oxygen control , each sponge was removed from the system and frozen in liquid N2 for subsequent RNA extraction ( discussed below ) .", "We analyzed the contraction data in R ( package lme4 ) using a generalized linear mixed-effects model ( GLMM ) with a Poisson distribution .", "To account for the fact that our setting required us to sample each individual multiple times , making our analysis a repeated measures one , we included each sponge as a random effect in the model .", "Significance of the Between and Within effects were assessed using the function Anova ( package car ) .", "Post-hoc comparisons for the factors Condition , Oxygen Level ( coded as Sampling Time in the model ) , and their interactions were tested with the function ghlt of package multcomp .", "Three sponges were placed into a non-circulating 100-mL blue cap bottle filled with artificial seawater degassed with N2 to anoxia ( <0 . 05% AS , 105 nM O2 ) .", "Adding the sponges re-introduced O2 to the system , reaching ca .", "10% AS , with anoxia returning 50 min later .", "The sponges were then kept under anoxia and still water for a total of 66 min , before they were removed and then frozen in liquid N2 for subsequent RNA extraction ( below ) .", "For protein trees , candidate proteins were identified by reciprocal BLAST alignment using blastp or tblastn using an e-value threshold of 10−5 .", "Datasets are listed in Supplementary file 3 , which included data from 26 sponge species and nine ctenophore species .", "All BLAST searches were done using the NCBI BLAST 2 . 2 . 29 + package ( Camacho et al . , 2009 ) .", "Because most functions were described for human , mouse , or fruit fly proteins , these served as the queries for all datasets .", "Candidate homologs were kept for analysis if they reciprocally aligned by blastp to a query protein , which was usually human .", "Alignments for protein sequences were created using MAFFT v7 . 029b , with L-INS-i parameters for accurate alignments ( Katoh and Standley , 2013 ) .", "Phylogenetic trees were generated using RAxML-HPC-PTHREADS v8 . 2 . 9 ( Stamatakis , 2014 ) , using the PROTGAMMALGF model for proteins and 100 bootstrap replicates with the ‘rapid bootstrap’ ( -f a ) algorithm and a random seed of 1234 .", "Domains were identified in bHLH-PAS proteins using the hmmsearch program of the HMMER package ( Eddy , 2011 ) , with a e-value cutoff of 0 . 1 and bitscore threshold of 10 .", "PFAM-A was used as the query database .", "Total RNA was extracted using Trizol reagent .", "Briefly , sponges were homogenized in Trizol and extracted using Chloroform followed by an isopropanol precipitation .", "The RNA extractions were quantified using a Nanodrop 1000 and quality controlled using a Bioanalyzer 2100 .", "Strand-specific libraries were prepared using Lexogen’s SENSE mRNA-Seq Library Prep Kit V2 , pooled and sequenced ( 50 bp SE ) on two lanes of an Illumina HiSeq-1500 .", "The resulting single-end reads were mapped against the T . wilhelma filtered gene set with Bowtie2 ( Langmead and Salzberg , 2012 ) with default parameters , allowing only single-mapping .", "To determine the set of differential expressed genes , the count matrix was analyzed using the R package DESeq2 .", "Differentially expressed genes , protein alignments , and the scripts used to analyse the data are available at the project repository ( https://bitbucket . org/molpalmuc/sponge-oxygen ) .", "Raw reads have been deposited at NCBI SRA under BioProject accession PRJNA380886 ." ] ]

[ "Animals have a carefully orchestrated relationship with oxygen .", "When exposed to low environmental oxygen concentrations , and during periods of increased energy expenditure , animals maintain cellular oxygen homeostasis by enhancing internal oxygen delivery , and by enabling the anaerobic production of ATP .", "These low-oxygen responses are thought to be controlled universally across animals by the hypoxia-inducible factor ( HIF ) .", "We find , however , that sponge and ctenophore genomes lack key components of the HIF pathway .", "Since sponges and ctenophores are likely sister to all remaining animal phyla , the last common ancestor of extant animals likely lacked the HIF pathway as well .", "Laboratory experiments show that the marine sponge Tethya wilhelma maintains normal transcription under oxygen levels down to 0 . 25% of modern atmospheric saturation , the lowest levels we investigated , consistent with the predicted absence of HIF or any other HIF-like pathway .", "Thus , the last common ancestor of all living animals could have metabolized aerobically under very low environmental oxygen concentrations ." ]

[ "Almost all animals need oxygen to live .", "This is because they use oxygen to release much of the energy locked up in their diets .", "Oxygen may have also played a crucial role in the early evolution of animal life .", "Animals evolved from single-celled ancestors in the ocean over 800 million years ago .", "Before then , it is debated whether the atmosphere and ocean had enough oxygen to permit animals to evolve .", "Oxygen levels are much higher now , but oxygen availability still varies in some environments .", "If oxygen becomes limited ( a condition known as hypoxia ) , almost all animals react using a specific set of molecules known as the HIF pathway .", "This pathway – which is named after proteins called “hypoxia-inducible factors” – triggers changes that help the animal to maintain a stable level of oxygen in its cells .", "Yet it was not clear if the capacity to sense hypoxia and regulate oxygen demands within the body evolved in the ancestor of all animals , or if it evolved more recently .", "When trying to understand early evolution , scientists often turn to some living species that sit on the oldest branches of a group’s family tree .", "In the animal kingdom , sponges and comb jellies occupy those branches .", "Mills , Francis et al . have now searched the genomes of several of these animals to ask how oxygen sensing evolved .", "The genomes of the sponges and comb jellies surveyed lack key components of the HIF pathway , suggesting that the last common ancestor of living animals lacked the HIF pathway as well .", "This also implies that the ancestor of all animals probably did not respond to oxygen stress or used unknown mechanisms to deal with it instead .", "In laboratory experiments , Mills , Francis et al . saw that a marine sponge named Tethya wilhelma does not alter its gene activity even when the oxygen levels are reduced to 0 . 25% of modern levels .", "This is consistent with the predicted absence of a HIF pathway or anything similar .", "Together these finding may indicate that the last common ancestor of all living animals maintained normal gene activity even at very low concentrations of oxygen .", "These findings help scientists understand how life and the global environment have shaped each other since the origin of life over 3 . 5 billion years ago .", "This fundamental knowledge may provide the context needed to help society navigate through current and on-going environmental changes , including the dropping oxygen levels in the world’s oceans ." ]

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

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

[ [ "The Rho family GTPases , including Rho , Rac and Cdc42 , are essential signaling proteins that mediate morphological changes in cells by directing local cytoskeletal rearrangements ( Bishop and Hall , 2000; Kimura et al . , 1996 ) .", "These rearrangements are generally initiated at and confined to specific subcellular regions .", "For example , a narrow , concentrated zone of Rho activity directs the formation of a ring of actin filaments and myosin-2 at the equatorial cortex that drives cytokinesis ( Bement et al . , 2005; Yonemura et al . , 2004; Yüce et al . , 2005 ) .", "Similarly , Rho , Rac and Cdc42 are activated near the leading edge of crawling cells in patterns that correspond to local cycles of protrusion , adhesion and retraction ( Machacek et al . , 2009; Martin et al . , 2016 ) .", "Because tight spatiotemporal regulation of the GTPases is a fundamental feature of these cellular processes , considerable effort has been invested in studying GTPase regulation .", "The RhoGTPases are classically characterized as cycling between membrane-associated , active states and soluble , inactive states as a result of interactions with three classes of regulatory proteins: guanine nucleotide exchange factors ( GEFs ) , which activate GTPases by promoting exchange of GDP for GTP ( Rossman et al . , 2005 ) ; GTPase activating proteins ( GAPs ) , which inactivate GTPases by promoting GTP hydrolysis ( Moon and Zheng , 2003 ) ; and guanine nucleotide dissociation inhibitors ( GDIs ) , which solubilize GTPases to generate a large reservoir of heterodimeric GTPase:GDI complexes in the cytoplasm ( Garcia-Mata et al . , 2011 ) .", "In the canonical model of GTPase regulation , GTPase cycling is thought to proceed as follows: a GTPase is activated by a GEF at the plasma membrane following its release from GDI , is subsequently inactivated by a GAP , and is then returned to the soluble pool by GDI .", "Thus , the lifetime of GTPase activity at the plasma membrane is generally thought to be controlled entirely by GEFs and GAPs , with GDIs essentially serving as passive shuttles that interact exclusively with inactive GTPases .", "The notion that RhoGDIs work as passive shuttles rests largely on two findings .", "First , when GTPase:GDI complexes are purified from cell lysates , the great majority of GTPase within the complex is in the inactive , GDP-bound form ( Abo et al . , 1994 ) , as expected if GDI solubilizes GTPases after inactivation by a GAP .", "Second , binding of GTPases by GDI strongly suppresses GTP hydrolysis ( Hart et al . , 1992 ) , indicating that hydrolysis must precede extraction from the membrane .", "However , work by several labs has shown that GDIs bind both inactive and active GTPases with relatively high affinity in vitro ( Hancock and Hall , 1993; Hart et al . , 1992; Nomanbhoy and Cerione , 1996; Tnimov et al . , 2012 ) , leading to the suggestion that GDIs may interact with active as well as inactive GTPases in vivo .", "As such , GDIs might have the potential to exert a more direct role in the regulation of GTPase activity than currently appreciated .", "In contrast to GEFs and GAPs , the biochemical activities of GDIs are not well understood .", "Presently , no consensus exists as to ( 1 ) the mechanism by which GDIs extract GTPases from membranes ( Johnson et al . , 2009; Zhang et al . , 2014 ) , ( 2 ) whether GDIs interact with GTPases in a nucleotide-specific manner ( Nomanbhoy and Cerione , 1996; Tnimov et al . , 2012 ) , or ( 3 ) how GDI activity is coordinated with GEFs or GAPs ( Garcia-Mata et al . , 2011 ) .", "This uncertainty stems in part from experimental limitations in studying GTPase:GDI dynamics .", "First , fusion with fluorescent proteins at the amino terminus can impair GTPase localization ( Yonemura et al . , 2004; Yüce et al . , 2005 ) and function ( Watson et al . , 2014; Freisinger et al . , 2013; Howell et al . , 2012; Bendezú et al . , 2015; Lee et al . , 2015; Coll et al . , 2007 ) while fusion with the carboxyl-terminus prevents prenylation ( Howell et al . , 2012 ) .", "In the absence of direct visualization , GTPase dynamics had to be inferred from activity probes .", "Second , with a few important exceptions ( Johnson et al . , 2009; Nomanbhoy et al . , 1999 ) , in vitro studies of GTPase:GDI dynamics have utilized unprenylated GTPases , omitted membranes , or both .", "Additionally , nearly all of these reconstitution experiments focused on the effect of GDI on membrane-associated or soluble GTPases at thermodynamic equilibrium ( Zhang et al . , 2014 ) .", "Thus , we do not currently understand how GDI affects the transitions between membrane and soluble GTPase states kinetically .", "This is especially true under conditions which mimic the cellular environment , which is far from equilibrium due to the constant dissipation of energy .", "To overcome these limitations , we developed two distinct methods to directly visualize vertebrate RhoGTPases in vivo and on supported lipid bilayers in vitro .", "Using these tools , we identify co-existing pools of active and inactive GTPases associated with the plasma membrane and provide additional evidence that GDI can extract both inactive and active GTPases in vitro , as suggested by previous steady-state affinity measurements ( Hancock and Hall , 1993; Leonard et al . , 1992; Nomanbhoy and Cerione , 1996; Tnimov et al . , 2012 ) .", "Finally , we show that the extraction of active GTPase also occurs in vivo and that this contributes to the spatial regulation of GTPase activity .", "Collectively , these results indicate that GDI itself can indeed directly mediate the spatiotemporal regulation of GTPase activity ." ], [ "Fusion of fluorescent proteins with the carboxy-terminus of RhoGTPases prevents prenylation , while fusion with the amino-terminus can impair their localization and function .", "This is likely due to occlusion of the switch I/II regions of the GTPase , which is the binding interface for all of its protein:protein interactions ( Dvorsky and Ahmadian , 2004 ) .", "Regardless of the explanation , it has been demonstrated that GFP- and YFP-Rho fail to concentrate at the cell equator during cytokinesis in mammalian cells ( Yonemura et al . , 2004; Yüce et al . , 2005 ) .", "Further , in gene replacement studies in yeast , amino-terminally tagged Cdc42 fails to localize properly , yields temperature sensitivity and/or aberrant morphology ( Bendezú et al . , 2015; Coll et al . , 2007; Freisinger et al . , 2013; Howell et al . , 2012; Lee et al . , 2015; Watson et al . , 2014 ) .", "We have also found that mCh-Rho , Rac and Cdc42 are not properly recruited to wounds in Xenopus laevis oocytes .", "Specifically , the zones of recruitment defined by amino-terminally tagged GTPases are much less focused and much less intense than those obtained with either the activity reporters or the internally-tagged GTPases ( Figure 1—figure supplement 1; see below for functional analysis ) .", "To overcome this problem , we first adapted an approach described by Bendezú et al . ( 2015 ) for labeling of yeast Cdc42 .", "We inserted GFP into a solvent-exposed external loop of the X . laevis GTPases ( see Methods ) .", "To test the internally-tagged ( IT ) GTPases in vivo , we exploited the cell wound repair model in X . laevis oocytes where wounding elicits a robust accumulation of active Rho and Cdc42 in discrete , concentric zones at the cortex as previously indicated by GTPase activity reporters ( Figure 1A; Benink and Bement , 2005 ) .", "It is important to note that ( 1 ) IT-GTPases were co-expressed with wild-type ( WT ) GDI to avoid disrupting the GTPase:GDI stoichiometric ratio , thereby preventing GTPase aggregation ( Boulter et al . , 2010 ) , and ( 2 ) IT-GTPases were expressed at the minimal level necessary to detect signal around the wound ( 36% above endogenous Rho , based on proteomic data from Wühr et al . , 2014 ) to avoid potential overexpression phenotypes ( see Materials and methods; Figure 1—figure supplement 2 ) .", "Both IT-Rho and IT-Cdc42 were recruited to concentric rings around the wound ( Figure 1B , C ) .", "Comparison of IT-Rho to a Rho activity reporter ( mRFP-2xrGBD; Davenport et al . , 2016 ) revealed that IT-Rho spatially overlapped with the Rho activity zone .", "Comparison of IT-Cdc42 to a Cdc42 activity reporter ( mRFP-wGBD; Benink and Bement , 2005 ) revealed that IT-Cdc42 localized throughout the active Cdc42 zone , as well as extended slightly beyond it towards the wound center ( see also below ) .", "We also tested the behavior of IT-Rac and found that it concentrated around wounds in the same region as IT-Cdc42 , as expected from previous experiments ( Figure 1—figure supplement 3; Abreu-Blanco et al . , 2014; Benink and Bement , 2001 ) .", "As an alternative approach , and as a means to obtain labeled RhoGTPases that could be used both in vivo and in vitro , purified recombinant Rho and Cdc42 were prenylated , coupled to Cy3 via a short N-terminal peptide by sortase-mediated ligation , and bound to GDI ( see Materials and methods ) .", "Cy3-Rho and Cy3-Cdc42 , bound to GDI for stabilization , were microinjected into oocytes 41% and 53% above endogenous levels , respectively .", "Both Cy3-Rho and Cy3-Cdc42 localized to wounds ( Figure 1D , E ) , in a manner indistinguishable from their IT counterparts expressed in the oocyte ( Figure 1F , G ) .", "As observed with IT-Rho and IT-Cdc42 , Cy3-Rho completely overlapped with the zone of Rho activity while Cy3-Cdc42 localized throughout and slightly interior to the active Cdc42 zone .", "These results indicate that the IT- and Cy3-tagged GTPase variants faithfully mimic endogenous GTPases during cell wound repair .", "To further test the behavior of the IT- and Cy3-labeled RhoGTPases , we sought to determine if they localize to the plasma membrane in other cellular processes .", "This is important because these processes likely depend on different regulators from those that operate during cell wound repair .", "IT-Rho and Cy3-Rho localized to the cytokinetic apparatus and epithelial junctions in Xenopus embryos , consistent with previous results obtained with a Rho activity reporter ( Figure 2A–C; Bement et al . , 2005 ) .", "Similarly , IT-Cdc42 localized to exocytosing cortical granules ( Figure 2D ) , consistent with previous results obtained with a Cdc42 activity reporter ( Yu and Bement , 2007 ) .", "IT-Cdc42 was also recruited to cell-cell junctions and enriched there upon wounding ( Figure 2E ) , a behavior previously revealed using a Cdc42 activity reporter ( Clark et al . , 2009 ) .", "Next , we wanted to determine whether IT-RhoGTPases can functionally substitute for the endogenous GTPases .", "The X . laevis oocyte system is not conducive to traditional knockdown approaches due to its large stores of maternal protein and relatively slow protein turnover .", "Therefore , we employed C3-exotransferase , a Rho-specific toxin , to inhibit endogenous Rho activity , and expressed an IT-Rho in which the C3 ribosylation site ( N41 ) is mutated to a residue that cannot be ribosylated ( N41V; Sekine et al . , 1989 ) .", "In control oocytes expressing the probe for active Rho , Rho activity around wounds was suppressed by C3 ( Figure 2F , G ) , while cells expressing IT-Rho-N41V generated a spatially defined zone of Rho activity around the wound that closed over similar timescales as the control .", "While there is a slight decrease in Rho activity in IT-Rho-N41V cells exposed to C3 versus without , we attribute the difference to the loss in endogenous Rho activity .", "In contrast , cells expressing amino-terminally tagged Rho ( mCh-Rho-N41V ) failed to rescue Rho activity upon inhibition of endogenous Rho by C3 ( Figure 2H , Figure 2—figure supplement 1 ) .", "Collectively , these results indicate that , unlike amino-terminally tagged GTPases , both IT- and Cy3-labeled GTPases are faithful reporters of the distribution of GTPases and show that IT-Rho can functionally substitute for its endogenous counterpart .", "The observation that Cdc42 extends slightly interior to its zone of activity suggests that there may be a pool of inactive , membrane-bound Cdc42 at this location .", "This notion is consistent with the previous observation that Abr , a Cdc42-GAP thought to regulate Cdc42 activity at wounds , also localizes interior to the Cdc42 zone ( Vaughan et al . , 2011 ) .", "To understand the relationship between activity and membrane-association of Cdc42 , we overexpressed Abr , a manipulation previously shown to decrease Cdc42 activity around wounds ( Vaughan et al . , 2011 ) .", "Remarkably , this resulted in a dose-dependent loss of active Cdc42 at wounds while having far less effect on Cy3-Cdc42 ( Figure 3A , B ) .", "These results further demonstrate that the IT- and Cy3-GTPases are functional .", "More importantly , they demonstrate that substantial pools of both active and inactive GTPases can be dynamically maintained at the plasma membrane .", "Efforts to visualize RhoGDI at the plasma membrane have generally failed ( Ngo et al . , 2017 ) , likely because GDI only transiently interacts with GTPases upon release into or extraction from the membrane .", "However , we reasoned it might be possible to detect GDI at wound sites due to the high local concentration of Rho and Cdc42 .", "Indeed , we found that 3xGFP-GDI is enriched at wounds and forms a broad zone that peaks between active Rho and active Cdc42 ( Figure 4A , B ) .", "To confirm that endogenous GDI also localizes to wounds , antibodies were raised against X . laevis GDI ( Figure 4—figure supplement", "1 ) and used to immunolabel wounded oocytes .", "Consistent with the results obtained with 3XGFP-GDI , endogenous GDI accumulated at wounds ( Figure 4C ) .", "These results demonstrate that GDI accumulation occurs at discrete regions of the plasma membrane that are enriched with its GTPase clients .", "The localization of RhoGDI around wounds suggests that it might play an active role in delivery to or extraction of GTPases from the membrane and thus their spatiotemporal patterning .", "As an initial test of this possibility , we overexpressed GDI via mRNA microinjection .", "This manipulation potently suppressed both Rho and Cdc42 activity , as well as Cy3-Rho and Cy3-Cdc42 localization , suggesting that GDI exerts its effects via extraction of the GTPases ( Figure 5A ) .", "To obtain a more quantitative understanding of the relationship between GDI and GTPase activity , we microinjected purified GDI ( Figure 5—figure supplement", "1 ) at increasing concentrations prior to wounding .", "High concentrations of microinjected GDI suppressed both Rho and Cdc42 activity at wounds ( Figure 5B , C ) , consistent with the results obtained from GDI via mRNA-mediated overexpression .", "However , more modest increases revealed differential effects on Rho and Cdc42 .", "Specifically , slight increases ( 15 . 8% ) in GDI levels resulted in a greater reduction of Cdc42 activity compared to Rho ( Figure 5C , D ) .", "We found the same to be true for bovine GDI ( Figure 5—figure supplement 2 ) .", "These results show that GDI differentially impacts Rho and Cdc42 activity in vivo and that this effect does not require gross overexpression .", "To directly probe the mechanism by which RhoGDI inhibits Rho and Cdc42 activity in vivo , we established a real-time GTPase dissociation assay on supported lipid-bilayers ( SLBs ) ( Figure 6A , Figure 6—figure supplement 1; see Materials and methods ) .", "Cy3-Cdc42 was added to SLBs and binding was detected by total internal reflection microscopy ( TIRF ) .", "Binding was dependent on its C-terminal prenyl moiety , as expected ( Figure 6B ) .", "We then studied the time course of Cdc42 release from SLBs under buffer flow which continuously flushed out unbound proteins from solution .", "Spontaneous release of inactive , GDP-bound Cdc42 from the membrane was rather slow ( t1/2 = 37 . 24 ± 3 . 05 s ) , however the addition of excess GDI lead to a dramatic acceleration of dissociation ( ca . 20-fold; Figure 6C , Figure 6—figure supplement 2 ) .", "To determine whether this was the result of either simple sequestration in solution or , alternatively , direct extraction of Cdc42 from membranes , we performed assays in the presence of an alternative solubilizer ( RabGGTase beta ) that sequesters the GTPase prenyl moiety ( Figure 6C ) .", "Sequestration alone only marginally affected the rate of dissociation , demonstrating that GDI directly extracts GTPases from membranes ( Figure 6D , E ) .", "To characterize membrane extraction more quantitatively , we carried out experiments over a wide range of RhoGDI concentrations using either inactive ( GDP-bound ) or active ( GTP-bound , constitutively-active ) forms of Cdc42 or Rho .", "We observed that WT GTPases hydrolyze even GTP analogs such as GTPγS over the long time period ( hours ) required for performing a full titration in our SLB assay ( data not shown ) , which led to them accumulating in the inactive , GDP-bound form during the course of the experiment .", "We therefore turned to two constitutively-active GTPase variants: Q61L and G12V for Cdc42 ( Q63L and G14V for Rho ) .", "While often used interchangeably , the biochemical properties of these two mutants are not entirely equivalent ( Smith et al . , 2013 ) .", "The Q61L substitution directly and strongly impairs spontaneous nucleotide hydrolysis , whereas the G12V mutation only moderately inhibits spontaneous GTPase activity .", "The constitutive activity of G12V variants rather originates from defects in GAP-induced stimulation of GTP hydrolysis .", "Previous work suggests that RhoGDI interacts much more weakly with Q61L variants compared to G12V in cells ( Hodgson et al . , 2016; Michaelson et al . , 2001; Pertz et al . , 2006 ) .", "Whether this is due to these mutations differentially weakening affinity for GDI directly or , alternatively , indirect effects resulting from differences in the nucleotide states these variants assume in the cytoplasm is unknown .", "We therefore tested the extraction of both of these variants in an active state from membranes by GDI .", "In line with the known catalytic differences of these RhoGTPase variants ( Smith et al . , 2013 ) , we found that they , when purified recombinantly , assume different nucleotide states as determined by HPLC analysis: Q61L GTPases were exclusively GTP-bound , whereas G12V mutants purified as a mix of GTP and GDP bound states ( Figure 7—figure supplement 1 ) .", "To mimic a homogenously active state of G12V variants in vitro , we exchanged their bound GTP and GDP nucleotides for GTPγS directly before the extraction experiment .", "Remarkably , RhoGDI was able to extract both inactive ( GDP-bound ) and active ( GTPγS-bound , G12V/G14V ) Cdc42 and Rho in a concentration-dependent manner ( Figure 7A , B ) .", "Although GDI extracted GDP-bound GTPase more efficiently than GTPγS-bound , G12V/G14V GTPases ( 1 . 5 fold difference ) , it was still able to effectively facilitate the dissociation of the latter ( Figure 7C , F ) .", "We made qualitatively similar observations for the GTP-bound Q61L/Q63L variants , which were also extracted by GDI , albeit with reduced rates compared to their G12V/G14V counterparts ( Figure 7—figure supplements 2 and 3 ) .", "The affinities of GDI for the active and inactive GTPases on membranes , determined by hyperbolic fits to the extraction rates , were surprisingly similar ( Figure 7G , H , Supplementary file 1 ) .", "On the other hand , the maximal rates of extraction were not , indicating that the rate-limiting step of membrane extraction depends on the activity state of GTPases .", "To investigate whether extraction of active and inactive GTPases is a conserved ability among GDI proteins , we also studied mammalian GDI .", "Similar to its Xenopus ortholog , bovine GDI1 was able to extract both GDP- as well as GTP-bound Cdc42Q61L and RhoQ63L variants ( Figure 7—figure supplement 4 ) .", "These data clearly demonstrate that GDIs can directly extract both inactive and active GTPase from membranes in vitro .", "The canonical RhoGTPase cycle assumes that GDI does not extract GTPase without its prior inactivation by a GAP ( Garcia-Mata et al . , 2011 ) .", "However , the above results suggest that GDI might directly attenuate GTPase activity at the plasma membrane via extraction of active GTPase .", "If this hypothesis is correct , then expression of an extraction-deficient GDI would influence the spatiotemporal patterning of GTPase activity .", "We therefore sought to generate an extraction-deficient GDI that would still initiate contact with GTPases but fail to extract them from the plasma membrane .", "Mutants were screened by quantifying their recruitment to wounds relative to WT GDI , based on the rationale that mutants capable of binding but not extracting should remain at the membrane longer and thus accumulate at wounds more than WT GDI .", "Using this screen , we first tested RhoGDI mutants that were previously reported to be deficient in extraction ( Dransart et al . , 2005; Ueyama et al . , 2013 ) .", "None of these extraction-deficient mutants were recruited to wounds more strongly than WT GDI , suggesting that they were impaired in binding to GTPases at wounds rather than extracting GTPases from wounds ( Figure 8—figure supplement 1 ) .", "We therefore designed three novel mutants: ( 1 ) the isolated regulatory arm of GDI that initiates contact with the GTPase but lacks a binding pocket for the hydrophobic prenyl group ( Δ51–199 ) and ( 2 , 3 ) mutation of residues E158/9 previously hypothesized to be responsible for GTPase extraction ( Hoffman et al . , 2000 ) .", "GDI mutants Δ51–199 , E158/9A and E158/9Q were halo-tagged , expressed in oocytes and their recruitment to wounds was quantified relative to WT GDI .", "GDI Δ51–199 showed minimal recruitment to wounds , however both GDI E158/9A and E158/9Q showed a significant increase in recruitment to wounds relative to WT GDI , with GDI E158/9Q ( GDI-QQ ) having the greatest increase ( Figure 8A , B ) .", "To directly test whether RhoGDI E158/9Q ( QQ ) is deficient in extraction , its functional capabilities were tested in vitro in the SLB assay .", "WT GDI was able to extract both inactive ( GDP-bound ) Cdc42 as well as active ( GTPγS-bound G12V or GTP-bound Q61L ) Cdc42 from the supported lipid bilayers ( Figure 7 , Figure 7—figure supplements 2 and 3 ) .", "In contrast , GDI-QQ retained most of its ability ( less than two-fold reduction ) to extract inactive ( GDP-bound ) Cdc42 but was completely deficient in extracting active ( both GTPγS-bound G12V and GTP-bound Q61L ) Cdc42 ( Figure 8C , Figure 8—figure supplement 2 , Figure 7—figure supplement 3C , D ) .", "Similar observations were made for Rho , however GDI-QQ retained some ability to extract active GTPγS-bound G14V , but not GTP-bound Q63L Rho ( Figure 8C , Figure 8—figure supplement 2 , Figure 7—figure supplement 3 ) .", "The corresponding mutant of bovine GDI1 ( E163/4Q ) shows an equivalent behavior to its Xenopus ortholog ( Figure 8—figure supplement 3 ) .", "These results confirm that the GDI-QQ mutant is indeed extraction deficient: modestly deficient for inactive GTPases , moderately deficient for active Rho and completely deficient for active Cdc42 .", "We sought to directly test whether RhoGDI can extract active GTPases in vivo by employing GDI-QQ .", "First , we compared the effects of WT vs . QQ GDI overexpression on wounded oocytes expressing constitutively-active Cdc42 ( G12V ) ( Q61L could not be used as it failed to elevate Cdc42 levels around wounds; see Figure 9—figure supplement 1 ) .", "While WT GDI significantly reduced the amount of Cdc42 ( G12V ) around wounds , GDI-QQ did not ( Figure 9A , B ) .", "Second , we compared the effects of WT vs . QQ GDI overexpression on wounded oocytes microinjected with Cy3-Cdc42 bound to GTPɣS .", "WT GDI significantly reduced the amount of Cy3-Cdc42 ( GTPɣS ) around wounds while GDI-QQ did not ( Figure 9C , D ) .", "Collectively , these data suggest that GDI can extract active GTPase from the plasma membrane in vivo .", "The above results imply that the extraction of active RhoGTPase by GDI might contribute to its spatiotemporal patterning in vivo .", "To test this hypothesis , we expressed GDI-QQ and monitored the consequences on Rho and Cdc42 activity following wounding .", "Strikingly , Cdc42 activity around wounds was significantly elevated , in contrast to Rho which was unaffected ( Figure 9E , F ) .", "To assess whether the increase in activity was due to an increase of total Cdc42 around wounds as opposed to competition between GDI-QQ and the Cdc42 activity probe , we repeated the experiment with Cy3-Cdc42 .", "Similar to the results obtained with the activity reporter , expression of GDI-QQ elevated Cy3-Cdc42 levels around wounds relative to controls ( Figure 9G , H ) .", "Cumulatively , these data suggest that GDI directly extracts active Cdc42 throughout the Cdc42 zone , and that extraction of active Cdc42 is necessary for its regulation around wounds ." ], [ "Direct visualization of the RhoGTPases in living cells is essential for the understanding of their complex spatiotemporal dynamics .", "We have established two methods to fluorescently label vertebrate GTPases that localize properly: internal tagging with a fluorescent protein and sortase-mediated labeling with a fluorescent dye .", "This now provides us with widely applicable reagents to analyze GTPase function .", "These probes faithfully mimic the distribution of the endogenous GTPases based on their comparison to activity reporters in several processes: cell wound repair , cytokinesis , junctional integrity and epithelial wound repair .", "Further , the successful rescue of Rho function at wounds in the presence of C3 by a C3-insensitive mutant of IT-Rho indicates that these proteins are capable of replacing their endogenous counterparts .", "It will be important to assess the ability of IT- or Cy3-labeled GTPases to substitute for their endogenous counterparts in other cellular processes in the future via gene replacement approaches , although we note that IT-Cdc42 has been shown to be functional in fission yeast ( Bendezú et al . , 2015 ) .", "In any case , the combination of the two labeling approaches is powerful as it permits side-by-side comparison of results obtained in vivo and in vitro , as demonstrated here .", "Visualization of labeled RhoGTPases in combination with activity reporters in living cells led to an unexpected observation: pools of inactive Cdc42 at the plasma membrane .", "To the best of our knowledge , this is the first time that inactive GTPases have been detected on membranes under conditions other than gross GTPase overexpression .", "Notably , the pool of inactive Cdc42 spatially coincides with a local Cdc42-GAP , Abr ( Vaughan et al . , 2011 ) .", "This pool of inactive Cdc42 expands with overexpression of Abr , further demonstrating that locally-inactivated Cdc42 can remain associated with the plasma membrane .", "This finding has important mechanistic implications for the regulation of GTPase activity .", "Namely , it suggests that GTP hydrolysis and extraction of GTPases , while they are likely linked , are not necessarily tightly coupled .", "This raises the possibility that GTPases might cycle through multiple rounds of activation and inactivation by GEFs and GAPs while remaining associated with the membrane .", "Remarkably , in addition to the RhoGTPases themselves , GDI also localized to the plasma membrane in proximity to wounds .", "The localization of GDI in the same place where the GTPases are especially abundant implies that its accumulation reflects interaction with is GTPase clients .", "It will be important to investigate the detailed mechanism of GDI localization and the control of its turnover at sites of high GTPase activity in the future .", "The most significant result of this study is that RhoGDI can extract active GTPase in vivo , particularly active Cdc42 during cell wound repair .", "This finding is based on two complementary lines of evidence .", "First , in vitro assays show that GDI directly extracts active GTPases from supported lipid bilayers .", "This ability is shared between GDI orthologs from distinct species and can be robustly observed for different GTPase mutants trapped in an active configuration ( G12/14V and Q61/63L ) .", "Their moderate differences in extraction kinetics might nonetheless be biologically meaningful .", "Previous studies suggest that Q61/63L variants interact more weakly with GDI compared to their G12/14V counterparts ( Hodgson et al . , 2016; Michaelson et al . , 2001; Pertz et al . , 2006 ) ; we observe a similar trend concerning membrane extraction ( Figure 7—figure supplement 3 ) .", "Second , we also demonstrate extraction of active GTPase in vivo .", "WT , but not GDI-QQ , extracts Cdc42 ( G12V ) and GTPɣS-Cdc42 from the plasma membrane .", "These results support previous findings that GDI binds both inactive and active GTPase with relatively high affinity in vitro ( Hancock and Hall , 1993; Hart et al . , 1992; Nomanbhoy and Cerione , 1996; Tnimov et al . , 2012 ) .", "Although we report greater extraction of inactive versus active GTPase , we demonstrate that the extraction of active GTPase is important for their spatiotemporal patterning during cell wound repair .", "We thus conclude that GDI has the capacity to extract active GTPases and that this ability is harnessed to limit the level of Cdc42 activity during cell wound repair .", "This finding has the virtue of explaining previous results in the oocyte cell wound repair system .", "Based on an indirect approach involving photoactivatable Rho and Cdc42 activity reporters , it was found that Cdc42 activity is lost throughout its zone , while Rho activity is preferentially lost at the trailing edge of its zone ( Burkel et al . , 2012 ) .", "The results presented here suggest that GDI is responsible for the removal of active Cdc42 throughout the Cdc42 zone , while Rho is inactivated by a trailing edge GAP prior to extraction ( Figure 10—figure supplement 1 ) .", "This may also explain why a mild overexpression of GDI significantly reduced Cdc42 activity but had no effect on Rho activity: loss of active Cdc42 can be controlled at the level of GDI while Rho inactivation is controlled at the level of a GAP .", "However , there is an alternative explanation for why the expression of GDI-QQ causes an increase in Cdc42 activity but not Rho activity: while GDI-QQ is utterly deficient in extraction of active Cdc42 , it retains a modest ability to extract active Rho ( Figure 7 ) .", "The broader implications of RhoGDI’s ability to extract active GTPase are two-fold .", "First , it suggests that a new branch should be added to the canonical GTPase cycle in which active GTPase can directly extracted from the plasma membrane by GDI ( Figure 10 ) .", "Further , because GDI binding strongly inhibits GTP hydrolysis and nucleotide exchange ( Hart et al . , 1992; Ueda et al . , 2001 ) , active GTPase may exist in its soluble form in complex with GDI .", "However , complementary evidence from biochemical and biological studies suggest that active GTPases are less stably bound to GDI compared to their inactive form ( Hodgson et al . , 2016; Slaughter et al . , 2009; Tnimov et al . , 2012 ) .", "As such , this secondary extraction branch may actually represent a loop through which active GTPases are not only removed from cell membranes , but rapidly returned to them ( Figure 10 ) .", "Such a mechanism might enhance the spatial reach of GTPase activity within the plasma membrane or even mediate its spreading between different membrane compartments ( Palamidessi et al . , 2008 ) .", "Second , RhoGDI’s ability to extract active GTPase forces us to reassess its role in GTPase regulation in different cellular processes .", "While the field primarily studies local GTPase regulation at the level of GEFs and GAPs , we should reconsider GDI’s role in regulation , as well as the regulation of GDI itself .", "Consistent with this idea , several studies have reported that the differential phosphorylation of GDI promotes global increases in the activity of Rho , Rac or Cdc42 by modulating the affinity of GDI for different GTPases ( reviewed by Garcia-Mata et al . , 2011 ) .", "The results presented here suggest that it will be of considerable interest to assess the potential for phosphorylation and GDI-dependent regulation on a local level .", "Indeed , results from empirical ( Vaughan et al . , 2014 ) and modeling ( Holmes et al . , 2016 ) studies not only indicate that different isoforms of protein kinases C act at different regions around cell wounds , but also have different impacts on Rho and Cdc42 activity zones .", "When taken with the results of this current study , a picture emerges in which a high level of precision in GTPase patterning is achieved by a complex regulatory network comprising kinases , GEFs , GAPs , and GDI ." ], [ "The active RhoGTPase probes , mRFP-wGBD , eGFP-wGBD , eGFP-2xrGBD , BFP-2xrGBD and mRFP-2xrGBD in pCS2+ were generated as previously described ( Sokac et al . , 2003; Benink and Bement , 2005; Davenport et al . , 2016 ) .", "mCh-Rho , mCh-Rac , mCh-Cdc42 ( Benink , 2005 ) , untagged Rho , Rac , and Cdc42 ( wild-type ( WT ) and constitutively-active ( G14/2V ) ) in pCS2+ were made as previously described ( Benink and Bement , 2005 ) .", "For expression and purification from E . coli , codon-optimized Cdc42 and Rho ( Eurofins Genomics Germany GmbH , Ebersberg , Deutschland ) were subcloned into a pETMz2 vector via Gibson assembly cloning ( Gibson et al . , 2009 ) , with primers GTPase ( GeneStrand ) fwd and -rev and pETfwd and -rev ( all primer sequences in Supplementary file 2 ) .", "A pentaglycine for sortase-mediated labeling was added onto the 5’ of the GTPases .", "Constitutively-active Cdc42 Q61L and Rho Q63L mutants were generated by Quickchange mutagenesis with primers Cdc42 ( Q61L ) fwd and -rev and Rho ( Q63L ) fwd and -rev , respectively .", "X . laevis IT-Cdc42 in pCS2+ was generated according to Bendezú et al . ( 2015 ) : a linker -SGGSACSGPPG- was cloned into Cdc42 after Q134 .", "The linker encodes for BamH1 and Asc1 restriction sites for digestion and insertion of GFP into the linker region .", "The 5’ end of Cdc42 was amplified with primers Cdc42 ( 1 ) and Cdc42 ( 2 ) ; the 3’ end was amplified separately with primers Cdc42 ( 3 ) and Cdc42 ( 4 ) .", "The two products were joined by PCR stitching with primers Cdc42 ( 1 ) and Cdc42 ( 4 ) .", "The single product was digested with EcoR1 and Xho1 and ligated into pCS2+ .", "The resulting construct was mutated by Quickchange with primers pCS2+-Cdc42 ( 1 ) and pCS2+-Cdc42 ( 2 ) to remove the BamH1 restriction site upstream of the insertion in the multiple cloning site .", "eGFP was amplified from eGFP-wGBD with primers eGFP ( 1 ) and eGFP ( 2 ) .", "Both the Quickchanged construct and eGFP were digested with BamH1 and Asc1 , and eGFP was ligated into the linker region internal to the Cdc42 coding sequence .", "X . laevis Rho and Rac were similarly tagged internally after residues Q136 and L134 , respectively .", "To make C3-insensitive mCh-Rho and IT-Rho , constructs were Quickchanged to N41V using primers Rho ( N41V ) fwd and -rev ( Sekine et al . , 1989 ) .", "X . laevis RhoGDI Clone ID:7010361 ( GE-Healthcare Dharmacon , Lafayette , CO ) was subcloned into pCS2+ with Cla1 and Xho1 , and into N’3xGFP and N’Halo ( Promega , Madison , WI ) pCS2+ with BspE1 and Xho1 .", "A FLAG-tag was added by PCR onto the 5’ of RhoGDI , and the product was subcloned into pFast-Bac1 with Cla1 and Not1 .", "The following mutations were made by Quickchange mutagenesis to untagged and N’3xGFP RhoGDI in pCS2+: E158/9A , E158/9Q , D40A , D40N , D180A and D180N ( Dransart et al . , 2005 ) .", "Mutant RhoGDI 8 ( A ) had the first eight charged amino acids to mutated to alanine ( D3/5 , E11-13 , E15-17A ) by sequential PCR ( Ueyama et al . , 2013 ) .", "The 3’ end of RhoGDI was amplified with primers 8 ( A ) F1 and R1 .", "The product was amplified and added to at its 5’ end with primers 8 ( A ) F2 and R1 , and for a third time with 8 ( A ) F3 and R1 .", "The final PCR product was subcloned into pCS2+ by Infusion PCR ( Takara Bio , Kusatsu , Japan ) .", "For subcloning into N’3xGFP-pCS2+ , the third PCR from above was repeated with 8 ( A ) F4 and R2 .", "Mutant RhoGDI helix replacement ( HR ) had alpha helix D39-Q48 replaced with a glycine linker GGGGSGGGGS .", "This was done by sequential PCR as described above with four rounds of PCR: HR1 and R1 , HR2 and R1 , HR3 and R1 , then either HR4 and R1 for subcloning into pCS2+ or HR5 and R2 for subcloning into N’3xGFP-pCS2+ .", "RhoGDI mutant Δ51–199 was generated by adding a stop codon after L50 by Quickchange mutagenesis .", "To make RhoGDI Δ1–19 , the 3’ end of RhoGDI was amplified with primers ( - ) 20 F1 and R for subcloning by infusion into pCS2+ , and primers ( - ) 20 F2 and R for N’3xGFP-pCS2+ .", "Primers ( −55 ) F1 , F2 and R were used to generate RhoGDI Δ1–54 as described above ( Hoffman et al . , 2000; Ueyama et al . , 2013 ) .", "For expression and purification in E . coli , X . laevis RhoGDI WT and E158/9Q were subcloned into a pGEX-6P-2 vector via Gibson assembly cloning ( Gibson et al . , 2009 ) using XlRhoGDIfwd and -rev .", "A cysteine for labeling was added by PCR onto the 5’ of RhoGDI .", "Bovine RhoGDI1 in pGEX-6P was a gift of Dr . Tomotaka Komori .", "E163/4Q mutation was made by Quickchange mutagenesis with the E163/4Qfwd and -rev primers .", "Mutant bovine RhoGDI Δ1–22 and Δ1–59 were subcloned with BamHI and NotI into a pGEX-6P-2 with Δ1-22fwd and -rev and Δ1-59fwd and -rev , respectively .", "Mutant bovine RhoGDI HR was generated via Gibson assembly cloning ( Gibson et al . , 2009 ) , with primers HRfwd and -rev and pGEXHRfwd and -rev , for amplification of RhoGDI and the pFASTBacH10 vector , respectively .", "RabGGTase two beta in a pGATEV vector was kindly provided by Dr . Konstantin Gavriljuk .", "Rosetta ( DE3 ) chemically competent E . coli cells were transformed with WT or mutant RhoGDIs , induced with 250 µM IPTG and incubated at 18°C ON .", "Bacteria cells were harvested , centrifuged at 4000xg for 20 min , and pellets flash frozen in liquid nitrogen and stored at −80°C .", "Frozen pellets were resuspended in a 3x volume of lysis buffer ( 50 mM KPi pH 8 , 400 mM KCl , 1 mM EDTA , 5 mM βME , 1 mM PMSF , 1 mM benzamidine ) and lysed with a high pressure homogenizer at 4°C .", "Lysate was clarified by centrifugation at 100 , 000xg for 1 hr and applied to a glutathione sepharose four fast flow column bed ( GE-Healthcare , Chicago , IL ) equilibrated with wash buffer ( 50 mM KPi pH 8 , 400 mM KCl , 1 mM EDTA , 5 mM βME , 1 mM benzamidine ) .", "The column was washed with wash buffer , and protein was eluted with elution buffer ( 50 mM KPi pH 8 , 400 mM KCl , 1 mM EDTA , 5 mM βME , 1 mM benzamidine , 10 mM reduced L-glutathione ) .", "Peak fractions were pooled , protein concentration was estimated with Bradford assay ( Bio-Rad Laboratories , Inc , Hercules , CA ) , and PreScission protease was added at 1:30 .", "After ON incubation on ice , the sample was concentrated using 5 , 000 MWCO Vivaspin15R centrifugal concentrators ( Sartorius AG , Göttingen , Germany ) , buffer exchanged in wash buffer on a HiPrep 26/10 desalting column ( GE-Healthcare ) and recirculated on the same glutathione sepharose four fast flow column bed re-equilibrated in wash buffer .", "Flow-through was collected , concentrated , spun down and gel filtered on a HiLoad Superdex 75 pg column ( GE-Healthcare ) in storage buffer ( 20 mM HEPES pH 7 . 5 , 150 mM KCl , 0 . 5 mM TCEP , 20% Glycerol ) .", "Peak fractions were pooled , concentrated , flash frozen in liquid nitrogen and stored at −80°C .", "Protein purification and purity were determined by Coomassie stain of 12% SDS-PAGE , protein concentration measuring absorbance at 280 nm .", "WT and mutant GTPases were expressed and purified similarly to GDIs with the following differences: ( 1 ) L21 ( DE3 ) chemically competent E . coli cells were used and proteins were expressed with 1 mM IPTG at 37°C for 4 hr; ( 2 ) the affinity step was performed on HiTrap Chelating HP columns loaded with cobalt and equilibrated in 50 mM HEPES pH 7 . 5 , 50 mM NaCl , 5 mM MgCl2 , 0 . 5 mM βME , 100 µM ATP and 100 µM GDP/GTP; ( 3 ) protein were gel filtered in storage buffer ( 50 mM HEPES pH 7 . 5 , 50 mM NaCl , 2 mM MgCl2 , 2 mM DTT , 20% Glycerol ) .", "RabGTTase Beta was expressed and purified as described before ( Gavriljuk et al . , 2013 ) .", "Before each membrane extraction reaction , nucleotide bound to Cdc42 G12V was exchange to GTPγS , incubating the protein with 10-fold excess EDTA and GTPγS for 30 min on ice .", "The new nucleotide state was stabilized by adding 20-fold excess MgCl2 .", "Exchange of nucleotide was confirmed by reversed phase chromatography using a C18 column under isocratic conditions ( 50 mM potassium phosphate pH 6 . 6 , 10 mM tetrabutylammonium bromide , 16% ( v/v ) ACN ) as described previously ( Müller et al . , 2010 ) .", "Nucleotide peaks were quantified spectrometrically measuring absorbance at 254 nm .", "RhoGTPases were labeled at the N-t with Cy3 using a sortase-mediated reaction and in vitro prenylated as previously described ( Gavriljuk et al . , 2013; Popp et al . , 2007 ) .", "In brief , RhoGTPases were incubated with sortase and Cy3 N-t labeled LPETGG peptide at 3:1:15 ratio in labeling buffer ( Tris pH 8 . 0 , 150 mM KCl , 6 µM CaCl2 , 0 . 5 mM TCEP ) and incubated ON at 16°C .", "The entire reaction was mixed with geranylgeranyltransferase type one and geranylgeranyl diphosphate at 10:1:30 ratio in prenylation buffer ( 50 mM HEPES pH 7 . 5 , 50 mM NaCl , 2 mM MgCl2 , 2 mM DTT , 30 µM GDP/GTP , 2% CHAPS ) , and incubated ON on a rotating mixer at 4°C .", "The sample was spun in a TLA-100 rotor ( Beckman Coulter , Brea , CA ) at 80 , 000 rpm for 30 min at 4°C and gel filtered on a HiLoad Superdex 75 pg column ( GE-Healthcare ) equilibrated with prenylation buffer with 0 . 5% CHAPS .", "Peak fractions were pooled , concentrated using 5 , 000 MWCO Vivaspin4 centrifugal concentrators ( Sartorius AG ) and buffer exchanged in prenylation buffer without CHAPS on a NAP-5 column ( GE-Healthcare ) .", "Residual detergent was removed by Pierce Detergent Removal Spin Column ( Thermo Fisher , Carlsbad , CA ) .", "After sortase-mediated labeling with Cy3 , unprenylated proteins were directly spun down and gel filtered in absence of CHAPS .", "DH10Bac-competent E . coli ( Thermo Fisher ) were transformed with FLAG-WT RhoGDI or E158/9Q in pFast-Bac1 and positive clones were identified by blue/white screening .", "Bacmid was purified and transfected into Sf9 cells using Cellfectin II reagent ( Thermo Fisher ) .", "High-expressing clones were identified and baculovirus was generated for two additional passages .", "Sf9 cells , 22 × 106 per 15 cm plate , were infected with high-titer baculovirus and incubated 27°C for 72 hr .", "Sf9 cells were harvested , centrifuged at 500xg for 5 min , and pellets were stored at −80°C .", "Frozen pellets were resuspended in a 5x volume of solubilization buffer ( 1xPBS pH 7 . 5 , 1% Triton X-100 , 0 . 5 µg/mL leupeptin , 0 . 5 µg/mL aprotinin , 0 . 5 µg/mL Pepstatin A , 40 µg/mL PMSF , 100 µg/mL benzamidine , 0 . 5 µg/mL E64 ) and incubated at 4°C with end-over-mixing for 1 hr .", "Lysate was clarified by centrifugation at 21 , 000xg for 15 min and applied to an anti-FLAG M2 agarose column bed ( MilliporeSigma , Burlington , MA ) .", "The column was washed 3x with wash buffer ( 1xPBS , 0 . 5 µg/mL leupeptin , 0 . 5 µg/mL aprotinin , 0 . 5 µg/mL Pepstatin A , 40 µg/mL PMSF , 100 µg/mL benzamidine , 0 . 5 µg/mL E64 ) .", "A buffer exchange was performed with 1:1 wash buffer:HEPES ( 25 mM HEPES pH 7 . 5 , 100 mM KCl ) , and the column washed 2x with HEPES .", "Protein was eluted with 1M Arginine pH 4 . 4 into an equal volume of collection buffer ( 50 mM HEPES pH 7 . 5 , 200 mM KCl ) .", "Fractions were analyzed by coomassie stain of a 12% SDS-PAGE .", "Peak fractions were pooled and concentrated using a 10 MW Amicon Ultra-15 Centrifugal filter ( MilliporeSigma ) .", "A buffer exchange was performed during concentrating with HEPES such that the final Arginine concentration was less than 1 mM .", "Protein purification and purity was determined by comparison to a BSA standard curve by Coomassie stain of a 12% SDS-PAGE .", "Ovarian tissue was harvested from adult X . laevis via surgical procedures approved by the University of Wisconsin-Madison Institutional Animal Care and Use Committee .", "Oocytes were stored in 1x modified Barth’s solution ( 88 mM NaCl , 1 mM KCl , 2 . 4 mM NaHCO3 , 0 . 82 mM MgSO4 , 0 . 33 mM NaNO3 , 0 . 41 mM CaCl2 , 10 mM HEPES , pH 7 . 4 ) with 100 µg/mL gentamicin sulfate , 6 µg/mL tetracycline and 25 µg/mL ampicillin at 16°C .", "Prior to manual defolliculation with forceps , oocytes were treated with 8 mg/mL type I collagenase ( Life Technologies , Grand Island , NY ) in 1x modified Barth’s solution for 1 hr at 16°C on an orbital shaker .", "mRNA was generated in vitro using the mMessage mMachine SP6 transcription kit ( Thermo Fisher ) and purified using the RNeasy Mini Kit ( Qiagen , Hilden , Germany ) .", "Transcript size was verified on a 1% agarose/formaldehyde denaturing gel relative to the Millennium Marker ( Life Technologies ) RNA molecular weight standard .", "Oocytes were microinjected with a 40 nL injection volume using a p-100 microinjector ( Harvard Apparatus , Holliston , MA ) .", "mRNA encoding probes for active Rho ( 2xrGBD ) and active Cdc42 ( wGBD ) were injected at a final needle concentration of 30 µg/mL and 100 µg/mL , respectively .", "IT-Rho , Rac and Cdc42 were each injected at a final needle concentration of 125 µg/mL , with 63 µg/mL of WT RhoGDI to stabilize the exogenous GTPase and maintain stoichiometric ratio of GTPase:GDI ( Boulter et al . , 2010 ) .", "mCh-Rho , Rac and Cdc42 were each injected at a final needle concentration of 125 µg/mL .", "mRNA encoding Abr was injected at a final needle concentration of 25–500 µg/mL .", "3xGFP-WT GDI and mutants at 333 µg/mL , untagged RhoGDI at 300 µg/mL , Halo-WT GDI and mutants at 200 µg/mL , Cdc42 G12V or Q61L at 28 µg/mL , and untagged GDI E158/9Q at 1 . 5 mg/mL .", "For purified protein , Cy3-Rho and Cy3-Cdc42 , in vitro prenylated and complexed with RhoGDI , were injected at a final needle concentration of 4 . 56 µM .", "C3 exotransferase was injected at a final needle concentration of 1 . 1 µg/mL in 1 mM DTT and WT GDI at 3 . 5–114 µM for the standard curve .", "For wounding experiments , all mRNA was injected 20–24 hr before imaging , and purified protein was injected at least 2 hr before imaging , except for C3 which was injected 30 min prior to imaging .", "For imaging cortical granule exocytosis , oocytes were injected 16 hr before imaging and matured ON in progesterone .", "Two-cell embryos were microinjected with a 5 nL injection volume at a final needle concentration of 167 µg/mL for IT-Rho and IT-Cdc42 mRNA , and 18 . 24 µM Cy3-Rho and Cy3-Cdc42 .", "Whole cell lysates ( WCL ) were generated from oocytes with or without IT-Rho expression .", "Oocytes were washed 3x with wash buffer ( 10 mM imidazole , 50 mM KCl , 2 . 5 mM MgCl2 , 1 mM EGTA , 10 mM EDTA ) .", "5 µL/oocyte of lysis buffer ( wash buffer supplemented with 1 mM DTT , 0 . 5% TritonX-100 , 50 µg/mL E64 , 4 mM Peflabloc , 60 µg/mL chymostatin , 5 µg/mL leupeptin , 1 µg/mL pepstatin , 4 µg/mL aprotinin , 2 µM calpeptin , 2 µM ALLN ) was added and oocytes were homogenized with a 200 µL pipet tip .", "Homogenates was added to a 3/16 × 25/32 Ultra-clear centrifuge tube ( Beckman Coulter , Ref:344718 ) , centrifuged at max speed for 5 min at 4°C and the cytoplasmic fractions were extracted by piercing the side of the tube with a syringe .", "The cytoplasmic fractions were transferred to 1 . 5 mL tubes , centrifuged at max speed for 5 min at 4°C , and the cytoplasmic fractions was isolated using a pipet .", "6X LSB was added , samples were boiled for 10 min and separated by 12% SDS-PAGE .", "IT-Rho expression was determined by western blot of the equivalent of 1 or two oocyte volumes ( lanes specified ) alongside a standard curve of purified GFP-UtrCH 261 , generously provided by Kevin Sonnemann .", "The blot was probed with anti-GFP ( B-2 ) ( 1:1000 , sc-9996 , Santa Cruz , Dallas , TX ) primary and goat anti-mouse IR-Dye 800CW ( 1:10 , 000 , 926–32210 , LI-COR , Lincoln , NE ) secondary antibodies .", "Visualization was achieved with a LI-COR Odyssey Fc imaging system .", "BL21 pLysS cells ( Thermo Fisher ) were transformed with GST-RhoGDI in pGEX6p . 1 .", "A positive clone was used to inoculate 12 mL of lysogeny broth ( LB ) supplemented with 25 µg/mL ampicillin and cultured ON .", "The 12 mL culture was added to 1L of LB with ampicillin and shaken at 37°C until OD600 ~0 . 6 .", "The culture was induced by adding a final concentration of 0 . 1 mM IPTG and shaken at 37°C for 2 hr .", "BL21 pLysS cells were pelleted at 5300 rpm for 10 min at 4°C , and the pellet resuspended in Buffer A ( 50 mM Tris-HCL , pH 7 . 6; 50 mM NaCl with 1 mM DTT in PBS ) .", "Pellets were stored at −80°C .", "Pellets were thawed at room temperature to promote cell lysis .", "Triton X-100 was added to a final concentration of 0 . 6% , PMSF at 500 uM , lysoszyme at 1 mM in 10 mM Tris pH 8 . 0 , 400 µM Peflabloc , 1 µg/mL aprotinin , 1 µg/mL leupeptin .", "Solubilate was incubated at RT for 30 min , DNAse1 was added to a final concentration of 10 ug/mL , incubated again for at RT for 30 min , and centrifuged at 16 , 000xg for 10 min at 4°C .", "The supernatant was collected and exposed to a column containing glutathione-sepharaose 4B ( MilliporeSigma ) .", "The column was washed 5x with Buffer A and the protein eluted with 20 mM Tris , pH 8 . 0 , 20 mM glutathione , 400 µM Peflabloc , 1 . 25 µg/mL aprotinin , 14 . 25 µg/mL leupeptin , 0 . 25 mM E-64 , 0 . 5 mM PMSF .", "Protein concentration was determined by Coomassie stain of a 12% SDS-PAGE alongside a BSA standard curve .", "FLAG-RhoGDI purified from Sf9 cells was used as an antigen for antibody production in rabbits ( Covance , Princeton , NJ ) .", "The serum was heat-inactivated at 56°C for 30 min , diluted 1:1 in 20 mM Tris , pH 7 . 5 , and filtered through a 0 . 22 µm syringe .", "The diluted , filtered serum was loaded onto a column containing GST-GDI coupled to Affi-Gel 15 , to minimize antibody cross-reactivity to the FLAG-tag on the antigen .", "The column was washed 20x with 20 mM Tris , pH 7 . 5 and 20x with 20 mM Tris , pH 7 . 5 , 500 mM NaCl .", "Antibody was first eluted with 100 mM glycine , pH 2 . 5 into 1M Tris , pH 8 . 8 for neutralization .", "The column was washed 20x with 20 mM Tris , pH 8 . 8 .", "Antibody remaining on the column was eluted with 100 mM Triethylamine , pH 11 . 5 into concentrated HCl and 1M Tris , pH 7 . 5 for neutralization .", "The concentration of each fraction was determined by A280 .", "The peak antibody fractions were pooled , dialyzed against PBS ( 2 × 2L ) ON , and concentrated using a 100 K MW Amicon Ultra-15 Centrifugal filter ( MilliporeSigma ) .", "An equal volume of glycerol was added and stored at −20°C .", "Antibody specificity was determined by western blotting of purified protein , X . laevis oocyte whole cell lysate ( WCL , described above ) , WCL of oocytes overexpressing GDI and WCL of oocytes expressing 3xGFP-GDI , separated by 12% SDS-PAGE .", "The blot was probed with anti-GDI ( 1:1000 ) primary and Alexa-Fluor 680 goat anti-rabbit ( 1:10 , 000 , A21076 , Molecular Probes , Eugene , OR ) secondary antibodies .", "Visualization was achieved with a LI-COR Odyssey Fc imaging system .", "Oocytes were wounded , allowed to heal for 2–3 min and fixed for 2 hr in 10 mM EGTA , 100 mM KCl , 3 mM MgCl2 , 10 mM HEPES , 150 mM sucrose ( pH 7 . 6 ) , 4% PFA , 0 . 1% glutaraldehyde , 0 . 1% Triton X-100 .", "Fixed oocytes were washed 5x in TBSN/BSA ( 5 mg/mL BSA in 1xTBS containing 0 . 1% NP-40 ) .", "Oocytes were bisected and blocked in TBSN/BSA for 4 hr at 4°C .", "Oocytes were stained with rabbit α-RhoGDI at 1:1000 in TBSN/BSA for 12 hr , washed 5x in TBSN/BSA over 12 hr , stained with chicken α-rabbit Alexa Fluor 647 ( Invitrogen , Carlesbad , CA ) at 1:10 , 000 for 12 hr in BSN/BSA at 4°C , and washed 5x in TBSN/BSA over 12 hr .", "Laser scanning confocal microscopy was performed using a Nikon Eclipse Ti inverted microscope with a Prairie Point Scanner confocal system ( Bruker , Middleton , WI ) .", "The microscope was fitted with a 440 nm dye laser pumped by a MicroPoint 337 nm nitrogen laser ( Andor , South Windsor , CT ) for wounding .", "Brightest-point projections , measurements of fluorescence intensities , area and distances were made in FIJI ( Schindelin et al . , 2012 ) .", "Bio-Formats Importer and De-Flicker plugins were used .", "Ring intensity corrected for background was calculated by quantifying the mean intensity of the ring and subtracting the mean intensity of the background .", "Total activity was calculated by multiplying the mean intensity of the zone ( corrected for background ) by the area of the zone , normalized for wound width .", "GraphPad Prism was used to plot quantifications and perform statistical analyses .", "An unpaired student’s T-test with a 2-tailed distribution and unequal variance was used to compare two conditions , one-way ANOVA with a Tukey post hoc analysis was used to analyze more than two conditions .", "*p<0 . 05 , **p<0 . 01 , ***p<0 . 001 , ****p<0 . 0001 .", "Small unilamellar vesicles and supported lipid bilayers were prepared with 100% 1 , 2-dioleoyl-sn-glycero-3-phosphocholine ( 18:1 DOPC; Avanti Polar Lipids , Inc , Alabaster , AL ) as described before ( Hansen et al . , 2019 ) .", "250µL Cy3 labeled GTPases were incubated on SLBs at 200nM final concentration until equilibrium was reached .", "A 0 . 5mm silicone tubing was attached drop-to-drop to the chamber with the equilibrated sample via a male luer connector ( ibidi GmbH , Gräfelfing , Germany ) .", "After acquisition of few frames in absence of flow , the chamber was flushed at 10µL/sec with imaging buffer ( 20mM HEPES pH 7 . 0 , 150mM KCl , 1 . 5mM MgCl2 , 0 . 5mM EGTA , 100µM GDP/GTP ) alone or in presence of a GTPase solubilizer until baseline was reached .", "Oxygen scavenger system ( 1 . 25mg/mL glucose oxidase , 0 . 2 mg/mL catalase , 400 mg/mL glucose ) was added fresh to each sample and buffer before imaging .", "TIRF was performed on a Nikon Eclipse Ti inverted microscope with a VisiScope TIRF-FRAP Cell Explorer system ( Visitron Systems GmbH , Puchheim , Germany ) using a 60× Apo TIRF oil-immersion objective ( 1 . 49 N . A . ) .", "Cy3-labeled proteins were excited with a 561-nm laser line , excitation light was passed through a ET-561nm Laser Bandpass Set ( Chroma Technology Corporation , Bellows Falls , VT ) before illuminating the sample .", "Fluorescence emission was detected on a Evolve 512 Delta EMCCD camera ( Teledyne Photometrics , Tucson , AZ ) .", "Measurements of fluorescence intensities were made in FIJI ( Schindelin et al . , 2012 ) .", "Plot Z-axis profile tool and Bio-Formats Importer plugins were used .", "Fluorescence intensity was corrected for background .", "To display multiple curves on the same graph , data from different experiments were aligned using the overshoot signal occurring after the flow was started and normalized dividing by the maximum intensity .", "Data from wash off experiments were fitted with a one component exponential decay function ( y=y0+A e-λ x; y0= y offset , A=amplitude , λ=exponential decay constant ) , choosing a fitting range that did not include the initial overshoot .", "This was possible because a monoexponential function can be fitted to a range of the data set without affecting the λ value obtained .", "Koff titration curves were fitted with a hyperbolic function ( y=y0+λmax xKd + x ) .", "Origin Pro ( OriginLab Corporation , Northampton , MA ) was used to analyze data , plot quantifications and perform statistical analyses .", "An unpaired student’s T-test with a 2-tailed distribution and equal variance was used to compare two conditions .", "**p<0 . 01 , ***p<0 . 001 , ****p<0 . 0001 ." ] ]

[ "The RhoGTPases are characterized as membrane-associated molecular switches that cycle between active , GTP-bound and inactive , GDP-bound states .", "However , 90–95% of RhoGTPases are maintained in a soluble form by RhoGDI , which is generally viewed as a passive shuttle for inactive RhoGTPases .", "Our current understanding of RhoGTPase:RhoGDI dynamics has been limited by two experimental challenges: direct visualization of the RhoGTPases in vivo and reconstitution of the cycle in vitro .", "We developed methods to directly image vertebrate RhoGTPases in vivo or on lipid bilayers in vitro .", "Using these methods , we identified pools of active and inactive RhoGTPase associated with the membrane , found that RhoGDI can extract both inactive and active RhoGTPases , and found that extraction of active RhoGTPase contributes to their spatial regulation around cell wounds .", "These results indicate that RhoGDI directly contributes to the spatiotemporal patterning of RhoGTPases by removing active RhoGTPases from the plasma membrane ." ]

[ "Organisms rely on many signaling molecules to control how their cells grow , divide and heal .", "For example , when the cell membrane is damaged , two signaling proteins , Rho and Cdc42 , are recruited to wounds and activated to promote repair .", "Active Rho and active Cdc42 form two concentric rings at the membrane to direct the closure of the wound .", "Rho and Cdc42 belong to the RhoGTPase family , a group of proteins that act as molecular switches and alternate between active and inactive forms .", "At the level of the cell , RhoGTPases are only active in the tiny patches of the membrane where they bind .", "However , individual proteins hop on and off membranes in a matter of seconds , only staying bound for short periods .", "This mechanism is controlled by a regulatory protein known as RhoGDI , and it allows RhoGTPases to form precise patterns of activity at membranes – such as the rings that surround a wound site .", "However , it was not known exactly how RhoGDI regulates the activity of RhoGTPases over space and time , partly because it is difficult to study these proteins in the laboratory .", "To fill this knowledge gap , Golding , Visco et al . developed new fluorescent probes to track Rho and Cdc42 in wounded cells from frogs and on artificial membranes .", "The experiments showed that pools of inactive Cdc42 accumulated on membranes , alongside the active form of the protein .", "RhoGDI removed both active and inactive RhoGTPases from artificial and frog cell membranes .", "In fact , removing active Rho and Cdc42 proteins from the cell membrane was necessary to form the spatial patterns of RhoGTPase activity observed in wounded frog cells .", "The findings of Golding , Visco et al . help to understand how RhoGDI proteins regulate RhoGTPases and provide new tools to further study these proteins .", "In humans , mutations in either RhoGDI or Cdc42 are responsible for severe conditions such as Nephrotic Syndrome Type 8 or Takenouchi-Kosaki syndrome .", "In the future , this work may aid the development of treatments and cures for these conditions ." ]

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

[ "structural biology and molecular biophysics", "computational and systems biology" ]

[ [ "Kinesin is an ATPase motor protein that walks along microtubules ( MTs ) , to carry out vital functions , which include intracellular transport and cell division ( Vale , 2003; Hirokawa and Noda , 2008 ) .", "As the smallest known motor that can walk processively , it also serves as the canonical motor protein ( Block , 2007; Hwang and Lang , 2009 ) .", "Kinesin families use variations in subdomains to harness nucleotide-dependent conformational changes of the conserved motor head to generate diverse motility characteristics ( Cochran , 2015 ) , such as: direction reversal ( Endow and Waligora , 1998; Endres et al . , 2006; Yamagishi et al . , 2016 ) , MT polymerization/depolymerization ( Wordeman , 2005; Hibbel et al . , 2015 ) , and motility with only a single head ( Kikkawa et al . , 2000 ) .", "To understand the mechanisms of the different kinesins , it is important to search for and elucidate the conserved features of the motor head that are involved in the nucleotide processing events of the motility cycle; that is , ATP binding , hydrolysis , and product release ( ADP and Pi , inorganic phosphate ) ( Figure 1A ) .", "One of the most intensely studied family member is Kinesin-1 ( Kin-1; hereafter we refer Kin-1 as kinesin ) .", "It forms a dimer to walk toward the MT plus-end using one ATP per step ( Figure 1B ) ( Svoboda et al . , 1993; Block , 2007 ) .", "It unbinds from the MT in the ADP state , and after making a step , it releases ADP and enters the nucleotide-free APO state with high MT-affinity ( Cross , 2016; Hancock , 2016 ) .", "Binding of an ATP triggers forward force generation ( the ‘power stroke’; Figure 1A ) by driving the cover strand ( CS ) and the neck linker ( NL ) , which are located respectively on the N- and C-terminal ends of the motor head , to fold into a β-sheet named the cover-neck bundle ( CNB; Figure 1C ) ( Rice et al . , 1999; Hwang et al . , 2008; Khalil et al . , 2008 ) .", "ATP hydrolysis completes a step ( Milic et al . , 2014; Andreasson et al . , 2015 ) .", "The pre- and post-stroke states differ in the motor head orientation relative to the MT . In the pre-stroke state , the head tilts rightward relative to the MT plus-end direction , and rotates clockwise when viewed from above ( wide arrows in Figure 1D ) .", "The head rotates in the opposite direction in the post-stroke state ( Sindelar and Downing , 2010 ) .", "The nucleotide pocket on the rear-left side of the motor consists of the phosphate loop ( P-loop ) , switch-I ( sw-I ) , and switch-II ( sw-II ) ( Figure 1E ) .", "These elements are conserved among different proteins including myosin and G-protein ( Vale and Milligan , 2000 ) .", "Compared to an isolated kinesin , a MT-bound kinesin has an at least 10-fold higher ATP hydrolysis rate ( Vale , 1996; Ma and Taylor , 1997 ) , which suggests that the nucleotide pocket is allosterically controlled by the interface with the MT . Among kinesin’s MT-facing domains ( Figure 1C ) , L11 and α⁢4 undergo large conformational changes upon binding to the MT . L11 is located after sw-II ( Figure 1E ) , followed by α⁢4 that N-terminally extends by a few turns when the motor head binds to the MT ( Supplementary file 1 ) ( Sindelar and Downing , 2010; Atherton et al . , 2014; Shang et al . , 2014 ) .", "However , substantial conformational variations are present in these conserved domains , notably in sw-I and L11 ( see Supplementary file 1 ) .", "Also , the extent of the kinesin-MT interface varies depending on experimental conditions ( Morikawa et al . , 2015 ) .", "Thus , it is necessary to identify core features of the motor head that are essential for nucleotide processing .", "Such information about a single head is a prerequisite for the atomic-level understanding of the motility of a dimer .", "We characterize these features via multi-microsecond molecular dynamics simulations on the Anton supercomputer ( Shaw et al . , 2009; Shaw et al . , 2014 ) of a motor head complexed with a tubulin dimer .", "Compared to previous all-atom simulations that used biasing potentials and were limited in time ( Li and Zheng , 2012; Shang et al . , 2014; Chakraborty and Zheng , 2015 ) , the unbiased simulations described here reveal the conformational changes of kinesin-MT complexes on a more realistic time scale .", "We find that the nucleotide binding pocket is conformationally the most dynamic part of the motor head , whose internal motions actively drive the nucleotide processing events .", "In particular , we show how ATP hydrolysis occurs in a fluctuating environment , and demonstrate the role of the kinesin-MT interface for this process .", "The dynamic nature of the kinesin mechanism elucidates how it robustly carries out its motility cycle despite significant conformational perturbations to the motor head and the MT in the crowded cellular environment ( Leduc et al . , 2012 ) .", "The source of the chemical energy and the mechanism involved in ‘walking on tracks’ are applicable to other translocating motors such as myosin on actin ( Vale and Milligan , 2000; Hwang and Lang , 2009 ) , making the present mechanistic results of general interest ." ], [ "We studied Kin-1 in different nucleotide or structural states .", "The names of the simulated systems are given below in italics .", "Simulation times and conformational states are in parentheses .", "We also carried out simulations of Eg5 ( Kin-5 family ) .", "However , currently only Kin-1 has atomic-resolution x-ray structures of the motor head complexed with the MT in both the pre- and post-stroke states ( Gigant et al . , 2013; Cao et al . , 2014 ) .", "Consequently , we focus our analysis on Kin-1 , and use Eg5 for comparison .", "We quantified the conformational motion by measuring the average displacement and root-mean-square deviation ( RMSD ) of Cα atoms relative to the first frame , during the first and the last 400 ns ( Figure 2A , B and Figure 2—figure supplement 1A–D ) .", "Displacements represent deformation from the initial structure , and RMSD shows the degree of conformational fluctuation .", "To find conformational changes over time , we plotted rolling averages of mean Cα displacements that are greater than 1 Å ( Figure 2C ) .", "To focus on changes in the core domains , the CS and NL , located at the termini of the motor head , were excluded from this calculation .", "Displacements saturate after 1–3 μs .", "Kin-only exhibits the greatest displacement , reflecting larger changes without the MT . ADP𝑝𝑟𝑒 had a large displacement between 2–3 μs , which is due to the release of ADP described below .", "Average displacements of other MT-bound structures were in the 2 . 5–3 Å range .", "Large displacements other than the CS and NL are localized around the nucleotide pocket ( sw-I , L11 , α⁢0 ) and the front end of the motor head ( L10 ) ( Figure 2D–H and Figure 2—figure supplement 1E–J ) .", "Sw-I ( R190–S204; Figure 1—figure supplement 1 ) is particularly flexible .", "Only in APOα , sw-I maintained the initial α-helical conformation with low displacement ( Figure 2—figure supplement 1D , F ) , which could be due to its relatively short simulation time .", "Sw-I in its hairpin-like state was also mobile during the first 400 ns ( Figure 2A ) , which is consistent with a previous 400-ns simulation study ( Chakraborty and Zheng , 2015 ) .", "L11 initially adopts an α-helical turn in all systems except for APO ( Figure 1E ) .", "It unfolded in APOα , becoming similar to APO ( Figure 2—figure supplement 1F ) .", "In addition to the MT , a nucleotide may thus be needed to stabilize the α-helical turn in L11 ( Yamada et al . , 2007 ) .", "In Kin-only , L11 and the N-terminal part of α⁢4 unfolded ( Figure 2E , star ) , which agrees with available x-ray structures of kinesin without MT ( Supplementary file 1 ) .", "The adenosine group of the nucleotide is close to α⁢0 and L5 ( Figure 1E ) .", "In Kin-5 , L5 is about 17–21-aa long and exhibits large nucleotide-dependent conformational changes ( Behnke-Parks et al . , 2011; Goulet et al . , 2012; Goulet et al . , 2014 ) .", "In Kin-1 , it is 9-aa long and fluctuates less compared to α⁢0 ( Figure 2A , B and Figure 2—figure supplement 1A–D ) .", "α⁢0 , which has not been considered previously , fluctuates mostly up-and-down ( arrow in Figure 2F ) .", "Below , we show that its mobility aids in binding of ATP .", "The front end of the motor head ( especially L10 ) also exhibits large deformation and fluctuation ( Figure 2G , H and Figure 2—figure supplement 1G–J ) .", "This region has a high temperature factor , is deformed , or exhibits low electron density in several Kin-1–MT structures ( Cao et al . , 2014; Atherton et al . , 2014; Shang et al . , 2014 ) and also in Kin-14 ( Hirose et al . , 2006 ) .", "The front end interacts with the C-terminal tail of a full-length kinesin when it is in an auto-inhibited state ( Kaan et al . , 2011 ) .", "Its compliance may thus be more relevant to tail binding rather than nucleotide processing ( Verhey and Hammond , 2009 ) .", "Another domain possessing relatively high flexibility is L8/β⁢5 on the frontal side of the interface with the MT ( Figure 1C ) , whose interaction with the MT varies ( Atherton et al . , 2014; Shang et al . , 2014; Morikawa et al . , 2015 ) .", "In Kin-only , L12 facing the MT also shows a large displacement , as expected without the MT ( Figure 2—figure supplement 1A ) .", "The curvature of the central β-sheet is another aspect of kinesin’s conformation .", "For the evolutionarily related myosin ( Vale and Milligan , 2000 ) , the corresponding β-sheet in the transducer domain exhibits large , nucleotide-dependent curvature changes ( Coureux et al . , 2004 ) .", "Its deformational energy has been speculated to partly drive force generation in myosin ( Sweeney and Houdusse , 2010 ) .", "For kinesin , the role of β-sheet curvature has been debated ( Arora et al . , 2014; Atherton et al . , 2014; Shang et al . , 2014 ) .", "For each coordinate frame , we measured the mean curvature M2 ( concaveness ) and the Gaussian curvature G ( saddle-point curvature ) of the central β-sheet , and calculated the curvature free energy ( potential of mean force; PMF ) for each simulation ( described in Materials and methods ) .", "These two curvatures quantify the bending and twisting of the β-sheet , respectively ( Sun et al . , 2003 ) .", "Pre-stroke states had generally higher curvature , especially in G ( Figure 2I and Figure 2—figure supplement 1K ) .", "Since ATP and ADP+Pi have very similar curvature , neither ATP hydrolysis nor Pi release ( see below ) is driven by the tendency of the β-sheet to adopt a higher curvature .", "Similarly , ATP and Kin-only had nearly the same curvature , indicating that binding to the MT does not impose any strain on the central β-sheet ( Figure 2—figure supplement 1K ) .", "We obtain information concerning the effect of curvature changes between pre- and post-stroke states by superposing the PMFs for ATP and APO ( Figure 2J ) .", "APO has a local free energy minimum that is 0 . 84 kB⁢T ( kB⁢T: thermal energy at 300 K ) higher than that of ATP .", "There is also a ∼1 . 7 kB⁢T energy barrier from ATP towards APO .", "The pre- and post-stroke states respectively have similar PMFs regardless of the details of individual simulations ( Figure 2—figure supplement 1K ) .", "Further , the PMF in Figure 2J does not directly represent the properties of the central β-sheet itself , but it implicitly reflects the energetics of the whole system , including domains surrounding the β-sheet , nucleotide and the MT , in controlling the curvature .", "These free energies are well below the 10-kB⁢T free energy ( 8 nm step×5 pN stall force ) used by kinesin , which can also be seen by the large overlap in individual curvature distributions between the pre- and post-stroke states ( Figure 2—figure supplement 1K ) .", "By comparison , the rotary motor F1-ATPase has about 5-kB⁢T curvature energy changes ( Sun et al . , 2003 ) .", "Therefore , curvature changes in kinesin are not substantial enough to drive ATP hydrolysis nor the transitions between pre- and post-stroke states .", "Next we studied the motion of the motor head relative to the MT . For positional and orientational reference , we used the central β-sheet , α⁢6 , α⁢4 , and β⁢5⁢a/b ( Figure 3A and Figure 3—figure supplement 1A ) .", "The central β-sheet , with its low RMSD , represents the overall position and orientation of the motor head .", "α⁢6 changes its orientation between pre- and post-stroke states ( Figure 1C , D ) .", "α⁢4 and β⁢5⁢a/b are MT-binding domains .", "For each domain , translations in longitudinal , transverse , and normal ( perpendicular to the MT surface ) directions , and rotations about these three directions were measured .", "Translational and orientational changes between pre- and post-stroke states captured various aspects of available x-ray and cryo-EM structures of kinesin-MT complexes .", "The central β-sheet shifts mostly leftward in the post-stroke state ( Figure 3A ) , and in all states , it fluctuates more in the transverse direction , indicating an anisotropic compliance ( Figure 3—figure supplement 1B ) .", "The shift in α⁢6 between pre- and post- stroke states agrees with its C-terminal end moving over α⁢4 ( Figure 1C , D vs . Figure 3A; Figure 3—figure supplement 1C ) .", "α⁢4 is nearly stationary , so that it serves as an anchor for binding to the MT ( Figure 3—figure supplement 1D ) .", "The vertical shifts of β⁢5⁢a/b ( Figure 3A and Figure 3—figure supplement 1E ) have been observed in cryo-EM structures of kinesin-MT complexes in both APO and ATP-analog states , depending on experimental conditions ( Atherton et al . , 2014; Morikawa et al . , 2015 ) .", "The shifts are thus likely non-essential for the operation of kinesin .", "The central β-sheet and α⁢6 rotate as observed in crystal structures ( Figure 1C , D vs . Figure 3B and Figure 3—figure supplement 1F , G ) .", "We also calculated the water density map for the kinesin-MT interface during the last 500 ns .", "The map was visualized with two different density cutoffs .", "When a cutoff equal to the bulk density ( 0 . 0333 Å-1 ) is used , globular hydration shells surround the interface ( Figure 3—figure supplement 1H ) .", "With a cutoff equal to three times the bulk density , a collection of blobs appear , which correspond to regions where water oxygens are found with high probability during the simulation ( Figure 3C ) .", "They are located within the kinesin-MT interface and crevices , for all simulations .", "Lack of any correlation between the extent of interfacial hydration and the conformational state can also be seen by the buried area within the kinesin-MT interface .", "It undulates with 80–720-ns correlation times and with instantaneous fluctuations of a few hundred Å2 ( e . g . , yellow trace in Figure 3D ) .", "We measured the binding energy between kinesin and the MT during the last 500 ns ( Figure 3—figure supplement 1I–L ) .", "Pre-stroke states interact less with the β-tubulin ( Figure 3—figure supplement 1L , squares ) , which is consistent with its front side ( β⁢5⁢a/b ) lifting from the MT ( Figure 3A ) .", "Interaction with α-tubulin differs more across simulations .", "Overall , ATP and APO have the strongest binding energy ( Figure 3—figure supplement 1L , circles ) , which are in line with experiments where the ATP and APO states have high MT affinity compared to the ADP state ( Woehlke et al . , 1997 ) .", "However , our binding energies do not include water-mediated interactions and entropic contributions , which are expected to be comparable to the binding energy in magnitude ( Zoete et al . , 2005 ) , so that the net binding free energy is much smaller than those in Figure 3—figure supplement 1L .", "Thus , the calculated binding energies , although they reflect the interaction between kinesin and the MT in different nucleotide states , do not correspond quantitatively to the experimental binding affinities .", "In any case , the mobility of the motor head and extensive hydration of the interface observed in all simulations suggest that the kinesin-MT interface is highly dynamic .", "This point is further explored in the analysis of kinesin-MT contacts below .", "To understand the conformational behavior of the system at the individual amino acid level , we traced all intra-kinesin and kinesin-MT contacts .", "Hydrogen bonds ( H-bonds; including salt bridges ) and nonpolar contacts were considered , majority of which form and break with less than 100% occupancy during the simulation ( example occupancy trajectories are in Figure 4—figure supplement 1A ) .", "Among intra-kinesin contacts , there were fewer H-bonds ( 1100–1500 ) than nonpolar contacts ( 1800–2400 ) .", "The occupancy distribution is U-shaped in logarithmic scale , majority of which have lower than 20% occupancy ( Figure 4A ) .", "The number of contacts with greater than 80% occupancy were 137–153 ( H-bond ) and 302–339 ( nonpolar ) .", "Among contacts showing irreversible transitions , we monitored those whose occupancy before breakage or after formation is greater than 80% ( Figure 4B; Supplementary file 2 ) .", "Post-stroke states had more contacts break than form , which occurred mainly within the first 3 μs ( Figure 4—figure supplement 1B ) .", "Locations at which changes occurred are clustered around the nucleotide pocket and the front side ( Figure 4B ) , that also had high RMSD ( Figure 2A ) .", "With a bound nucleotide , contacts involving sw-I undergo extensive changes , which is responsible for the greater number of changes in the post- than in the pre-stroke state ( Figure 4—figure supplement 1B ) .", "This is mainly because in the post-stroke state , contacts between the N- and C-terminal sides of sw-I forming the hairpin break .", "But contacts between sw-I with ATP and sw-II , necessary for the hydrolysis of ATP , remain intact ( Supplementary file 2A–C; see below ) .", "Fewer contacts formed between kinesin and the MT , 170–240 H-bonds and 250–390 nonpolar contacts , of which only 4–10 ( H-bond ) and 5–16 ( nonpolar ) had greater than 80% occupancy ( Supplementary file 3 ) .", "Majority of nonpolar contacts are by charged or polar residues so that a hydrated interface is maintained ( Figure 3C ) .", "In contrast to intra-kinesin contacts , very few contacts formed or broke irreversibly during the simulation ( Supplementary file 3 ) .", "Kinesin-MT contacts can be grouped into the rear ( mainly L11 and α⁢4 ) , middle ( L12 and α⁢5 ) , and front ( β⁢5⁢a-L8b , herein called L8/β⁢5 ) ( Figure 4C ) .", "The first two interact respectively with α and β-tubulins , and they are present in all nucleotide states .", "The front contacts are less robust in the pre-stroke states , lacking high-occupancy nonpolar contacts .", "This is consistent with the increase of its normal position ( Figure 3A ) , higher ( weaker ) binding energy with β-tubulin ( Figure 3—figure supplement 1L ) , and also with variations in its MT-binding mode in available structures ( Sindelar and Downing , 2007; Morikawa et al . , 2015; Shang et al . , 2014; Atherton et al . , 2014 ) .", "Moreover , our analysis agrees with previous alanine-scanning experiments ( Woehlke et al . , 1997 ) .", "Mutations in L8/β⁢5 ( H156 , E157 , R161 ) caused marginal changes in the MT binding affinity , while mutating K252 , Y274 , and R278 in the rear and middle parts of the interface affected the MT affinity more strongly , which form high-occupancy contacts in our simulations ( Supplementary file 3 ) .", "Variations in contact occupancy suggest that the kinesin-MT interface is maintained by an ensemble of contacts that do not need to be present simultaneously at any given time .", "In this way , kinesin may be able to bind to the MT quickly without needing to establish a precise combination of contacts .", "It also allows a certain degree of mobility of the motor head relative to the MT ( Figure 3A ) .", "Nevertheless , kinesin binds selectively to the cleft on the MT lattice with β-tubulin in front , but not with α-tubulin in front ( Figure 1B ) .", "Although the two tubulins have similar sequence and structure ( Löwe et al . , 2001 ) , we found that some of the residues making contacts with kinesin diverge .", "Especially , H12 of β-tubulin has several contact residues that are non-homologous to those of α-tubulin ( Figure 4—figure supplement 1C ) .", "Thus , the kinesin-MT interface is tuned so that it permits flexibility in binding , yet it is specific enough to recognize the MT binding site .", "Our RMSD and contact analyses show that sw-I is among the most mobile kinesin subdomains .", "In the ATP-state , although its pseudo-hairpin structure has been suggested to be hydrolysis-competent ( Kull and Endow , 2002 ) , in all simulations of the post-stroke state , it unfolded ( Figure 2D , E and Figure 2—figure supplement 1E; Video 1 ) .", "The unfolding occurred well after simulation began , at 1 . 83 μs ( ATP ) , 1 . 38 μs ( Kin-only ) , and 0 . 98 μs ( ADP+Pi ) .", "Even for a 1 . 73-μs simulation of an isolated Eg5 in the ATP state ( Parke et al . , 2010 ) , sw-I unfolded at 522 ns ( Video 1 ) .", "Prior to full unfolding , contacts within the hairpin partially broke ( Figure 5A , B ) , and other contacts within the surrounding domains or with sw-I broke or formed even earlier ( Supplementary file 2 ) .", "Thus , unfolding of the sw-I hairpin is a result of gradual changes that accumulate over time , rather than being an isolated event .", "After unfolding , α⁢3 at the N-terminal side of sw-I rotated outward , increasing the distance between the two ends of sw-I ( Figure 5A and Figure 5—figure supplement 1A ) .", "In the pre-stroke states , α⁢3 generally points outward ( larger θ3 in Figure 5C ) , suggesting a tendency to move outward in the absence of ATP that holds sw-I .", "To refold into a hairpin , inward rotation of α⁢3 is necessary .", "Such rotation requires a broader conformational motion of the motor head that may occur over a time scale longer than that of our simulation .", "The apparent instability of the sw-I hairpin is at odds with its presence in several crystal structures ( Supplementary file 1 ) .", "In fact , the hairpin is stabilized by crystal contacts in these structures ( Figure 5—figure supplement 1B–E ) .", "In comparison , the sw-I hairpin in myosin forms extensive contacts with the upper 50 kDa domain ( Figure 5—figure supplement 1F ) .", "However , unfolding of sw-I in our simulation is only partial , where its N-terminal ( outer ) side separates from the C-terminal side , while the latter maintains contact with ATP .", "This was also the case for Kin-only ( Figure 2E ) .", "In other x-ray structures of kinesin in the ATP ( analog ) states where sw-I does not adopt a clear hairpin conformation ( Supplementary file 1 ) , the C-terminal side in contact with the nucleotide is visible , which agrees with the partial unfolding in our simulation ( e . g . , Figure 5—figure supplement 1G ) .", "To further examine the partial unfolding of the sw-I hairpin , we aligned the initial structure of ATP and the structure after the hairpin unfolding to high-resolution cryo-EM maps in the ATP states ( Figure 6 ) .", "The central β-sheet and α⁢4 were used as alignment references since their conformation varied little during the simulation ( Figure 2A ) .", "To highlight differences between these and the cryo-EM structures , we rigidly docked the structures instead of performing flexible fitting .", "In both structures with the sw-I hairpin folded and unfolded , the N-terminal side deviates more from cryo-EM maps compared to the C-terminal side .", "Also , the outward rotation of α⁢3 ( ∼10∘; Figure 5C ) is not enough to show any significant deviation from cryo-EM maps .", "In ATP , the unfolded N-terminal side deviates less from its position in the hairpin state compared to Kin-only ( Figure 2D vs . E ) .", "Thus , at cryogenic temperatures , the N-terminal side is likely to settle to a hairpin-like state with the C-terminal side as a template , instead of landing in different configurations that lead to low electron density .", "These findings suggest the mobility of the N-terminal side of sw-I does not contradict existing cryo-EM data .", "What would be the functional role of sw-I’s mobility ?", "We first consider binding of an ATP molecule to kinesin in the APO state .", "For the APO system , we added a free Mg-ATP and performed another 2 . 04-μs simulation .", "To prevent ATP from diffusing away , we imposed a 32 Å radius spherical boundary on ATP around the center of mass of kinesin .", "During the simulation , ATP formed and broke contacts with various parts of kinesin ( Figure 5D; Video 2 ) .", "Nonpolar contacts were dominant , with the adenosine ring of ATP pointing toward kinesin and the charged phosphate moiety pointing away ( Figures 5D , 960 ns , and Figure 5—figure supplement 2A ) .", "Direct binding of an ATP with the phosphate moiety pointing inward will be unfavorable due to the desolvation penalty for the phosphate moiety and the hydrophobic attraction for the adenosine ring .", "ATP binding is more likely a multi-step process orchestrated by the surrounding domains .", "Among domains whose contact occupancy with ATP was high ( labeled in Figures 5D , 2040 ns; Figure 5—figure supplement 2A ) , sw-I , α⁢0 ( including L1a; Figure 1—figure supplement 1 ) , and L5 take the shape of a funnel with the P-loop in the middle .", "During the simulation , the three domains transiently made contacts with ATP or even held it for a while ( Figure 5D; Video 2 ) .", "This α0/L5/sw-I ‘trio’ act like an antenna that captures nearby ATP and delivers it to the P-loop .", "Sw-I , the most mobile member of the trio ( higher RMSD than the other two; Figure 2B ) , may be particularly important .", "When it moves away from the P-loop , it may form contacts with the adenosine ring , so that the phosphate moiety of ATP points towards the P-loop .", "A closing motion of sw-I will then bring the phosphate moiety in contact with the P-loop .", "Sw-I’s opening and closing motions have been observed in other simulations described below ( cf . , Figure 5E , F ) .", "Since ATP is amphiphilic , and since the trio domains closely surround the P-loop , their dynamic role should hold even though a complete binding event was not observed in our simulation – alternative scenarios such as ATP approaching kinesin with the phosphate moiety pointing towards the P-loop , or the trio domains not interacting with the incoming ATP despite their proximity , are physically unlikely .", "A critical question regarding the mobility of sw-I is whether it can support ATP hydrolysis .", "As noted above , even when the sw-I hairpin is unfolded , its inner side maintains contact with ATP .", "In its conserved SSR motif ( S201-S202-R203 ) ( Vale and Milligan , 2000; Kull and Endow , 2002 ) , S201 and S202 contact Mg-ATP with higher than 99% occupancy in both ATP and Kin-only .", "Furthermore , R203 contacts E236 of sw-II ( Figure 7A and Figure 7—figure supplement 1 ) .", "We investigated whether this organization is sufficient to coordinate the catalytic water molecules .", "A previous ab initio calculation on Eg5 suggested a two-water mechanism: The ‘lytic’ water next to Pγ donates an OH group necessary for hydrolysis , and the released H atom travels through the second ‘transfer water , ’ arriving at E236 ( McGrath et al . , 2013 ) .", "Formation of a two-water bridge between Pγ and E236 is thus necessary for hydrolysis .", "For ATP and Kin-only , we calculated the water density map around the phosphate moiety during the last 500 ns .", "In ATP , high-density blobs corresponding to the two catalytic water molecules were found , whereas in Kin-only , the density for the transfer water broadened ( Figure 7A , B ) .", "This is because in Kin-only , the absence of the support by MT leads to a downward shift of L11 and R203-E236 , so that the channel leading to Pγ widens ( Figure 7C ) .", "Furthermore , during the simulation period , two-water bridges formed with higher frequency in ATP than in Kin-only ( Figure 7D ) .", "These suggest that ATP hydrolysis is carried out in a dynamically fluctuating environment where contacts between ATP and sw-I ( S201/S202 ) , and between sw-I and sw-II ( R203-E236 ) create a narrow channel that facilitates formation of the two-water bridge between Pγ and E236 .", "Our result also explains the higher ATP hydrolysis rate when kinesin is bound to the MT: Without support from α-tubulin that keeps L11 ordered , R203-E236 move downward and broaden the channel ( Figure 7C ) , thereby reducing the two-water bridge formation .", "This agrees with the lower but non-zero ATP hydrolysis rate of kinesin in the absence of the MT ( Vale , 1996 ) .", "In general , binding of a substrate to an enzyme is believed to induce mechanical strain , thereby lowering the activation energy for cleavage ( Williams , 1993; Bustamante et al . , 2004 ) .", "To check for strain on the kinesin-bound ATP , we measured the internal coordinates of the phosphate moiety in ATP and Kin-only .", "For comparison , we performed a 4-ns simulation of an isolated Mg-ATP in water ( named ATP-only ) .", "Between isolated and kinesin-bound ATP , the length of the cleaved Oβ–Pγ bond increased from 1 . 576±0 . 032 Å ( ATP-only; avg±std ) to 1 . 586±0 . 032 Å ( ATP ) and 1 . 586±0 . 033 Å ( Kin-only ) .", "In the case of the Ras protein , increase in bond length by 0 . 01 Å has been shown to affect catalysis of GTP ( Klähn et al . , 2005 ) .", "However , for myosin , although the Oβ–Pγ bond elongates in the active site , an energetic analysis reveals no significant destabilization of ATP ( Yang and Cui , 2009 ) .", "Furthermore , in the CHARMM param36 force field ( Pavelites et al . , 1997 ) , the equilibrium length of the Oβ–Pγ bond is longer , 1 . 68 Å .", "Without an ab initio calculation of the energetics , it is difficult to assess the impact of stretching the bond on hydrolysis .", "Internal angles of ATP had greater changes ( Figure 7E , F ) .", "In particular , the dihedral angle ϕγ increased by 21∘–29∘ when ATP is bound to kinesin .", "This places the O atoms of Pγ in an ‘eclipsed’ ( cis ) position compared to ATP-only , where they are in a more relaxed , ‘staggered’ ( trans ) position ( Figure 7E ) .", "The different torsional states of Pγ also affects contact with Mg2+ .", "In ATP and Kin-only , Mg2+ forms bidentate contacts with two O atoms each from Pβ and Pγ .", "In ATP-only , it forms tridentate contacts with two O atoms of Pγ and one from Pβ ( Figure 7E , dotted lines ) .", "Since the increase in the dihedral energy is not substantial ( 1 . 2 kcal/mol ) , torsional angles of the phosphate moiety should readily change when ATP binds to kinesin ( calculation using a modified force field that worked well for certain ATP-bound protein structures ( Komuro et al . , 2014 ) , also yielded only marginal changes in the dihedral energy ) .", "But holding only one Oγ atom by Mg2+ will result in a greater electron withdrawal effect compared to the case when the contact is shared between two oxygens .", "This permits the lytic water to more easily attack Pγ momentarily on the opposite side .", "Similar dihedral transition and charge redistribution in the phosphate group of GTP upon binding to Ras/GTPase activating protein have been observed ( Rudack et al . , 2012 ) .", "For myosin , the eclipsing is present in both pre-powerstroke and post-rigor states .", "The latter state is incapable of hydrolyzing ATP since critical residues in the switch domains are displaced ( Lu et al . , 2017 ) .", "Although a torsion-based ATPase mechanism may hold across nucleotide triphosphatases , there are likely multiple hydrolysis pathways , whose relative energetics may be determined collectively by the ATP conformation , catalytic water coordination , and conformations of residues immediately surrounding ATP as well as remote domains of the motor ( Lu et al . , 2017 ) .", "ADP+Pi models the state after ATP hydrolysis , and Pi was pulled out by sw-I as it moved away from the P-loop ( Figure 5E; Video 3 ) .", "The gap created between sw-I and ADP was sufficient for Pi to exit above ( Figure 5E , 739 . 92 ns ) .", "In reality , there may be multiple Pi release paths due to the mobile sw-I .", "In a similar 2 . 04-μs simulation of the Eg5-MT complex , Pi released at 701 ns in a rearward direction ( Figure 5—figure supplement 2B; Video 3 ) .", "Recent experiments suggest that the duration of the ADP+Pi state affects the processivity of a kinesin dimer ( Milic et al . , 2014; Andreasson et al . , 2015; Mickolajczyk et al . , 2015; Hancock , 2016 ) .", "In the above simulations , Pi was monovalent ( H2PO-4 ) .", "In two simulations ( 3 . 7 μs and 3 . 8 μs each ) of the Eg5-MT complex with a divalent phosphate ( HPO2-4; P2-i ) , P2-i formed an extensive network of contacts with Mg-ADP and sw-I , and did not release ( Figure 5—figure supplement 2C , D ) .", "P2-i is a high-energy transition state where the proton released after hydrolysis is added to convert it to Pi ( McGrath et al . , 2013 ) .", "Since the proton can instead release into bulk water , the time of conversion from divalent to monovalent phosphate may depend on the time scale of proton transfer and other factors such as conformational fluctuation of kinesin .", "The phosphate release time also depends on the orientation of the phosphate in the nucleotide pocket .", "In another 2-μs simulation of Kin-1 with a monovalent Pi ( as in ADP+Pi ) , its lone oxygen atom formed a contact with Mg2+ , and release did not happen until the end of the simulation , analogous to the situation in Figure 5—figure supplement 2D .", "While various factors affect the time scale of Pi release , since sw-I firmly contacts Pi and is mobile , its outward motion is expected to be involved in driving the release .", "Sw-I also facilitates ADP release , which was observed in ADP𝑝𝑟𝑒 ( Figure 5F ) .", "At 201 ns , it swung toward ADP and its conserved N198 formed nonpolar contact with the adenosine ring ( Figure 5F ) .", "This contact was transient and broke again .", "After a number of attempts , ADP was gradually pulled out ( Figure 5—figure supplement 2E; Video 4 ) .", "Sw-I then lost its α-helical conformation ( Figures 5F , 2910 ns ) .", "It is unclear whether the α-helical state of sw-I is required for ADP release .", "A possible advantage is that the helix is more rigid than a disordered state , and it can exert a lever action for pulling ADP out of the P-loop .", "In the simulation of ATP binding , sw-I spontaneously formed an α-helical turn ( Figures 5D , 2040 ns ) , which indicates that it can transition between disordered and helical states unless the SSR motif is stabilized by a bound ATP ." ], [ "The present results and previous findings lead us to propose a detailed model of kinesin dimer motility in which subdomain dynamics plays an essential role ( Figure 8 and Video 5 ) .", "It begins with the hydrolysis of the bound ATP in the rear head , which involves the sw-I–II connection , dynamic water coordination , and torsional strain in catalyzing ATP hydrolysis ( Figure 8A ) .", "After ATP hydrolysis , the rear head changes its position and orientation slightly , which allows the front head to release ADP and fully bind to the MT ( Figure 8B ) .", "Until the rear head releases Pi and detaches , ATP binding to the front head is prevented ( ‘gated’; Figure 8C ) .", "Once the rear head unbinds , possibly coupled with unfolding of α⁢4 ( see below ) , ATP binding to the front head occurs , assisted by the α⁢0/L5/sw-I trio domains ( Figure 8D ) .", "The resulting formation of the CNB in the front head generates the power stroke in which the rear head is thrown forward and begins a diffusive search for the next binding site .", "The E-hook of the MT helps with capturing what is now the front head ( Figure 8E ) .", "The present work focuses on the properties of a single kinesin head that is likely to form the basis for diverse motility characteristics of different kinesins .", "In this regard , while the above model of dimer motility describes how it may be achieved by subdomain dynamics , the present model cannot address aspects pertaining specifically to the dimer motility , which would require knowledge of the communication between the two heads .", "Nevertheless , several general conclusions can be made .", "Our model highlights the active nature of nucleotide processing , where binding of ATP , hydrolysis , and release of hydrolysis products are mediated by concerted motions of mobile subdomains .", "The inherent mobility of sw-I is consistent with an experiment based on fluorescence resonance energy transfer ( Muretta et al . , 2015 ) .", "It was shown in the paper that sw-I in both Kin-1 and Kin-5 bound to the MT stayed in an ‘open’ state more than 50% ( mole fraction ) , even in the ATP state , as opposed to the ‘closed’ state that was assumed to take the hairpin conformation .", "The higher mobility or deformation of sw-I for an isolated kinesin compared to the MT-bound kinesin with ATP ( Figure 2D , E ) is also consistent with a previous electron paramagnetic resonance experiment ( Naber et al . , 2003 ) .", "In the ATP state , the mobility of sw-I is limited such that its C-terminal side maintains contacts with ATP and sw-II that are necessary to support the hydrolysis .", "This was the case even for an isolated kinesin , whose lower ATP hydrolysis rate is likely due to the widening of the nucleotide pocket that reduces coordination of the catalytic water molecules ( Figure 7 ) .", "The conformational fluctuation of the outer ( N-terminal ) side of sw-I may allosterically affect the catalytic water coordination and the precise orientation of residues of the SSR motif on the C-terminal side .", "In the case of myosin , these factors have been shown to affect the energetics of ATP hydrolysis ( Lu et al . , 2017 ) .", "The hairpin conformation of sw-I could be more advantageous for hydrolysis than the unfolded state .", "However , since the hydrolysis reaction itself proceeds over a picosecond time scale ( McGrath et al . , 2013 ) , the hairpin does not need to stay stably folded .", "Thus , variations in the sequence of the distal part of sw-I may provide an allosteric mechanism to fine-tune ATP hydrolysis and other kinetic rates by controlling its flexibility .", "This idea is supported by the behavior of the R190A/D231A mutant ( Cao et al . , 2014 ) , which is expected to destabilize or prevent the hairpin state ( Figure 5—figure supplement 1A ) .", "Rather than abolishing hydrolysis , its catalytic rate is 22% of the wild-type value .", "This demonstrates that the hairpin state may promote hydrolysis , but it is not required .", "Another illuminating feature of the dynamic role of sw-I is that the ATP hydrolysis rate depends on whether kinesin is bound to the MT filament or to unpolymerized tubulin ( Alonso et al . , 2007; Gigant et al . , 2013 ) .", "Since sw-I is the most labile element within the nucleotide pocket , its conformational motion may be affected the most when kinesin is bound to an unpolymerized tubulin , so as to influence the catalytic rate .", "We propose that α⁢0 is the structural element responsible for ATP gating in the front head ( Figure 8C ) .", "For the gating , the rearward orientation of the NL , rather than its tension , is essential ( Clancy et al . , 2011; Andreasson et al . , 2015 ) .", "In x-ray structures of kinesins with a rearward-docked NL , it interacts with the 3-stranded β⁢1 domain , which is linked to the C-terminal end of α⁢0 ( Figure 1—figure supplement", "1 ) ( Sablin and Fletterick , 2004; Guan et al . , 2017 ) .", "This raises the possibility that the positional fluctuation of α⁢0 ( Figure", "2 ) is controlled by the interaction between the NL and β⁢1 , thereby affecting the ATP binding .", "Further tests are needed to validate this proposal .", "The relatively low specificity of the hydrated kinesin-MT interface ( Figure 3C , Figure 4C , Supplementary file", "3 ) is suited for rapid interaction with MTs in vivo ( Leduc et al . , 2012 ) .", "In ADP+Pi , we did not observe unbinding of the motor head after Pi release , as is expected for the ADP-state kinesin .", "It is significant that ADP+Pi had more high-occupancy contacts with the MT than the other states ( Supplementary file 3; this was the case even when only the last 500 ns , well after Pi release in ADP+Pi , were considered ) , and its MT-binding energy was among the strongest ( Figure 3—figure supplement 1L ) .", "It appears to us unlikely that extension of the simulation time will lead to detachment of the motor head .", "To disrupt the kinesin-MT interface , we suggest that a conformational transition must occur .", "A likely subdomain for this transition is the N-terminus of α⁢4 .", "It unfolded transiently in ADP+Pi and more extensively in Kin-only ( Figure 2E ) .", "Conformational fluctuation of the unfolded α⁢4 can disrupt the rear part of the kinesin-MT contact ( Figure 4C ) , leading to detachment .", "This picture agrees with the recent finding that Pi release is required for unbinding of kinesin from the MT ( Milic et al . , 2014 ) .", "The presence of Pi in the nucleotide pocket suppresses the unfolding of α⁢4 , thereby keeping the motor head bound to the MT . This is an interesting subject for studies beyond the present simulations .", "Compared to a static mechanism that requires a specific structure , the dynamic mechanism for nucleotide processing and MT binding found here , provides greater flexibility in fine-tuning time scales and affinities .", "For example , the conserved residues that contact ATP cannot account for differences in catalytic rates among various kinesins .", "Altering residues that do not directly contact ATP can affect the conformational dynamics , which could in turn influence the frequency of 2-water bridge formation and charge fluctuation around Pγ , thereby controlling the catalytic rate .", "Of interest are experimental means to test the dynamic roles of subdomains by introducing mutations .", "Since the N-terminal side of sw-I does not contact ATP directly , it may be possible to mutate it to alter the dynamical properties of sw-I without severely impairing motility .", "For example , a more flexible sw-I may enhance rates for all phases of nucleotide processing .", "However , caution must be exercised here , since if sw-I is made too flexible , it may disrupt contacts with the nucleotide .", "Similarly , elongating α⁢0 by lengthening L1a and L1b ( Figure 1—figure supplement 1E ) may increase the ATP binding rate , but it may also affect the gating behavior ( Figure 8C ) .", "The design of mutants and their predicted behavior will require careful analysis and simulations .", "We also showed that the elastic energy of the curvature of the central β-sheet or deformation of the MT , are unlikely to drive motility ( Figure 2 and Figure 2—figure supplement 1 ) .", "The aspect ratio of the motor head is too small to store any significant deformational energy .", "Even for myosin V that has a larger aspect ratio , there is little evidence that the twist of its β-sheet has any strong energetic role ( Cecchini et al . , 2008 ) .", "Furthermore , the hydrated and dynamic kinesin-MT interface is unlikely to induce substantial strain in the motor head .", "Instead , small amount of strain may play a role in fine-tuning kinetic rates .", "The free energy change upon NL docking in the absence of load ( 0 . 7–2 . 9 kcal/mol ) ( Rice et al . , 2003; Muretta et al . , 2015 ) is much smaller than the maximum work done near the stall force ( 5 . 8–8 . 1 kcal/mol ) .", "Additional energy is likely to originate from the CNB formation ( Hwang et al . , 2008; Khalil et al . , 2008 ) and binding of the front head to the MT ( Figure 8E ) .", "Since the hydrolysis energy of ATP thermalizes rapidly ( on the picosecond time scale ) , it is unlikely to have any direct role .", "However , the differential binding energy of ATP and its hydrolysis products are likely to be important in triggering the large transitions of the motility cycle .", "Thus , while the net free energy change after a motility cycle may be that of hydrolyzing an ATP , there is a large free energy flow between kinesin and the environment during each phase of the cycle .", "In this regard , kinesin’s mobile subdomains are ‘free energy transducers . ’ The present results provide the basis for understanding the role of local subdomain dynamics in the kinesin motility cycle .", "We emphasize that the extension of the simulations to multiple microseconds for each state , made possible by the use of Anton , played an important role in obtaining converged results .", "Suggestions are made concerning elements that will have to be tested by future experiments and simulations .", "This is important because the variation in structural rigidity , hydration , and protein-protein interaction found in the simulations provide a dynamic description of how kinesin works that is significantly different from conclusions based solely on static crystal and cryo-EM structures .", "Given the commonality among translocating motor proteins ( Hwang and Lang , 2009 ) , it is likely that local subdomain dynamics plays active roles for driving conformational changes and reactions , more generally ." ], [ "Among the above , PDB 4HNA and 4LNU are x-ray structures of kinesin-MT complexes respectively in ATP ( ATP analogue ) and APO states .", "Although the tubulin dimers in these structures are slightly curved , it has been shown not to affect the kinesin-MT interface ( Gigant et al . , 2013; Cao et al . , 2014 ) .", "We thus did not straighten the tubulin structure .", "For Kin-5 , we used the following structures: We constructed the kinesin structure up to the NL ( M1–A337 ) , excluding the α-helical stalk .", "The C-terminal end of a tubulin has 13-aa glutamate-rich E-hook that are invisible in x-ray structures due to its flexibility .", "For α and β tubulins , we omitted the last 9 ( E443–Y451 ) and 4 ( E452–A455 ) residues of E-hooks , respectively .", "These truncations render the system size to fit within Anton .", "The E-hook of α tubulin is located on the minus end side of a tubulin dimer ( at the left end of αH12 in Figure 1C ) and is away from the kinesin motor head .", "The E-hook of β tubulin locates on the right side of the motor head ( at the end of βH12 in Figure 1C ) .", "Being negatively charged and flexible , E-hooks are known not to affect kinesin in the MT-bound state , and it is more important for making non-specific electrostatic contacts with an unbound head ( Figure 8E ) ( Lakämper and Meyhöfer , 2005; Sirajuddin et al . , 2014 ) .", "Truncations of E-hooks are thus unlikely to affect our result for MT-bound kinesins .", "For each system , the protein structure was placed in a cubic TIP3P water box of linear size 113–119 Å ( for kinesin-MT complex; 88 Å for Kin-only ) and it was made electrically neutral by adding ions to about 50 mM concentration .", "The number of atoms in our systems were in the 150 , 000–170 , 000 range ( 65 , 000 for Kin-only ) .", "A periodic boundary condition was applied to the box .", "In preparation for simulations on Anton , the solvated system was simulated using CHARMM ( Brooks et al . , 2009 ) on a conventional computer cluster .", "Initially , a series of energy minimization procedure was done with harmonic constraints applied to proteins and nucleotides , which were gradually reduced to zero in successive 200-step minimization cycles .", "Next , the system underwent heating ( 100 ps ) and equilibration ( 200 ps ) runs under 1-atm pressure .", "During heating to 300 K , harmonic constraints were applied to backbone heavy atoms of proteins except for the 4-aa N-terminal end of kinesin’s CS and the C-terminal E-hook domains of MT that are flexible .", "The spring constant of the harmonic constraint was 1 kcal/mol⋅Å2 during heating , and 0 . 5 kcal/mol⋅Å2 during equilibration .", "It was further reduced to 0 . 25 kcal/mol⋅Å2 , with only Cα atoms restrained ( excluding those of the flexible domains noted above ) , and simulation continued for 2 ns using the constant temperature ( 300 K ) and pressure ( 1 atm ) ( CPT ) dynamics method implemented in CHARMM .", "The final phase of the preparatory run lasted 2 ns with 0 . 5-kcal/mol⋅Å2 harmonic constraints applied to Cα atoms of the loops of tubulins that are near the interface with neighboring MT protofilaments ( aa 57–61 , 83–88 , and 279–286 , for both tubulins ) .", "They are located on the bottom in Figure 1B , and restraining them mimics the effect of the tubulin dimer embedded within a polymerized MT . For Kin-only that lacks the MT , we harmonically restrained the Cα atoms of L229–D231 of β⁢7 ( Figure 1—figure supplement 1 ) with a 0 . 1-kcal/mol⋅Å2 spring constant .", "These atoms are located approximately at the center of mass of the motor head , and the weak restraint suppresses translational diffusion of kinesin .", "For simulation , the CHARMM param36 force field was used .", "For ADP+Pi , the monovalent form of Pi ( H2PO-4 ) was constructed based on phosphate parameters in the param22 force field .", "The SHAKE algorithm was applied to fix the length between hydrogen and its base heavy atom .", "The integration time step was 2 fs .", "We wrote a Python script to convert the CHARMM restart file at the end of the preparatory run to the the Desmond Maestro format file , which was further processed using the Anton software .", "The CHARMM param36 force field was used through the Viparr utility of Anton .", "Harmonic restraints of spring constant 0 . 25 kcal/mol⋅Å2 were applied to Cα atoms of the same residues of the MT loops as in the last phase of the preparatory simulation .", "SHAKE was applied to hydrogen atoms , with a 2-fs integration time step .", "The multigrator integration method of Anton was used under a CPT ( 300 K , 1 atm ) condition .", "Coordinates were saved every 0 . 24 ns .", "After simulation , coordinate trajectories were converted to CHARMM DCD format files by using VMD ( Humphrey et al . , 1996 ) , for analysis using CHARMM .", "All simulations were carried out on Anton ( Shaw et al . , 2009 ) except for APO , which was on the newer Anton-2 machine ( Shaw et al . , 2014 ) .", "We considered seven strands within the central β-sheet of kinesin: β⁢1 ( V11–F15 ) , β⁢3 ( T80–G85 ) , β⁢4 ( I130–Y138 ) , β⁢5 ( I142–D144 ) , β⁢6 ( S206–K213 ) , β⁢7 ( K226–L232 ) , and β⁢8 ( T296–C302 ) ( Figure 1—figure supplement 1 ) .", "This choice excludes regions of β⁢4 , β⁢6 , and β⁢7 at the front end of the motor head that deformed in some simulations ( Figure 2G , H and Figure 2—figure supplement 1G–J ) .", "To calculate curvature , the central β-sheet in each coordinate frame was oriented to a reference kinesin structure whose least-square-fit plane was oriented to the x⁢y-plane of the Cartesian coordinate system and the center of mass positioned at the coordinate origin .", "In this configuration , z-coordinates of Cα atoms within the central β-sheet were parameterized by their x and y coordinates and were fit using the quadratic expansion ( Sun et al . , 2003 ) ( 1 ) z⁢ ( x , y ) =a0+a1⁢x+a2⁢x2+a3⁢y+a4⁢x⁢y+a5⁢y2 , where {ai} ( i=0 to 5 ) are fitting parameters that vary among coordinate frames .", "Fitting was done using the SciPy package of Python .", "Examples of fitting surfaces are in Figure 2I .", "For a given frame , the mean and Gaussian curvatures are given by M=2⁢ ( a2+a5 ) and G=a42-4⁢a2⁢a5 ( Sun et al . , 2003 ) .", "To calculate the potential of mean force ( PMF ) versus the curvature , we calculated a 2-dimensional histogram of M2 and G normalized by the maximum count , ρ⁢ ( M2 , G ) .", "PMF is given by -kB⁢T⁢ln⁡ρ ( Figure 2—figure supplement 1K ) .", "To align PMFs for ATP and APO ( Figure 2J ) , we identified bins of the histogram that have nonzero counts in both simulations .", "We determined the constant free energy shift Δ that needs to be added to the PMF for APO so that the mean-square difference of the two PMFs in the overlapping bins is minimized .", "The mean-square difference was calculated weighted by the histogram counts of respective PMFs .", "Denote the histogram values of ATP and APO in the k-th bin within the overlap region by ρk𝐴𝑇𝑃 and ρk𝐴𝑃𝑂 , respectively , and similarly denote their PMFs by Ek𝐴𝑇𝑃 and Ek𝐴𝑃𝑂 .", "Minimizing ∑kρk𝐴𝑇𝑃⁢ρk𝐴𝑃𝑂⁢ ( Ek𝐴𝑇𝑃-Ek𝐴𝑃𝑂-Δ ) 2 yields ( 2 ) Δ=∑kρk𝐴𝑇𝑃⁢ρk𝐴𝑃𝑂⁢ ( Ek𝐴𝑇𝑃-Ek𝐴𝑃𝑂 ) ∑kρk𝐴𝑇𝑃⁢ρk𝐴𝑃𝑂 where the sum is for bins in the overlap region .", "We added Δ to the PMF for APO , and the merged PMF Ek for the k-th bin in the overlap region was set to ( 3 ) Ek=ρk𝐴𝑇𝑃⁢Ek𝐴𝑇𝑃+ρk𝐴𝑃𝑂⁢ ( Ek𝐴𝑃𝑂+Δ ) ρk𝐴𝑇𝑃+ρk𝐴𝑃𝑂 .", "To calculate the position and orientation of kinesin relative to the microtubule ( Figure 3A , B , Figure 3—figure supplement 1A–G ) , we used the Cα atoms of the following domains: Each coordinate frame was aligned to the first frame of ATP , with the Cα atoms of H12 helices ( aa417–432 ) of α- and β-tubulins as reference for alignment .", "Let Rα−tub , and Rβ−tub be the centers of masses of H12 in respective tubulins .", "An orthonormal triad {uL , uN , uT} was constructed in the following way ( Figure 3—figure supplement 1A ) : Let rβ be the center of mass of the central β-sheet .", "We projected ( rβ−Rα-tub ) onto the three directions of the triad .", "Differences of these projections from those of the reference structure were defined respectively as the longitudinal ( ΔL ) , normal ( ΔN ) , and transverse ( ΔT ) displacements .", "Displacements of α⁢4 , Q320 , and β⁢5⁢a/b were measured similarly .", "To calculate the orientation of the motor head , we used an approximately rectangular section of β⁢4 ( I130–E136 ) , β⁢6 ( S206–V212 ) , and β⁢7 ( K226–D231 ) .", "Using their Cα atoms , we calculated the major and normal axes of the least-square-fit plane , vβ1 and vβ2 , respectively .", "We projected vβ1 onto the plane spanned by uL and uN , and measured the forward tilt angle θβ as the angle between this projection and uL .", "The azimuthal angle ϕβ was measured as the angle between the projection of vβ1 onto the plane spanned by uL and uT , with uL .", "The transverse tilt angle ωβ was between the projection of vβ2 onto the plane spanned by uN and uT , with uN .", "Increase in ϕβ is associated with clockwise rotation of the motor head when viewed from top , and for ωβ , it is counterclockwise rotation when viewed from the MT plus end .", "Since the central β-sheet is tilted to the left , ωβ is typically negative , and in Figure 3—figure supplement 1F , -ωβ was plotted .", "A larger ( less negative ) ωβ indicates that the motor head tilts more to the right .", "Azimuthal ( ϕα ) and transverse tilt ( ωα⁢6 ) angles of α⁢6 were similarly measured using the projection of the axis of α⁢6 on respective planes .", "The orientation angle ψα⁢4 of α⁢4 was measured between its axis and uT .", "To calculate the water density map for the kinesin-MT interface ( Figure 3C ) , we adopted a method that we developed previously ( Ravikumar and Hwang , 2011 ) .", "Coordinate frames were aligned to the first frame of ATP with Cα atoms of the reference domains consisting of α⁢4 ( E250–E270 ) , and parts of H11–H12 of α-tubulin ( F395–E420 ) and H12 of β-tubulin ( M425–Y435 ) .", "A search box was set whose boundary is at least 15 Å away from any atom in the above domains .", "The box was divided into a cubic grid of linear size 0 . 7 Å .", "For each cell in the grid , the fraction of frames where a water oxygen is found was calculated and divided by the volume of the cell ( 0 . 73 Å3 ) .", "The map was saved into an MRC electron density map format file and visualized using UCSF Chimera ( Pettersen et al . , 2004 ) .", "The water density map around the phosphate moiety of ATP ( Figure 7A , B ) was calculated similarly , with Cα atoms of the P-loop ( G85–G90 ) and Pγ as positional reference for aligning coordinate frames .", "To calculate the buried area in the kinesin-MT interface ( Figure 3D ) , for each coordinate frame , we used a 1 . 4 Å probe radius to calculate the solvent accessible surface area of kinesin and the MT , together and separately , and measured their difference .", "Calculation of the binding energy was done based on a previously developed method ( Zoete et al . , 2005 ) .", "Briefly , for each coordinate frame during the last 500 ns , the following energies were measured: van der Waals ( Lennard-Jones ) , electrostatic , generalized Born solvation free energy , and the nonpolar energy .", "Calculation of energy terms was done using the Generalized Born with a simple SWitcthing ( GBSW ) module of CHARMM ( Im et al . , 2003 ) .", "To find the binding energy , we used the kinesin motor head , α-tubulin , and β-tubulin , individually or in combination .", "For example , between the motor head and the α-tubulin , we measured EK for kinesin , Eα for the α-tubulin , and EK⁢α for them together ( E denotes an energy term ) .", "Their binding energy was defined as EK⁢α-EK-Eα .", "Similar calculations were done for the binding energy between kinesin and the β-tubulin , and kinesin and the whole tubulin dimer .", "For each coordinate frame in a simulation , hydrogen bonds ( H-bonds ) were identified with the donor-acceptor distance cutoff of 2 . 4 Å .", "A residue pair was considered to form a nonpolar contact if the pair has neutral atoms ( partial charge less than 0 . 3e; e=1 . 6×10-19 C ) that are closer than 3 . 0 Å .", "The occupancy of a bond is the fraction of frames over which the bond is formed during the simulation , and its occupancy trajectory is the rolling ( running ) average with a 96-ns ( 400 frames ) window .", "To identify formation and breakage of a bond during simulation , we calculated its occupancy for the first and last 200 frames ( 48 ns ) , respectively .", "If the initial occupancy is less than 0 . 05 , and the final one is greater than 0 . 5 , the bond was regarded to have formed during the simulation , and vice versa for identifying bonds that broke .", "We traced the trajectory forward ( bond formation ) or backward ( bond breakage ) in time and located the time point where the local occupancy became greater than 0 . 5 , as the corresponding transition time ( Figure 4—figure supplement 1A ) ." ] ]

[ "The microtubule ( MT ) -associated motor protein kinesin utilizes its conserved ATPase head to achieve diverse motility characteristics .", "Despite considerable knowledge about how its ATPase activity and MT binding are coupled to the motility cycle , the atomic mechanism of the core events remain to be found .", "To obtain insights into the mechanism , we performed 38 . 5 microseconds of all-atom molecular dynamics simulations of kinesin-MT complexes in different nucleotide states .", "Local subdomain dynamics were found to be essential for nucleotide processing .", "Catalytic water molecules are dynamically organized by the switch domains of the nucleotide binding pocket while ATP is torsionally strained .", "Hydrolysis products are 'pulled' by switch-I , and a new ATP is 'captured' by a concerted motion of the α0/L5/switch-I trio .", "The dynamic and wet kinesin-MT interface is tuned for rapid interactions while maintaining specificity .", "The proposed mechanism provides the flexibility necessary for walking in the crowded cellular environment ." ]

[ "Motor proteins called kinesins perform a number of different roles inside cells , including transporting cargo and organizing filaments called microtubules to generate the force needed for a cell to divide .", "Kinesins move along the microtubules , with different kinesins moving in different ways: some ‘walk’ , some jump , and some destroy the microtubule as they travel along it .", "All kinesins power their movements using the same molecule as fuel – adenosine triphosphate , known as ATP for short .", "Energy stored in ATP is released by a chemical reaction known as hydrolysis , which uses water to break off specific parts of the ATP molecule .", "The site to which ATP binds in a kinesin has a similar structure to the ATP binding site of many other proteins that use ATP .", "However , little was known about the way in which kinesin uses ATP as a fuel , including how ATP binds to kinesin and is hydrolyzed , and how the products of hydrolysis are released .", "These events are used to power the motor protein .", "Hwang et al . have used powerful computer simulation methods to examine in detail how ATP interacts with kinesin whilst moving across a microtubule .", "The simulations suggest that regions ( or 'domains' ) of kinesin near the ATP binding site move around to help in processing ATP .", "These kinesin domains trap a nearby ATP molecule from the environment and help to deliver water molecules to ATP for hydrolysis .", "Hwang et al . also found that the domain motion subsequently helps in the release of the hydrolysis products by kinesin .", "The domains around the ATP pocket vary among the kinesins and these differences may enable kinesins to fine-tune how they use ATP to move .", "Further investigations will help us understand why different kinesin families behave differently .", "They will also contribute to exploring how kinesin inhibitors might be used as anti-cancer drugs ." ]

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

[ "developmental biology" ]

[ [ "Nodal , a member of the TGFβ superfamily of cell-cell signaling ligands , is essential in the establishment of vertebrate axis patterning , both the primary embryonic axis during gastrulation and the orthogonal left-right ( LR ) asymmetries in heart , brain and gut development .", "vg1 , another member of the TGFβ family , was prototypically described as the first-known regionally localized RNA in a vertebrate oocyte , localized to the vegetal pole of the egg , and subsequently in early embryos ( Rebagliati et al . , 1985 ) .", "Humans and other mammals have two vg1 orthologues , GDF1 and GDF3 , and zebrafish have a single orthologue , gdf3 , previously known as dvr1 .", "Although gdf3 ( vg1 ) has been a target of considerable study for many years , genetic mutations for functional analysis have not been reported in zebrafish .", "Studies in Xenopus have suggested that maternal Vg1 is required for early zygotic expression of anterior mesendodermal genes ( Birsoy et al . , 2006 ) .", "Vg1 has been implicated in early Left-Right patterning , as overexpression of Xenopus Vg1 fusion proteins or mouse Gdf1 fusion proteins in specific early cell lineages can fully invert the LR axis ( Hyatt et al . , 1996; Hyatt and Yost , 1998; Wall et al . , 2000 ) .", "Vg1 is the only known member of the TGFβ family with this early LR patterning capability .", "At later stages of development , overexpression or grafts into lateral plate mesoderm that activate TGFβ family member Xenopus Nodal ( Xnr1 ) in right lateral plate can also invert asymmetry ( Ohi and Wright , 2007 ) .", "In mice , homozygous knockout mutations have altered asymmetry ( Rankin et al . , 2000 ) .", "In humans , genetic variants in GDF1 have been implicated in complex congenital heart defects ( Karkera et al . , 2007; Zhang et al . , 2015 ) , likely to be effects of upstream altered LR patterning .", "Similar to vg1 in Xenopus , gdf3 RNA and protein are abundantly stored in the oocyte and early embryo before zygotic gene activation in zebrafish ( Helde and Grunwald , 1993; Peterson et al . , 2013 ) .", "In zebrafish , Kupffer’s vesicle ( KV ) , the ciliated organ of asymmetry ( Essner et al . , 2005; Essner et al . , 2002 ) , contains motile cilia that generate asymmetric fluid flow and LR patterning information that is transmitted to lateral plate mesoderm ( LPM ) , which then conveys LR patterning information to the brain , heart and gut primordia ( Dasgupta and Amack , 2016 ) .", "Zebrafish gdf3 is expressed in tissues implicated in LR patterning including cells adjacent to the KV , in the LPM and in heart primordia .", "Morpholino knockdown of gdf3 results in normal KV structure , KV cilia length and motility , and normal asymmetric KV fluid flow , but disruption of downstream LR patterning in the LPM .", "Thus , zebrafish Gdf3 was proposed to transmit LR information generated by KV cilia flow to LPM ( Peterson et al . , 2013 ) , and the reasonable expectation was that mutants of gdf3 in zebrafish would have LR patterning defects .", "We engineered mutants in zebrafish to test the roles of gdf3 .", "Surprisingly , homozygous zygotic mutants develop normally and do not show alterations in LR patterning .", "On the other hand , zebrafish mutants in which the maternal stores of Gdf3 are eliminated have a much more dramatic embryonic lethal phenotype , which we analyzed for a wide range of tissue specification and embryonic patterning pathways .", "Results from a series of epistasis experiments and cell-lineage targeted rescues of gdf3 mutants indicate that Gdf3 functions as an obligate co-factor of Nodal , and that Nodal functions as an obligate cofactor of Gdf3 .", "These results indicate that neither Nodal nor Gdf3 can function without the other in fundamental patterning of the vertebrate embryonic axis ." ], [ "We generated mutant alleles of gdf3 by using TALENs designed to target genomic sequences encoding the first few amino acids of the pro-domain of Gdf3 ( Figure 1A; Figure 1—figure supplement 1 ) .", "High resolution melt analysis ( HRMA ) ( Parant et al . , 2009 ) was used to screen for mutations in genomic DNA from embryos of G0 founders and later , from fin clips from adults derived from those founders ( Figure 1B ) .", "Among several mutants identified , three lines were selected and propagated .", "The results presented here utilize gdf3zy51 and gdf3zy52 alleles; both mutations result in reading frame shifts which encode missense amino acids and early termination codons , suggesting they may be functional nulls ( Figure 1C ) .", "The phenotypes are indistinguishable between gdf3 mutants carrying either the zy51 or zy52 allele and these mutants are used and described interchangeably .", "A third mutant line , gdf3zy53 , has a 6 bp in-frame deletion that removes two amino acids but leaves the predicted protein otherwise unchanged and had no observable phenotype .", "Surprisingly , homozygous recessive zygotic mutants generated by in-crosses of heterozygous carriers of either gdf3zy51 or gdf3zy52 had no morphological differences from wild-type ( WT ) embryos ( N > 100 ) ( Figure 1D , E ) .", "Homozygous mutants survive to adulthood at normal wild-type rates .", "In contrast to previous results with morpholinos in zebrafish , zygotic mutants of gdf3 had normal LR patterning as assessed by scoring heart looping in clutches of embryos derived from heterozygous gdf3 in-crosses at 48 hr post-fertilization ( hpf ) .", "As in WT , clutches of Zgdf3 embryos showed about a 2% heart reversal rate ( Zgdf3zy51 64/65 normal heart looping , 2 clutches scored; Zgdf3zy52 154/157 normal heart looping , 3 clutches scored; WT 164/168 normal heart looping , 3 clutches scored ) .", "Consistent with the heart looping data , an examination of the expression of the early Left-Right marker gene spaw at the 18–20 somite-stage in embryos derived from WT parents or from an incross of gdf3zy51 heterozygotes showed clutches of WT and Zgdf3 with similar percentages of normal left-sided gene expression ( 93% in WT clutches; 90% in Zgdf3 clutches ) ( Figure 1—figure supplement 2 ) .", "One quarter of the adults raised from in-crosses of gdf3 heterozygotes were homozygous for the mutant alleles as assayed by HRMA , indicating no embryonic or adult lethality of Zgdf3 .", "Homozygous zygotic gdf3 mutant adults are fertile .", "In-crosses of homozygous mutants yielded Maternal Zygotic ( MZgdf3 ) embryos that exhibit a pronounced phenotype at 24 hpf , with 100% penetrance ( N > 100 ) ( Figure 1F ) .", "At 24–26 hpf , MZgdf3 embryos lack a notochord and spinal cord and structures associated with lateral plate mesoderm including the heart , and have greatly reduced anterior neural development reflected in the narrowed neural tube , un-folded brain and reduced tissue between the eyes resulting in cyclopia ( Figure 1—figure supplement 3 ) .", "To confirm that the MZgdf3 phenotype was due to a loss of wild-type gdf3 gene product , we asked whether MZgdf3 embryos could be rescued by gdf3 mRNA injections ( Figure 1G ) .", "To assess the dose dependence of rescue , we devised a phenotype classification system ( Figure 1—figure supplement", "4 ) somewhat analogous to the DAI ( dorso-anterior indices ) in Xenopus ( Kao and Elinson , 1988 ) that has been useful for studies of axis formation .", "Injection of 100 pg gdf3 mRNA fully rescued over 90% of the MZgdf3 embryos , and these fully rescued embryos could survive until adulthood and survivors were fertile .", "For example , of 22 rescued MZgdf3 embryos selected for an inflated swim bladder at 6 days post-fertilization and raised to adulthood , 14 survived .", "From those , 5 mating pairs produced clutches of embryos with the MZgdf3 phenotype at 24 hpf , demonstrating fertility of both males and females that were derived from Gdf3 null embryos that received only a pulse of WT gdf3 mRNA shortly after fertilization .", "To assess whether both maternal and paternal contributions to the zygote were necessary for the MZgdf3 phenotype , we performed reciprocal pairwise crosses of male and female MZgdf3 homozygotes to either Zgdf3 heterozygotes or to wild-type fish and found that the mutant phenotype of the derived embryos was solely dependent on a gdf3−/− maternal genotype; the paternal genome did not alter the phenotype .", "Embryos derived from gdf3−/− males showed no mutant phenotype when derived from a heterozygous female with a wildtype allele of gdf3 ( n = 4 mating pairs ) .", "Strikingly , paternal contribution of a wild-type gdf3 allele could not rescue embryos derived from MZgdf3- females ( n = 7 mating pairs with gdf3+/− male; n = 3 mating pairs with WT +/+ males ) .", "In other words , gdf3+/− heterozygotes that were derived from gdf3 null eggs and wildtype sperm displayed the MZgdf3 phenotype .", "To assess whether the paternal genome was transcriptionally activated following zygotic gene activation ( ZGA ) in embryos from a cross of MZgf3 female by WT male cross , we carried out RT-PCR to compare cDNA samples prepared from pre-ZGA 1000 cell-stage and post-ZGA 90% epiboly , and 18 somite-stage embryos , and compared those to samples at the same time points derived from embryos from a MZgdf3 in-cross and a WT cross ( Figure 1—figure supplement 5 ) .", "There was a substantial decrease in the amount of PCR product from the 1000 cell stage to the 18-somite stage in each genotype , but we were unable to detect any substantial differences among samples prepared from the same time points across the three genotypes indicating that the mutant maternal RNA from MZgdf3 embryos was as stable as that from WT embryos ( Figure 1—figure supplement 5A , B ) .", "To resolve whether the paternal genome was activated in a gdf3- maternal background , we designed primers specific to the WT allele of gdf3 and carried out RT-PCR on the same series of stages and genotypes ( Figure 1—figure supplement 5C , D ) .", "WT gdf3 transcript was detected in all stages of WT embryos ( lanes C1-3 ) and was absent from cDNA derived from homozygous MZgdf3 embryos ( serving to indicate that the WT primers do not amplify the maternal or paternal mutant allele; lanes C7-9 ) .", "WT transcript was also undetectable in pre-ZGA stage embryos ( 1000 cell stage , lane", "4 ) derived from a cross of a MZgdf3 mutant female by WT male , but was present in cDNA derived from post-ZGA stages ( lanes C4 , 5 ) .", "The fact that there is less transcript detected at 90% epiboly and 18 somite stages of embryos derived from a MZ mutant female versus a WT female may reflect perdurance of maternal WT transcript from the WT female , that there is significantly less mesoderm in MZ mutant embryos in which to express gdf3 , or that the paternal genome is expressed to a lesser extent in embryos lacking WT maternal transcript ( or a combination thereof ) .", "Although the paternal WT genome is clearly activated in MZgdf3 embryos , this post-ZGA expression of paternal WT gdf3 does not rescue the MZ phenotype .", "To further characterize the phenotypes of MZgdf3 embryos , we carried out whole mount in situ hybridization ( WISH ) in wild-type and MZgdf3 embryos using several tissue-specific gene expression markers at 24hpf .", "The T-box transcription factor ta ( ntl ) is expressed in the notochord , a derivative of the axial mesoderm , and in the tailbud mesenchyme ( Figure 1H ) .", "In MZgdf3 embryos , the notochord was absent as evidenced by the lack of ta expression in the midline; expression in the tailbud remains ( Figure 1I ) .", "Expression of another T-box transcription factor tbx16 ( spt ) , expressed in paraxial and ventral mesoderm , was maintained in the tailbud of MZgdf3 embryos but tbx16 expression in a subset of spinal cord neurons in WT embryos was not detected in MZgdf3 mutants ( Figure 1J , K ) .", "Similarly , the ventral mesoderm marker eve1 was expressed in the tailbud of both WT and MZgdf3 embryos ( not shown ) .", "The myogenic transcription factor myod1 was expressed in chevron-shaped somites extending the length of the trunk and tail of embryos .", "In similar stage MZgdf3 embryos , myod1 expression occurred in fewer , smaller , u-shaped somites restricted to the caudal trunk and tail ( Figure 1L , M ) .", "In WT embryos hand2 was expressed in the heart tube , a derivative of the anterior lateral plate mesoderm , in the pharyngeal mesoderm of the branchial arches and in the pectoral fin bud .", "All of these tissue-specific expression domains were absent in MZgdf3 ( Figure 1N , O ) .", "The forkhead box transcription factor foxa2 ( axial ) was expressed in the midline in the ventral brain , the spinal floorplate and the chorda-neural hinge as well as laterally in the pharyngeal endoderm of the branchial arches .", "All of these expression domains were absent in MZgdf3 mutant embryos ( Figure 1P–S ) .", "The rostral-most regions of the neural tube were relatively unaffected in MZgdf3 embryos .", "Expression domains of otx2 in the forebrain and midbrain , and egr2b ( krox20 ) in hindbrain rhombomeres 3 and 5 were present though somewhat misproportioned and misshapen in MZgdf3 mutant embryos versus WT embryos ( Figure 1T–W ) .", "Together , these results indicate that maternally provided Gdf3 is essential in the patterning of mesoderm , endoderm and in some aspects of neural development .", "To investigate roles of Gdf3 during germ layer formation and early tissue patterning , we assessed the consequences of loss of Gdf3 function , gain of function and functional rescue on the patterns of tissue-specific gene expression markers by injecting WT and MZgdf3 embryos with gdf3 RNA at the 1–2 cell stage .", "Injected or un-injected embryos were analyzed by WISH with tissue-specific markers at 90% epiboly ( Figure 2 ) .", "The MZgdf3 phenotypes described above are strikingly similar to phenotypes of mutant embryos lacking maternal Nodal signaling components such as in double homozygous mutants for ndr1;ndr2 ( sqt;cyc ) , MZtdgf1 ( MZoep ) and MZfoxh1 ( Dougan et al . , 2003; Gritsman et al . , 1999; Slagle et al . , 2011 ) , or from overexpression of the Nodal antagonists lft1 or lft2 ( Bisgrove et al . , 1999; Thisse and Thisse , 1999 ) .", "In all cases , these mutants or treated embryos exhibit a loss of notochord and spinal cord and reduction and disorganization in trunk and tail somites as well as a severe reduction in anterior and neural structures that manifest as a loss of brain patterning and synophthalmia as observed in MZgdf3 ( see Figure 1—figure supplement 3 ) .", "Thus , we examined expression of Nodal signaling pathway genes ndr2 , lft1 and lft2 ( Figure 2A–L ) .", "The Nodal family member ndr2 was expressed in anterior and posterior axial mesoderm and anterior axial neurectoderm ( Figure 2A ) .", "This WT expression pattern was unchanged in embryos injected with gdf3 RNA ( Figure 2B ) .", "In contrast , MZgdf3 embryos were void of any detectable ndr2 expression , but the normal expression pattern could be completely restored by injection of gdf3 RNA ( Figure 2C , D ) .", "Since a nodal-deficiency phenotype can be obtained by overexpression of Nodal antagonists lft1 or lft2 ( Bisgrove et al . , 1999 ) , we asked whether the MZgdf3 phenotype was due to hyperexpression of lft1 or lft2 .", "The normal expression patterns of lft1 in marginal mesendoderm , the axial chordamesoderm , prechordal plate mesoderm and anterior ventral neurectoderm ) and lft2 in posterior axial neurectoderm and prechordal plate mesoderm ( Figure 2E , I ) were absent in MZgdf3 embryos ( Figure 2G , K ) .", "Neither gene expression pattern was altered in WT embryos injected with gdf3 RNA ( Figure 2F , J ) , and gdf3 RNA injection fully rescued expression of both of these Nodal response genes in MZgdf3 ( Figure 2H , L ) .", "These results , and the observation that lft1 and lft2 expression are absent from MZgdf3 embryos at shield stage during mesendoderm formation ( Figure 2—figure supplement 1 ) , indicate that the MZgdf3-deficient phenotype was not due to hyperexpression of endogenous lft1 and lft2 .", "We next examined expression of mesodermal genes that are not Nodal ( TGFβ ) family members ( Figure 2M–X ) .", "The transcription factors ta and gsc are direct targets of Nodal signaling and overlap with the expression of ndr2 in the midline .", "( Chen and Schier , 2001; Thisse et al . , 1994 )", "In WT embryos , ta was expressed in posterior axial chordamesoderm that gives rise to the notochord as well as in the marginal mesendoderm , while gsc was expressed in the anterior prechordal plate mesoderm , which gives rise to the polster , and in anterior ventral neurectoderm ( Figure 2M ) .", "gsc and ta expression patterns were unchanged in embryos injected with gdf3 RNA ( Figure 2N ) .", "In MZgdf3 embryos midline gsc and ta expression domains were absent while ta expression in the marginal mesendoderm was unaffected ( Figure 2O ) .", "Injection of gdf3 RNA fully rescued the axial expression domains of both ta and gsc ( Figure 2P ) .", "The t-box transcription factor tbx16 was expressed in a broad domain of paraxial mesendoderm at the margin as well as in the midline in the prechordal plate mesoderm ( Fig . Q ) .", "In MZgdf3 mutant embryos axial tbx16 expression in the prechordal plate was absent and the width of the paraxial expression domain was reduced ( Figure 2S ) .", "Injection of gdf3 RNA into WT embryos did not alter normal tbx16 domains but was capable of fully rescuing expression in the prechordal plate and expanding the paraxial expression domain to a normal width ( Figure 2T ) .", "The homeobox transcription factor eve1 was expressed in a broad domain at the ventral margin of WT embryos and like other genes with margin expression was unaffected by ectopic expression of gdf3 ( Figure 2U , V ) .", "In MZgdf3 embryos the width of this expression domain was reduced , but was fully restored to normal width by the injection of gdf3 at the 1–2 cell stage ( Figure 2W , X ) .", "The roles of Gdf3 in non-mesodermal germ layers were examined using probes to the transcription factors sox17 , foxa2 and otx2 ( Figure 2Y–J’ ) .", "Sox17 is normally expressed in presumptive endoderm cells scattered throughout the blastoderm and in dorsal forerunners , a group of non-involuting cells at the dorsal margin ( Figure 2Y ) that form the KV .", "Neither of these cell types is affected by the injection of gdf3 into WT embryos ( Figure 2A’ ) .", "In MZgdf3 , no sox17-expressing cells were detected in the blastoderm and in most embryos ( 14 of 16 ) no expression was detected in cells at the dorsal margin .", "In the remaining few cases , one or two sox17 positive cells were detected at the dorsal margin ( Figure 2A’ ) .", "sox17 expression in the presumptive endoderm and dorsal forerunner cells of MZgdf3 was restored by injection of gdf3 ( Figure 2B' ) .", "Like sox17 , foxa2 was also expressed in presumptive endoderm , and additionally in axial mesendoderm and neurectoderm ( Figure 2C’ ) .", "None of these WT expression domains was affected by overexpression of gdf3 ( Figure 2D’ ) .", "In MZgdf3 embryos foxa2 expression in the endoderm was absent and in the majority of embryos , expression in the axis was absent .", "In a few embryos ( 3 of 15 ) one or two foxa2-expressing cells were present at the dorsal margin ( Figure 2E’ ) .", "foxa2 expression in MZgdf3 embryos was rescued to a normal pattern by injected gdf3 RNA ( Figure 2F’ ) .", "The anterior neural plate , marked by triangle-shaped expression domain of otx2 that extends from the midpoint of the dorsal axis over the animal pole , was unchanged in WT embryos by overexpression of gdf3 ( Figure 2G’ , H’ ) .", "In MZgdf3 embryos a triangular otx2 expression domain was present but greatly reduced in size , failing to extend to the animal pole ( Figure 2I’ ) .", "Like all of the marker genes examined here , otx2 expression was fully restored to a normal pattern in MZgdf3 embryos by expression of exogenous gdf3 RNA ( Figure 2J’ ) .", "Together , these results indicate that Nodal signaling response pathways are dependent on Gdf3 function .", "To investigate the position of Gdf3 in the Nodal signaling pathway , we expressed other members of that pathway in WT and MZgdf3 by injecting RNAs encoding those proteins at the 1–2 cell stage and assaying the resultant phenotypes at shield stage and at 24 hpf ( Figure 3 ) .", "In WT shield-stage embryos gsc was expressed in a dorsal crescent of cells that occupied about 15% of the circumference of the germ ring , while ta was expressed throughout the germ ring ( Figure 3A , B ) .", "Shield-stage embryos of MZgdf3 lacked gsc expression and ta expression was reduced dorsally ( Figure 3D , E ) compared to WT embryos .", "Injection of gdf3 in WT embryos had no effect on the expression of gsc or ta and did not alter morphological phenotype at 24 hpf ( Figure 3G-I ) .", "Injection of gdf3 into MZgdf3 embryos rescued dorsal expression of gsc and ta ( Figure 3J , K ) and morphology at 24 hpf ( Figure 3L ) .", "Exogenous expression of RNAs encoding Nodal family members ndr1 and ndr2 in WT embryos caused ectopic expression of both gsc and ta in the margin germ ring and blastoderm at shield stage and resulted in dorsalized phenotypes at 24 hpf ( Figure 3M–O , S–U ) .", "This serves as a positive control for the power of these ligands to activate Nodal signaling .", "In contrast , injection of ndr1 or ndr2 RNA into MZgdf3 embryos did not rescue gsc or ta expression in the dorsal germ ring of shield stage embryos , did not rescue axial tissues in embryos at 24 hpf , and did not induce a dorsalized phenotype ( Figure 3P–R , V–X ) .", "These results indicate that Nodal signaling ligands cannot activate the nodal signaling pathway in the absence of Gdf3 , and gives genetic evidence that Nodal and Gdf3 function at the same step in the signaling pathway .", "Lefty proteins are negative regulators of the Nodal signaling pathway ( Bisgrove et al . , 1999; Thisse and Thisse , 1999 ) .", "Injection of lft1 RNA into WT embryos resulted in a loss of gsc expression and a reduction in dorsal expression domain of ta ( Figure 3Y , Z ) , and lacked midline tissues including notochord , pharyngeal mesoderm and endoderm and ventral brain at 24 hpf ( Figure 3A’ ) .", "These phenotypes are indicative of Nodal deficient mutants and shared by MZgdf3 .", "Importantly , injection of lft1 RNA into MZgdf3 did not exacerbate their defective patterns or phenotypes ( compare Figure 3B’ , C’ , D’ with Figure 3D , E , F ) , indicating that there no residual Nodal signaling in MZgdf3 embryos that can be further antagonized by exogenous Lft1 .", "Given that MZgdf3 embryos do not respond to overexpression of Nodal family ligand agonists and antagonists , we tested whether the receptors and other components of the downstream pathway were intact .", "The TGFβ ligand Activin signals through Type I and Type II Activin Receptors; subsets of which are also utilized in the propagation of Nodal signaling .", "To determine if loss of Gdf3 function impacted other TGFβ signaling pathways , we injected embryos with RNA encoding Xenopus Activin .", "Ectopic expression of Activin in both WT and MZgdf3 embryos resulted in massive ectopic expression of gsc and ta at shield stage ( Figure 3E' , F' , G' , H' , I' ) .", "At this concentration of injected RNA , the majority of injected WT and mutant embryos died prior to 24 hpf .", "Surviving WT embryos exhibited slightly dorsalized phenotypes with narrower trunk and tail and kinked notochord , and a slight increase in anterior tissues ( Figure 3G’ ) .", "Exogenous expression of Activin in MZgdf3 embryos partially rescued the mutant phenotype at 24 hpf ( Figure 3J’ ) .", "Embryos had a thin trunk and tail with a notochord present in the anterior tail and trunk .", "Ventral brain tissues were also rescued as evidenced by the increase width of the head and nearly normal size eyes .", "Different TGFβ ligands signal through various combinations of Type I and Type II receptors including the Type I receptors Alk2 ( Acvr1 ) and Alk4 ( Acvr1b ) ( zebrafish homologues acvr1l and acvr1ba , respectively ) .", "Alk4 functions as a Type I receptor for multiple TGF beta-related ligands to regulate dorsal mesoderm induction and left-right axis determination in Xenopus ( Chen et al . , 2004 ) .", "Alk2 is important in the BMP signaling pathway ( Branford et al . , 2000 ) , and in zebrafish ( acvr1l ) is implicated in the specification of ventral mesoderm ( Mintzer et al . , 2001 ) .", "To examine whether TGFβ signaling components downstream of these receptors were functional in MZgdf3 , we injected RNAs encoding constitutively active ( CA ) constructs of Xenopus Alk4 and Alk2 .", "Injection of CA-Alk4 RNA into either WT or MZgdf3 embryos caused ectopic expression of both gsc and ta at shield stage ( Figure 3K’ , L’ , N’ , O’ ) .", "At 24hpf , injected WT embryos were partially dorsalized , and injected MZgdf3 embryos were partially rescued having some notochord present and largely normal anterior morphology ( Figure 3M’ , P’ ) .", "Exogenous expression of CA-Alk2 had the effect of slightly reducing the extent of the dorsal margin gsc expression domain in WT embryos while the ta expression pattern was not noticeably altered ( Figure 3Q’ , R’ ) .", "At 24 hpf , CA-Alk2 caused a severe ventralization of the embryo characterized by loss of dorsal structures such as the notochord , expanded and misshapen somites and a reduction in anterior structures including the brain and eyes ( Figure 3S’ ) .", "The absence of gsc expression in shield stage MZgdf3 was not rescued in embryos injected with CA-Alk2 , but ta expression was expanded into the dorsal domain of the margin that normally lacked expression , indicating an expansion of more lateral or ventral mesoderm domains ( Figure 3T’ , U’ ) .", "Similar to CA-Alk2 injected WT embryos , MZgdf3 embryos at 24 hpf showed a phenotype consistent with ventralization , having further reduced anterior mesoderm and neural structures including the complete loss of eyes ( Figure 3V’ ) .", "A marker for ventral mesoderm specification , the transcription factor eve1 , is expressed in ventral mesoderm during early gastrula stages in zebrafish .", "Expression of eve1 in shield stage embryos of WT or MZgdf3 injected with CA-Alk2 was expanded from the ventral domain into more dorsal regions of the margin ( Figure 3—figure supplement 1 ) .", "These results indicate that the downstream components of the Nodal signaling pathway are intact and capable of activation in MZgdf3 mutants , and suggest that Gdf3 acts at the same level in the pathway as Nodal , as an agonist for the activation of Alk4 ( Acvr1ba ) .", "We asked whether there was a lineage-specific requirement for the function of Gdf3 in the Nodal pathway by injecting gdf3 or ndr2 RNAs into a single cell at the 4–8 cell stage in WT and MZgdf3 embryos , with co-injection of eGFP RNA as a lineage tracer .", "We designate the injected cells as ‘targeted cells , ’ recognizing that this retrospective lineage tracing allows analysis of the embryonic locations of the cells that expressed the injected exogenous gdf3 or ndr2 .", "Successfully injected embryos with targeted cells confined to limited sectors of the blastoderm were selected at 50% epiboly ( Figure 4A ) .", "Morphological phenotypes of injected embryos were assessed at 24 hpf by transmitted light microscopy and with eGFP epifluorescence to detect fates of targeted cells , or were fixed at shield stage and processed for WISH to identify gsc expressing cells ( purple ) and IHC with anti-GFP ( brown ) to identify targeted cells .", "Consistent with results from ubiquitous expression of gdf3 ( Figure 3I ) , expression of exogenous gdf3 in subsets of cell lineages had no effect in WT embryos , irrespective of whether targeted cells were in midline-derived tissues ( 21 of 84 embryos ) ( Figure 4B ) , non-midline tissues ( 20 of 84 ) ( Figure 4C ) or more broadly distributed ( 43 of 84 ) .", "Strikingly , MZgdf3 embryos were only fully rescued when gdf3-expressing targeted cells had contributed to midline tissues including the notochord and prechordal plate mesoderm ( 37 of 131 embryos ) ( Figure 4D ) , or in midline and non-midline tissues ( 62 of 131 ) .", "When targeted cells were confined to non-midline tissues ( 32 of 131 ) , ectopic gdf3 expression rescued only the posterior structures of MZgdf3 embryos , including posterior notochord , but did not rescue anterior structures such as the ventral brain and eyes ( Figure 4E ) .", "Results from analysis of shield stage embryos ( Figure 4J–Q ) were consistent with the results presented above for 24 hpf embryos , and those presented for 1–2 cell injections ( Figure 3G ) .", "Ectopic gdf3 in wild-type embryos had no effect on expression of the dorsal midline marker gsc , irrespective of whether targeted cells were in the dorsal midline ( 4 of 69 embryos ) , contiguous and adjacent to the midline gsc domain ( 40 of 69 ) , or not associated with the midline ( 25 of 69 ) ( Figure 4J–M ) .", "The ability of ectopic expression of gdf3 to rescue gsc expression in presumptive shield stage MZgdf3 embryos was dependent on the location of the targeted cells .", "gsc expression was rescued when targeted cells were either in the midline ( 10 of 74 embryos , Figure 4O ) or contiguous and adjacent to the midline ( 42 of 74 , Figure 4P ) .", "In contrast , when targeted cells were confined to regions outside the dorsal midline , gsc expression was not rescued by ectopic gdf3 expression ( 22 of 74 , Figure 4Q ) .", "The lineage-tracing rescue data analyed at shield stage and at 24 hpf correlate well .", "The proportion of non-rescued embryos is about 1/3 in both cases .", "The proportion of embryos with targeted cells in the midline at 24 hpf is greater than the proportion of embryos with targeted cells in the dorsal midline at shield stage , most likely due to the convergence of cells toward the midline during gastrulation ( Schier and Talbot , 2005 ) .", "In WT embryos , confinement of ndr2-expressing targeted cells to midline tissues ( 34 of 118 embryos ) gave a relatively mild phenotype that included a kinked notochord ( Figure 4F ) .", "This is in striking contrast to hyperdorsalization phenotypes with extremely disorganized tissues in WT embryos in which targeted cells contributed to non-midline tissues ( 32 of 118 ) ( Figure 4G , Figure 4—figure supplement 1 ) , or from ubiquitous ndr2 expression ( Figure 3U ) .", "Distribution of ndr2 targeted cells to subsets of both midline and non-midline tissues ( 52 of 118 ) resulted in intermediate phenotypes .", "When analyzed at the shield stage , in WT embryos ndr2 induces ectopic expression of gsc in sectors that correspond to the targeted cells , regardless of whether targeted cells are located at the dorsal midline ( 11 of 64 embryos , Figure 4S ) , adjacent to the midline ( 31 of 64 , Figure 4T ) , or at a location distant from the midline ( 22 of 64 , Figure 4U ) .", "In contrast to the effects of ndr2 ectopic expression in WT embryos , targeted expression of ndr2 in MZgdf3 embryos by injection at the 4–8 cell stage had no effect , that is , could not rescue , irrespective of whether targeted cells localized to prospective midline or non-midline tissues ( n = 53 embryos: 11 midline , 17 non-midline , 25 broad expression ) ( Figure 4H , I ) .", "When analyzed at the shield stage , overexpression of ndr2 in spatially restricted clones of cells in MZgdf3 embryos failed to induce gsc expression , regardless of the location of targeted cells ( n = 47 , Figure 4W–Y ) .", "Thus , in the absence of gdf3 , regardless of lineage , ndr2 had no apparent function; it could not induce shield-stage expression of gsc and could not rescue the MZgdf3 phenotype .", "The ability of ectopic gdf3 to fully rescue MZgdf3 embryos only when it is expressed in lineages that co-express Nodal , and the observation that MZgdf3 embryos are refractory to Nodal ( ndr2 ) overexpression phenotypes seen in WT embryos , indicate that Gdf3 and Nodal must be co-expressed to have function , and that either ligand alone cannot function without the other ." ], [ "Maternally supplied Gdf3 is required during early embryonic development .", "Zygotic gdf3 homozygous mutant embryos have no apparent morphological phenotype , and can grow to viable and fertile adults .", "In contrast , embryos generated from fertilization of a gdf3-deficient mutant egg by a wildtype gdf3+ sperm display the same embryonic lethal MZgdf3 mutant phenotype as those fertilized with sperm from a homozygous gdf3- mutant father .", "This indicates that zygotic expression of wildtype gdf3 , confirmed by allele-specific RT-PCR , from the paternal genome after zygotic gene activation ( ZGA , 4 . 2 hpf ) ( Lee et al . , 2014 ) , is insufficient rescue the MZgdf3 mutant phenotype .", "The inability to rescue the MZ phenotype may reflect the timing and/or levels of zygotic expression .", "The embryonic lethal MZgdf3 phenotype can however , be rescued to grow to viable fertile adults when injected with gdf3 RNA at the 1 cell to 2 cell stages , supporting the idea that Gdf3 is essential during early embryogenesis .", "Although Vg1/Gdf3 has been implicated in left-right patterning by specific cell lineage-targeting overexpression experiments in Xenopus ( Hyatt and Yost , 1998 ) and by morpholino knockdown in zebrafish ( Peterson et al . , 2013 ) , the zygotic gdf3 mutants had no discernable alterations in LR patterning .", "There are several ways to consider the divergent zygotic mutant and morpholino LR phenotypes .", "First , it is possible that gdf3 morpholinos had off-target effects .", "However , the LR phenotype in gdf3 morphants could be rescued by manipulation of downstream components of the LR pathway .", "For example , lowering the doses of Charon , thought to be an inhibitor of the transfer of LR information from Kupffer’s vesicle to LPM , rescued gdf3 morphants ( Peterson et al . , 2013 ) .", "If the agent of LR information is an obligate heterodimer of Gdf3 and Nodal , then lowering the dose of the inhibitor of this agent would be expected to rescue the partial knockdown of a component of this agent , Gdf3 .", "Second , it is possible that in this case morpholinos provides a more acute disruption of Gdf3 function during early development , revealing roles for Gdf3 in LR patterning that are masked in the more chronic disruption of function in genetic mutants .", "The ability of mutants to acquire ‘genetic compensation’ by which some of the functions of a mutation-targeted gene are masked , has been proposed ( El-Brolosy and Stainier , 2017 ) and is a topic of vigorous discussion .", "Third , it is possible that maternal stores of Gdf3 persist late enough into embryogenesis to participate in LR patterning and to mask loss of zygotically encoded gdf3 .", "The dramatic phenotype of the MZgdf3 mutants which includes a loss of lateral plate mesoderm and other tissues involved in the transfer of LR patterning signals and the inability of post-ZGA expressed paternal WT gdf3 to rescue the MZ phenotype preclude ready assessment of this possible role of gdf3 in LR patterning .", "The loss of gene expression domains marking , in particular , neural mesodermal and endodermal tissues originating in or near the embryonic shield indicates a central role of Gdf3 in maintaining/promoting embryonic organizer function during gastrulation and is consistent with a role for Gdf3 function in the Nodal signaling pathway ( Andersson et al . , 2007; Chen et al . , 2006; Cheng et al . , 2003 ) .", "The results of exogenous expression of the receptor-promiscuous Activin ligand and constitutively active Alk4 receptors , and their partial rescue of the MZgdf3 phenotype , indicates that the TGFβ response pathway is intact in MZgdf3 , and that Gdf3 functions upstream of the Alk4 receptor , genetically placing its function at the same level as Nodal .", "The fact that exogenous Nodal ( Ndr1 and Ndr2 ) could not activate transcription of the mesodermal transcription factors gsc or ta , nor rescue the loss of midline tissues in MZgdf3 indicates that Gdf3 function is an essential factor in the activation of the Nodal signaling pathway , and that addition of Nodal homodimers cannot overcome the deficit of Gdf3 .", "Our results indicate that Gdf3 and Nodal must be co-expressed in the prospective dorsal sector of the embryo to drive embryonic patterning .", "Strikingly , our genetic , epistasis and lineage targeting experiments indicate that either ligand cannot function without the other ligand , so that Gdf3 is an obligate cofactor of Nodal , and Nodal is an obligate co-factor of Gdf3 .", "Members of the Nodal and Gdf families have been shown to co-immunoprecipitate , depending on the assay and the cells in which they are co-expressed ( Fuerer et al . , 2014; Peterson et al . , 2013 ) , and co-expression in Xenopus animal cap assays induces a stronger and more distant response than either ligand alone ( Andersson et al . , 2007; Tanaka et al . , 2007 ) .", "Combining our genetic results and these previous biochemical experiments , we suggest that the functional ligand for the Nodal signaling pathway during early zebrafish development is an obligate heterodimer of Gdf3/Nodal ( Gdf3/Ndr1 and/or Gdf3/Ndr2 ) , and importantly , that homodimers of either Nodal/Nodal or Gdf3/Gdf3 are not functional in early embryonic patterning .", "There is precedent for this idea from studies of the role of the TGFβ family members bmp2 and bmp7 during zebrafish dorsoventral ( DV ) patterning .", "While homodimers of both are found in the embryo , only the heterodimer possess sufficient receptor activity to elicit the signaling required for DV patterning ( Little and Mullins , 2009 ) .", "There is a single Nodal locus in mammals , whereas there are three Nodal-related loci in zebrafish , ndr1 , which is maternally expressed , and ndr2 and spaw which are zygotic ( Long et al . , 2003; Rebagliati et al . , 1998a ) .", "The early embryonic functions of mouse Nodal , in germ layer and axial development ( Brennan et al . , 2001; Conlon et al . , 1994; Varlet et al . , 1997 ) , are reflected by the functions of zebrafish paralogs Ndr1 and Ndr2 during cleavage and gastrulation ( Feldman et al . , 1998; Rebagliati et al . , 1998a , 1998b; Sampath et al . , 1998 ) .", "The role of murine Nodal in Left-Right patterning ( Brennan et al . , 2002; Saijoh et al . , 2003 ) appears to be accomplished by Spaw and Ndr2 during somitogenesis in zebrafish ( Long et al . , 2003; Rebagliati et al . , 1998a ) .", "In an inversion of the usual comparisons between the mammalian and duplicated zebrafish genomes , there is a single locus of the gdf gene family in zebrafish , gdf3 , whereas there are two related loci in mammalian genomes , ancestral Gdf3 and more recently derived Gdf1 .", "Murine Gdf3 mutants ( Chen et al . , 2006 ) phenocopy early germ layer and axial phenotypes of Nodal mutants while Gdf1 mutants ( Rankin et al . , 2000; Wall et al . , 2000 ) phenocopy the laterality phenotypes of node-specific Nodal mutants .", "To our knowledge , double mutants of Gdf1 and Gdf3 have not been reported , nor is it known whether there is a maternal contribution of either Gdf1 or Gdf3 that participates in murine embryonic patterning .", "As we have proposed in this study , and as proposed by Peterson et al . ( 2013 ) in zebrafish , Nodal-Gdf heterodimers are thought to be the functional entity in both germ layer and LR patterning in mice ( Andersson et al . , 2006; Tanaka et al . , 2007 ) .", "While the varying number of Gdf and Nodal family members between fish and mammals may complicate comparing genetic and biochemical outcomes between mammals and zebrafish , it provides an interesting opportunity to explore the evolution of these co-factors and their signaling pathways ." ], [ "TALEN nucleases targeting gdf3 were designed and constructed in conjunction with Timothy Dahlem at the Mutation Generation and Detection Core at the University of Utah ( Figure 1—figure supplement 1 ) .", "Sequences encoding left ( L ) and right ( R ) TALENs were cloned into the pCS2 +expression vector and TALEN RNAs were synthesized using the Message Machine SP6 transcription kit ( Ambion ) .", "TALEN RNAs ( 25 pg each L and R ) were pressure injected into 1–2 cell AB strain embryos derived from a pool of adults that had previously been sequenced to ensure genomic homogeneity ( ‘gdf3 clean’ ) around the TALEN target site .", "Injected G0 embryos were harvested at 24 hpf and assayed by HRMA ( Parant et al . , 2009 ) to determine the frequency of TALEN-induced mutations in gdf3 .", "Sibling embryos to those showing high frequency of gdf3 mutations were raised to adulthood and outcrossed to ‘gdf3 clean’ AB adults .", "Clutches of F1 embryos were screened by HRMA to identify G0 parents carrying gdf3 mutations , and sibling embryos were raised to adulthood as potential gdf3 F1 founders , which were subsequently identified by HRMA of tailfin genomic DNA .", "Wild-type and mutant zebrafish lines were maintained at the Central Zebrafish Animal Resource ( CZAR ) at the University of Utah .", "Embryos for experiments were collected from natural spawnings , cultured and staged by developmental time and morphological criteria ( Westerfield , 1995 ) .", "Capped , synthetic RNAs for injection were generated from expression constructs encoding gdf3 ( Dohrmann et al . , 1996 ) , ndr , ndr2 ( Rebagliati et al . , 1998a , 1998b ) , lft1 , lft2 ( Bisgrove et al . , 1999 ) , Xenopus activin βb ( inhbb ) ( Thomsen et al . , 1990 ) and CA-alk2 ( Hemmati-Brivanlou and Thomsen , 1995 ) and human CA-alk4 ( ACVR1B ) ( Willis et al . , 1996 ) using Message Machine .", "Wild-type AB strain or MZgdf3 mutant embryos at the 1–2 or 4–8 cell stages were injected with a single or combinations of RNA .", "Following injection embryos were allowed to develop to desired stages , then photographed or fixed for in whole mount in situ hybridization ( WISH ) .", "In all cases injection experiments were carried out on two independent clutches of embryos and a minimum of 15 embryos were examined for each experimental condition and in situ probe .", "For WISH , embryos were fixed and processed as described previously ( Bisgrove et al . , 1999 ) .", "Anti-sense riboprobes were synthesized from linearized DNA templates using T3 or T7 polymerases and digoxigenin labeling mixes ( Roche ) .", "Probes used in this study included gsc ( Stachel et al . , 1993 ) , ta ( ntl ) ( Schulte-Merker et al . , 1994 ) , ndr2 ( cyc ) ( Rebagliati et al . , 1998a ) , lft1 , lft2 ( Bisgrove et al . , 1999 ) , tbx16 ( spt ) ( Ruvinsky et al . , 1998 ) , foxa2 ( axial ) ( Strähle and Jesuthasan , 1993 ) , otx2 ( Li et al . , 1994 ) , erg2b ( krox20 ) ( Oxtoby and Jowett , 1993 ) .", "In all cases , a minimum of 15 embryos were examined for each experimental condition and in situ probe .", "To label clones of cells expressing eGFP by IHC , embryos were re-fixed following WISH and incubated with peroxidase-conjugated GFP antibody diluted 1/500 ( Rockland Immunochemicals Inc . #600-103-215 ) .", "3 , 3'-diaminobenzidine was used as color development substrate .", "Following WISH and IHC , embryos were cleared in 70% glycerol/PBS and photographed with a Leica M165FC or Leica M205FA microscope Using Leica Application Suite digital acquisition software .", "Images were processed using Adobe Photoshop to correct color balance , exposure , brightness and contrast only ." ] ]

[ "Zebrafish Gdf3 ( Dvr1 ) is a member of the TGFβ superfamily of cell signaling ligands that includes Xenopus Vg1 and mammalian Gdf1/3 .", "Surprisingly , engineered homozygous mutants in zebrafish have no apparent phenotype .", "Elimination of Gdf3 in oocytes of maternal-zygotic mutants results in embryonic lethality that can be fully rescued with gdf3 RNA , demonstrating that Gdf3 is required only early in development , beyond which mutants are viable and fertile .", "Gdf3 mutants are refractory to Nodal ligands and Nodal repressor Lefty1 .", "Signaling driven by TGFβ ligand Activin and constitutively active receptors Alk4 and Alk2 remain intact in gdf3 mutants , indicating that Gdf3 functions at the same pathway step as Nodal .", "Targeting gdf3 and ndr2 RNA to specific lineages indicates that exogenous gdf3 is able to fully rescue mutants only when co-expressed with endogenous Nodal .", "Together , these findings demonstrate that Gdf3 is an essential cofactor of Nodal signaling during establishment of the embryonic axis ." ]

[ "All vertebrates – animals with backbones like fish and humans – have body plans with three clear axes: head-to-tail , back-to-front and left-to-right .", "Animals lay down these plans as embryos , when signaling molecules bind to receptors on the surface of their cells .", "These signaling molecules include related proteins called “Nodal” and “Growth and Differentiation Factors” .", "However , there has been much debate in the field of developmental biology about whether these proteins work together or independently during the early development of vertebrates .", "Zebrafish are often used to study animal development , and Bisgrove et al . decided to test whether these fish need a Growth and Differentiation Factor known as Gdf3 by deleting it using genome editing .", "It turns out that zebrafish can survive and develop as normal without the gene for Gdf3 , just as long as their mothers still had a working copy of the gene .", "Yet , when the offspring of mutant females did not inherit the instructions to make Gdf3 from their mothers , they died within a couple of days .", "This was true even if the offspring inherited a working copy of the gene from their fathers .", "Bisgrove et al . then went on to show that embryos from a mutant mother could be saved with an injection of short-lived RNA molecules that include the instructions to make some Gdf3 proteins .", "The injected mutant embryos could live to adulthood .", "This shows that Gdf3 is only needed during the embryo’s early development .", "Further experiments suggested that Gdf3 does cannot activate its receptors on its own .", "Instead , it is likely that Gdf3 interacts with Nodal to form a two-protein complex that activates the receptors .", "Two other groups of researchers have independently reported similar findings .", "Mutations affecting proteins very similar to Gdf3 have been found in people with congenital heart defects .", "By revealing the interaction between Gdf3 and Nodal , these new findings could help scientists to understand the genetic causes of this condition in more detail .", "Further studies using the mutant zebrafish could also be used to explore the causes of other developmental diseases ." ]

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

[ "biochemistry and chemical biology", "physics of living systems" ]

[ [ "Ribosomes possess three binding sites for tRNA: the aminoacyl ( A ) , peptidyl ( P ) , and exit ( E ) sites , each of which is shared between the 30S and 50S ribosomal subunits .", "Following codon recognition and peptide bond formation , the ribosome has a deacylated tRNA in the P site and a peptidyl-tRNA in the A site .", "In order to start a new elongation cycle , the A site must be emptied to allow binding of the next aminoacyl-tRNA .", "To this end , the tRNAs and mRNA must move relative to the ribosome .", "This movement occurs in two steps ( Moazed and Noller , 1989b ) : first , the 3′ ends of the tRNAs in the A and P sites move , with respect to the 50S subunit , into hybrid A/P and P/E states , respectively .", "In vitro , formation of these states can occur spontaneously , reversibly , and independently of elongation factor G ( EF-G ) ( Moazed and Noller , 1989b; Sharma et al . , 2004; Cornish et al . , 2008; Munro et al . , 2010 ) and is coupled to rotation of the 30S body ( Moazed and Noller , 1989b; Frank and Agrawal , 2000; Agirrezabala et al . , 2008; Julian et al . , 2008; Dunkle et al . , 2011 ) .", "In the second step , which is irreversible and EF-G-dependent ( Moazed and Noller , 1989b; Savelsbergh et al . , 2003 ) , the mRNA is translocated by one codon , along with movement of the associated anticodon ends of the tRNAs to the classical P and E sites , coupled to an orthogonal rotation of the 30S subunit head domain ( Ratje et al . , 2010; Dunkle et al . , 2011; Ermolenko and Noller , 2011; Guo and Noller , 2012; Zhou et al . , 2013 ) .", "The translocation process also involves other large-scale conformational changes in the ribosome , including reverse rotational movements of the 30S subunit body and head ( Ermolenko and Noller , 2011; Guo and Noller , 2012 ) , and movement of the large subunit L1 stalk into the intersubunit space ( Fei et al . , 2008; Cornish et al . , 2009 ) .", "Translocation is therefore a highly coordinated and complex process composed of inter- and intra-molecular , force-generating mechanical movements .", "During translation , the ribosome must also overcome significant mechanical barriers posed by structured portions of the mRNA .", "These structures are exploited by the cell to create diverse strategies for translation regulation; for example , pseudoknots and hairpins are used to induce programmed frameshifting ( Tsuchihashi , 1991; Namy et al . , 2006 ) , whereas synonymous mutations that can alter the local structure of RNA and codon usage are employed to control protein expression levels ( Duan et al . , 2003; Nackley et al . , 2006 ) .", "Hence , force is not only a product of the chemical reactions during translation , but also an important player in the regulation of this process ." ], [ "We have designed an experiment to monitor the movement of individual ribosomes against an opposing force during translation ( Figure 1A ) .", "A gene fusion encoding ribosomal protein S16 linked via its C-terminus to the biotinylation domain of the biotin carboxyl carrier protein ( BCCP ) was introduced ( Link et al . , 1997 ) into the chromosome of Escherichia coli , and biotinylated ribosomes were then purified .", "In the presence of initiation factors , initiator tRNA and GTP , a biotinylated ribosome is assembled at the AUG start site of an mRNA , whose 3′ end had been previously annealed to a complementary DNA handle harboring a 5′ digoxigenin .", "The complex is then tethered between a pair of 2 . 1 µm diameter polystyrene beads: a streptavidin-coated bead , which binds to the ribosome and is held by suction on the end of a micropipette , and an anti-digoxigenin antibody-coated bead , which binds to the DNA handle and is held in an optical trap .", "Next , a mixture containing elongation factors , aminoacyl-tRNAs and GTP is introduced into the experimental chamber .", "The tension in the tether , stabilized by an automated feedback routine , produces a constant opposing force as translocation proceeds .", "Translation is followed in real time as a decrease in the tether length between the beads ( Figure 1B–C , Figure 1—figure supplement 1 ) .", "No translation signals were detected in the absence of GTP and EF-G .", "( Figure 1E , F ) . 10 . 7554/eLife . 03406 . 003Figure 1 . Following translation by a single ribosome on a single mRNA .", "( A ) Geometry for single-molecule translation experiments .", "A biotinylated ribosome is loaded onto a single-stranded mRNA and attached to a streptavidin-coated polystyrene bead fixed to a micropipette .", "The 3′ of the message is anchored to a second bead through a 1460 bp DNA/RNA hybrid handle .", "Calibrated forces can be applied to the ribosome by manipulating the second bead with an optical trap , while the translation progress of the ribosome is determined by the change in extension of the tether .", "( B–D )", "Typical translation events recorded under 4 , 6 and 8 pN of constant tension .", "The upper panels show the codons translated as a function of time , and indicate that translation proceeds not in a continuous manner , but in a series of translational bursts separated by long pauses .", "The gray line shows the raw ( 1 kHz ) data , while green ( translocation ) and red ( pause ) are filtered down to 1 Hz .", "The lower panels show the instantaneous velocities calculated from the traces above .", "( E and F )", "Control experiments , under 8 pN of tension , showing that in the absence of GTP or EF-G , no translation signals were detected . DOI: http://dx . doi . org/10 . 7554/eLife . 03406 . 00310 . 7554/eLife . 03406 . 004Figure 1—figure supplement 1 . A partial translation trace showing an unusually low noise level , and a sequence of presumptive single-codon translocation steps . The data was sampled at 2 kHz and averaged down to 5 Hz . DOI: http://dx . doi . org/10 . 7554/eLife . 03406 . 004 Ribosomes do not translate the message in a continuous manner , but in bursts of translation separated by long pauses ( Figure 1B ) that do not appear to be correlated with template position .", "As in all single-molecule experiments , there is a distribution of the noise level in the different single ribosome trajectories .", "As a result , some of the trajectories observed during translation bursts exhibit a particularly low noise level , and clearly show that the ribosome moves in periods of stationary dwells that are followed by translocation events corresponding to single codon steps ( three nucleotides ) along the mRNA ( Figure 1 , Figure 1—figure supplement 1 ) .", "However , in most trajectories the noise prevents us from unambiguously identifying the individual steps , and we therefore base our analysis on the average properties of the translation bursts .", "We separate these pauses from active translation bursts using a velocity threshold ( see ‘Materials and methods’ ) , and calculate the ‘pause-free’ velocity of the ribosome ( Figure 1C ) .", "We find that as the opposing force , F , is increased , the mean pause-free velocity , v , decreases monotonically ( Figure 2 ) , indicating that mRNA translocation is rate limiting under the conditions of the experiment .", "Note that the applied force acts between the 3′ end of the mRNA and the small 30S subunit; there is no directly applied force between the mRNA and the large 50S subunit or between the 30S and the 50S subunits .", "In addition , the position of the attachment point ( protein S16 , in the ‘back’ of the 30S subunit ) was chosen because it is remote of any known functional site and is not known to exhibit conformational dynamics during translocation .", "As a result , force only affects directly the mechanical step in which the anticodon loops of the tRNAs move from the A and P sites to the canonical P and E sites of the 30S subunit , respectively , together with the concomitant movement of the mRNA with respect to the ribosome .", "Hence , the position of the ribosome relative to the mRNA provides a convenient reaction coordinate to follow the translocation reaction during translational elongation .", "We can , thus , fit our data to an Arrhenius expression ( 1 ) v ( F ) =v0exp ( −F·x˜kBT ) , where x˜ is the typical distance over which the force acts , v0 is the zero-force translocation velocity , kB is Boltzmann's constant and T = 296 K is the absolute temperature .", "The fit yields a zero-force velocity v0 = 2 . 9 codons/s ( 1 . 8 , 4 . 0 ) and a distance x˜ = 1 . 4 nm ( 0 . 9 , 1 . 8 ) .", "The numbers in parenthesis indicate 95% confidence bounds . 10 . 7554/eLife . 03406 . 005Figure 2 . Pause-free translational velocity as a function of opposing force . Data points are the mean velocities for all measured traces at each force ( N = 54 ) .", "Error bars represent the standard error of the mean .", "The solid line is an exponential fit of the form v ( F ) =v0exp ( −F·x˜kBT ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 03406 . 005 Notably , these results have implications for the intrinsic step size during translocation: crystal structures ( Jenner et al . , 2010 ) show that the distance between A- and P-site mRNA codons equals 1 . 48 nm .", "Clearly , at the end of translocation the mRNA has moved by this distance from its pre-translocation position; however , this value can either reflect a single , one-codon mechanical movement ( a single barrier crossing in the free energy surface landscape ) or successive smaller substeps ( multiple barrier crossings ) that sum to one codon , for example , three one-nucleotide substeps .", "Directly observing these potential substeps requires a temporal and spatial resolution that is not possible in our present experiment because the flexible nature of the mRNA and the relatively low forces involved give rise to high levels of thermally-induced fluctuations .", "However , because the distance x˜ determined here is similar to the measured one-codon translocation step , we can rule out one- and two-nucleotide translocation substeps and conclude that codon translocation is performed by the ribosome in a single step .", "In addition , our measurements shed light on the mechanism of translocation .", "Mechano-enzymes in general act by coupling a mechanical task ( translocation , force generation , work ) to a downhill chemical reaction ( i . e . , a reaction that lowers the total free energy of the system ) ( Bustamante et al . , 2004 ) .", "Clearly , given the diversity of conformational changes and chemical events associated with translocation by the ribosome , a complete description of the process should involve diffusion on a free-energy hypersurface with high dimensionality .", "However , given that our attachment geometry ensures that we affect and probe a single and well-defined mechanical coordinate , we can reduce this description to a simplified two-dimensional picture .", "In this two-dimensional free energy landscape one axis represents the mechanical coordinate that describes the movement of the mRNA relative to the 30S subunit , and the other axis the chemical coordinate that describes all binding , hydrolysis and dissociation processes ( Figure 3 ) and , for the sake of simplicity , also conformational changes that have a reaction coordinate orthogonal to the reaction coordinate probed in our experiments .", "The most likely path for the reaction occurs along a minimum energy channel on this surface and the different events involved in translocation can now be described as diffusive transitions between minima of this ( reduced ) energy surface .", "Thus , for example , the classical-to-hybrid transitions and the associated ribosomal intersubunit rotations are assigned as movements along the chemical coordinate over a rather shallow activation energy that accounts for their reversible nature ( Munro et al . , 2007; Cornish et al . , 2008; Fei et al . , 2008 ) .", "The three-dimensional energy surface depicted in Figure 3 naturally explains how transition rates are affected when a mechanical force is applied .", "The effect is equivalent to tilting the potential energy surface by rotating the diagram around its chemical axis ( Bustamante et al . , 2004 ) , hence affecting the rate and equilibrium constants of reactions along the mechanical coordinate , for example making translocation more ( force applied in the aiding or ‘pushing’ direction ) or less ( force applied in the opposing or ‘pulling’ direction ) favorable . 10 . 7554/eLife . 03406 . 006Figure 3 . Reduced energy landscape for mRNA translocation . The mechanical coordinate describes the movement of the mRNA relative to the 30S subunit , while the chemical coordinate describes all binding , hydrolysis and dissociation processes , in addition to conformational changes with a reaction coordinate orthogonal to the translocation coordinate probed in our experiments .", "Translocation proceeds by diffusive transitions between minima of this reduced energy surface .", "A Power Stroke mechanism involves a diagonal transition , with simultaneous progress in the chemical and mechanical axis ( dashed , purple line ) .", "Alternatively , a Brownian Ratchet ( full , red lines ) is composed of two orthogonal transitions: a fast equilibrium between pre- and post-translocated states along the mechanical coordinated , followed by a ‘rectifying’ chemical transition . DOI: http://dx . doi . org/10 . 7554/eLife . 03406 . 006 Translocation of mRNA and its two associated tRNA anticodon stem-loops from the A and P sites to the P and E sites of the 30S subunit ( a movement along the ‘mechanical’ axis ) must then be coupled to a downhill progress along the ‘chemical’ axis .", "Fundamentally , there are two ways in which this coupling can occur: one possibility is that the energy released by the ‘chemical’ transition is directly harnessed to produce the change .", "In this case , usually called a ‘Power Stroke ( PS ) ’ mechanism , the system moves diagonally in the energy landscape .", "Alternatively , it is possible that the system moves back-and-forth spontaneously , driven by thermal energy , along the mechanical coordinate , until a chemical transition , that occurs when the system is in the post-translocated state , prevents the back-translocation and ‘rectifies’ this random motion into directed motion .", "This second mechanism , in which the system moves on the energy landscape in two orthogonal steps , is called a ‘Brownian Ratchet ( BR ) ’ .", "Importantly , although both these different molecular mechanisms will result in a velocity which depends exponentially on the force , as in the Arrhenius equation above , they will differ in the identity of the distance x˜ ( Wang et al . , 1998 ) : in the PS case , as the post-translocation state is achieved in a single ( diagonal ) transition , the force-dependence of the reaction rate will be given by an Arrhenius expression in which x˜=x†† , that is , the distance along the mechanical coordinate to the transition state during translocation ( Bustamante et al . , 2004 ) .", "Alternatively , in the BR case , the post-translocation state is achieved via two transitions , and the rate will be given by the product of the ( force-dependent ) probability of spontaneously populating the post-translocation state and the ( force-independent ) rate of the chemical reaction .", "As a result , the force dependence of the velocity is dictated by the force-dependent equilibrium constant between the pre- and post-translocated states , and hence described by an Arrhenius expression in which x˜=xstep , that is , equal to the distance between these states , or the step size of the motor ( Bustamante et al . , 2004 ) .", "While these are two idealized cases and a real system is likely to combine features of these two models , the distance determined here ( 1 . 4 nm ) is very close to the full 1 . 48 nm step size and indicates that , while we cannot fully rule out a PS mechanism , the ribosome likely functions as a BR during translocation .", "Notably , recent crystallographic studies of the ribosome bound to EF-G in a translocation intermediate ( Chen et al . , 2013; Pulk and Cate , 2013; Tourigny et al . , 2013; Zhou et al . , 2013 ) , and a previous structure of the ribosome bound to EF-G in the post-translocation state ( Gao et al . , 2009 ) can lend support for this result and help clarify the identity of the reaction that rectifies translocation in this ratchet mechanism: the structures suggest that domain IV of EF-G could prevent back-translocation of the P-site tRNA by occupying the A-site , and that intercalation of two highly conserved bases of 16S rRNA into mRNA could prevent its back-translocation .", "These conformational changes could thus act as ‘pawls’ in the BR mechanism .", "Interestingly , the fact that both tRNA and mRNA movements would be locked could contribute to prevent frame shifting .", "The force at which the velocity approaches zero ( the stall force ) represents the maximum force that can be intrinsically generated by the motor in a cycle .", "Our results indicate that the ribosome can generate forces as high as 13 ± 2 pN .", "Remarkably , the ribosome stall force is very close to that required to unwind the strongest secondary structure motifs typically present in mRNA ( Tinoco et al . , 2004 ) .", "The comparable magnitude between the mechanical strengths of RNAs and the stall force of the ribosome indicates that RNA secondary structures can have a strong effect on the rate of translation ( and hence on phenomena such as frameshifting and cotranslational folding of the protein ) and explains how these structures , while not being insurmountable barriers , can fulfill a regulatory role in the cell .", "The work generated by the ribosome near stalling , that is , the product of the stall force and the step size , 21 . 2 pN · nm = 5 . 2 kBT or about 3 . 1 Kcal/mol , represents the maximal mechanical work generated by the motor during the translocation step .", "What is the energetic source for this mechanical work ?", "The free-energy difference between peptide bond and ester bond hydrolysis is approximately −3 . 7 ± 1 . 2 kcal/mol , equivalent to 6 . 3 ± 2 kBT per bond exchange in our experimental conditions ( ‘Materials and methods’ ) .", "Hence , the maximal mechanical work that can be generated by the ribosome is ∼80% of the total energy available from transpeptidation and , in principle , it is possible to power translocation from this energy without the need to invoke an energetic contribution from the hydrolysis of GTP .", "In fact , studies have shown that the ribosome can translocate in the absence of EF-G ( Gavrilova et al . , 1976 ) or in the absence of GTP ( Pestka , 1969; Rodnina et al . , 1997; Fredrick and Noller , 2003 ) .", "However , spontaneous forward translocation is unfavorable in many contexts ( Shoji et al . , 2006 ) , and efficient and rapid translocation does require EF-G and GTP hydrolysis .", "Furthermore , 80% thermodynamic efficiency for conversion of chemical energy to mechanical motion is higher than occurs in most molecular motors ( Bustamante et al . , 2004 ) , and , moreover , it is not clear how the energy available from peptide bond formation could be stored and transmitted from the 50S to the 30S subunit .", "Instead , in view of our results , a mechanism in which EF-G binding and GTP hydrolysis account for the energy of translocation and resetting ( including EF-G–GDP dissociation ) appears to be more likely .", "Not surprisingly , the mechanism of action of the ribosome as a mechano-enzyme during translocation is more complicated than that of a typical molecular motor .", "One possible scenario that emerges from this and previous studies is that translocation is achieved by two consecutive BRs: during the first step , the ribosomal subunits rotate back and forth relative to each other along an axis perpendicular to the subunits interface .", "This process , reversible and thermally activated , is accompanied by the repositioning of the 3′-acceptor ends of the tRNAs initially in the classical A and P states into the hybrid A/P and P/E states and movement of the L1 stalk .", "Binding of EF-G–GTP stabilizes the tRNAs in their hybrid states and the counter-clockwise rotation of the 30S subunit relative to the 50S subunit .", "Hence , binding of EF-G functions as a rectifying reaction for the first BR .", "The ribosome acts as a GTPase activator for EF-G , and rapid GTP hydrolysis catalyzes conformational changes in the ribosome ( e . g . , swiveling of the head , which opens the way for the passage of the P-site tRNA anti-codon stem-loop to the E site ) that result in allowing spontaneous and thermally activated transitions between the pre- and post-translocation state .", "This second BR is likely to be rectified by the movement of domain IV of EF-G into the A-site and the intercalation of two conserved bases of 16S rRNA into mRNA .", "Pi release then induces a relaxation of EF-G , destabilizing the contacts between domains III and V and the ribosome and resulting in EF-G dissociation .", "Finally , we turn our attention now to the pauses observed during translation .", "The duration of the translation bursts ( and hence the effective pause entry rate , kp ) is independent of the force opposing translocation ( Figure 4A ) .", "Moreover , the full distribution of burst durations is well described , at all forces , by a single exponential function with nearly identical parameters ( Figure 4—figure supplement 1 ) .", "Likewise , Figure 4B shows that the duration of the pauses ( and hence the pause exit rate , k-p ) is independent of force .", "Since the pause duration is also well described by single exponential functions with the same parameters at all the tested forces ( Figure 4—figure supplement 1 ) , we conclude that entering into the paused state and exiting from it are both governed by single , force-independent steps .", "Hence , we can cluster all our measurements into one data set and calculate the entry and exit rates: kp = 0 . 16 s−1 ( 0 . 14 , 0 . 18 ) and k−p = 0 . 14 s−1 ( 0 . 1 , 0 . 18 ) , where the numbers in parenthesis indicate 95% confidence intervals . 10 . 7554/eLife . 03406 . 007Figure 4 . Pause entry and exit rates .", "( A ) Pause entry rate , calculated as the inverse of the mean duration of the translation bursts in between the pauses .", "( B ) Pause exit rate , equal to the inverse of the mean pause duration .", "Both rates are essentially independent of the applied opposing force . DOI: http://dx . doi . org/10 . 7554/eLife . 03406 . 00710 . 7554/eLife . 03406 . 008Figure 4—figure supplement 1 . Distribution of the translation bursts and pauses durations for all the measured opposing forces . Left panels: distribution of the bursts durations measured at the forces specified in each graph .", "Right panels: distributions of the pauses durations .", "With the exception of the pause duration distribution at 2 pN , for which the amount of data is limited ( N = 6 ) , all the distributions are well fitted by a single exponential function .", "The rates calculated from the distributions ( k1 , k−1 ) are indicated together with their 95% confidence interval .", "These rates are in good agreement with the rates calculated from the mean burst and mean pause durations ( Figure 4 ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 03406 . 008 What is the origin of the observed pauses ?", "It is well known that translating ribosomes tend to pause at specific secondary structure motifs ( such as hairpins and pseudoknots ) .", "However , if that were the case for the observed pauses , their occurrence would be correlated with the position on the template ( which we do not observe ) and their density would depend strongly on the applied force , as this force will destabilize the secondary structure ahead of the ribosome in the geometry used in these experiments .", "Thus , the force independence of the entry and exit rates rules out this possibility .", "Hence , we favor a model in which these pauses represent events that are off-pathway from the main incorporation and translocation cycle .", "The rate limiting transitions into these off-pathway states are steps along a non-mechanical reaction coordinate , or alternatively a mechanical coordinate that is orthogonal to the translocation movement , and hence not affected by the externally applied force .", "Interestingly , the pause entry and exit rates are very similar , indicating that in the conditions of our experiments the time spent by the ribosome in the productive translation states and the unproductive paused state are nearly the same .", "The geometry of our experiments defines a single reaction coordinate .", "A more complete description of the translation process will require a multidimensional energy space .", "Nonetheless , the assay presented here opens the way for additional experiments where , by choosing different attachment points on the ribosome—across the small and large subunits for example—it may be possible to mechanically probe the internal degrees of freedom associated with ribosome translocation .", "Such experiments should reveal how this motor coordinates its internal dynamics with its translocation and helicase activities ." ], [ "A gene fusion encoding S16 linked via its C-terminus to the biotinylation domain of the biotin carboxyl carrier protein ( BCCP ) was introduced into the chromosome of E . coli , using the allelic replacement method of Church et al . ( Link et al . , 1997 ) .", "SDS-PAGE analysis of 30S subunits purified from these strains indicated the absence of native S16 and the presence of S16-BCCP in stoichiometric amounts .", "Addition of excess avidin prior to electrophoresis resulted in complete shift of the S16-BCCP band to that of a high molecular weight complex , indicating highly efficient biotinylation of the fusion protein in vivo .", "Cells expressing the BCCP-ribosome construct ( E . coli strain KLF203 ) have no detectable phenotype .", "Typically , 1 liter of cells grown to mid-log at 37°C in LB broth , was pelleted , resuspended in buffer A ( 20 mM Tris-Cl [pH 7 . 0] , 100 mM NH4Cl , 10 mM MgCl2 , and 5 mM βME ) , lysed , layered onto a 35 ml cushion containing 1 . 1 M sucrose , 20 mM Tris-Cl ( pH 7 . 0 ) , 500 mM NH4Cl , 10 mM MgCl2 , and 5 mM βME , and centrifuged in a Beckman Ti45 rotor at 36 , 000 rpm for 21 hr at 4°C .", "The ribosome pellet was dissolved in 0 . 5 ml buffer A , layered onto two 38 ml 10–35% sucrose gradients containing buffer A , and centrifuged in a Beckman SW28 rotor at 19 , 000 rpm for 16 hr at 4°C .", "The ribosomes in the 70S peak were collected and centrifuged in a Beckman Ti60 rotor at 38 , 000 rpm for 20 hr at 4°C .", "The ribosome pellet was dissolved in 0 . 2 ml buffer A , aliquoted , quick-frozen in liquid N2 , and stored at −80°C .", "A DNA oligomer called TTC17 , CAACCATGGTCTCG ( TTC ) 17 GTCTTCCTAGGAAC , was synthesized with 17 repeats of the TTC triplet in the center , a BsaI site on the 5′ end and dual BbsI-AvrII sites on the 3′ end .", "TTC17 was first converted to a double-stranded duplex .", "Half of TTC17 was cut by BbsI and half by BsaI to remove the sequences after and before the TTC repeats , respectively .", "Ligation of both restriction fragments ( with complementary cohesive ends ) resulted in TTC32 , which contains 32 TTC repeats with the same flanking sequences as in TTC17: CAACCATGGTCTCG ( TTC ) 32GTCTTCCTAGGAAC .", "The procedure was repeated to generate a sequence ( TTC62 ) with 62 TTC repeats .", "TTC62 was finally cut with BsaI and AvrII before being inserted into the vector pRC4a , a derivative of pRC4 ( Wen et al . , 2008 ) .", "pRC4a was then cut with NcoI and AvrII and ligated to an adaptor ( CATGCGCTAGCTTACCATGGGTCTCG ) to convert the cohesive end of NcoI to BsaI and thus to allow ligation to TTC62 .", "The plasmid ( with 62 TTC repeats ) was cut at BspHI and transcribed ( Megascript T7 kit , Ambion , Austin , TX ) into RNA with a length of 1827 nt .", "A region ( 1453 nt ) on the 3′ side of the RNA was annealed to a complementary DNA strand ( as a ‘handle’ ) containing two digoxigenin tags at the end .", "To make initiation complex ( ICs ) the following components were mixed in buffer TL ( 40 mM HEPES-KOH [pH = 7 . 5] , 60 mM NH4CL , 10 mM Mg [OAc]2 , 1 mM DTT , 3 . 6 mM β-ME ) and incubated at 37°C for 15 min: GTP ( 1 . 0 mM ) , mRNA ( 0 . 2 µM ) , initiation factors ( IF1 , 4 . 0 µM , IF2 , 3 . 7 µM , IF3 , 3 . 9 µM ) , fMet-tRNAfMet ( 3 . 9 µM ) and biotinylated ribosomes ( 1 . 0 µM ) .", "Finally , 1 µl aliquots of the mixture were prepared , quick-frozen in liquid N2 , and stored at −80°C .", "Total tRNA mixtures ( Sigma ) were aminoacylated using S-100 enzymes ( Moazed and Noller , 1989a ) and extracted with phenol/chloroform .", "To make a large-scale preparation of EF-Tu·aa-tRNAaa·GTP ternary complex , the following components were mixed in a total of 1 ml buffer TL-DTT ( buffer TL without DTT ) : 1 mM GTP , 5 mM PEP , 24 µM EF-Tu , and 0 . 04 mg/ml pyruvate kinase .", "The mixture was incubated at 37°C for 15 min .", "Then , 20 µl ( 1 U/µl ) total aa-tRNAs were added , incubated at 37°C for 5 min , and on ice for 10 min .", "Since free tRNAs , which are not productive in translation , tend to increase the noise in the translation traces ( probably by binding to the single-stranded mRNA ) , we developed a procedure to purify the ternary complexes using the 6xHis-tag present in EF-Tu .", "Briefly: The above reaction was bound to a Ni-NTA resin ( 30 min incubation at 4°C ) , and the resin washed three times with TL , with the addition of 20 mM imidazole .", "Ternary complexes were eluted with 600 µl elution buffer ( TL + 250 mM imidazole ) and dialyzed into Buffer TL for a total of 4 hr .", "Next , 50 µl purified ternary complexes ( containing 0 . 2 U total aa-tRNA ) were diluted with 390 µl ( total 440 µl ) buffer TL containing 1 mM GTP , 1 mM ATP , 40 U RNAguard ( GE Healthcare , Piscataway , NJ ) and 1 µM EF-G ( final concentrations ) .", "Finally , the mixture was filtered with a 0 . 22 µm low protein- binding MILLEX-GV Durapore membrane , ( Millipore , Billerica , MA ) and kept on ice .", "1 µl of ICs were first diluted with 50 µl TL , and then 1–10 µl mixed with 2 . 1 µm diameter polystyrene beads coated with anti-digoxigenin antibodies , and incubated on ice for 10 min .", "During this step , initiation complex attach to the beads through recognition between the complementary DNA handle ( harboring a 5′ digoxigenin ) that has been annealed to the mRNA and the anti-digoxigenin antibody coated on the beads .", "The initiation complex beads were then flowed into the chamber of the optical tweezers .", "After trapping one initiation complex bead in the optical trap , a streptavidin-coated bead , which binds to the ribosome and is held by suction on the end of a micropipette was moved to approach to the initiation complex bead .", "Binding of the biotinylated ribosome on the initiation complex to the streptavidin bead , results in an initiation complex tethered between two beads .", "Next , the translation mixture is flown into the chamber , and translation is followed in real time as a decrease in the tether length between the beads .", "No translation signals were detected before the introduction of the mixture .", "The experiments were conducted using force-measuring dual-beam optical tweezers , similar in concept to the instrument described by Smith et al . ( 2003 ) , but with an improved design that allows for better spatial and temporal resolution .", "Briefly , the two counter-propagating , orthogonally polarized laser beams that form the trap are coupled into two single-mode optical fibers and focused by two high numerical aperture objectives at a common position .", "The location of the optical trap can be controlled by tilting the tips of the optical fibers using two piezoelectric crystals , controlled by a feedback loop that maintains the respective focal points at a common position .", "The position of the trap is measured by a pair of position sensitive detectors ( PSDs ) that measure the tilting of the beams before entering the objectives , and the force is assessed from a second pair of PSDs to which the light distribution at the back focal planes of the objectives is imaged .", "All signals are sampled at 1 kHz .", "The 1 kHz raw data of tether extension at constant force was first averaged using a moving Savitzky-Golay filter with a span of 4000 data points .", "Then , instantaneous velocities were calculated using the averaged data .", "We further calculated the standard deviation of the instantaneous velocities for the part of the tether extension before elongation factors were injected into the chamber , and noted it as σpause .", "To distinguish between pausing and translocation , we use 2 . 5 σpause as a threshold .", "All the absolute instantaneous velocities that are smaller than the threshold are attributed to pauses .", "All the absolute instantaneous velocities that are greater than or equal to the threshold are attributed to ribosome translocation ( Figure 1B–D ) .", "The mean pause-free translocation velocity and translocation distance were calculated for each ribosome that actively translated the mRNA at constant forces .", "The mean translocation velocity for all the ribosomes that translate at specific constant forces was weighted by the total distance translocated by each ribosome .", "The mean translation velocities in nm/s were then converted to codon/s using the worm-like chain ( WLC ) model with a rise-per-base for ssRNA of 0 . 59 nm and a persistence length of 1 nm ( Liphardt et al . , 2001 ) .", "The translation rate vs force data was fitted with an exponential function using a weighted nonlinear least squares algorithm .", "Taking into account the possibility of a residual drift in the instrument , the stall-force was calculated as the force required to slow down the translation to a rate of 0 . 1 nt/s .", "Using the fitted exponential dependence parameters , this results in Fstall = 13 ± 2 pN .", "The free energy of ester hydrolysis was obtained as an average from the published values ( Fasman and Chemical Rubber Company , 1976 ) of glycine ethyl ester ( −8 . 40 kcal/mol ) , valyl RNA ( −8 . 40 kcal/mol ) , and ethyl acetate ( −4 . 72 kcal/mol ) , yielding −7 . 2 ± 1 . 2 kcal/mol .", "Similarly , the free energy of hydrolysis of amides was obtained as the average of published values ( Fasman and Chemical Rubber Company , 1976 ) for asparagine ( −3 . 60 kcal/mol ) , glutamine ( −3 . 4 kcal/mol ) , to yield −3 . 5 ± 0 . 1 kcal/mol .", "Thus , the average energy available for mechanical work as a result of transpeptidation is ∼3 . 7 ± 1 . 2 kcal/mol ." ] ]

[ "A detailed understanding of tRNA/mRNA translocation requires measurement of the forces generated by the ribosome during this movement .", "Such measurements have so far remained elusive and , thus , little is known about the relation between force and translocation and how this reflects on its mechanism and regulation .", "Here , we address these questions using optical tweezers to follow translation by individual ribosomes along single mRNA molecules , against an applied force .", "We find that translocation rates depend exponentially on the force , with a characteristic distance close to the one-codon step , ruling out the existence of sub-steps and showing that the ribosome likely functions as a Brownian ratchet .", "We show that the ribosome generates ∼13 pN of force , barely sufficient to unwind the most stable structures in mRNAs , thus providing a basis for their regulatory role .", "Our assay opens the way to characterizing the ribosome's full mechano–chemical cycle ." ]

[ "Producing a protein first requires its gene to be transcribed into a long molecule called a messenger RNA ( mRNA ) .", "A complex molecular machine called the ribosome then translates the mRNA code by reading it three letters at a time .", "Each triplet of letters—known as a codon—tells the ribosome which amino acid to add next into the protein .", "After adding an amino acid , the ribosome moves along the mRNA molecule to read the next codon and add another amino acid into the protein chain .", "While researchers understand how protein chains are formed , how the ribosome shifts along the mRNA strand—a process called translocation—is still unclear .", "It is known that this process involves many force-generating movements and changes to the shape of the ribosome .", "However , it is only recently that researchers have been able to measure these forces .", "Using optical tweezers—an instrument that uses a highly focused laser beam to hold and manipulate microscopic objects—Liu , Kaplan et al . followed individual ribosomes as they translated an mRNA and measured the effect that applying an opposing force has on the rate of translation .", "The results shed new light on the mechanism of translocation .", "First , Liu , Kaplan et al . found that ribosomes jump directly from one triplet to the next in the mRNA sequence , rather than moving there in a series of smaller steps .", "Next , the results indicate that translocation occurs spontaneously , driven by thermal energy , while chemical reactions prevent the reverse movement , in a mechanism known as a ‘Brownian Ratchet’ .", "Measurements of the maximum force generated by the ribosome also give insights into how translation is regulated .", "Strands of mRNA can fold into certain structures that slow down translation , because the mRNA must first be unfolded before the ribosome can translate it .", "Liu , Kaplan et al . found that the maximum force generated by a ribosome is only just enough to unwind these mRNA structures , making the translation rate highly sensitive to the existence of such structures , and the structures themselves of high importance for regulating transcription .", "Given its importance as the ultimate decoder of the genetic information , understanding the ribosome's function and regulation has broad implications .", "The work of Liu , Kaplan et al . opens the way for a full characterization of the role of mechanical forces in the translation process ." ]

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

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

[ [ "The activity of RNA Polymerase ( Pol ) II is responsible for transcription of mRNAs and many noncoding RNAs .", "Essential for Pol II function is the carboxy-terminal domain ( CTD ) of its largest subunit Rpb1 that consists of a highly conserved YSPTSPS heptad repetition ( Buratowski , 2009; Heidemann et al . , 2012 ) .", "Post-translational modifications ( PTMs ) of the CTD coordinate both transcription cycle transitions and loading of RNA processing complexes .", "In the recent years , novel PTMs were described in addition to the well-known Ser5P and Ser2P associated with early transcription and elongation , respectively .", "These include Ser7P , involved in snRNA gene transcription ( Chapman et al . , 2007; Egloff et al . , 2007 ) , Thr4P associated to transcription elongation in mammals ( Hintermair et al . , 2012 ) and to histone gene transcription in chicken ( Hsin et al . , 2011 ) , and Tyr1P that in yeast is found at gene body locations , consistent with a role in transcription elongation ( Mayer et al . , 2012 ) .", "This latter modification remains however so far uncharacterized in mammalian cells and we aimed at deciphering its function in human cells using biochemical and genome-wide approaches ." ], [ "To analyze expression and pattern of Tyr1P modified Pol II , we took advantage of our previously developed Tyr1P specific antibodies ( 3D12 ) ( Mayer et al . , 2012 ) .", "We investigated various mouse and human cells and could detect Tyr1P in western blots for all examined lines , in most cases associated with the hyper-phosphorylated IIO form of Pol II ( Figure 1—figure supplement 1 ) .", "To address the function of Tyr1P , we next generated Raji cell lines expressing Pol II resistant to α-amanitin ( Chapman et al . , 2004 ) and carrying either wild-type ( WT ) or a mutant Rpb1 gene with substitution of tyrosine to phenylalanine ( Y1F ) in CTD repeats 4 to 51 ( Figure 1—figure supplement 2 ) .", "After expression of the mutant , we observed that Y1F yielded a truncated Rpb1 ( Pol IIB , Figure 1A ) and was unable to form the hyper-phosphorylated IIO Pol II .", "After disruption of the activity of endogenous Pol II by α-amanitin ( Figure 1B ) and soon after disappearance of WT Rpb1 , cells became rapidly inviable .", "This phenotype reveals an essential function of the Y1 residue that appears more drastic than T4A or S7A mutations , but comparable with that of S5A ( Chapman et al . , 2007; Hintermair et al . , 2012 ) .", "We conclude that Tyr1P very likely contributes to stabilization of CTD and may occur early within the transcription cycle . 10 . 7554/eLife . 02105 . 003Figure 1 . Y1F mutations of the CTD heptads yield a truncated Pol IIB Rpb1 . ( A ) Rpb1-Y1F mutant was expressed after removal of tetracycline and in the presence of endogenous Rpb1 .", "Probing with Rpb1 Ab reveals both endogenous and recombinant Rpb1 whereas HA reveals only recombinant Y1F mutant .", "( B ) Protein expression of the Y1F mutant after shut-down of endogenous Rpb1 following treatment with α-amanitin . DOI: http://dx . doi . org/10 . 7554/eLife . 02105 . 00310 . 7554/eLife . 02105 . 004Figure 1—figure supplement 1 . Tyr1P is expressed in various human and mouse cell lines .", "( A ) Western blot analyses of antibody recognition in mouse and human cell lines of Rpb1 , CTD ( 8WG16 ) , and CTD isoforms including Tyr1P ( 3D12 ) .", "MEF , mouse embryo fibroblasts; Raji , Burkitt-Lymphoma; U2OS , osteosarcoma cell line; HEK293; human embryonic kidney cells; H9 , human embryonic stem cells; HFB , human skin fibroblasts; Neural Pre , human neural precursor cells .", "( B ) Western blot , as in ( A ) showing the specificity of 3D12 in Hela whole cell extracts over a wider range of proteins . DOI: http://dx . doi . org/10 . 7554/eLife . 02105 . 00410 . 7554/eLife . 02105 . 005Figure 1—figure supplement 2 . Sequence of the CTD heptads for the Tyr1 to Phe mutant ( Y1F ) .", "Amino-acid composition of the C-terminal domain of the Y1F mutant ( as described in the ‘Materials and methods–Construction of the CTD Y1F mutant’ ) used for phenotypic and western blot analyses ( Figure 1 ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 02105 . 005 To gain further insight into the involvement of Tyr1P in the transcription cycle , we performed co-immunoprecipitation experiments in human cells using antibodies directed against various CTD modifications reflecting 5′ transcriptionally engaged ( Ser5P , Ser7P ) or elongating forms ( Ser2P , Thr4P ) of Pol II ( Chapman et al . , 2007; Hintermair et al . , 2012; Mayer et al . , 2012 ) .", "Our experiments indicated clearly that Tyr1P co-immunoprecipitated with Ser5P and Ser7P but not Ser2P or Thr4P ( Figure 2A ) .", "Consistently , signals for Tyr1P were observed in Ser5P and Ser7P but not in Ser2P co-immunoprecipitations .", "Thus , overall , this data points out an association of Tyr1P with early transcribing isoforms of human Pol II . 10 . 7554/eLife . 02105 . 006Figure 2 . CTD Tyrosine 1 is phosphorylated mainly at TSS and is dominant in antisense transcription .", "( A ) Co-immunoprecipitation with specific CTD isoforms in Raji B-cells reveals Tyr1P ( 3D12 ) association with Ser5P and Ser7P but not with Ser2P and Thr4P .", "( B ) ChIP-seq example illustrating Tyr1P ( 3D12 ) association around the promoter of RPL22L1 gene .", "( C ) Composite average profiling of ChIP-seq data at coding genes locations for Pol II ( 1433 genes ) , Tyr1P ( 3D12 , 2462 genes ) , Ser5P ( 1464 genes ) , and Ser7P ( 2186 genes ) in Raji B-cells and based on selections described in Figure 2—figure supplement 1B .", "Less stringent selections with more genes gave equivalent profiling ( Figure 2—figure supplement 4A ) .", "( D ) Profiling of Pol II , Tyr1P ( 3D12 ) , Ser5P , Ser7P , nucleosomes midpoint and short strand specific RNAs ( ssRNAs ) around TSS locations with same selections described in ( C ) .", "( E ) Boxplots on 3201 genes without outliers showing mean levels of Pol II ( 2986 genes ) , Tyr1P ( 2964 genes ) , Ser5P ( 2909 genes ) , and Ser7P ( 2948 genes ) ChIP-seq signal on regions representing each transcription orientation .", "The p-values ( parametric two sided paired t test ) of the difference of AS vs S signal are Pol II = 0 . 5 , Tyr1p=3 . 4 × 10−15 , Ser5p=0 . 6 , Ser7p=3 . 5 × 10−2 . DOI: http://dx . doi . org/10 . 7554/eLife . 02105 . 00610 . 7554/eLife . 02105 . 007Figure 2—figure supplement 1 . Reproducibility of ChIP-seq experiments and selection of relevant signals used for analyses .", "( A ) Correlation plots of biological replicates ( for all but H3K36me3 i . e . , a technical replicate ) of ChIP-seq experiments used in this study at gene locations ( ‘Materials and methods–Correlation of biological replicates and cross-correlation’ ) .", "Spearman correlation coefficient is indicated on the top left of the plots .", "( B ) Distribution and threshold of background-subtracted signal used for profiling of significantly bound gene ( Total , i . e . , whole genic regions ) in Figure 2 , Figure 2—figure supplement 5A , and Figure 2—figure supplement 7C .", "The mean values used for distribution were computed on [TSS-1000 bp:TES+2000 bp] ( TSS: transcription start site; TES: transcription end site ) .", "Note that the thresholds were set to the mean of the second Gaussian of the distribution ( ‘Materials and methods–Gene selection and average binding profiles’ ) .", "Numbers of genes selected for Pol II , Ser2P , Ser5P , Ser7P , Tyr1P 3D12 , and Tyr1P 8G5 are 1521 , 1536 , 1543 , 2382 , 2652 , and 2608 , respectively .", "( C ) Distribution and threshold of Pol II significantly bound promoters ( TSS ) as in ( B ) .", "The selection is used in Figure 3 , Figure 3—figure supplement 1 , and Figure 3—figure supplement 2 .", "2044 genes were selected based on their mean values on TSS −/+ 500 bp . DOI: http://dx . doi . org/10 . 7554/eLife . 02105 . 00710 . 7554/eLife . 02105 . 008Figure 2—figure supplement 2 . Pol II and CTD PTMs correlate positively with expression . Based on microarray expression data , three groups of genes with low ( L , 3414 genes ) , medium ( M , 1238 genes ) , and high ( H , 1007 genes ) expression were used to profile Pol II isoforms and short ssRNA at promoters .", "( A ) Heatmaps of signal densities for the three defined groups .", "( B ) Average profiles of Pol II phospho-isoforms and ssRNA at the three defined groups .", "( C ) Boxplots of the mean values retrieved at TSS −/+ 500 bp in the three classes for Pol II ( 3095 , 1169 , 957 genes ) , Tyr1P ( 3159 , 1150 , 958 genes ) , Ser5P ( 3072 , 1157 , 956 genes ) , and Ser7P ( 3184 , 1130 , 942 genes ) .", "( D ) Boxplot of regions representing each transcription orientation as in Figure 2E for each class divided by Pol II binding values .", "p-value ( parametric two sided paired t test ) are respectively: 2 . 3 × 10−13; 5 × 10−4; 6 × 10−3 ( low ) , 2 . 4 × 10−13; 6 × 10−3; 2 × 10−4 ( medium ) , 7 × 10−6; 0 . 02; 0 . 8 ( high ) .", "Represented number of genes are 3175 , 3126 , 3074 , 3051 , 3123 , 3134 ( low ) ; 1154 , 1079 , 1154 , 1125 , 1139 , 1084 ( medium ) ; 955 , 930 , 941 , 941 , 935 , 913 ( high ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 02105 . 00810 . 7554/eLife . 02105 . 009Figure 2—figure supplement 3 . Examples of Tyr1P binding patterns at genic locations . EIF1B and SNHG8 are mainly bound by Tyr1P ( 3D12 ) at TSS as for RPL22L1 gene of Figure 2B . DOI: http://dx . doi . org/10 . 7554/eLife . 02105 . 00910 . 7554/eLife . 02105 . 010Figure 2—figure supplement 4 . Average profiling of Pol II and phospho-isoforms at genic and promoter locations using wide relaxed threshold selections .", "( A ) Composite and TSS focused average profiling of ChIP-seq data as in Figure 2C , D , for a selection threshold of 0 as described in Figure 2—figure supplement 1B , at coding genes locations for Pol II ( 2714 genes ) , Tyr1P ( 3D12 , 2987 genes ) , Ser5P ( 2697 genes ) , and Ser7P ( 3002 genes ) in Raji B-cells .", "( B ) Boxplots on 4749 genes as in Figure 2E for the less stringent selection showing mean levels of Pol II , Tyr1P , Ser5P , and Ser7P ChIP-seq signal on regions representing each transcription orientation .", "The p-values ( parametric two sided paired t test ) of the difference of AS vs S signal are Pol II = 0 . 2 , Tyr1p=3 . 5 × 10−16 , Ser5p=0 . 2 , Ser7p=0 . 03 .", "Boxplots do not show outliers for Pol II ( 3933 genes ) , Tyr1P ( 3897 genes ) , Ser5P ( 3920 genes ) , and Ser7P ( 3878 genes ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 02105 . 01010 . 7554/eLife . 02105 . 011Figure 2—figure supplement 5 . Ser2P average profile at genic locations and examples of Tyr1P signal at promoter locations .", "( A ) Ser2P average profile on 1415 genes selected on mean values distribution shown in Figure 2—figure supplement 1B and represented as for Figure 2C .", "( B ) Examples of Tyr1P ( and other isoforms , short ssRNAs ) at promoters of 5 coding genes .", "These genes show a dominance of Tyr1P ( 3D12 ) signal upstream ( AS direction ) relatively to downstream TSSs and as compared to Pol II and isoforms . DOI: http://dx . doi . org/10 . 7554/eLife . 02105 . 01110 . 7554/eLife . 02105 . 012Figure 2—figure supplement 6 . Tyr1P presents a specific pattern of phosphorylation along genes compared to Pol II .", "( A ) Genome-wide profiling of Pol II ( N20 ) and CTD isoforms ( as in Figure 2 ) for different classes of binding levels indicate a distribution of Tyr1P more prominent at promoters vs gene bodies as compared to Pol II and Ser7P , but comparable to that of Ser5P .", "The indicated signal rank of the values is over an area encompassing TSS , GB , and 3′ ends of genes as indicated in the ‘Materials and methods–Gene selection and average binding profiles’ .", "Note that more Tyr1P signal is found at 3′ ends as compared to Ser5P .", "( B ) Spearman correlation plots of significantly enriched areas for Pol II and phospho-isoforms ( genes size >2 kb ) indicate that Tyr1P relates more to Pol II and early transcription marks at promoters than it does at gene bodies or 3′ends .", "Mean values for Spearman correlation were computed at [TSS-500 bp;TSS+500 bp] , [TSS+1000 bp; 3′end-500 bp] , and [3′end-500 bp; 3′end+1000 bp] ( ‘Materials and methods–Correlation of biological replicates and cross-correlation’ ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 02105 . 01210 . 7554/eLife . 02105 . 013Figure 2—figure supplement 7 . Tyr1P specific antibodies with distinct peptide recognition patterns show similar genome-wide profiling at TSS .", "( A ) CTD peptide recognition patterns of 3D12 and 8G5 Tyr1P Abs used in this study .", "Note that 8G5 shows a wider range of peptide recognition compared to 3D12 .", "( B ) Specificity and reactivity of mAbs were tested in ELISA experiments towards the peptides CTD-1 to -19 .", "( C ) Genome-wide profiling of ChIP-seq experiments performed with 8G5 at TSSs ( left panel ) or at gene body locations on 2365 genes .", "As for 3D12 Ab , the AS peak is over-represented when compared to Pol II . DOI: http://dx . doi . org/10 . 7554/eLife . 02105 . 013 To assess its relation to transcription genome-wide , we next performed Tyr1P ChIP-seq , using 3D12 mAb , and compared it to Pol II and the other phospho-isoforms .", "We isolated significantly associated regions based on the signal distribution of the background-subtracted data ( Figure 2—figure supplement 1B ) and found that Pol II and all isoforms , including Tyr1P , correlated with transcription levels of genes ( Figure 2—figure supplement 2 ) .", "At many gene locations , a predominant signal of Tyr1P at promoters was observed ( Figure 2B , Figure 2—figure supplement 3 ) .", "We further confirmed this by genome-wide profiling of Pol II isoforms at coding-gene locations ( Figure 2C , Figure 2—figure supplement 4 , Figure 2—figure supplement 5A for Ser2P profile ) .", "Our profiling analysis shows that Tyr1P signal is predominantly found at promoters similarly to Ser5P , weak or essentially absent at gene bodies and weak at 3′ends in contrast to Ser2P elongating mark and Ser7P ( associated to both promoters and gene bodies ) .", "These observations are further supported by quantification of signals at various genic sections ( Figure 2—figure supplement 6 ) and reinforce our conclusion that human Tyr1P is mainly associated to promoters in an early , post-initiation step of transcription .", "Although we did not further investigate this possibility , in the accompanying manuscript , Hsin et al show that Chicken Tyr1 is found phosphorylated in the nucleoplasm , raising the possibility that Tyr1P is also associated with recruitment of the enzyme and transcription initiation .", "Genomic profiling at the vicinity of the transcription start site ( TSS ) indicates two main peaks of Pol II upstream and downstream of the TSS ( Figure 2D , left panel and Figure 2—figure supplement 4 ) .", "These peaks most likely reflect sense and antisense paused transcription as evidenced by our short strand specific ( ssRNA ) sequencing analysis , as previously described ( Core et al . , 2008; Preker et al . , 2008; Seila et al . , 2008; Fenouil et al . , 2012 ) for mammalian promoters .", "This transcription results in short promoter-associated transcripts production and might relate to pervasive transcription of promoters in sequence context lacking strong elements imposing directionality .", "By comparing the signals of these two peaks with that of the Ser5P and Ser7P isoforms , Tyr1P showed a clearly distinct pattern with a more pronounced upstream peak ( Figure 2D , Figure 2—figure supplement 4 and examples in Figure 2—figure supplement 5B ) .", "We confirmed this result with an independent Tyr1P antibody ( 8G5 ) harboring wider range of CTD peptide recognition ( Figure 2—figure supplement 7 ) , and by using statistical analysis showing that antisense/sense ( AS/S ) difference was significant for Tyr1P as compared to other isoforms ( Figure 2E , Figure 2—figure supplement 4B ) .", "Together , our analyses indicate that Tyr1P is predominantly associated with upstream polymerases , mostly reflecting AS transcription at mammalian promoters .", "We previously showed that mammalian promoters associated with Pol II can be grouped in three main classes in mouse T-lymphocytes ( Fenouil et al . , 2012 ) , based on ranking of the main Pol II signal from the most upstream to the most downstream of the TSS .", "We reproduced this result and the main features of the three groups in human Raji B-cells by ranking the signal of Tyr1P ( Figure 3A , Figure 3—figure supplement 1A , B ) .", "The first class ( the majority of genes ) , with Pol II signals most upstream of TSSs , harbors strongly paused Pol II at promoters with high GC content and CpG islands ( CGIs ) and is associated with the highest level of bidirectional and AS transcription .", "The second class , with a sharper Pol II peak centered close to the TSS and lower GC content , contains mostly mono-directional sense paused transcription whereas the third class contains more downstream Pol II with less pause .", "We then focused our attention on class I that contains most AS short RNAs .", "In this class , Tyr1P is essentially observed in AS while Ser5P , Ser7P , or total Pol II generally show a second peak around the TSS reflecting sense and therefore bidirectional transcription ( Figure 3C , Figure 3—figure supplement 1C ) .", "This indicates that AS Tyr1P relates to one specific class of promoters and suggests that in AS orientation , Tyr1P associates mainly with the leading edge of Pol II .", "Pleading for this hypothesis , the location of the AS Tyr1P in class I is found more downstream on average as compared to Pol II or Ser5P , and locates just after the −2 nucleosome midpoint ( Figure 3B , C ) .", "A more detailed investigation of the individual positions of phospho-isoforms further shows that for the majority of promoters significantly associated with AS short RNAs in class I , Tyr1P is either located at the immediate proximity or after the main Pol II peak ( Figure 3—figure supplement 2 ) suggesting that it might play a role in early elongation .", "Although Ser7P displayed similar characteristics , its influence on transcription of coding genes is likely to be minor , as Ser7 mutations do not show significant phenotype ( Chapman et al . , 2007 ) or transcriptome impairment ( JCA and DE , unpublished observations ) .", "We overall conclude that Tyr1P is a CTD PTM that associates with the 5′ end of genes and shows a stronger linkage to paused Pol II at promoters with bidirectional and AS transcription . 10 . 7554/eLife . 02105 . 014Figure 3 . Tyr1 is preferentially phosphorylated in antisense orientation on a particular subset of genes .", "( A ) Heatmaps of Tyr1P ( 3D12 ) , Pol II , Ser5P , Ser7P , nucleosome midpoints ( positioning ) and short strand specific RNAs ( red for AS and blue for S signal ) at promoters with a significant level of Pol II .", "The genes were ordered by position of the main Tyr1P accumulation area from the most 5′ to the most 3′ within −1000 bp and +1000 bp around TSS .", "Three main classes are defined by Tyr1P occupancy: class I most 5′ ( red bar , 1066 genes ) , class II TSS-proximal ( green bar , 579 genes ) , and class III most 3′ ( blue bar , 209 genes ) .", "( B ) Average profiling of short ssRNAs and nucleosomes positions in class I . Positions of the nucleosome midpoints are indicated by a dashed line ( nucleosome −3 , −2 , −1 , and +1 from left to right ) .", "( C ) Profiles of Pol II and CTD isoforms in class I . Red , blue , orange , and green dashed lines indicate the average position of the maximum values of Pol II , Tyr1P ( 3D12 ) , Ser5P , and Ser7P signals , respectively .", "The distance between Pol II leading edge and isoforms is indicated below each graph .", "The borders of nucleosomes −3 , −2 , and +1 ( from left to right ) are shown as pink rectangles whereas the red , blue , orange , and green circles represent Pol II , Tyr1P , Ser5P , and Ser7P , respectively with indication of directionality based on the short ssRNA signals . DOI: http://dx . doi . org/10 . 7554/eLife . 02105 . 01410 . 7554/eLife . 02105 . 015Figure 3—figure supplement 1 . Three classes of Pol II-bound promoters ordered by Tyr1P location in human Raji cells .", "( A ) Heatmaps of a selection of Pol II-bound promoters for ssRNAs , nucleosome and AT , GC contents ordered by Tyr1P ( 3D12 ) maximum signal from the most upstream to the most downstream of the annotated TSSs ( as previously described in mouse lymphocytes , Fenouil et al . , 2012 ) .", "Note that Pol II main accumulation areas occur at proximity of the main nucleosome position for each promoter class .", "As described before ( Fenouil et al . , 2012 ) , GC content and CpG islands correlate with nucleosome depletion .", "( B ) Profiles of ssRNAs ( sense and antisense ) and nucleosome in the three groups .", "( C ) Profiles of Pol II and CTD isoforms in the three classes of promoters as indicated . DOI: http://dx . doi . org/10 . 7554/eLife . 02105 . 01510 . 7554/eLife . 02105 . 016Figure 3—figure supplement 2 . CTD isoforms and nucleosome distribution around Pol II upstream of TSSs in class I promoters .", "( A ) 3D plots of Tyr1P , Ser5P , Ser7P and nucleosomes midpoints ( MP ) maximum signal locations as compared to Pol II ChIP-seq maxima for genes of group 1 of Figure 3A .", "Only genes with a significant signal of antisense ssRNA and higher than sense ssRNA were taken into account ( see ‘Materials and methods–CTD isoforms and nucleosomes midpoint maximal peaks spatial organization analysis’ for details ) .", "The positive values of the distance to Pol II axis ( in bp ) indicate that maximum signals are located after Pol II in opposite direction of TSSs whereas negative values are in the inverse orientation .", "The number of maximal peaks before , after or colocalized with Pol II for Tyr1P , Ser5P , and Ser7P are 90/265/174 , 99/152/278 , 125/234/170 , respectively .", "Note that most of the Tyr1P max values are located after Pol II whereas Ser5P is mainly found around Pol II main signal .", "( B ) 2D Boxplots of the maximum values shown in ( A ) ( upper panel ) and for an independent analysis using Tyr1P max signal as reference ( lower panel ) .", "In both cases Tyr1P locates at or after the leading edge of Pol II .", "( C ) Distance to Pol II distribution of Tyr1P , Ser5P , and Ser7P for class I promoters selected as described in ( A ) .", "Data is represented in bins of 10 ( ‘Materials and methods–Processing of sequenced tags’ ) .", "The difference of distribution with the whole set of genes ( black line ) was assessed by a nonparametric Kolmogorov-Smirnov test .", "p-values are indicated at the top-right of each panel . DOI: http://dx . doi . org/10 . 7554/eLife . 02105 . 016 Many groups including ours have shown that highly active and tissue-specific enhancers are transcribed by Pol II in various tissues ( De Santa et al . , 2010; Kim et al . , 2010; Koch et al . , 2011; Natoli and Andrau , 2012 ) .", "These enhancers can also be hallmarked by the occurrence of H3K4me1high/H3K4me3low epigenetic marks combination ( Koch et al . , 2011; Pekowska et al . , 2011 ) .", "To investigate if Tyr1P can be detected at enhancers , we first isolated intergenic regions ( IGRs ) with stringent criteria in B-cells using Pol II , H3K4me1 , and me3 signals .", "These were further discriminated from noncoding promoters using the relative ratio of H3K4me1/me3 ( Pekowska et al . , 2011; Li et al . , 2012 ) , and from both unannotated coding and some long intergenic noncoding genes using the absence of H3K36me3 that marks gene bodies ( Guttman et al . , 2009 ) .", "Using these criteria , we isolated 390 B-cells enhancers ( Figure 4—figure supplement 1A–D ) .", "Our selection was further validated using tissue-specificity analyses ( Figure 4—figure supplement 1E ) indicating IGRs associated with genes specific to B-cells .", "We next performed profiling of the various Pol II isoforms at these enhancers .", "As before ( Koch et al . , 2011 ) , we observed that these IGRs were associated with Ser5P ( Figure 4A ) but not with Ser2P Pol II ( not shown ) as well as with short transcripts ( reflecting paused transcription ) and a discrete nucleosome depleted region .", "Consistent with early elongating Pol II at enhancers , we found signal for both Ser7P and Tyr1P at these IGRs .", "Importantly , Tyr1P appeared more bound to enhancers as compared to promoters and total Pol II ( Figure 4B , C , Figure 4—figure supplement 2 ) , suggesting that Tyr1 is more phosphorylated than Ser5 or Ser7 at enhancers and represent a hallmark of these essential areas of the genome .", "Additionally , Tyr1P also displayed the best correlation with Pol II at isolated enhancers ( Figure 4D ) .", "Finally , using an independent selection for active enhancers based on H3K27ac brought very similar results ( Figure 4—figure supplement 3 ) .", "Together , our investigations showed that Tyr1P is a strong signature of Pol II-transcribed active enhancers associated with tissue-specific gene expression . 10 . 7554/eLife . 02105 . 017Figure 4 . Tyr1P is a hallmark of enhancers relative to Pol II and promoters signal .", "( A ) Average profiling of Pol II , Tyr1P ( 3D12 ) , Ser5P , Ser7P , nucleosomes occupancy , and short ssRNAs .", "390 active putative enhancers ( red ) and 4618 control promoters ( blue ) were detected in human Raji B-cells ( ‘Materials and methods–Selection of enhancers and promoters using Pol II’ ) .", "Profiles are centered on Pol II ChIP-seq maximal signal and are not oriented .", "( B ) Boxplots of mean ChIP-seq signal on selected enhancer and control promoter regions for Ser5P ( 371/4378 values ) , Ser7P ( 368/4257 values ) , and Tyr1P ( 372/4266 values ) .", "Signals were normalized by the mean ChIP-seq signal of Pol II on the same regions .", "All marks show a significant difference ( nonparametric Mann-Whitney-Wilcoxon test , p-values <10−10 ) .", "( C ) Example of Tyr1P at promoter and putative enhancer .", "( D ) Spearman cross-correlation between Pol II , Ser5P , Ser7P , Ser2P , and Tyr1P ( 3D12 ) at intergenic putative enhancers .", "Tyr1P and Pol II best correlate with each other . DOI: http://dx . doi . org/10 . 7554/eLife . 02105 . 01710 . 7554/eLife . 02105 . 018Figure 4—figure supplement 1 . Pol II-bound enhancer selection procedure and features .", "( A ) Workflow of the enhancers ( 390 ) and control promoters ( 4618 ) selection based on ChIP-seq of H3K36me3 , H3K4me3 , H3K4me1 , and Pol II .", "Details of procedure and number of regions isolated at each step ( E1-4 and P1-3 ) are indicated in ‘Materials and methods–Selection of enhancers and promoters using Pol II’ .", "( B ) Plot of H3K4me3/me1 mean values ratios of selected intergenic regions at step E2 ( in red ) , promoter regions at step P1 ( in blue ) , and Hg19 RefSeq annotated promoters ( in black ) .", "To stringently select isolated promoters and intergenic regions shown in ( A ) and attribute their putative enhancer and control promoter status , a threshold was defined ( in dashed green line ) .", "( C ) Nonoriented profiling of epigenetic marks associated with putative enhancers ( in red ) and control promoters ( in blue ) selected at steps P3 and E4 of procedure described in ( A ) and centered on the main Pol II peak as in Figure 4A .", "( D ) Boxplots of H3K4me3 ( 363/4325 genes plotted ) and H3K4me1 ( 375/4259 genes plotted ) signals at putative enhancers ( in red ) and control promoters ( in blue ) .", "Nonparametric Mann-Whitney-Wilcoxon test gave p-values <10−152 .", "( E ) Tissue specificity analysis of the genes associated with putative enhancers ( closest genes on each side of the isolated genomic loci ) compared to genes of HGU133 array ( whole genes , see ‘Materials and methods–Tissue specificity analysis’ ) .", "The isolated tissues are ranked by p-values ( indicated on the left ) from top to bottom .", "This analysis indicates that both WT ( CD19 ) and Raji human B-cells are among the most significant tissues thus validating the putative enhancer regions identified in our analysis and as described in mouse lymphocytes ( Li et al . , 2012 ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 02105 . 01810 . 7554/eLife . 02105 . 019Figure 4—figure supplement 2 . Examples of Tyr1P enhancer association upstream or downstream of CXCR4 , DUSP2 , and IER5 genes . As in Figure 4 , light orange and blue rectangles highlight enhancer and promoter locations with higher H3K4me3 at promoters and higher H3K4me1 at enhancers .", "Relative amount of Tyr1P is higher at enhancers as compared to Pol II and to promoters .", "H3K4me3 level at CXCR4 IGR was observed but is not visible due to the scale used and because of high level of signal at promoter . DOI: http://dx . doi . org/10 . 7554/eLife . 02105 . 01910 . 7554/eLife . 02105 . 020Figure 4—figure supplement 3 . Selection of enhancers using H3K27ac also shows a dominance of Tyr1P on active and tissue specific enhancers .", "( A ) Workflow of a complementary selection of enhancers ( 927/2598 active ) , and control promoters ( 5946/6057 active ) based on ChIP-seq of H3K36me3 , H3K4me3 , and H3K4me1 .", "H3K27ac was used to extract specifically active enhancers from the whole set .", "Details of procedure and number of regions isolated at each step ( E1'-6' and P1'-6' ) are indicated in ‘Materials and method–Selection of active enhancers and promoters using H3K27ac’ .", "( B ) Average profiles of Pol II and isoforms for active enhancers/promoters and the whole set of enhancers/promoters .", "( C ) Active ( H3K27ac selection ) enhancers show increased enrichment over Pol II and tissue-specific gene expression .", "As in Figure 4 , Spearman correlation , boxplots of comparison of levels of Pol II isoforms , and tissue specificity analyses indicate Tyr1P to be over-enriched at active enhancers as compared to Pol II and promoters .", "Nonparametric two-sided Mann-Whitney-Wilcoxon test for boxplots of Ser5P ( 780/5068 values ) , Ser7P ( 752/4953 values ) , and Tyr1P ( 739/5233 values ) yields p-values of 5 . 1 × 10−56 , 7 . 05 × 10−4 , and 2 . 1 × 10−30 , respectively .", "( D ) Whole enhancer set ( H3K4me1/3 ) analysis as in ( C ) .", "Nonparametric two sided Mann-Whitney-Wilcoxon test for boxplots of Ser5P ( 2220/5141 values ) , Ser7P ( 2186/5005 values ) , and Tyr1P ( 2112/5306 values ) yields p-values of 2 . 6 × 10−139 , 6 . 8 × 10−4 , and 8 . 09 × 10−4 , respectively . DOI: http://dx . doi . org/10 . 7554/eLife . 02105 . 020 Here , we described that Tyr1P associates with 5′ Pol II and AS transcription at promoters and is a signature of active , tissue-specific enhancers in human B-cells .", "These findings contrast with features of Tyr1P in yeast , which is located at gene bodies and proposed to play a role in elongation by impairing termination factor recruitment ( Mayer et al . , 2012 ) .", "These apparent discrepancies thus provide an interesting paradigm whereby a conserved PTM has evolved to display specialized functions specific to metazoans .", "However , S . cerevisiae genes are very compact , mostly devoid of introns and promoters structure is also extremely divergent in both length and sequence between yeast ( around 100–200 nt , AT-rich ) and mammals ( around 1000 nt , GC-rich ) .", "Furthermore , enhancers do not exist per se in yeast .", "In an accompanying manuscript , Hsin et al . ( 2014 ) describe similar observations regarding stability of Y1F mutant in chicken cells and involvement of Tyr1P in AS transcription at promoters , thus providing further evidence that our observations are conserved in vertebrates .", "We therefore speculate that differential CTD PTMs might not only reflect , but also play a role in regulating the directionality of transcription .", "How would Tyr1P behave in organisms with less prominent bidirectional transcription at promoters such as Drosophila ( Core et al . , 2012 ) thus represents an interesting evolutionary question to be addressed in future studies .", "Based on the spatial location of Tyr1P in class I promoters , mostly found at the leading edge of Pol II in opposite orientation of the gene , it is tempting to speculate that this PTM might be involved in a transcriptional state marking the transition between early and productive elongation , providing a checkpoint for transcriptional complexes to proceed in productive elongation .", "Depending on the level of Tyr1P at promoters , Pol II might become competent for elongation as well as for overcoming the nucleosomal barrier both in sense and antisense orientation .", "Since less Pol II molecules are able to effectively enter elongation in AS orientation , more accumulation of the Tyr1P could be observed upstream of the TSS toward the leading edge of Pol II .", "This could also explain degradation of Y1F mutant that is due to absence of Tyr1P checkpoint signal , would accumulate around the edge of the promoters and become degraded .", "Finally there could also be a link between hyperphosphorylation of Tyr1 in AS orientation and exosome machinery recruitment to degrade nascent RNA prior release of the Pol II enzyme ( Preker et al . , 2008; Andersen et al . , 2013 ) .", "We believe our work will thus provide a new frame of investigation to decipher the complexity of mechanisms leading to transcriptional activation , at the heart of gene regulation ." ], [ "Generation and validation of modification specific mAbs have been described before: Tyr1P mAb ( 3D12 , Mayer et al . , 2012 ) and 8G5 ( see Figure 2—figure supplement 7 ) , Ser2P ( 3E10 ) , Ser5P ( 3E8 ) , and Ser7P ( 4E12 , Chapman et al . , 2007 ) , Thr4P ( 6D7 , Hintermair et al . , 2012 ) .", "For further characterization of specificity , the 3D12 and 8G5 Tyr1P antibodies were analyzed in ELISA experiments using CTD-like peptides with different modification patterns ( Peptide Specialty Laboratories GmbH , Heidelberg , Germany ) coupled to 96-well maleimide plates ( Thermo Fisher Scientific Inc . , Rockford , IL USA ) as antigen ( Figure 2—figure supplement 7 ) .", "Peptides were incubated with the monoclonal antibodies and biotinylated , subclass-specific antibodies , respectively .", "After incubation with horseradish peroxidase ( HRP ) -coupled avidin , H2O2 and TMB ( 3 , 3' , 5 , 5'-tetramethylbenzidine ) were added .", "Absorbance of each well was measured at 650 nm after color change and quantitated with an ELISA reader .", "ChIP-seq and MNase-seq experiments were performed essentially as described before using same standard and QC for experiments ( Fenouil et al . , 2012 ) .", "Experimental details of individual experiments , including replicates when applicable , are also indicated in Table 1 . 10 . 7554/eLife . 02105 . 021Table 1 . Summary of ChIP conditions and bioinformatics treatment for each experiment ( NR = not relevant , NA = not available ) DOI: http://dx . doi . org/10 . 7554/eLife . 02105 . 021ChIP antibodies and conditions used ( * For ChIP-QPCR ) Peak detectionExperimentAntibody ( clone ) OriginReference AntibodyNumber of cellsAntibody/BeadsWashes ( RIPA/TE ) Replicates NumberTags Not Aligned/Multiple Alignment ( × 106 ) Tags Used ( × 106 ) Lanes NumberExtension Size ( bp ) ThresholdMax GapPol IITotal ( N-20 ) Rabbit polyclonalSanta Cruz ( sc-899x ) 1 × 10820 µg/200 µl8x/1x18 . 9319 . 83117680350217 . 9433 . 022166316 . 5428 . 481156H3K4me1H3K4me1Rabbit polyclonalAbcam ( ab8895 ) 5 × 1062 µg/20 µl8x/1x19 . 357 . 8311766070027 . 5920 . 931226H3K4me3H3K4me3Rabbit polyclonalAbcam ( ab8580 ) 5 × 1062 µg/20 µl8x/1x17 . 122 . 611186504002NA14 . 141123H3K36me3H3K36me3Rabbit polyclonalAbcam ( ab9050 ) 2 × 1078 µg/80 µl8x/1x1NA21 . 2111964010002NA5 . 571316H3K27acH3K27acRabbit polyclonalAb47295 × 1062 µg/20 µl5x/1x15 . 3352 . 501197100750Tyr1PTyr1P ( 3D12 ) Rat monoclonalMayer et al . ( 2012 ) 1 × 10810 µg/100 µl5x/1x112 . 3015 . 561206NRNR29 . 9815 . 551276Tyr1P ( 8G5 ) Rat monoclonalThis article1 × 10810 µg/100 µl5x/1x130 . 2628 . 781187NRNRSer2PSer2P ( 3E10 ) Rat monoclonalChapman et al . ( 2007 ) 2 × 10880 µg/400 µl5x/1x19 . 3111 . 281192NRNR29 . 8515 . 941286Ser5PSer5P ( 3E8 ) Rat monoclonalChapman et al . ( 2007 ) 1 . 2 × 108 ( 2 . 5 × 107* ) 24 µg/240 µl ( 5 µg/50 µl* ) 8x/1x1NA13 . 981146NRNR2NA3 . 571216Ser7PSer7P ( 4E12 ) Rat monoclonalChapman et al . ( 2007 ) 1 × 10810 µg/100 µl5x/1x1NA16 . 461156NRNR2NA1 . 921226Short-RNA-seqNRNRNR1 × 107NRNR1NA9 . 871NRNRNRMNase-seqNRNRNR2 × 107NRNR190 . 00289 . 601152/NR midpoints**NRNRInputNRNRNRNRNRNR120 . 1018 . 181126NRNR2NA29 . 741146315 . 4124 . 931118411 . 2028 . 321196**For MNase-seq , the experiment was performed and processed in pair-end .", "For nucleosome density , tags were not elongated but connected and the indicated sequence average length is withdrawn by our analysis pipeline using the pair-end information .", "For midpoints analyses , elongation does not apply and data treatment is indicated earlier in ‘Materials and methods–Processing of sequenced tags’ .", "Details of the data pre-processing are described in Fenouil et al . ( 2012 ) ." ] ]

[ "In mammals , the carboxy-terminal domain ( CTD ) of RNA polymerase ( Pol ) II consists of 52 conserved heptapeptide repeats containing the consensus sequence Tyr1-Ser2-Pro3-Thr4-Ser5-Pro6-Ser7 .", "Post-translational modifications of the CTD coordinate the transcription cycle and various steps of mRNA maturation .", "Here we describe Tyr1 phosphorylation ( Tyr1P ) as a hallmark of promoter ( 5′ associated ) Pol II in mammalian cells , in contrast to what was described in yeast .", "Tyr1P is predominantly found in antisense orientation at promoters but is also specifically enriched at active enhancers .", "Mutation of Tyr1 to phenylalanine ( Y1F ) prevents the formation of the hyper-phosphorylated Pol IIO form , induces degradation of Pol II to the truncated Pol IIB form , and results in a lethal phenotype .", "Our results suggest that Tyr1P has evolved specialized and essential functions in higher eukaryotes associated with antisense promoter and enhancer transcription , and Pol II stability ." ]

[ "When a gene is expressed , the DNA is first transcribed to produce an intermediate molecule called a messenger RNA ( mRNA ) , which is then translated to produce a protein .", "RNA Polymerase II is an enzyme that makes mRNA molecules in organisms as diverse as plants , animals , and yeast .", "RNA Polymerase II is a complex made of a number of proteins .", "The largest protein in this complex includes a ‘carboxy-terminal domain’ that has multiple repeats of seven amino acids one after the other .", "The first amino acid in each repeat , a tyrosine , is referred to as tyrosine-1 .", "Adding various chemical tags to the amino acids in these repeats co-ordinates the steps involved in the transcription of genes .", "In yeast , for example , adding a phosphate group to tyrosine-1 seems to help the polymerase to proceed to make long mRNA molecules .", "However , it is not known what these chemical tags do in humans or other animals .", "Now Descostes , Heidemann et al . ( and independently Hsin et al . ) have shown that the same phosphate groups on tyrosine-1 perform functions in vertebrates ( animals with backbones ) that are different to those performed in yeast .", "These functions include protecting the carboxy-terminal domain from being broken down inside cells , and transcribing the DNA that is upstream of genes .", "Descostes , Heidemann et al . found that in human cells , RNA Polymerase II with phosphate groups on tyrosine-1 tends to bind to the beginning of genes .", "However , rather than moving along each gene and transcribing it , the polymerase then moves in the opposite ( or ‘antisense’ ) direction to transcribe the DNA that is upstream of the gene .", "In most cases , however , the transcription of these ‘upstream antisense RNAs’ does not make a functional RNA molecule and transcription is paused .", "Furthermore , Descostes , Heidemann et al . found that when RNA Polymerase II that is not tagged with these phosphate groups is degraded in human cells , these cells rapidly die .", "Descostes , Heidemann et al . also found that RNA Polymerase II with phosphate tags on tyrosine-1 also binds to , and transcribes , sections of DNA called ‘enhancers’ , which are outside of the genes but that help to activate nearby genes .", "Importantly , these transcribed enhancers are those that work to define the type of cell and tissue—for example a white blood cell—that any given cell will become .", "Future studies should help to answer remaining questions such as: how do these chemical tags affect the transcription of genes that are specific to certain tissue types ?", "And do these tags on RNA Polymerase II help to direct cells to become specific cell types ?" ]

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

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

[ [ "Interactions between marine phytoplankton and bacteria constitute an important ecological linkage in the oceans ( Cole , 1982 ) , controlling chemical cycling and energy transfer to higher trophic levels ( Azam and Malfatti , 2007; Falkowski et al . , 2008 ) .", "The cycling of sulfur , an essential element for living organisms , depends on the metabolic interactions between these two Kingdoms ( Sievert et al . , 2007 ) .", "A striking example is the production of the sulfur compound dimethylsulfoniopropionate ( DMSP ) by phytoplankton and its degradation by marine bacteria ( and phytoplankton themselves ) into the climate-active gas dimethylsulfide ( DMS ) ( Alcolombri et al . , 2015; Ayers and Gras , 1991; Howard et al . , 2006; Todd et al . , 2007 ) .", "The subsequent release of DMS into the atmosphere contributes 90% of biogenic sulfur emissions and initiates the formation and growth of aerosols , thereby enhancing cloud formation and sunlight scattering ( Ayers and Gras , 1991 ) .", "This highlights how chemical interactions occurring between marine microorganisms across micrometre-scales can ultimately have large-scale impacts on climate ( Sievert et al . , 2007; Simó , 2001 ) .", "However , direct measurements of these metabolic interactions , critical to the global sulfur cycling , have not previously been possible at the scale where they occur , the sub-cellular level .", "In the surface ocean , the largest quantities of sulfur are present as dissolved sulfate , which constitutes the main sulfur source for phytoplankton ( Sievert et al . , 2007; Stefels , 2000 ) .", "Most of the sulfur derived from sulfate uptake is converted by these organisms into sulfur-based amino acids , and a fraction is ultimately used to synthesise DMSP ( Stefels , 2000 ) ( Figure 1 ) .", "Globally , more than a billion tons of DMSP are produced every year , which has been estimated to represent up to 10% of the amount of carbon fixed by phytoplankton ( Archer et al . , 2001; Simó et al . , 2002 ) .", "However , despite the central role played by DMSP in the marine sulfur cycle , a mechanistic understanding of the biochemistry at the heart of DMSP cycling is currently lacking .", "Previous studies in higher plants provided strong evidence that DMSP biosynthesis starts in the cytosol and ends in the chloroplast ( Trossat et al . , 1996 , 1998 ) .", "However , DMSP biosynthesis occur through a different route in phytoplankton ( Stefels , 2000 ) , and we still do not know: ( 1 ) where this compound is produced and stored in phytoplankton cells; ( 2 ) what are its functions; and ( 3 ) how efficiently it is transferred from phytoplankton producers to bacterial degraders . 10 . 7554/eLife . 23008 . 003Figure 1 . DMSP biosynthetic pathway targeted in this study . Sulfate ( SO42- ) taken up from seawater by Symbiodinium is converted to sulfite ( SO32- ) , sulfur-based amino acids and finally DMSP .", "Some DMSP molecules are then exuded from Symbiodinium cells and can be degraded by a variety of marine bacteria ( sulfur atoms ( S ) and bacterial cells that have taken up sulfur are in red ) .", "The biosynthetic pathway presented here is simplified , for more details see Stefels ( Stefels , 2000 ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 23008 . 00310 . 7554/eLife . 23008 . 004Figure 1—source data 1 . ASP-8A supplement composition used for Symbiodinium cultures modified from Blank ( 1987 ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 23008 . 00410 . 7554/eLife . 23008 . 005Figure 1—figure supplement 1 . Sampling design showing the four different culture treatments . Symbiodinium cells were incubated into artificial sea water ( ASW ) containing either 34SO42- ( red , 34S-ASW ) or natSO42- ( green , natS-ASW control ) .", "In treatments ( 1–3 ) , Symbiodinium cells were incubated in 34S-ASW; after 18 days all treatments were rinsed three times with natS-ASW ( in order to remove all residual 34SO42- from the medium ) and inoculated with different bacterial strains for six hours .", "Treatment ( 1 ) was inoculated with the DMSP-degrading bacterium Pseudovibrio sp P12; treatment ( 2 ) with Escherichia coli W ( ATCC 9637 ) , a bacterium incapable of utilizing DMSP; treatment ( 3 ) acted as a negative control with no bacteria added .", "Furthermore , an additional control was used ( 4 ) , where the Symbiodinium cells were never in contact with enriched levels of 34S nor inoculated with bacteria . DOI: http://dx . doi . org/10 . 7554/eLife . 23008 . 00510 . 7554/eLife . 23008 . 006Figure 1—figure supplement 2 . Growth kinetics of Symbiodinium cells ( strain C1; mean ± SE; n = 8 ) incubated at 27°C in artificial seawater containing either 34SO42- ( red ) or natSO42- ( green ) as the sole sulfur source . The round symbols present the number of cells alive while the square symbols represent the number of dead cells ( as determined with Evans Blue stain ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 23008 . 006 We used the dinoflagellate Symbiodinium , a taxon that includes some of the most prodigious DMSP producers on the planet ( Caruana and Malin , 2014; Saltzman and Cooper , 1989 ) .", "Symbiodinium cells can be free-living in the water column , but are primarily known for the endosymbiotic associations they form with tropical cnidarians that fuel the extremely high productivity of coral reef ecosystems ( Dubinsky , 1990 ) .", "Populations of reef-building corals are major DMSP production hotspots ( Broadbent et al . , 2002; Raina et al . , 2013 ) and their contribution to the marine sulfur cycle is disproportionately large given their relatively restricted distributions ( Raina et al . , 2013; Fischer and Jones , 2012 ) .", "In this ecosystem , DMSP constitutes an important source of carbon and sulfur for the diverse and highly abundant bacterial communities harboured by corals ( Raina et al . , 2010 ) .", "Here we tracked and quantified the incorporation of a stable isotope of sulfur into Symbiodinium and its subsequent transfer to associated bacteria .", "To provide the first sub-cellular imaging and quantification of DMSP , we used a unique suite of analytical techniques , taking advantage of:", "( i ) the spatial resolution afforded by nano-scale secondary ion mass spectrometry ( NanoSIMS ) ,", "( ii ) the molecular characterization enabled by Time-of-Fight secondary ion mass spectrometry ( ToF-SIMS ) , and", "( iii ) the precise quantification allowed by nuclear magnetic resonance ( NMR ) and liquid chromatography-mass spectrometry ( LC-MS ) ." ], [ "We used the rare isotope 34S as a tracer to follow the exchange of sulfur between marine micro-organisms at the single-cell level .", "Symbiodinium cells were incubated for 18 days in artificial seawater containing 34S-labelled sulfate as the sole sulfur source ( 34S-ASW; Figure 1—figure supplement 1 ) .", "We relied exclusively on the Symbiodinium cellular machinery to biosynthesise and exude 34S-labelled DMSP following incubation with the 34S-sulfate precursor .", "To prevent direct uptake of 34S-sulfate by bacteria , all Symbiodinium cultures were rinsed thoroughly and re-inoculated into ASW containing sulfate in natural isotopic abundance ( natS-ASW ) before addition of bacterial cells .", "Two different bacterial strains were added to the rinsed cultures and co-incubated for six hours:", "( i ) Pseudovibrio sp .", "P12 , a DMSP-degrading bacterium isolated from healthy corals ( Raina et al . , 2016 ) , selected because of its worldwide distribution in coastal waters ( Shieh et al . , 2004 ) and its abundance in benthic invertebrate communities ( Bondarev et al . , 2013 ) ; and", "( ii ) a control , Escherichia coli W ( ATCC 9637 ) , a widely studied and fully sequenced strain , able to grow in seawater and not capable of degrading DMSP .", "To precisely localise bacterial cells , both strains were pre-grown in a medium enriched in the rare stable isotope 15N ( in amino-acids and ammonium form ) .", "The cellular incorporation of the stable isotope tracers ( 34S and 15N ) was identified by an increase in the sulfur ( 34S/32S ) and/or nitrogen ( 15N/14N ) ratio above their natural abundance values ( 0 . 043 and 0 . 0037 , respectively ) .", "Symbiodinium cell numbers doubled during the incubation period in the medium containing 34S-labelled sulfate , reaching approximately 2 . 9 million cells ml−1 after 18 days ( Figure 1—figure supplement 2 ) .", "LC-MS analyses carried out at the end of the experiment on extracted Symbiodinium cells confirmed that all cultures initially incubated with 34S-sulfate were highly enriched in 34S-DMSP , which represented up to 46% of the DMSP molecules present in samples analysed ( Figure 2 , Figure 2—source data 1 ) .", "This result confirms that sulfur atoms used by dinoflagellates to synthesise DMSP can originate from the uptake of inorganic sulfate derived from seawater ( Stefels , 2000 ) .", "In addition to 34S-DMSP , unexpectedly high levels of 32S-DMSP ( ranging from 54% to 66% of total DMSP ) were recorded in Symbiodinium cultures ( Figure 2—source data 1 ) .", "The presence of these high levels of 32S-DMSP can be explained by a combination of two factors:", "( i ) Symbiodinium cells density only doubled during the incubation phase in 34S-ASW , retaining a large fraction of the natural pool of 32S initially present in the starting culture prior to the incubation;", "( ii ) new 32S-DMSP might have been synthesised during the six hours immediately preceding sampling , when Symbiodinium cells were incubated in natS-ASW medium .", "Although high concentrations of DMSP were present in the methanolic Symbiodinium cells extract ( Figure 2—source data 1 ) , sulfur containing amino acids ( methionine and cysteine ) were not detected by LC-MS or 1H NMR . 10 . 7554/eLife . 23008 . 007Figure 2 . Representative HPLC-MS spectra showing the presence and relative abundance of 32S-DMSP ( green peak ) and 34S-DMSP ( red peak ) in methanol extracts derived from Symbiodinium culture ( particulate fraction ) .", "( a ) incubated with natS ( treatment 4 , see Figure 1—figure supplement 1 ) ;", "( b ) incubated with 34S ( treatment 3 , see Figure 1—figure supplement 1 ) .", "For more detailed spectra , see Figure 2—figure supplement 2; for absolute DMSP abundance , see Figure 2—source data 1 .", "( c ) Positive-ion ToF-SIMS spectrum of Symbiodinium incubated with 34S ( treatment 3 , see Figure 1—figure supplement 1 ) after resin embedding ( 34S-DMSP represented 46% of total DMSP counts ) .", "For comparison between treatment and control spectra , see Figure 2—figure supplement 1;", "( d ) Negative-ion ToF-SIMS images showing the distribution of CN- , HS- and 34S- species over a Symbiodinium cell ( treatment 3 , see Figure 1—figure supplement 1 ) enriched in 34S .", "Field of view is 20 × 20 μm2 ( lateral resolution is ~300 nm ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 23008 . 00710 . 7554/eLife . 23008 . 008Figure 2—source data 1 . DMSP in methanol extracts derived from the four different Symbiodinium culture treatments ( particulate fraction ) , as measured by quantitative NMR ( n = 3 biological replicates for cultures inoculated with Pseudovibrio sp . ) and HPLC-MS ( 32S-DMSP and 34S-DMSP fractions , n = 3 ) .", "Note , when the samples were collected , the Symbiodinium densities were not significantly different between the different treatments ( T-Test , n = 8 , t = 0 . 589 , p=0 . 565 ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 23008 . 00810 . 7554/eLife . 23008 . 009Figure 2—figure supplement 1 . Representative positive-ion spectra of", "( a ) Araldite 502 resin , and Symbiodinium", "( b ) incubated with natS ( treatment 4 ) and", "( c ) incubated with 34S ( treatment 3 ) after resin embedding . Spectra in", "( b ) and", "( c ) were arbitrarily scaled such that the 32S-DMSP peaks have similar intensities .", "The areas under the peaks of 34S-DMSP ( red ) normalised to that of 32S-DMSP ( green ) are 0 . 26 and 0 . 91 in", "( b ) and", "( c ) , respectively . DOI: http://dx . doi . org/10 . 7554/eLife . 23008 . 00910 . 7554/eLife . 23008 . 010Figure 2—figure supplement 2 . Representative HPLC-MS spectra showing the presence and relative abundance of 32S-DMSP ( mass 135 . 04 ) and 34S-DMSP ( mass 137 . 04 ) in methanol extracts:", "( a ) DMSP standard containing natural abundance of 34S-DMSP;", "( b ) Symbiodinium cells incubated with natS ( treatment 4 ) ;", "( c ) Symbiodinium cells incubated with 34S ( treatment 3 ) .", "For each spectrum , the number on the right hand side refer to:", "( i ) TOF MS ES+ time-of-flight mass spectrometer and electrospray ionisation positive mode;", "( ii ) 137 . 04 or 135 . 04 being the mass of the ion investigated ( with a range of ±0 . 3 Da ) ;", "( iii ) the ion count detected for the ion investigated .", "Note: the slight difference in retention time between ( a , b and", "c ) would be an effect of temperature change in the laboratory . DOI: http://dx . doi . org/10 . 7554/eLife . 23008 . 010 Up to 10% of the carbon fixed by photosynthetic algae is used for the production of DMSP ( Sievert et al . , 2007; Archer et al . , 2001; Simó et al . , 2002 ) , which represents a major energy investment for these organisms and strongly suggests that this compound plays a central function in algal cells .", "To understand more precisely the functional role of DMSP , we used two SIMS approaches to infer its location within cells .", "To effectively prevent the loss of DMSP from the cells , the entire sampling procedure leading to SIMS analyses had to be water-free , with all steps performed under strict anhydrous conditions .", "For this , we used cryopreservation techniques followed by freeze substitution in an acrolein-ether mixture .", "This method has routinely been used to successfully preserve cellular ions and compounds in a variety of systems ( Altus and Canny , 1985; Ashford et al . , 1999; Kaiser et al . , 2015; Marshall et al . , 2007; Mostaert et al . , 1996 ) , with the acrolein stabilizing and preserving cellular proteins , nucleic and fatty acids through cross linking , while the low temperature , anhydrous conditions ensure preservation and retention of diffusible ions and water-soluble molecules ( such as DMSP ) .", "The inclusion of acrolein ensures excellent cell structural preservation at a low temperature , which is required for high resolution NanoSIMS analyses ( Kaiser et al . , 2015; Marshall , 1980 ) .", "ToF-SIMS revealed that 34S-DMSP was present and abundant in the preserved cells following resin embedding , with a ratio of 34S-DMSP/32S-DMSP matching the bulk analyses carried out with LC-MS prior to embedding ( Figure 2c–d , Figure 2—figure supplement 2 ) .", "NanoSIMS analysis revealed that Symbiodinium exposed to 34S-labelled sulfate were nine times more enriched in 34S than the cells in the control ( 34S/32S ratio in 34S-ASW treatments: 0 . 391 ± 0 . 046 , compared to natS-ASW controls 0 . 044 ± 0 . 001 [Figure 4—figure supplement 1] ) .", "Furthermore , substantial spatial variability in 34S enrichment was detected within Symbiodinium cells .", "Relatively low level of enrichments were detected in the nucleus ( 34S/32S: 0 . 087 ± 0 . 004 ) which might correspond to the presence of 34S-labelled amino-acids in the histone-like proteins that condense Symbiodinium DNA into chromosomes ( Shoguchi et al . , 2013 ) ( Figure 3 ) .", "Much higher enrichment levels were detected in vacuoles ( 34S/32S: 0 . 337 ± 0 . 011 ) , chloroplasts ( 34S/32S: 0 . 384 ± 0 . 020 ) and cytoplasm ( 34S/32S: 0 . 451 ± 0 . 025 ) ; which means that the enrichment in these cellular structures was 7 . 7 , 8 . 8 and 10 . 3 times over the natural abundance levels ( Figure 3 ) .", "However , the largest 34S enrichment was observed in small hotspots often observed near the Symbiodinium cell periphery ( 34S/32S: 0 . 971 ± 0 . 059; Figure 3 ) , reaching more than 22 times the natural abundance level .", "Based on their small size and their high 34S enrichment , these hotpots are likely storage droplets containing sulfolipids , a group of sulfur compounds known to accumulate in Symbiodinium ( Garrett et al . , 2013; Yuyama et al . , 2016 ) .", "Lipid droplets of similar sizes and locations can be observed in these cells using electron microscopy ( Figure 3—figure supplement 1 ) .", "We were not able to detect methionine or cysteine using LC-MS or ToF-SIMS , which suggest that the intracellular concentration of these sulfur based amino-acids was relatively low .", "In contrast , DMSP is known to be by far the most abundant organic sulfur compound present in dinoflagellate cells ( Matrai and Keller , 1994 ) , representing more than 50% of the total organic sulfur in these organisms ( Matrai and Keller , 1994 ) .", "DMSP was the only organic sulfur compound we were able to detect in the Symbiodinium cells ( through LC-MS , 1H NMR and ToF-SIMS ) , suggesting that most of the remaining 34S signal measured in Symbiodinium cells with NanoSIMS is highly likely originating from DMSP . 10 . 7554/eLife . 23008 . 011Figure 3 . Representative NanoSIMS ion images of Symbiodinium cells showing the sub-cellular distribution of 34S .", "( a and", "b ) 12C14N/12C2 mass images showing cellular structures .", "( c and", "d ) 34S/32S ratio images of the same cells , shown as Hue Saturation Intensity ( HSI ) images where the colour scale indicates the value of the 34S/32S ratio , with natural abundance in blue , changing to pink with increasing 34S levels .", "( e ) Isotope ratio of 34S/32S in different cellular regions ( nucleus n = 10; vacuole n = 3; chloroplast n = 35; cytoplasm n = 12; hotspot n = 20; error bar: SE; source data available: Figure 3—source data 1 ) .", "The dashed blue line represents the natural 34S abundance recorded in the control samples .", "nu: nucleus; ch: chloroplast; py: pyrenoid; ua: uric acid storage; v: vacuole; cy: cytoplasm; li: sulfolipids .", "Scale bars: 1 µm . DOI: http://dx . doi . org/10 . 7554/eLife . 23008 . 01110 . 7554/eLife . 23008 . 012Figure 3—source data 1 . 32S and 34S measured in the different cellular region depicted in Figure 3e . DOI: http://dx . doi . org/10 . 7554/eLife . 23008 . 01210 . 7554/eLife . 23008 . 013Figure 3—figure supplement 1 . Representative electron micrographs of Symbiodinium cells after OsO4 staining showing the position and size of intracellular lipid droplets . nu: nucleus; py: pyrenoid; ua: uric acid storage; li: lipids .", "Scale bars: 1 µm . DOI: http://dx . doi . org/10 . 7554/eLife . 23008 . 013 DMSP is an effective scavenger of reactive oxygen species ( ROS ) , particularly hydroxyl radicals ( •OH ) ( Sunda et al . , 2002 ) .", "The in vivo half-life of •OH is 10−9 seconds ( Sies , 1993 ) , which implies that these highly reactive molecules can damage lipids , nucleic acids , amino-acids or carbohydrates present in their direct vicinity .", "To be an effective antioxidant , a molecule needs not only to be able to scavenge ROS , but also to be located close to their source .", "Although the capacity of DMSP to detoxify ROS is established ( Sunda et al . , 2002 ) , it has not been previously possible to ascertain its specific cellular function because its location is still unknown .", "If some DMSP is located in the cytoplasm , as suggested by our NanoSIMS data , it will be ideally localised to act as an osmolyte ( Kiene et al . , 1996 ) .", "Furthermore , the presence of strong 34S signals in and around chloroplasts , where ROS are formed , support its role as an antioxidant ( Sunda et al . , 2002 ) .", "Following synthesis by phytoplankton , DMSP constitutes an important carbon and sulfur source for heterotrophic marine bacteria , which can either demethylate the compound and incorporate its sulfur into proteins or cleave it to produce DMS ( Curson et al . , 2011 ) .", "At the termination of the experiment , total DMSP concentrations in Symbiodinium cells inoculated with the DMSP-degrading bacterium Pseudovibrio sp .", "P12 were 31% lower relative to those containing no bacteria or bacteria unable to degrade DMSP ( Figure 2—source data 1 ) .", "As Symbiodinium abundance did not differ between the treatments ( Figure 1—figure supplement 2 ) , the lower DMSP concentrations recorded are likely a consequence of the presence of Pseudovibrio cells able to degrade this compound .", "We sequenced the genome of Pseudovibrio sp .", "P12 , revealing that this bacterium harbours a complete DMSP cleavage pathway , including a DMSP acyl-CoA transferase ( encoded by dddD ) , a DMSP transporter ( dddT ) and the downstream catabolic enzymes ( dddB-C ) ( Todd et al . , 2007; Raina et al . , 2016 ) .", "Further analyses using NMR revealed that this DMSP degradation pathway was functional , enabling this strain to convert high concentrations of DMSP into DMS ( Raina et al . , 2016 ) .", "In addition , Pseudovibrio sp .", "P12 harbours homologues of genes involved in the demethylation pathway ( dmdA-B-C-D ) , though these genes have a relatively low sequence identity ( 24% , 30% , 43% and 32% , respectively ) ( Raina et al . , 2016 ) to the genes originally identified in Ruegeria pomeroyi DSS-3 ( Reisch et al . , 2011 ) .", "Bacteria-sized 15N hotspots localised outside Symbiodinium cells in NanoSIMS images were accurately identified as inoculated bacterial cells based on their unique nitrogen isotopic signatures ( 1151-fold increase on average over natural abundance , n = 79 , Figure 4—figure supplement 1 ) .", "Notably , within the Pseudovibrio treatment , the position of these 15N hotspots correlated exactly with 34S hotspots ( Figure 4 ) , which were characterised by a 3 . 3-fold increase in the 34S/32S ratio over natural abundance ( n = 60 , Figure 4h ) .", "These observations confirmed that Pseudovibrio cells assimilated 34S-labelled Symbiodinium-derived metabolites .", "A 34% increase was also recorded in the mean 34S/32S ratio of E . coli cells ( 0 . 058 ± 0 . 002; n = 19 ) , which are unable to degrade DMSP ( compared to controls: 0 . 0438 , Figure 4h ) .", "This enrichment , significantly higher than the expected natural abundance levels ( t-Test , n = 19 , t = 9 . 227 , *p<0 . 001 ) , can be explained by:", "( i ) the capacity of E . coli to uptake small quantities of DMSP through betaine transporters to use as an osmoprotectant ( Cosquer et al . , 1999 ) ;", "( ii ) the exudation of small quantities of other sulfur-containing substrates by Symbiodinium , such as methionine , which occur at a ratio of 8 . 2 ± 2 . 6 per 1000 amino acid residues in these dinoflagellates ( Markell and Trench , 1993 ) .", "In contrast , the high 34S enrichment recorded in Pseudovibrio cells , together with the significant decrease of particulate DMSP recorded in Pseudovibrio-inoculated treatments ( Figure 4i ) , are likely due to the incorporation and degradation of DMSP .", "A comparison of 34S uptake between the two bacterial strains further highlights differences in their capacity to metabolise DMSP; Pseudovibrio incorporated seven times more sulfur than E . coli during the six-hours incubation ( Pseudovibrio: specific uptake of 6 . 4 ± 0 . 3 ng S mg−1 of dry weight , n = 60; E . coli: 0 . 9 ± 0 . 1 ng S mg−1 of dry weight , n = 19 ) .", "However , enzymatic cleavage of 34S-DMSP into volatile 34S-DMS , which diffuses out of Pseudovibrio cells and is therefore not captured by our NanoSIMS measurements , are likely to have caused an underestimation of the amount of sulfur cycled by this bacterium . 10 . 7554/eLife . 23008 . 014Figure 4 . Representative NanoSIMS ion images of Symbiodinium cells exposed to 34S- or natS-artificial seawater ( ASW ) for 18 days and subsequently inoculated with two different bacterial strains for six hours .", "( a ) Timeline of the experiment .", "( b , c and", "d ) 12C15N/12C14N mass images showing the presence of 15N enriched bacterial cells .", "( e , f and", "g ) 34S/32S ratio image of the same regions .", "These mass images are shown as HSI images where the colour scale indicates the value of the stable isotope ratios , with natural abundance in blue , changing to pink with increasing 15N or 34S levels .", "( b , c , e and", "f ) Symbiodinium cultures were inoculated with the DMSP-degrading bacterium Pseudovibrio sp .", "P12 ( treatment 1 ) .", "( d and", "g ) Symbiodinium cultures were inoculated with Escherichia coli ( treatment 2 ) .", "White arrows indicate bacteria .", "( h ) Isotope ratio of 34S/32S in bacteria , Pseudovibrio cells were significantly more enriched than E . coli ( t-Test , n = 60 , t = 9 . 021 , *p<0 . 001 , error bars: SE ) .", "The dashed blue line represents the natural 34S abundance recorded in the control samples .", "( i ) Total particulate DMSP concentration in Symbiodinium inoculated with Pseudovibrio sp .", "or E . coli ( t-Test , n = 3 , t = 9 . 908 , *p<0 . 001 , error bar: SE ) .", "Source data available: Figure 4—source data 1 .", "Note: two regions of interest were merged to create Figure 4c due to stage-shifting errors during sequential acquisition of N and S data .", "Scale bars = 3 µm . DOI: http://dx . doi . org/10 . 7554/eLife . 23008 . 01410 . 7554/eLife . 23008 . 015Figure 4—source data 1 . 12C15N , 12C14N , 32S and 34S measured in the different organisms and treatments depicted in Figure 4h and Figure 4—figure supplement 1 . DOI: http://dx . doi . org/10 . 7554/eLife . 23008 . 01510 . 7554/eLife . 23008 . 016Figure 4—figure supplement 1 . Isotope ratio of", "( a ) 15N/14N and", "( b ) 34S/32S in Symbiodinium and bacteria cells measured by NanoSIMS in the different treatments ( values were extracted from the images ) .", "The dashed lines represent the natural 15N and 34S abundance measured in the controls ( 0 . 0037 and 0 . 0438 , respectively ) .", "15N/14N of the inoculated bacterial cells was 4 . 2243 ± 0 . 1886 on average , compared to 0 . 0037 ± 8 . 29E-06 recorded in the controls .", "Source data available: Figure 4—source data 1 .", "Note: only inoculated bacteria were incubated in 15N .", "Error bars = SE . DOI: http://dx . doi . org/10 . 7554/eLife . 23008 . 016 The marine sulfur cycle is a fundamental driver of atmospheric chemistry and climatic processes , yet its global influence is the product of unquantified cellular interactions between microorganisms .", "Here we used two SIMS approaches to directly visualise the accumulation and subsequent transfer of DMSP between marine microalgae and bacteria with unprecedented sub-cellular resolution .", "We applied a method that enables the preservation of water-soluble compounds , such as DMSP , in samples .", "This procedure , applicable to any system , may serve as a template to study the sub-cellular localization and identification of other small and highly diffusible molecules .", "In addition , similarly to other recent stable isotope approaches ( Stefels et al . , 2009 ) , our method may be used to quantify the production rate of DMSP at the single cell level .", "We confirmed that 34S-DMSP was the main organic sulfur compound within the algal cells and we subsequently localised large quantities of the sulfur tracer 34S in algal vacuole , cytoplasm and chloroplasts .", "This strongly indicates that the relative concentrations of DMSP are higher in these key cellular locations , providing corroborative evidence for its functional role in mitigating both osmotic and oxidative stresses .", "Taken together , we have demonstrated that it is possible to image and quantify DMSP in phytoplankton and their associated bacteria at the sub-cellular scale .", "These methods open the way to further studies resolving the role of DMSP in phytoplankton and its contribution to phytoplankton-bacteria interactions ." ], [ "Cells of Symbiodinium type C1 ( confirmed by sequencing of the ITS1 gene ) used in this study were isolated from air-brushed tissues of the coral Acropora tenuis , which had been collected from Magnetic Island , Great Barrier Reef , Australia ( latitude 19°10’S; longitude 146°50’E ) .", "Cells were sequentially washed three times ( 5 min at 1600 g ) with 0 . 2 µm filtered seawater .", "Clean Symbiodinium cells were inoculated into 24 well plates with sterile IMK medium ( Wako Chemicals , Richmond , VA , USA ) with the antibiotics penicillin ( 100 μg ml−1 ) , neomycin ( 100 μg ml−1 ) , streptomycin ( 100 μg ml−1 ) , nystatin ( 100 μg ml−1 ) , amphotericin ( 2 . 5 μg ml−1 ) , and Germanium dioxide ( 50 μM ) ) for 15 days at 27°C , 50 µE and 14:10 light:dark cycle .", "After this initial incubation , cells from uncontaminated wells were pooled and re-inoculated in new 24-well plates with IMK medium plus antibiotics as above , and incubated for 20 days at the same temperature and lighting conditions .", "Finally , uncontaminated cells were pooled and inoculated into 25 mL of sterile IMK without antibiotics until the start of the experiment ( Santos et al . , 2011 ) .", "Cultures were genotyped by single-strand conformation polymorphism ( SSCP ) of the ITS1 region ( van Oppen et al . , 2001 ) .", "A coral-associated bacterium , Pseudovibrio sp .", "P12 , was isolated from healthy colonies of the reef-building coral Pocillopora damicornis .", "This bacterial strain is commonly associated with reef-building corals ( Bondarev et al . , 2013; Nissimov et al . , 2009; Radjasa et al . , 2008; Ritchie , 2006; Rypien et al . , 2010; Sulistiyani et al . , 2010 ) and capable of metabolizing DMSP as a sole carbon source ( Garren et al . , 2014 ) .", "Coral colonies were collected from Davies Reef , Great Barrier Reef , Australia ( latitude 18°51’S; longitude 147°41’E ) and maintained in aquaria at the Australian Institute of Marine Science ( Townsville , Queensland , Australia ) prior to strain isolation .", "A dilution series of coral tissue slurries was inoculated on minimal marine agar plates ( 1% bacteriological agar; 0 . 3% casamino acids; 0 . 4% glucose; in 1 litre of artificial seawater ) ( Hjelm et al . , 2004 ) .", "After 2 days of incubation at 28°C , single colonies were transferred into Marine Broth ( Difco ) and grown overnight .", "Liquid cultures were re-plated on minimal marine agar and the procedure was repeated iteratively until pure cultures were obtained .", "A laboratory strain of Escherichia coli ( E .", "coli W ( ATCC 9637 ) ) was chosen as a control strain based on its ability to grow in the artificial seawater used in this study ( see medium composition below ) , and its lack of DMSP degradation and subsequent sulfur assimilation pathways ( unlike many marine or coral bacterial isolates [Raina et al . , 2010; Howard et al . , 2008] ) .", "High molecular weight DNA from a pure culture of the Pseudovibrio sp .", "P12 strain was obtained using a miniprep phenol/chloroform based DNA extraction ( Ausubel et al . , 1987 ) .", "A paired-end library was prepared using the Illumina Truseq protocol ( Illimina , San Diego , CA , USA ) , with an insert size of 169 bp and a read size of 150 bp .", "The library was sequenced on an Illumina MiSeq instrument at Monash University ( Melbourne , Australia ) .", "The genome was assembled with the SPAdes assembler ( v2 . 4 . 0 ) ( Bankevich et al . , 2012 ) and annotated with the Prokka software ( v1 . 5 . 2 ) ( Seemann , 2014 ) , providing a draft genome assembly of Pseudovibrio sp .", "P12 .", "The presence of the genes involved in DMSP metabolism was investigated by searching for homologs of the corresponding genes using reciprocal best BLAST hits .", "Magnesium sulfate ( Mg34SO4 ) was synthesised from pure sulfur 34S ( purity >99% , Cambridge Isotope , MA ) following a two-step reaction: Elemental sulfur 34S ( 0 . 1069 g ) was ground into a fine powder and transferred to a pear-shaped flask .", "Nitric acid ( 65% , 4 ml ) was added to the flask , heated to 80°C and refluxed for 5 hr .", "The temperature was subsequently raised to 130°C and refluxed for an additional 24 hr in order to completely oxidise remaining nitric acid .", "The resulting sulfuric acid ( H234SO4 ) was then converted to Mg34SO4 by the addition of magnesium carbonate ( MgCO3 ) ( 0 . 2643 g ) , giving a yield of 0 . 3780 g .", "The solution was subsequently heated to 100°C until all water had completely evaporated .", "Elemental analysis of the dried crystals was carried out with an electron probe microanalyser ( EPMA , Jeol JXA8200 ) , equipped with an energy dispersive spectrometer ( EDS ) , to confirm the synthesis of Mg34SO4 .", "Symbiodinium C1 cells were inoculated into artificial seawater ( ASW; starting density: 1 . 5 × 106 cells ml−1 ) and incubated at 27°C for 18 days ( based on results from a pilot study ) .", "LED lights were mounted above the culture , providing an average light intensity of 50 μE over a 14:10 hr light/dark cycle ( AI Super Blue LED module 1003 , IA , USA ) .", "Temperature and light intensities were monitored every 2 min for the duration of the experiment ( using a HOBO UA-002-64 , 64K temperature/light data logger ) .", "The ASW contained 24 . 72 g of NaCl , 0 . 67 g of KCl , 1 . 36 g of CaCl2·2H2O , 4 . 66 g of MgCl2·6H2O , 0 . 18 g of NaHCO3 , and 3 . 8 ml of modified ASP-8A solution ( Figure 1—source data 1 ) in 1 litre of MilliQ water .", "Magnesium sulfate ( MgSO4·7H2O , 6 . 29 g L−1 ) was used as the sole sulfur source , with either 34S ( 99% 34S , hereafter called 34S-ASW ) or natural abundance of sulfur ( 95% 32S , 0 . 7% 33S , 4 . 2% 34S; hereafter called natS-ASW ) .", "Symbiodinium cells were incubated in 34S-ASW , whereas a batch incubated only in natS-ASW acted as a control .", "Both growth media were replaced every 5 days in order to actively remove dead and floating cells from the cultures .", "Symbiodinium cell numbers were monitored every 3 days for both 34S-ASW and natS-ASW treatments , using a light microscope and haemocytometer ( depth 0 . 1 mm; eight replicates were averaged per time point ) and cell mortality assessed using a 0 . 05% ( w/v ) Evans Blue solution ( Morera and Villanueva , 2009 ) .", "After 18 days , the medium in both 34S-ASW and natS-ASW Symbiodinium cultures , was decanted and discarded .", "The Symbiodinium cells were thoroughly rinsed three times with natS-ASW and subsequently resuspended in natS-ASW ( 5 mins ) prior to the addition of bacteria ( Figure 1—figure supplement 1 ) .", "This medium exchange ( from 34S-ASW to natS-ASW ) was carried out in order to prevent any potential direct bacterial uptake of 34SO42- .", "The two bacterial strains ( Pseudovibrio sp . P12 and E . coli W ) were grown overnight at 28°C in ASW medium enriched with 15N ( in the form of amino-acids and NH4+; Celtone Base Powder; Cambridge Isotope Laboratories , Tewksburry , MA ) .", "The bacterial cells were subsequently washed three times in ASW before inoculation .", "Symbiodinium cells in treatment 1 were subsequently inoculated with the DMSP-degrading bacterium Pseudovibrio sp .", "P12; treatment 2 with E . coli; treatment 3 acted as a control without bacteria added; and treatment 4 , which had no contact with 34S , acted as negative control for sulfur isotope incorporation ( Figure 1—figure supplement 1 ) .", "The two bacterial strains were inoculated at a density of 106 cells ml−1 and all samples were collected six hours after bacterial inoculation ( based on results from a pilot study ) .", "We used high-pressure freezing ( Smart et al . , 2010 ) , followed by a water-free embedding procedure to effectively prevent the loss of highly soluble compounds such as DMSP from our samples .", "This method does retain elements in solution ( Altus and Canny , 1985; Ashford et al . , 1999; Kaiser et al . , 2015; Marshall et al . , 2007; Mostaert et al . , 1996 ) by effectively replacing the ‘solution’ with resin , without displacing the ions and osmolytes .", "Symbiodinium cultures pre-incubated with bacteria ( 20 μl ) were dropped onto Thermanox strips ( Thermo Fisher Scientific , Waltham , MA , USA , 4 × 18 mm ) and then placed in humidified chambers .", "After 15 min , the cells settled onto the strips and the excess medium was carefully removed with filter paper before being frozen by immersion into liquid nitrogen slush ( liquid nitrogen placed under low-vacuum in order to lower its temperature ) .", "Samples for structural imaging by electron microscopy ( 2 µl ) were also collected .", "These were deposited in a gold planchet and high-pressure frozen using an EMPACT2 high-pressure freezer ( Leica Microsystems , Wetzlar , Germany ) .", "Both sample types were stored in liquid nitrogen until required .", "Frozen samples for NanoSIMS were freeze-substituted in anhydrous 10% acrolein in diethyl ether , and warmed progressively to room temperature over three weeks in an EM AFS2 automatic freeze-substitution unit ( Leica Microsystems , Wetzlar , Germany ) based upon the original method of Marshall ( Marshall , 1980 ) , and as described recently in step-by-step detail by Kilburn and Clode ( Kilburn and Clode , 2014 ) .", "The samples were subsequently infiltrated and embedded in anhydrous Araldite 502 resin , after which the Thermanox strip was removed and the sample re-embedded and stored in a desiccator .", "Although it is possible that not 100% of cellular DMSP may be preserved by this procedure , any losses would affect all samples equally; not impacting the validity of our comparisons between treatments .", "Furthermore , as 15N was only used as a tag to visualise the bacteria , dilution by processing and resin embedding ( Musat et al . , 2014 ) is of no concern here .", "For 34S analyses , dilution can be expected to be negligible as there is no sulfur contained in processing or resin components .", "Resin sections ( 1 µm thick ) of embedded Symbiodinium cells were cut dry using a Diatome-Histo diamond knife on an EM UC6 Ultramicrotome ( Leica Microsystems , Wetzlar , Germany ) , mounted on a silicon wafer and coated with 5 nm of gold .", "The NanoSIMS-50 ( Cameca , Gennevilliers , France ) at the Centre for Microscopy , Characterisation and Analysis ( CMCA ) at The University of Western Australia was used for all subsequent analyses .", "The NanoSIMS-50 allows simultaneous collection and counting of multiple isotopic species , which enables the determination of 15N/14N and 34S/32S ratios .", "Enrichments of the rare isotopes 34S and 15N were confirmed by an increase in the sulfur ( 34S/32S ) and/or nitrogen ( 15N/14N ) ratio above natural abundance values recorded in controls ( equal to 0 . 0438 and 0 . 00367 , respectively ) .", "NanoSIMS analysis was undertaken by rastering a 2 pA Cs+ beam ( ~100 nm diameter ) across defined 20 μm2 sample areas ( 256 × 256 pixels ) .", "The NanoSIMS-50 was tuned to achieve mass resolution at levels where the isobaric species 12C15N and 13C14N could be separated .", "The isotope ratio values are represented hereafter using a colour-coded transform ( hue saturation intensity ( HSI ) ) showing natural abundance levels in blue , and grading to high enrichment in pink .", "Images were processed and analysed using Fiji ( http://fiji . sc/Fiji ) ( Schindelin et al . , 2012 ) with the Open-MIMS plug-in ( http://nrims . harvard . edu/software ) .", "All images were dead-time corrected ( Hillion et al . , 2008 ) .", "Quantitative data were extracted from the mass images through manually drawn regions of interest .", "Ratio data were tested for QSA ( quasi-simultaneous arrivals ) by applying different beta values from 0 . 5 to 162 .", "No differences in the data were observed , indicating that the secondary ion count rates were too low to be affected by QSA .", "During ToF-SIMS analysis the sample surface is sputtered with a focused primary ion beam to produce ionic species ( secondary ions ) of the atoms , molecules and molecular fragments from the uppermost monolayers of the surface .", "The secondary ions are extracted into a flight column ( time-of-flight analyser ) and their masses determined by the exact time at which they arrive at the detector .", "The data collected can provide:", "( i ) mass spectral information in the form of an accumulated mass spectrum , and", "( ii ) image information in the XY dimensions showing the intensity distribution of the specific secondary ions from the area analysed .", "The mass resolution of the ToF-SIMS analysis is determined by the temporal pulse width of the primary ions hitting the sample surface; whereas the spatial resolution is determined by the spot size of the primary ion beam .", "ToF-SIMS analyses are conducted with the instruments optimised either for high mass resolution or for high spatial resolution , as achieving both short pulses ( for mass resolution ) and narrow focus ( for spatial resolution ) simultaneously will greatly reduce the primary ion current density .", "In this study , ToF-SIMS analyses were conducted using the TOF . SIMS five instrument ( ION-TOF GmbH , Münster , Germany ) at the Mark Wainwright Analytical Centre ( MWAC ) , University of New South Wales .", "The instrument is equipped with a bismuth liquid metal cluster ion gun for analysis and an electron flood gun for charge compensation .", "Analysis was performed using a 30 keV Bi3+ cluster ion beam on resin sections ( 1 µm thick ) mounted on silicon wafers .", "The ‘spectrometry’ mode was used to acquire high-mass resolution spectra ( m/△m > 4000 ) and ‘fast-imaging’ mode was used to acquire high spatial resolution images ( lateral resolution ~300 nm , m/Δm ~ 200 ) .", "In a typical analysis , a positive ion spectrum was acquired over a defined area of 20 × 20 μm2 or 50 × 50 μm2 .", "The area of interest was identified by negative ion images acquired over areas of 20 × 20 μm2 ( 64 × 64 pixels ) to 200 × 200 μm2 ( 128 × 128 pixels ) , where maps of CN- ( m/z 26 ) , S- ( m/z 32 ) , HS- ( m/z 33 ) and 34S- ( m/z 34 ) were generated to locate the position of cells and the presence of sulfur-containing compounds within the sample .", "Care was taken to ensure the ion dose density was kept below the static SIMS limit ( 1012–1013 primary ions per cm2 ) ( Lindgren et al . , 2014 ) when acquiring imaging data , e . g . no more than 5–10 scans over areas of 20 × 20 μm2 .", "Keeping the static limit in the imaging mode prevents any significant damage to the sample structure or chemistry ( Vickerman and Briggs , 2013 ) , and enables further analyses of the same area in the positive polarity in this case .", "In the positive spectrum , the molecular ion [M + H]+ peak of both the 32S-and 34S-containing DMSP molecules ( C5H1132SO2+ and C5H1134SO2+ , respectively ) are closely spaced with peaks arise from the resin ( Figure 2—figure supplement 2 ) .", "To maximise signal-to-noise ratio , data acquisition over a relatively small area encompassing the cell was desired , allowing unambiguous identification of the C5H1132SO2+ and C5H1134SO2+ peaks .", "High mass resolution positive spectra were calibrated using the masses of CH2+ , C2H4+ , C4H8+ and C6H12+ molecules .", "Data processing and evaluation were conducted using the SurfaceLab six software package ( ION-TOF GmbH , Münster , Germany ) .", "Prior to the analyses of the resin sections , the mass spectrum of dimethyl-β-propiothetin standard ( Research Plus Inc . , USA ) was recorded to provide spectral information of DMSP generated by ToF-SIMS analysis .", "The molecular ion [M + H]+ peak ( C5H11SO2+ , m/z 135 . 05 ) was observed to be the most intense peak in the spectrum , and was used as the mass peak position when determining the presence of DMSP molecules in the samples .", "The mass spectrum of a mixture of methionine and cysteine ( Sigma-Aldrich , USA ) was also acquired to serve as a reference standard .", "Both methionine and cysteine were not detected or the amounts were below the detection limit of the instrument ( ppm range ) .", "High-pressure frozen samples for structural imaging were freeze-substituted in 1% OsO4 in acetone over two days and similarly infiltrated and embedded as described above .", "Sections 90 nm thick were cut on water using a diamond knife , collected on copper grids and imaged unstained at 120 kV in a JEOL 2100 TEM ( Tokyo , Japan ) fitted with a Gatan ORIUS camera ( California , USA ) .", "Please note: the high solubility of DMSP in water prevented the coupling of NanoSIMS with TEM images ( Clode et al . , 2009 ) to identify the location of small organelles such as mitochondria , as ultrathin sections cannot be prepared without exposing the samples to water .", "After samples were collected for NanoSIMS analysis , all Symbiodinium cultures were centrifuged ( 3000 g ) for 5 min , the medium was discarded and pelleted cells were extracted with 5 mL of HPLC-grade methanol .", "Crude methanol extracts were then analysed by reverse-phase ( RP18 ) HPLC-MS in triplicate along with pure DMSP and amino acid standards .", "A 10 µL aliquot of the methanol extract was diluted with an equal volume of acetonitrile and chromatographed using a Waters Alliance 2695 HPLC system comprising a quaternary pump , autosampler and photodiode array detector ( 200–400 nm ) coupled to a Waters Micromass LCT Premier orthogonal acceleration time-of-flight ( oa-TOF ) mass spectrometer .", "Separation was achieved on an Alltima HP HILIC column ( 250 × 4 . 6 mm with a particle size of 5 µm ) at 27°C and a flow rate of 0 . 75 ml min-1 .", "The gradient was: acetonitrile ( 90% ) :0 . 1% formic acid ( 10% ) at 0 min; acetonitrile ( 60% ) :0 . 1% formic acid ( 40% ) at 0 . 4 min; acetonitrile ( 10% ) :0 . 1% formic acid ( 90% ) at 12 min; acetonitrile ( 90% ) :0 . 1% formic acid ( 10% ) at 12 . 25 min .", "TOF-MS accurate mass measurements ( scan-range m/z 100–1000 at 4 GHz , resolution = 9500 ) were acquired using an electrospray ionization ( ESI ) source in W positive mode with the following operation parameters: capillary voltage: 3000 V; cone voltage: 80V; ion source temperature: 80°C; desolvation temperature: 350°C; cone gas flow: 10 l hr−1; desolvation gas flow: 750 l hr−1; ion energy: 33 V; acceleration voltage: 100 V . MassLynx software ( version 4 . 1 , Waters ) was used for operating the HPLC-MS , as well as for data acquisition and processing .", "Leucine Enkephalin was used as the external reference .", "The MeOH extracts remaining after HPLC-MS analysis was dried using a vacuum-centrifuge and dissolved in a mixture of deuterium oxide ( D2O , D 99 . 8% , 250 μl ) and deuterated methanol ( CD3OD , D 99 . 8% , 750 μl ) ( Cambridge Isotope Laboratories , Andover , MA , USA ) .", "A 700 µl aliquot of the particulate-free extract was transferred into a 5 mm Norell 509-UP-7 NMR tube ( Norell Inc . , Landisville , NJ , USA ) and analysed immediately by 1H NMR .", "1H NMR spectra were recorded on a Bruker Avance 600 MHz NMR spectrometer with TXI 5 mm probe and quantification performed using the ERETIC method ( Tapiolas et al . , 2013 ) .", "This technique generates an internal electronic reference signal , calibrated using stock solutions of DMSP .", "Bacterial strains and Symbiodinium were counted ( Becton Dickinson LSR II flow cytometer ( BD Biosciences , Franklin Lakes , NJ , USA ) , and pellets were subsequently freeze-dried and weighed in order to determine their total sulfur content ( equal to 5390 ng S mg−1 ) .", "Samples were analysed on a Thermo Scientific FLASH 2000 Series ( Thermo Scientific , Waltham , MA , USA ) .", "The sulfur uptake per mg of bacterial cells ( ρ ) was expressed in ng S mg−1 and was calculated by normalizing the 34S-incorporation measured using NanoSIMS to the average sulfur content ( % of dry mass ) according to the equation of Dugdale and Wilkerson ( 1986 ) , presented in Pernice et al . ( 2012 ) .", ": ρ = ( ( Smes - Snat ) / ( Senr - Snat ) ) × Scontent ×103 Where: Smes: 34S/32S measured in labelled samples by NanoSIMS Snat: natural abundance of 34S/32S measured in unlabelled samples by NanoSIMS Senr: 34S-enrichment of the Symbiodinium cells measured by NanoSIMS Scontent: average sulfur content ( % ) measured by Thermo Scientific FLASH 2000 Series .", "The calculated uptake ( in nmol S mg−1 ) was then converted into an estimate uptake rate per day ( nmol S l−1 day−1 ) , based on: the bacterial exposure to 34S ( 6 hr ) , and the bacterial cell density for a given dry weight ( acquired through flow cytometry; equal to 7 . 12 × 10−7 g for 5 × 105 bacterial cells ) ." ] ]

[ "Phytoplankton-bacteria interactions drive the surface ocean sulfur cycle and local climatic processes through the production and exchange of a key compound: dimethylsulfoniopropionate ( DMSP ) .", "Despite their large-scale implications , these interactions remain unquantified at the cellular-scale .", "Here we use secondary-ion mass spectrometry to provide the first visualization of DMSP at sub-cellular levels , tracking the fate of a stable sulfur isotope ( 34S ) from its incorporation by microalgae as inorganic sulfate to its biosynthesis and exudation as DMSP , and finally its uptake and degradation by bacteria .", "Our results identify for the first time the storage locations of DMSP in microalgae , with high enrichments present in vacuoles , cytoplasm and chloroplasts .", "In addition , we quantify DMSP incorporation at the single-cell level , with DMSP-degrading bacteria containing seven times more 34S than the control strain .", "This study provides an unprecedented methodology to label , retain , and image small diffusible molecules , which can be transposable to other symbiotic systems ." ]

[ "Sulfur is an essential element for many organisms and environmental processes .", "Every year , organisms including microalgae produce more than one billion tons of a sulfur-containing compound called DMSP .", "Some of this DMSP is released into seawater , where it acts as a key nutrient for microscopic organisms and as a foraging cue to attract fish .", "DMSP is also the precursor of a gas that helps to form clouds .", "Despite DMSP’s potential large-scale effects , it is still not clear what role it plays in the organisms that produce it , or how it is transferred from the microalgae that produce it to the bacteria that use it .", "It is thought that DMSP could potentially protect the cells from sudden changes in the amount of salt in the seawater ( salinity ) or from other damage , such as oxidative stress – a build-up of harmful chemicals inside cells .", "In a controlled setting using artificial seawater , Raina et al . used high-resolution imaging and chemical analysis to track the journey of DMSP from microalgae to recipient bacteria .", "The results show that similar to land plants , algae store DMSP in the compartments that regulate cell pressure and photosynthesis .", "The presence of DMSP in these locations also supports its proposed role in protecting cells from changes in salinity or oxidative damage .", "A future step will be to identify the genes involved in producing DMSP in microalgae .", "This knowledge could be used to create mutants that are either incapable of producing this molecule or that overproduce it .", "In combination with the high-resolution imaging techniques described here , this will allow researchers to fully understand the role that DMSP plays in these organisms ." ]

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

[ "neuroscience" ]

[ [ "Dystonia is a common , debilitating movement disorder caused by co-contraction of agonist-antagonist muscle pairs ( Frucht , 2013 ) .", "The underlying neurological causes of dystonia are not fully understood and there are few effective therapeutic interventions .", "The most common inherited dystonia is early onset generalized torsion dystonia , referred to as DYT1 .", "The onset of symptoms in DYT1 patients occurs before the age of 26 with a focal dystonia that often generalizes , resulting in severe disability ( Bressman et al . , 2000 ) .", "The majority of DYT1 patients share a single amino acid deletion in torsinA ( Ozelius et al . , 1997; Risch et al . , 1995; Warner and Jarman , 1998 ) , resulting in loss of function of the protein ( Goodchild et al . , 2005 ) .", "Studies in DYT1 patients have implicated several brain areas in this disorder , including the cerebellum ( Eidelberg et al . , 1998 ) and basal ganglia ( Panov et al . , 2013 ) .", "How torsinA disruption causes dystonia and which brain areas play a key role remain outstanding questions .", "Recent studies in an animal model of Rapid-onset Dystonia Parkinsonism ( RDP ) have suggested that in this rare genetic dystonia , abnormal cerebellar output alters basal ganglia activity and likely contributes to dystonic postures ( Calderon et al . , 2011; Chen et al . , 2014; Fremont et al . , 2014 ) .", "Examining the role of these brain regions in a symptomatic model of DYT1 may provide insight into its pathophysiology .", "Unfortunately , mice with complete knockout of torsinA exhibit early lethality and cannot be studied ( Goodchild et al . , 2005 ) .", "Further , no viable rodent models of DYT1 show overt dystonia ( Dang et al . , 2005; Goodchild et al . , 2005; Grundmann et al . , 2012 , 2007; Page et al . , 2010; Sharma et al . , 2005; Shashidharan et al . , 2005 ) .", "The same is true of brain region-specific genetic models of torsinA dysfunction ( Liang et al . , 2014; Pappas et al . , 2015; Weisheit and Dauer , 2015; Yokoi et al . , 2011 , 2008; Zhang et al . , 2011 ) .", "While two recently generated mouse models of DYT1 exhibited some twisting movements ( Liang et al . , 2014 ) , these mice do not exhibit the overt sustained postures common in dystonic patients .", "Recapitulating the major symptoms and disease progression seen in DYT1 patients has been challenging in mouse models and therefore it has been difficult to address how loss of torsinA causes dystonia .", "Genetic models of DYT1 to date involve manipulation of torsinA during embryogenesis .", "But there is evidence that while there is high embryonic expression of the protein in rodents ( Vasudevan et al . , 2006; Xiao et al . , 2004 ) , it is only expressed postnatally in humans ( Siegert et al . , 2005 ) .", "This suggests that compensation of torsinA is highly likely in rodents which could explain discrepancies between current DYT1 rodent models and patients with DYT1 .", "We hypothesized that regional , acute knockdown of torsinA in the adult mouse , when expression is similar in mice and humans might better replicate DYT1 symptoms .", "In fact , we previously showed in a mouse model of RDP that acute knockdown of the α3 isoform of the Na+/K+ - ATPase pump in the cerebellum of adult rodents was sufficient to induce dystonia , whereas genetic mouse models targeting the gene early in development did not produce dystonia .", "This provides further support for compensatory mechanisms in the rodent following the loss of certain genes early in development .", "We found that in adult mice , knockdown of torsinA in the cerebellum but not the basal ganglia resulted in severe and persistent dystonia .", "Symptoms were associated with abnormal erratic cerebellar output caused by dysfunctional intrinsic pacemaking activity of both Purkinje cells and neurons of the deep cerebellar nuclei .", "In contrast , mice with cerebellar knockdown of torsinA during the early post-natal period had mild symptoms with no dystonia .", "This observation suggests that indeed compensation of torsinA in early rodent brain development can explain the major differences in phenotype between adults and juveniles after torsinA knockdown .", "It is tempting based on these and previous findings ( Fremont et al . , 2015 ) to hypothesize that abnormal cerebellar activity may be a common mechanism in some hereditary dystonias .", "In fact , imaging studies have implicated the cerebellum in another inherited dystonia ( Carbon et al . , 2010a , 2013 ) .", "Overall , by utilizing acute knockdown in adult rodents , we identified a mechanism whereby disruption of torsinA produces aberrant firing in the cerebellum and results in dystonic symptoms and provide evidence that the cerebellum may initiate symptoms in the most common inherited dystonia , DYT1 ." ], [ "Since dystonia is often associated with changes in the basal ganglia , we first explored whether torsinA KD in this brain region results in symptoms .", "AAVs expressing a shRNA against torsinA with a green fluorescent protein marker ( AAV-TorsinAshRNA-GFP , Figure 1A ) were stereotaxically injected into the basal ganglia ( striatum and globus pallidus ) of adult mice .", "Significant knockdown of torsinA ( average torsinA levels after KD: 0 . 187 ± 0 . 06 ) was achieved and post-mortem histology demonstrated robust expression throughout the injected areas ( Figure 1B , C ) .", "The behavior of these mice in the open field was assessed by observers blinded to the animals’ condition on a previously published dystonia scale ( Calderon et al . , 2011 ) .", "Since shRNAs take approximately two weeks to be expressed in vivo ( Fremont et al . , 2015 ) , the behavior of the animal can be compared before and after expression of the shRNA .", "Animals injected with a shRNA against torsinA in the basal ganglia did not develop dystonia , as assessed by the dystonia scale .", "Moreover , the behavior of the mice was similar to controls injected with a non-targeted shRNA ( AAV-NTshRNA-GFP ) ( Figure 1D , Video 1 ) .", "Multiple parameters of open field behavior quantified in animals with knockdown of torsinA in the basal ganglia , including average velocity , steps taken in 30 s , distance travelled in 5 min and step size , were unchanged throughout the post-injection period ( Figure 1—figure supplement 1 ) .", "Injection of a second , non-overlapping shRNA against torsinA ( AAV-TorsinAshRNA2-GFP ) yielded similar results ( Figure 1D ) .", "These data suggest that torsinA KD in the basal ganglia does not result in dystonia . 10 . 7554/eLife . 22775 . 003Figure 1 . Although knockdown of torsinA in the striatum and globus pallidus of adult mice does not cause dystonia , its knockdown in the cerebellum does .", "( A ) Schematic of the construct delivered to cells via AAV9 containing either non-targeted shRNA or shRNA against torsinA .", "Arrows are used to show promoters .", "( B ) There was strong GFP expression in the basal ganglia ( left image ) of mice injected with the shRNA against torsinA .", "The right image is a sagittal section of a representative mouse injected with AAV-TorsinA shRNA-GFP .", "The dotted line delineates the separation between basal ganglia and cerebral cortex .", "CX = cerebral cortex , BG = basal ganglia , V = ventricle .", "Scale bar = 2 . 5 mm in left image , 500 µm in right image .", "( C ) Example western blot ( left ) and quantification ( right ) from three animals in which AAV9 containing shRNA against torsinA was injected into the basal ganglia of adult mice .", "There is an average knockdown of 81 . 3% ± 6 . 2% ( Mean ±S . E . M ) .", "( D ) Observers blinded to the condition of the animals rated the basal ganglia injected animals on a previously published dystonia scale .", "On the graph each point represents an individual animal and the bars represent the average of all animals .", "All animals up to 13 weeks post-injection had an average score <1 ( Mean ± S . E . M , NT shRNA , N = 8; Tor1A KD BG , N = 6; Tor1A KD BG2 , N = 8 ) .", "( E ) There was strong GFP expression throughout the cerebellum ( left image ) of mice injected with the shRNA against torsinA .", "The right image is a sagittal section of a representative mouse injected with AAV-TorsinA shRNA-GFP .", "The dotted line delineates the cerebellum ( CB ) .", "Scale bar = 2 . 5 mm in left image , 500 µm in right image .", "( F ) Representative Western blot ( left ) and quantification ( right ) showing knockdown of torsinA from cerebellar lysates prepared from animals injected with a shRNA against torsinA when compared to WT animals or animals injected with a non-targeted ( NT ) shRNA .", "TorsinA KD 77 . 9% ± 4 . 7% ( Mean ± S . E . M , Tor1A KD N = 6 ) .", "( G ) Adult mice with knockdown of torsinA in the cerebellum showed symptoms consistent with dystonia as rated by observers blinded to the condition of the animals .", "On the dystonia scale , a score of ≥2 is considered dystonic .", "Adult mice with knockdown of torsinA , using two shRNAs targeted to different regions of the protein , exhibited dystonic symptoms beginning around 7–9 weeks .", "( *=p<0 . 05 , **=p<0 . 01 , ***=p<0 . 001 , ****=p<0 . 0001 , Mean ± S . E . M , NT shRNA , N = 10; Tor1A KD CB , N = 20; Tor1A KD CB2 , N = 15 ) .", "By 13 weeks post-injection animals on average exhibited symptoms consistent with dystonia on this scale .", "( H ) An example mouse 13 weeks after the injection of shRNA against torsinA into the cerebellum exhibiting an abnormal hind limb posture ( arrow ) .", "( I ) EMGs performed in three animals confirmed that abnormal postures are due to co-contraction of agonist and antagonist muscle pairs .", "In this example trace , the gastrocnemius ( Ga , red ) and anterior tibialis ( AT , black ) are shown during a dystonic co-contraction ( arrow ) .", "Scale bar represents 5 s", "( x ) by 100 μV", "( y ) .", "( J ) The severity of motor symptoms , quantified by the dystonia scale 13 weeks after injection of torsinA in the cerebellum , is correlated with the level of shRNA mediated knockdown of torsinA .", "As the percent of torsinA knockdown increases , so does the severity of the motor phenotype ( Torsin A KD CB ( N = 22 , purple ) and torsin A KD CB2 ( N = 15 , pink ) .", "Black symbols show the average of the binned combined data from both shRNAs against torsinA ( Spearman r = 0 . 63 , 95% CI 0 . 3759 to 0 . 7960 , p value < 0 . 0001 ) .", "knockdown is presented as within-blot loading control-normalized values .", "The data are presented as mean ±S . E . M . DOI: http://dx . doi . org/10 . 7554/eLife . 22775 . 00310 . 7554/eLife . 22775 . 004Figure 1—figure supplement 1 . Knockdown of torsinA in the basal ganglia of adult mice does not change open field behavior . To determine whether or not torsinA knockdown in the basal ganglia affected motor behavior in the open field , quantification of average velocity , average distance traveled in 5 min , number of steps taken in 30 s , and step size were quantified using ethovision ( N = 5 first shRNA; 5 s shRNA ) over a 13 week period post-injection .", "Because there was no significant difference between the effect of the two shRNAs , the data shown is a combined analysis ( N = 10 ) .", "There was no significant effect of shRNA knockdown on any of these parameters ( p>0 . 05 compared using Kruskal-Wallis test ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 22775 . 00410 . 7554/eLife . 22775 . 005Figure 1—figure supplement 2 . Symptoms in adult mice with simultaneous knockdown of torsinA in the basal ganglia and the cerebellum are not significantly different from symptoms seen in mice with knockdown in the cerebellum alone . The figure shows a direct comparison of dystonia scores for animals with torsinA knockdown in the cerebellum alone ( TorsinA CB , N = 20 ) compared with mice with knockdown in the cerebellum and basal ganglia ( TorsinA CB+BG , N = 3 ) at three representative time points; three weeks ( TorsinA CB average dystonia score: 0 . 11 ± 0 . 04 , TorsinA CB+BG: 0 . 17 ± 0 . 17 , p=0 . 82 Mann-Whitney test ) , 11 weeks ( TorsinA CB: 1 . 95 ± 0 . 26 , TorsinA CB+BG: 1 . 67 ± 0 . 67 , p=0 . 80 ) , and 13 weeks post-injection ( TorsinA CB: 2 . 43 ± 0 . 24 , TorsinA CB+BG: 2 . 32 ± 0 . 32 , p=0 . 88 ) .", "At no time point was there a significant difference in dystonia score between mice with torsinA knockdown in the cerebellum alone compared to those with knockdown in the cerebellum and basal ganglia . DOI: http://dx . doi . org/10 . 7554/eLife . 22775 . 00510 . 7554/eLife . 22775 . 006Video 1 . shRNA-mediated knockdown of torsinA in the basal ganglia of adult mice does not produce dystonic postures . A representative video showing a mouse 13 weeks after injection , injected with a shRNA against torsinA in the cerebellum at 6 weeks of age .", "In the open-field no motor abnormalities were observed . DOI: http://dx . doi . org/10 . 7554/eLife . 22775 . 006 The cerebellum has also been implicated in DYT1 ( Argyelan et al . , 2009; Eidelberg et al . , 1998 ) and there is evidence that it plays a role in other hereditary dystonias ( Calderon et al . , 2011; Carbon et al . , 2013; Oblak et al . , 2014 ) .", "Therefore , we tested whether torsinA KD in the cerebellum of adult rodents produces dystonia .", "Injection of AAV-torsinA shRNA-GFP resulted in generalized expression of the construct ( Figure 1E ) and significant knockdown of torsinA in the cerebellum ( average torsinA levels after KD: 0 . 2214 ± 0 . 05 ) ( Figure 1F ) .", "Mice with acute knockdown of torsinA in the cerebellum developed uncoordinated movements progressing to frequent dystonic postures ( Figure 1G and Video 2 ) .", "In separate cohorts of mice , injection of a second shRNA ( AAV-TorsinA shRNA2-GFP ) resulted in comparable symptoms , while injection of AAV-NTshRNA-GFP to the same region had no effect on motor behavior ( Figure 1F ) .", "In patients with dystonia , abnormal co-contraction of agonist and antagonist muscle pairs often underlies abnormal postures ( Frucht , 2013; Hughes and McLellan , 1985; Yanagisawa and Goto , 1971 ) .", "Electromyographic ( EMG ) recordings in mice with torsinA KD in the cerebellum demonstrated that abnormal posturing of the hind limb in these animals is due to muscle co-contraction , similar to what is seen in patients ( Figure 1H , I ) .", "It is generally postulated that dystonia may be a circuit-level disorder , resulting from the dysfunction of multiple motor-related areas .", "Therefore , we examined whether concurrent torsinA KD in the cerebellum and basal ganglia would result in more severe symptoms .", "We found that knockdown of torsinA in the cerebellum and basal ganglia did not enhance the severity of dystonia when compared to mice with torsinA KD in the cerebellum alone ( Figure 1—figure supplement 2 ) .", "These findings provide evidence that torsinA KD in the adult cerebellum is sufficient to induce dystonia . 10 . 7554/eLife . 22775 . 007Video 2 . shRNA-mediated knockdown of torsinA in the cerebellum of adult mice produces dystonic postures by 13 weeks after injection . A representative video showing the progression of symptoms in a mouse injected with a shRNA against torsinA in the cerebellum at 6 weeks of age .", "The video starts with the mouse walking in an open-field chamber at three weeks after injection displaying no obvious motor abnormalities .", "At the 24 s time-point in the video , the same mouse is shown at seven weeks after injection with an unsteady gait and jerky movements .", "At 1 min 10 s , the same mouse is seen at 13 weeks after injection with exacerbated symptoms including difficulty maintaining balance , frequent repetitive movements and dystonic postures . DOI: http://dx . doi . org/10 . 7554/eLife . 22775 . 007 An observation made during the course of our experiments was the variability in the severity of symptoms in animals with cerebellar knockdown of torsinA ( Figure 1G ) .", "To determine the source of the variability , the amount of knockdown of torsinA in each animal was compared to the Dystonia score .", "It was noted that the severity of dystonic symptoms correlated with the extent of knockdown , such that as knockdown increased so did the severity of symptoms .", "When torsinA protein level was knocked down by at least 60% dystonic symptoms were observed ( Figure 1J , p<0 . 0001 ) .", "From these data , it is clear that a substantial decrease in torsinA protein levels is necessary to produce dystonia .", "This may explain , at least in part , why heterozygous animal models of torsinA dysfunction have routinely lacked overt dystonic symptoms .", "Previous studies have suggested that abnormal cerebellar output in the rodent can result in dystonia ( Calderon et al . , 2011; Fremont et al . , 2014 , 2015; LeDoux et al . , 1993; Pizoli et al . , 2002 ) .", "To address whether a similar alteration underlies dystonia in this model , in vivo extracellular recordings were performed in dystonic torsinA KD animals .", "Recordings from deep cerebellar nuclei ( DCN ) neurons , which comprise the majority of the cerebellar output , revealed that these cells fired abnormally with bursts of action potentials , whereas DCN cells in controls ( NTshRNA ) fired tonically ( Figure 2A ) .", "Further , the average firing rate of DCN neurons from dystonic animals was nearly half that of controls ( Figure 2B , control average firing rate: 55 . 5 ± 4 . 1 spikes/second ( sp/s ) , average firing rate in dystonic mice: 22 . 4 ± 3 . 4 sp/s , p<0 . 00001 ) .", "It has been shown that an increase in the irregularity of cerebellar output can underlie ataxia ( Walter et al . , 2006 ) and in some cases dystonia in rodents ( Fremont et al . , 2014 , 2015 ) .", "The regularity of neuronal firing was calculated using the coefficient of variation of the interspike intervals ( CV ISI ) , with increasing CV ISI representing an increase in irregularity .", "Since the CV ISI is determined by dividing the standard deviation of the interspike interval ( ISI ) by the mean ISI of the cell , changes in the firing rate will affect the CV ISI .", "DCN neurons recorded from dystonic animals exhibited a lower firing rate than controls , and would thus be expected to have a lower CV ISI , even without a change in firing regularity .", "However , the CV ISI of DCN neurons recorded from dystonic animals was nearly triple that of controls , despite the decrease in average firing rate , suggesting that the activity of these neurons is highly erratic ( Figure 2C , control CV ISI: 0 . 55 ± 0 . 03 , dystonic CV ISI: 1 . 53 ± 0 . 13 , p<0 . 00001 ) .", "The CV ISI will increase if the cell begins to fire with long pauses , in high frequency bursts , or both .", "In a mouse model of the inherited dystonia RDP , high-frequency burst firing was shown to underlie severe dystonia ( Fremont et al . , 2014 , 2015 ) .", "The predominant firing rate ( PFR ) and the interspike interval ( ISI ) histogram can identify high frequency firing in dystonic animals .", "There was a trend towards an increase in the PFR of DCN cells recorded from dystonic mice with torsinA KD compared to controls , ( Figure 2D , control: 77 . 3 ± 5 . 7 sp/s , dystonic animals: 105 . 1 ± 22 . 3 sp/s , p=0 . 21 ) , and there was a peak in the ISI histogram of DCN neurons from dystonic mice at shorter interspike intervals around 2 ms ( Figure 2E ) .", "Taken together , these data suggest that dystonia in mice with torsinA KD is caused by abnormal and erratic cerebellar output . 10 . 7554/eLife . 22775 . 008Figure 2 . Purkinje cells and neurons of the deep cerebellar nuclei ( DCN ) fire abnormally in dystonic mice .", "( A ) Schematic showing recordings from DCN neurons in awake head restrained animals .", "To the right are example traces recorded from a non-dystonic mouse injected with non-targeted shRNA ( NTshRNA , black ) and from a dystonic mouse with cerebellar knockdown of torsinA ( torsinA KD , purple ) .", "Scale bar = 500 ms", "( x ) by 50 μV", "( y ) for both traces .", "( B ) Quantification of the average firing rate of DCN neurons revealed that DCN cells recorded from dystonic mice with knockdown of torsinA ( purple ) ( N = 3 , n = 25 ) exhibited a significantly decreased average firing rate compared to those from non-dystonic mice expressing NTshRNA ( gray ) ( N = 5 , n = 49 ) ( control average firing rate: 53 . 21 ± 2 . 998 spikes/second ( sp/s ) , average firing rate in dystonic mice: 22 . 4 ± 3 . 4 sp/s , p<0 . 0001 ) .", "( D ) DCN cells from dystonic mice ( purple ) also exhibited an increased coefficient of variation of the interspike intervals ( CV ISI ) compared to controls ( gray ) indicating that these cells fired more irregularly than controls ( control CV ISI: 0 . 63 ± 0 . 034 , dystonic CV ISI: 1 . 53 ± 0 . 13 , p<0 . 0001 ) .", "( C , E )", "There was no statistically significant difference between the predominant firing rate of control DCN cells and those from dystonic animals ( C ) ( control: 64 . 86 ± 4 . 056 sp/s , dystonic animals: 105 . 1 ± 22 . 3 sp/s , p=0 . 3044 ) , although the interspike interval ( ISI ) histograms of representative DCN neurons ( n = 20 per condition ) showed an additional peak at shorter ISIs in dystonic animals ( purple , ( E ) compared to controls ( black , ( E ) .", "On all bar graphs , bars represent mean ± S . E . M with points representing individual values ****= p<0 . 0001 .", "( F ) Schematic showing that Purkinje cells ( PCs ) were recorded from awake head restrained mice .", "Beneath are example raw traces of a PC recorded from a control mouse with expression of NTshRNA in the cerebellum ( black ) and one recorded from a dystonic mouse with knockdown of torsinA in the cerebellum ( purple ) .", "PCs recorded in dystonic animals exhibited abnormal burst firing .", "Both scale bars are 200 ms", "( x ) by 200 μV", "( y ) .", "( G ) Quantitatively , there was a significant difference between the average firing rate of control PCs ( gray; N = 5 , n = 38 ) and those from dystonic mice ( purple; N = 3 , n = 17 ) control: 64 . 31 ± 3 . 094 sp/s , dystonia: 36 . 45 ± 7 . 6 sp/s , ***p=0 . 0001 .", "( I ) In addition , PCs in dystonic mice ( purple ) exhibited an increase in the coefficient of variation of interspike intervals compared to controls ( gray ) suggesting that these cells fire more irregularly ( control: 0 . 5 ± 0 . 019 , dystonia: 1 . 26 ± 0 . 135 , p<0 . 0001 ) .", "( H , J )", "PCs in dystonic mice ( purple ) exhibited a trend towards an increased predominant firing rate PFR compared to controls ( gray ) ( control: 77 . 7 ± 3 . 76 sp/s , dystonia: 105 . 6 ± 20 . 52 sp/s , p=0 . 24 ) , and the ISI histograms of representative cells from each condition ( n = 15 per condition ) showed a peak ISI shifted to the left towards shorter ISIs in torsinA KD animals ( purple , ( J ) .", "On all graphs , bars represent mean ± S . E . M with points representing values from individual cells . DOI: http://dx . doi . org/10 . 7554/eLife . 22775 . 00810 . 7554/eLife . 22775 . 009Figure 2—figure supplement 1 . Neurons of the deep cerebellar nuclei ( DCN ) and Purkinje cells recorded in vivo from less severe mice injected with the shRNA against torsinA in the cerebellum exhibit abnormal activity . We recorded from mice with a dystonia score ranging from one to < 2 ( N = 4 , light purple ) .", "While these mice showed abnormal motor symptoms , the symptoms did not include dystonia .", "( A ) DCN neurons in these animals had a significant decrease in their average firing rate ( N = 4 , n = 22 , control average firing rate: 53 . 21 ± 2 . 998 spikes/second ( sp/s ) , average firing rate in less severe mice: 28 . 62 ± 1 . 86 sp/s , p<0 . 0001 ) .", "( B ) In addition , there was a decrease in their predominant firing rate compared to dystonic animals ( control: 64 . 86 ± 4 . 056 sp/s , less severe animals: 64 . 2 ± 17 . 09 sp/s , **p=0 . 0064 ) .", "( C ) However , there was only a slight , not statistically significant , increase in the CV ISI of these animals when compared to the control animals injected with the non-targeting shRNA ( control mice: 0 . 63 ± 0 . 034 , less severe mice: 0 . 7655 ± 0 . 1 , p=0 . 78 ) .", "( D ) The ISI histogram of the DCN neurons in these animals also shows that the majority of ISIs lie in the same range as the control animals , with some cells showing longer ISIs .", "( E ) Purkinje cells in these animals showed a significant decreased average firing rate ( control: 64 . 31 ± 3 . 094 sp/s , less severe mice: 37 . 33 ± 4 . 044 sp/s , ***p=0 . 0001 ) .", "( F ) There was also a decrease in the predominant firing rate compared to dystonic animals and controls ( control: 77 . 7 ± 3 . 76 sp/s , less severe mice: 72 . 28 ± 7 . 39 sp/s , **p=0 . 0099 ) .", "( G ) There was also an increase in the CV ISI of these animals when compared to the control animals injected with the non-targeted shRNA ( control: 0 . 5 ± 0 . 019 , less severe mice: 0 . 91 ± 0 . 08 , p<0 . 0001 ) .", "( H ) The ISI histogram of the DCN neurons in these animals shows that the majority of ISIs lie in the same range as the control animals , with some cells showing longer ISIs , which like the DCN neurons is shown in the decreased average firing rate . DOI: http://dx . doi . org/10 . 7554/eLife . 22775 . 009 To determine whether the irregularity of firing changes with the severity of symptoms , awake head-restrained recordings were also performed in mice which developed less severe symptoms not characteristic of dystonia when they were injected with the torsinA shRNA .", "On the dystonia scale these mice scored between 1 and 2 , corresponding to an approximately 30–60% knockdown of torsinA .", "On average , the changes in the activity of Purkinje cells and DCN neurons in these mice were in between those of the normal and dystonic mice ( Figure 2—figure supplement 1 ) .", "However , these data should be interpreted with caution as it is difficult to know whether the data obtained in the less severe mice represents the average behavior of two groups of neurons; namely those with and without knockdown of torsinA , or alternatively it primarily represents less knockdown in the majority of cells .", "We think the former possibility is the more likely scenario .", "Since DCN activity is strongly influenced by GABAergic input from Purkinje cells , the main computational neurons of the cerebellar cortex , the activity of Purkinje cells was also examined in vivo .", "Normally , Purkinje cells fire regularly in vivo but in dystonic animals Purkinje cells exhibited abnormal bursting activity ( Figure 2F ) .", "There was no significant difference between the average firing rate of Purkinje cells from dystonic mice ( torsinA KD ) and those from controls ( NTshRNA ) ( Figure 2G , control: 69 . 1 ± 5 . 3 sp/s , dystonia: 51 . 1 ± 12 . 1 sp/s , p=0 . 13 ) .", "However , the CV ISI of Purkinje cells from dystonic animals was increased by over two-fold compared to controls ( Figure 2I , control: 0 . 05 ± 0 . 03 , dystonia: 1 . 21 ± 0 . 12 , p<0 . 0001 ) .", "The PFR of Purkinje cells from torsinA KD animals was also significantly increased ( Figure 2H , control: 85 . 7 ± 7 . 1 sp/s , dystonia: 124 . 6 ± 22 . 5 sp/s , p=0 . 031 ) .", "The ISI histogram of Purkinje cells showed that , similar to the DCN neurons , some cells had short ISIs ( around 5 ms compared to a peak ISI at 15 ms for the NTshRNA injected animals , Figure 2J ) .", "These findings suggest that aberrent Purkinje cell output caused by knockdown of torsinA in the cerebellum could contribute to the abnormal activity of DCN neurons .", "Similar to that seen for DCN neurons , Purkinje cells in mice with less severe symptoms also showed abnormal aberrant firing which were , by and large , less altered than those in the dystonic animals ( Figure 2—figure supplement 1 ) .", "Purkinje cells are intrinsically active pacemaking neurons that fire regular action potentials even when deprived of synaptic input ( Batini and Kado , 1967; Bell and Grimm , 1969; Latham and Paul , 1971 ) .", "Since Purkinje cells have high expression of torsinA , alterations in this activity resulting from torsinA KD could contribute to the changes in Purkinje cell firing reported above ( Konakova et al . , 2001; Puglisi et al . , 2013 ) .", "To address this possibility , we performed extracellular recordings from Purkinje cells in cerebellar slices in the presence of GABAergic and glutamatergic synaptic blockers .", "Both Purkinje cells with torsinA KD , identified by their expression of GFP ( GFP+ torsinA KD ) , as well as neighboring neurons without GFP expression ( GFP-torsinA KD ) were recorded ( Figure 3A ) .", "The Purkinje cells from torsinA KD animals were then compared to those recorded from wild-type animals ( WT ) and GFP positive ( NT GFP+ ) and negative cells ( NT GFP− ) from animals injected with AAV9-NTshRNA-GFP .", "TorsinA KD altered the intrinsic activity of Purkinje cells , converting their pacemaking to irregular burst firing .", "Although Purkinje cells with torsinA KD had an average firing rate similar to NT and WT ( average firing rate of WT: 53 . 9 ± 6 . 7 , GFP+ TorsinA KD: 68 . 1 ± 13 . 1 sp/s , NT GFP+: 49 . 8 ± 7 . 0 sp/s , p=0 . 45 , Figure 3B ) , the PFR was nearly doubled ( PFR of WT: 53 . 7 ± 6 . 6 , GFP+ TorsinA KD: 93 . 2 ± 20 . 7 sp/s , NT GFP+: 49 . 5 ± 6 . 9 sp/s , p=0 . 048 , Figure 3C ) and the CV ISI was increased by ~10 fold ( CV ISI of WT: 0 . 110 ± 0 . 010 , GFP+ TorsinA KD: 1 . 151 ± 0 . 248 , NT GFP+: 0 . 096 ± 0 . 008 , p=0 . 0003 , Figure 3D ) .", "The ISI histogram also showed that torsinA KD Purkinje cells had a peak ISI shifted to the left similar to the in vivo recordings ( Figure 3E ) .", "GFP− Purkinje cells from dystonic animals had similar firing patterns compared to those from wild-type and AAV9-NTshRNA-GFP controls ( Figure 3B , C , D , E ) , providing evidence that torsinA knockdown affects the intrinsic pacemaking of these cells .", "Together , these findings suggest that knockdown of torsinA disrupts the intrinsic activity of Purkinje cells . 10 . 7554/eLife . 22775 . 010Figure 3 . Knockdown of torsinA alters the intrinsic activity of Purkinje cells and neurons in the deep cerebellar nuclei ( DCN ) .", "( A ) Schematic of extracellular recordings which were performed from acute brain slices in which fast glutamatergic and GABAergic synaptic transmission was blocked .", "Beneath are raw traces of adjacent Purkinje cells recorded from dystonic mice with torsinA knockdown in the cerebellum .", "The Purkinje cell on the top is not infected and exhibits regular firing characteristic of this cell type ( black ) .", "The one on the bottom had torsinA knockdown and exhibited erratic firing ( purple ) .", "Scale bar is 200 ms", "( x ) by 50 μV", "( y ) and is applicable to both traces .", "( B ) There is no significant change in the average firing rate of Purkinje cells with torsinA knockdown ( TorsinA KD ( GFP+ TorsinA KD , N = 5 , n = 12 ) compared to GFP− Purkinje cells ( GFP− TorsinA KD , n = 12 ) from the same animal and Purkinje cells from control animals WT , ( N = 5 , n = 21 ) and GFP positive ( NT GFP+ , N = 3 , n = 15 ) and negative cells ( NT GFP− , n = 10 ) from animals infected with AAV9-NTshRNA-GFP ( average firing rate of WT: 53 . 9 ± 6 . 7 , GFP+ TorsinA KD: 68 . 1 ± 13 . 1 sp/s , GFP+ NT: 49 . 8 ± 7 . 0 sp/s , p=0 . 45 ) .", "( C ) Purkinje cells with torsinA knockdown have a higher predominant firing rate than controls ( PFR of WT: 53 . 7 ± 6 . 6 , GFP+ TorsinA KD: 93 . 2 ± 20 . 7 sp/s , GFP+ NT: 49 . 5 ± 6 . 9 sp/s , p=0 . 048 ) .", "( D ) Purkinje cells with torsinA knockdown have an increased CV ISI compared to controls reflecting the irregularity of the firing of these neurons ( CV ISI of WT: 0 . 110 ± 0 . 010 , GFP+ TorsinA KD: 1 . 151 ± 0 . 248 , GFP+ NT: 0 . 096 ± 0 . 008 , p=0 . 0003 ) .", "On all graphs , bars represent mean ± S . E . M with points representing individual values .", "*=p<0 . 05 , ****= p<0 . 0001 .", "( E ) ISI histogram of all cells in each condition shows that cells with knockdown of torsinA ( purple ) have shorter ISIs compared to cells from all other conditions .", "( F ) Schematic showing extracellular recordings of DCN neurons in slices where fast glutamatergic and GABAergic synaptic transmission was blocked .", "Below are representative traces of DCN neurons recorded in slices taken from dystonic mice with knockdown of torsinA .", "The top trace is a neuron recorded from an area of DCN with no GFP expression which exhibits regular pacemaking characteristic of these cells .", "The bottom trace is from a neuron located in an area of DCN with high GFP expression and torsinA knockdown which exhibits irregular activity .", "Scale bar = 500 ms", "( x ) by 200 μV and is applicable to both traces .", "( G ) The average firing rate of DCN cells with torsinA knockdown ( torsinA KD N = 3; GFP+ cells n = 7 and GFP− cells n = 6 ) was significantly reduced compared to DCN cells recorded in control animals ( wild type = WT ( N = 3 , n = 8 ) , control animals injected with AAV-NTshRNA-GFP ( N = 3 ) : neurons from GFP+ DCN ( NT GFP+ DCN , n = 8 ) and neurons from GFP− DCN ( NT GFP− DCN , n = 9 ) ( average firing rate of GFP+ neurons from dystonic animals: 7 . 9 ± 2 . 3 sp/s , GFP+ neurons from control animals: 21 ± 2 . 1 sp/s **=p<0 . 01 .", "( H ) There was no significant difference between the predominant firing rates ( PFR ) of DCN neurons with torsinA knockdown compared to controls ( GFP+ TorsinA: 41 . 9 ± 14 . 2 sp/s , GFP+ NT DCN: 21 . 4 ± 2 . 2 sp/s , p=0 . 15 ) .", "( I ) DCN neurons with torsinA knockdown exhibited an increased coefficient of variation of interspike intervals compared to controls , reflecting the irregularity of firing of these neurons ( GFP+ neurons from dystonic animals: 1 . 21 ± 0 . 312 , GFP+ neurons from control animals: 0 . 1 ± 0 . 011 , **p=0 . 0021 ) .", "( J ) ISI histogram of all cells for each condition shows that cells with knockdown of torsinA ( purple ) have larger ISIs compared to cells from all other conditions .", "On all graphs , bars represent mean ± S . E . M with points representing individual values . DOI: http://dx . doi . org/10 . 7554/eLife . 22775 . 010 DCN neurons are also intrinsically active , pacemaking cells with high expression of torsinA ( Konakova et al . , 2001; Puglisi et al . , 2013 ) .", "In vitro recordings of the intrinsic activity of DCN neurons , with fast glutamatergic and GABAergic synaptic transmission blocked , demonstrated that knockdown of torsinA alters the normal tonic activity of these neurons , causing them to fire at lower rates and with bursts ( Figure 3F ) .", "Extracellular recordings were performed in regions of the DCN with clusters of GFP+ cells ( GFP+ DCN ) and areas with clusters of GFP− cells ( GFP− DCN ) .", "Cells were recorded from cerebellar slices of wild type animals , animals injected with AAV9-NTshRNA-GFP ( GFP+ and GFP− cells ) and animals injected with AAV9-TorsinA shRNA-GFP ( GFP+ and GFP− cells Figure 3G , H , I ) .", "DCN neurons from GFP+ areas of torsinA shRNA injected animals had a mean average firing rate nearly half that of the controls ( GFP+ neurons from dystonic animals: 7 . 9 ± 2 . 3 sp/s , p=0 . 0011 , GFP+ neurons from control animals: 21 ± 2 . 1 sp/s , Figure 3G ) .", "Further , DCN neurons recorded from GFP+ areas in slices from dystonic mice exhibited a ten-fold increase in the CV ISI compared to controls ( GFP+ neurons from dystonic animals: 1 . 21 ± 0 . 312 , GFP+ neurons from control animals: 0 . 1 ± 0 . 011 , p=0 . 0021 , Figure 3I ) .", "Although there was a trend towards an increased PFR in neurons with torsinA KD , it was not statistically significant ( GFP+ TorsinA: 41 . 9 ± 14 . 2 sp/s , GFP+ NT DCN: 21 . 4 ± 2 . 2 sp/s , p=0 . 15 , Figure 3H ) .", "The ISI histogram of DCN neurons in GFP+ areas showed that these cells had larger ISIs compared to all other conditions , which is reflected in their decreased average firing rate ( Figure 3J ) .", "These combined in vitro electrophysiological findings suggest that knockdown of torsinA in the adult mouse cerebellum affects the intrinsic activity of at least two populations of cerebellar neurons , Purkinje cells and neurons of the DCN , likely contributing to dystonia .", "Even though the function of torsinA in the nervous system remains elusive , it is plausible that the effects of torsinA KD in cerebellar neurons could also result in cell death .", "Although patients with DYT1 exhibit no gross morphological changes in the brain , there may be more subtle cell loss in certain brain areas ( McNaught et al . , 2004 ) .", "Further , rodent models of DYT1 suggest that loss of torsinA results in apoptosis and gliosis in particular regions including the cerebellum ( Liang et al . , 2014 ) .", "To examine whether dystonic mice with torsinA KD in the cerebellum exhibited cell death , TUNEL staining of apoptotic cells was performed post-mortem on the brains of AAV9-TorsinA shRNA-GFP and AAV9-NT shRNA-GFP injected animals ( Figure 4A ) .", "An increase in apoptotic cells was identified in dystonic mice compared to control animals injected with a non-targeted shRNA ( Figure 4A ) .", "Interestingly , even though both Purkinje cells and DCN neurons were found to intrinsically fire abnormally in the dystonic mice , cell death was comparable in the cerebellar cortex of control animals injected with the non-targeting ( NT ) shRNA and those with torsinA knockdown ( Percent cell death in torsinA KD mice: 3 . 53 ± 0 . 54 compared to NT shRNA mice: 4 . 3 ± 0 . 95 ) ( Figure 4B ) .", "However , there was noticeable cell death in the DCN of mice with torsinA KD ( Percent cell death in torsinA KD mice: 12 . 59 ± 1 . 54 compared to NT shRNA mice: 5 . 43 ± 1 . 14 ) .", "Closer inspection showed that at the injection site both NT- and torsinA- shRNA injected mice had significant cell death compared to age-matched wild-type mice both in the cerebellar cortex and deep cerebellar nuclei .", "This cell death was most likely caused by the injury associated with the injection as , for both the NT and torsinA injected mice , most of the cell death was seen at the injection site .", "Nonetheless , significant cell death in the DCN was observed up to 200 µm away from the injection site in the torsinA injected animals but not in the NT injected mice ( Figure 4C ) , suggesting that the DCN is sensitive to dysfunction of torsinA and that loss of torsinA can lead to DCN cell death . 10 . 7554/eLife . 22775 . 011Figure 4 . Apoptotic cells in the deep cerebellar nuclei ( DCN ) were observed in mice with torsinA knockdown in the cerebellum . At 15 weeks post-injection , dystonic mice injected with AAV-TorsinAshRNA-GFP ( N = 6 ) , age-matched mice injected with AAV-NTshRNA-GFP ( N = 6 ) and wild-type mice ( N = 4 ) were perfused , cryosectioned and stained using a TUNEL assay protocol .", "( A ) Sections were stained for TUNEL and Hoechst .", "Representative images compare the density of TUNEL positive cells in cerebellar cortex ( Cb Cx ) and the deep cerebellar nuclei ( DCN ) .", "Dashed lines outline the Cb Cx and dotted lines outline the DCN .", "Note the increase in TUNEL positive cells ( white ) in the DCN of AAV-TorsinA shRNA-GFP injected animals .", "Bottom panels are magnified images of the DCN .", "( B ) Quantification of percent cell death in the DCN and Cb Cx .", "There is a significant increase in apoptotic cells in the Cb Cx and DCN of AAV-TorsinA shRNA-GFP injected mice and non-targeted ( NT ) injected mice .", "While there was no difference in the cerebellar cortex between the percentage of apoptotic cells in AAV-NTshRNA-GFP mice compared to AAV-TorsinAshRNA-GFP mice , there was a significant increase in apoptotic cells in the DCN of AAV-TorsinAshRNA-GFP compared to AAV-NTshRNA-GFP mice .", "Quantification of percent cell death in relation to distance from the injection site shows that most of the apoptotic cells from the AAV-NTshRNA-GFP mice were located at the injection site .", "Mice injected with AAV-TorsinAshRNA-GFP displayed a significant increase in cell death up to 200 μm from the injection site when compared to the AAV-NTshRNA-GFP mice .", "( *p<0 . 05 , **p<0 . 01 , ***p<0 . 001 , ****p<0 . 0001 Mean ± SEM ) ( Scale bar = 500 μm in images in the top panel; scale bar = 100 μm in magnified images of the DCN , bottom panels ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 22775 . 011 The difference in symptoms reported for mice with embryonic knockout of torsinA in the hind brain ( Liang et al . , 2014 ) and those with acute knockdown of torsinA in the adult is notable .", "Mice with conditional knockout of torsinA and mice with a conditional knock-in of torsinA in the cerebellum and midbrain using an En1-cre mouse line were mildly ataxic ( Liang et al . , 2014 ) , whereas mice with torsinA KD in the adult mouse cerebellum developed overt dystonic postures .", "Interestingly , the motor abnormalities of the En1-cre selective knock-in mice attenuated over time ( Liang et al . , 2014 ) , suggesting a compensatory mechanism .", "Of particular importance is the continued development of the cerebellum postnatally in humans and rodents .", "Purkinje cells continue their maturation after birth up to 28 days postnatally and granule cells undergo enormous proliferation during this time ( Hatten and Heintz , 1995 ) .", "To determine whether the severity of symptoms in mice depends on the age at which torsinA is lost , a cohort of mice was injected with AAV-TorsinA shRNA-GFP during postnatal cerebellar development 7 days after birth ( KD Juveniles ) ( Figure 5A ) .", "These animals showed knockdown of torsinA ( average torsinA levels after KD: 0 . 16 ± 0 . 025 ) comparable to that seen in mice injected as adults ( Figure 5B—figure supplement 1 ) .", "However , unlike mice injected in adulthood , mice injected in the early postnatal period developed only mild motor symptoms and no dystonia , regardless of the level of knockdown ( Figure 5C , Video 3 ) .", "This finding suggests that mice compensate for knockdown of torsinA during development , and it may explain , at least in part , why the development of dystonic animal models of DYT1 has been challenging .", "Differences in compensation for knockdown between adult and developing animals has also been shown for other neuronal proteins ( Mallucci et al . , 2002; Mukherjee et al . , 2010; Nerbonne et al . , 2008; Yuan et al . , 2005 ) .", "It is interesting to note that in patients with DYT1 , onset of dystonia usually occurs in childhood/early adolescence , the average age of onset is 12 , ( Bressman , 2004 ) in which most of the brain development has occurred .", "Therefore , it is likely that our findings in dystonic mice with acute knockdown of torsinA could be relevant to patients . 10 . 7554/eLife . 22775 . 012Figure 5 . Knockdown of torsinA in the cerebellum of mice injected at early postnatal life produces mild motor symptoms not consistent with dystonia .", "( A ) Injection of AAV-TorsinAshRNA-GFP in the cerebellum of mice at postnatal day 7 ( P7 ) results in expression of the construct throughout the cerebellum .", "( B ) Injection of AAV-TorsinAshRNA-GFP in the cerebellum produces similar knockdown in mice injected at P7 ( torsin A KD Juveniles ) and in mice injected at 6–8 weeks of age ( torsinA KD Adults ) ( Adult KD: N = 17 , Juveniles KD: N = 8 , Mann-Whitney test , p=0 . 5294 , ns ) 13 weeks after injection .", "The left image displays a representative western blot of cerebellar lysates from a non-injected mouse and a mouse injected at P7 .", "The y-axis in the right graph represents within-blot loading control normalized values .", "The shRNA against torsinA decreases expression of torsinA in vivo by approximately 80% in both adults and juveniles compared to wild-type mice .", "( C ) shRNA mediated torsinA knockdown in the cerebellum produces dystonia in a dose-dependent manner when mice are injected in adulthood , in contrast to mice injected at early postnatal life that do not develop a severe phenotype independent of the knockdown level .", "The severity of motor symptoms quantified by the dystonia scale 13 weeks after injection is correlated with the level of shRNA mediated knockdown of torsinA in mice injected at 6–8 weeks of age , such that as the percent of torsinA knockdown increases , so does the severity of the motor phenotype ( Spearman r = 0 . 63 , 95% CI 0 . 3759 to 0 . 7960 , p value < 0 . 0001 ) .", "In contrast , mice injected at early postnatal life do not develop dystonia by 13 weeks after injection , independent of the level of torsinA knockdown ( Spearman r = 0 . 1228 , 95% CI −0 . 3803 to 0 . 5698 , p value = 0 . 6387 , ns ) .", "The x-axis represents within-blot loading control normalized values .", "The y-axis represents the average of the dystonia score 13 weeks after injection from four colleagues blinded to the animal’s condition .", "Mice were grouped in bins of 20% knockdown .", "Each point represents the average of the dystonia score and percent knockdown of the mice within the bin and the lines show SEM . DOI: http://dx . doi . org/10 . 7554/eLife . 22775 . 01210 . 7554/eLife . 22775 . 013Figure 5—figure supplement 1 . TorsinA dose response curve . shRNA mediated torsinA knockdown in the cerebellum produces dystonia in a dose-dependent manner when mice are injected in adulthood , in contrast with mice injected at early postnatal life that do not develop a severe phenotype independent of the knockdown level .", "The severity of motor symptoms quantified by the dystonia scale 13 weeks after injection is correlated with the level of shRNA mediated knockdown of torsinA in mice injected at 6-8 weeks of age , such that as the percent of torsinA knockdown increases , so does the severity of the motor phenotype ( Spearman r=0 . 63 , 95% CI 0 . 3759 to 0 . 7960 , p value < 0 . 0001 , N=37 ) .", "In contrast , mice injected at early postnatal life do not develop dystonia by 13 weeks after injection , independent of the level of torsinA knockdown ( Spearman r=0 . 1228 , 95% CI -0 . 3803 to 0 . 5698 , p value = 0 . 6387 , ns , N=17 ) .", "The x-axis represents within-blot loading control normalized values .", "The y-axis represents the average of the dystonia score 13 weeks after injection from four colleagues blinded to the animal’s condition . DOI: http://dx . doi . org/10 . 7554/eLife . 22775 . 01310 . 7554/eLife . 22775 . 014Video 3 . shRNA-mediated knockdown of torsinA in the cerebellum of juvenile mice does not produce dystonic postures by 13 weeks after injection . A representative video showing a mouse 13 weeks after injection , injected with a shRNA against torsinA in the cerebellum at 1 week of age .", "In the open-field the mouse displays an unsteady gait but no dystonic postures are observed . DOI: http://dx . doi . org/10 . 7554/eLife . 22775 . 014" ], [ "Although dystonias are the third most common movement disorder we currently have few , if any , effective therapies ( Balint and Bhatia , 2014; Stacy , 2006 ) .", "A sizable fraction of dystonias are hereditary and are caused by mutations in a variety of different genes ( Bressman , 2004; Charlesworth et al . , 2013; Klein et al . , 1999; Müller , 2009; Németh , 2002; Nygaard et al . , 1999; Paudel et al . , 2012; Risch et al . , 1995 ) .", "While giant strides were made when the implicated genes were identified for a number of different hereditary dystonias , by and large to date these discoveries have not translated into new therapeutics or even more effective management of the symptoms ( Balint and Bhatia , 2014 ) .", "Plausibly , two setbacks have slowed down progress;", "( a ) we do not have a clear understanding of the functions of most of the genes identified , and", "( b ) the rodent transgenic mouse models have not shown overt symptoms , thus making identification of the relevant affected brain regions and pathways difficult .", "In the case of DYT1 , arguably both of these factors have hampered progress .", "In most cases , DYT1 is caused by a 3 bp deletion in the torsin1A gene , resulting in the loss of a single glutamate residue in the C-terminus of the protein torsinA ( Ozelius and Lubarr , 1993; Ozelius et al . , 1997 ) .", "Because this mutation causes little , if any , notable neurodegeneration in DYT1 , it is thought that dystonia results from dysfunction of neurons ( Konakova et al . , 2001; McNaught et al . , 2004; Rostasy et al . , 2003 ) .", "However , unraveling how the mutation results in neuronal dysfunction has proven to be a challenge .", "TorsinA is a member of the AAA-ATPase family of proteins and is likely to have a number of different functions in the cell ( Neuwald et al . , 1999; Vale , 2000 ) .", "Because to date the transgenic mouse models of DYT1 have failed to show overt severe dystonia , it has been difficult to examine the neural , neuronal , and even molecular substrates of dystonia with certainty ( Dang et al . , 2005; DeAndrade et al . , 2016; Goodchild et al . , 2005; Grundmann et al . , 2012 , 2007; Page et al . , 2010; Sharma et al . , 2005; Shashidharan et al . , 2005 ) .", "Much research has been carried out on non-symptomatic transgenic mice , and in a few other animal models such as C . elegans and flies ( Beauvais et al . , 2016; Caldwell et al . , 2003; Dang et al . , 2005; Goodchild et al . , 2005; Muraro and Moffat , 2006; Tanabe et al . , 2012 ) .", "These studies have attributed a number of different functions to the protein , from being a cytoskeletal and nuclear envelope component to serving as protein quality control , contributing to synaptic transmission , and forming part of synaptic vesicle machinery ( Beauvais et al . , 2016; Caldwell et al . , 2003; Goodchild et al . , 2015 , 2005; Grillet et al . , 2016; Koh et al . , 2004; Lee et al . , 2009; Muraro and Moffat , 2006 ) .", "However , the absence of faithful symptomatic mouse models has prevented critical evaluation of the importance , or even the relevance , of the various proposed functions of torsinA for dystonia .", "A good animal model with both genetic and phenotypic validity , therefore , is cardinal for scrutiny of the underlying causes of DYT1 , and for design of rational therapies for the disorder .", "It is difficult to know with certainty why most transgenic mouse models of hereditary dystonias have often yielded asymptomatic animals .", "What is clear , however , is that the failure of the approach is not because rodents do not suffer from dystonia .", "This assertion is based on the fact that there are a number of spontaneous mutations in hamsters , rats and mice that have resulted in strains that exhibit overt and profound dystonia ( Duchen , 1976; Green and Sidman , 1962; Jinnah et al . , 2005; LeDoux , 2011; Liu et al . , 2015; Löscher et al . , 1989; Wilson and Hess , 2013 ) .", "Scrutiny of these models have shown that in some cases dysfunction of the implicated murine genes also generate dystonia , ataxia , or other forms of movement disorders also in humans , thus underlying the potential suitability of mice as a model for examination of dystonia ( Charlesworth et al . , 2013; Jinnah et al . , 2005; LeDoux , 2011 ) .", "Further , there is strong evidence that other movement disorders with dystonic features can be modelled in rodents .", "For example , the symptoms of paroxysmal nonkinesigenic dyskinesia was faithfully recapitulated by modifying the causative gene Pnkd in mice ( Lee et al . , 2012 ) .", "A very plausible explanation for the lack of symptoms in some of the transgenic mouse models of dystonia may be compensation .", "Genetically , humans are invariably more sophisticated than the lower species used to model disease , and humans often use the same proteins in a far more specialized and dedicated manner .", "Consequently , the lower species are better poised to compensate for the loss of function of a gene by taking advantage of the increased genetic and signaling plasticity present during embryonic and early postnatal development ( Blendy et al . , 1996; Chesselet and Carmichael , 2012; Dow and Lowe , 2012; Pietrobon , 2002 ) .", "To underscore this point , it is noteworthy that there are numerous examples where acutely disrupting the function of a protein in mice has major consequences , but knockout of the same gene manifests as few , if any , discernable outcomes ( Daude et al . , 2012; De Souza et al . , 2006; Hall et al . , 2013; Hommel et al . , 2003; Rossdeutsch et al . , 2012; Rossi et al . , 2015; White et al . , 2016 ) .", "In fact , there are now a number of studies which demonstrate that the effect of knockdown or knockout of a particular protein in the brain can be dependent on when that protein is lost ( Erdmann et al . , 2007; Mallucci et al . , 2002; Mukherjee et al . , 2010; Nerbonne et al . , 2008; Wang et al . , 2003; Yuan et al . , 2005 ) .", "One approach to overcome compensation is to prevent or reduce the engagement of the potential compensatory mechanisms in mice by acutely targeting the causative gene in the mature animal .", "Such an approach was successfully employed to generate a faithful animal model of RDP , a movement disorder caused by loss of function mutations in the Na/K pump ( Calderon et al . , 2011; Fremont et al . , 2014 , 2015 ) .", "In RDP , subjects carrying the mutation often lead a relatively healthy life until they are exposed to a very stressful event , at which point they rapidly develop severe dystonia and Parkinsonism-like symptoms ( Brashear et al . , 2007 , 1996 ) .", "Similar to other mouse models of hereditary dystonias , transgenic mice harboring loss of function mutations in the Na/K pump have generally failed to exhibit overt dystonia ( Clapcote et al . , 2009; DeAndrade et al . , 2011; Moseley et al . , 2007 ) .", "However , in contrast to prior dystonia-related proteins , the function of the Na/K pump as an ion transporter is well-understood and the availability of an exquisitely selective blocker allowed Calderon et al . to generate a pharmacologic mouse model of RDP which closely paralleled the disorder in humans ( Calderon et al . , 2011 ) .", "While partially blocking the function of the Na/K pump in the basal ganglia and the cerebellum of adult mice resulted in mild motor dysfunction , stressing these mice precipitated severe dystonia and Parkinsonism-like symptoms that persisted thereafter ( Calderon et al . , 2011 ) .", "Further scrutiny of this mouse model surprisingly revealed that dystonia was instigated by the dysfunction of the cerebellum , whereas the Parkinsonism-like features were of basal ganglia origin ( Calderon et al . , 2011; Fremont et al . , 2014 ) .", "The success of the pharmacologic model of RDP in replicating the human disorder was recapitulated in vivo in adult mice using short hairpin RNAs to reduce the expression of the Na/K pump in select brain regions ( Fremont et al . , 2015 ) .", "Reassuringly , the shRNA approach fully corroborated the findings of the pharmacologic model , underscoring a potentially causal role for the cerebellum in generation of dystonia in RDP .", "The relevance of this finding to the human disorder has been strengthened by the fact that recent postmortem histological studies have identified cerebellar degeneration in RDP ( Sweadner et al . , 2016 ) .", "Moreover , it has also been recently reported that in some patients mutations in the Na/K pump can result in Adult Rapid Onset Ataxia rather than dystonia ( Sweadner et al . , 2016 ) , a finding that highlights the role of the cerebellum in the disorder and further validates the pharmacologic and shRNA mouse models .", "The success of the acute knockdown models of RDP in replicating the relevant features of the human disorder prompted us to generate a comparable rodent model of DYT1 to explore the possibility that compensation might have hindered efforts in generating faithful transgenic mouse models of DYT1 ( Dang et al . , 2005; DeAndrade et al . , 2016; Goodchild et al . , 2005; Grundmann et al . , 2012 , 2007; Page et al . , 2010; Sharma et al . , 2005; Shashidharan et al . , 2005 ) .", "Differential compensation in mice compared to humans might be a particularly significant problem in the case of torsinA .", "This is because expression studies demonstrate that the level of torsinA transcript and protein in neural tissue is markedly different between the developing mouse and human brain ( Siegert et al . , 2005; Vasudevan et al . , 2006; Xiao et al . , 2004 ) .", "In humans torsinA protein is not detectable in neurons until one month after birth when expression abruptly increases to levels maintained throughout adulthood ( Siegert et al . , 2005 ) .", "In contrast , in the rodent brain torsinA is present at high levels embryonically and peaks during the early postnatal period , decreasing rapidly within a couple of weeks postnatally before stabilizing ( Vasudevan et al . , 2006 ) .", "Based on this expression pattern it could be argued that if torsinA is necessary for the development of CNS in the mouse but not the human brain , then the mouse brain might more aggressively engage compensatory mechanisms to overcome loss of torsinA during this crucial period than might the human brain .", "Indeed , the most symptomatic transgenic mouse models to date exhibiting mild abnormal twisting movements were generated using Nestin-cre mice ( Liang et al . , 2014 ) .", "Even though recombination begins at E11 in Nestin-cre mice , it increases gradually until early postnatal life , which temporally delays neuronal torsinA dysfunction during the gestation period ( Liang et al . , 2012 ) .", "To bypass a potentially powerful developmental compensation in mice , we knocked down torsinA in six weeks old adult animals , at a time when cerebellar and basal ganglia development is complete ( White and Sillitoe , 2013 ) .", "In agreement with the hypothesis that developmental compensation in mice had prevented generation of symptomatic transgenic mice , we found that acutely knocking down torsinA in the cerebellum of adult mice resulted in overt and severe dystonia .", "As evidence that compensation plays a major role in the lack of overt symptoms observed in most transgenic mouse models of DYT1 , we found that comparably knocking down torsinA in juvenile mice at postnatal day 7 , before cerebellar development is completed , did not produce dystonia .", "It is important to note that no animal model of a human disorder is ever without a flaw .", "While torsinA loss of function is present throughout development in human patients , the model described here acutely knocks down torsinA during adulthood , which clearly does not faithfully replicate the human disease .", "Moreover , it is also unknown whether in humans , a developmental disruption in basal ganglia function contributes to dystonia .", "However , given that in humans torsinA is not detectable in the brain until four weeks postnatal ( Vasudevan et al . , 2006; Siegert et al . , 2005 ) , the shRNA approach described might be a more appropriate mouse model of what occurs in humans than the models that reduce expression of torsinA developmentally when it is expressed at relatively high levels in the rodent brain .", "It is difficult to know what the potential developmental compensatory mechanisms are by which the rodent brain effectively tackles loss of torsinA .", "While it is possible that compensation engages a number of distinct and parallel processes , the most parsimonious possibility might be the presence of functional redundancy in the family of torsin proteins .", "In addition to torsinA , there are three other members of the torsin family ( Ozelius et al . , 1999 ) .", "One of these proteins , torsinB , is a related homologue to torsinA with 70% homology ( Ozelius et al . , 1999 ) .", "Both torsinA and torsinB proteins are localized to the endoplasmic reticulum and nuclear envelope membrane system in cells throughout the brain and non-neural cells ( Naismith et al . , 2004 ) .", "The first evidence of functional redundancy for torsinA was noted in studies performed in DYT1 animals .", "In DYT1 KI and KO mice , nuclear envelope budding was observed only in neurons in the CNS and no nuclear envelope budding was found in non-neuronal cells or in non-CNS tissue even though torsinA is expressed in all these tissues ( Goodchild et al . , 2005 ) .", "However , when torsinB was knocked down in fibroblasts from a DYT1 mutant background , nuclear envelope defects were seen in non-neuronal cells as well , demonstrating that torsinB can compensate for loss of torsinA ( Kim et al . , 2010 ) .", "To determine whether a similar compensation can occur in the CNS , Tanabe et al used embryoid bodies derived from DYT1 KI animals and showed that either torsinA or torsinB can individually rescue the abnormal nuclear envelope budding when the cells are differentiated into neural lineage ( Tanabe et al . , 2016 ) .", "Interestingly , torsin2A also appeared to rescue nuclear envelope budding to some extent ( Tanabe et al . , 2016 ) .", "Whether these redundancies can compensate for all the various functions assigned to torsinA , or whether the ability to compensate is shared amongst human torsin proteins is unknown .", "The presence of such forms of redundancies in rodents may explain , at least in part , the difficulty in recapitulating the human phenotypic symptoms of DYT1 in mice .", "It is also noteworthy that expression of torsinB like torsinA , differs temporally between mice and humans ( Siegert et al . , 2005; Tanabe et al . , 2016; Vasudevan et al . , 2006 ) adding yet another level of complication to modeling DYT1 in rodents .", "If compensation is indeed the mechanism by which the mouse brain can developmentally overcome the shortcomings associated with loss of torsinA function , it suggests that there might be a potentially powerful therapeutic opportunity – understanding the compensatory mechanisms in mice might shed light on strategies for management of torsinA dysfunction in humans .", "It is tempting to speculate that perhaps some of the same compensatory mechanisms , alas not as effectively or on every occasion , might be engaged in humans and might account for the partial ( 30–40% ) penetrance of DYT1 in humans ( Argyelan et al . , 2009; Eidelberg et al . , 1998; Martino et al . , 2013; Ozelius and Lubarr , 1993 ) .", "For a number of years neuroimaging studies in patients with DYT1 have hinted that the cerebellum is involved in dystonia ( Carbon et al . , 2010b , 2008a , 2008b; Eidelberg et al . , 1998; Trost et al . , 2002; Zoons et al . , 2011 ) .", "However , to date it has been difficult to examine the role of the cerebellum in DYT1 dystonia using animal models because region specific transgenic mice have generally failed to recapitulate overt dystonic symptoms ( Liang et al . , 2014; Pappas et al . , 2015; Weisheit and Dauer , 2015; Yokoi et al . , 2011 , 2008; Zhang et al . , 2011 ) , as discussed perhaps likely because of developmental compensation .", "Our approach allowed for targeted knockdown of torsinA in select brain regions including the cerebellum , and provided strong evidence that the cerebellum may be causative for instigating dystonia in DYT1 , thus supporting the findings of the neuroimaging studies done in patients .", "Based on patient studies , the notion that the cerebellum might be involved in some dystonias has been around for decades and a number of reports have unequivocally documented that the cerebellum is abnormal in a number of different dystonias ( Alarcón et al . , 2001; Carbon et al . , 2010a; Filip et al . , 2013; Jinnah and Hess , 2006; Neychev et al . , 2008; Prudente et al . , 2014; Simonati et al . , 1997; Trost et al . , 2002 ) .", "However , human studies are limited in their scope and have not been in a position to determine whether the cerebellum causally contributes to dystonia , or whether the noted abnormalities are compensatory in nature .", "Given the diversity of dystonias , it would be surprising if both cases are not rampant .", "Nonetheless there are clear examples of dystonia in patients that appear to be causally related to cerebellar dysfunction , particularly when dystonia manifests as a symptom in cerebellar-centric disorders such as spinocerebellar or episodic ataxias ( Bang et al . , 2003; Kawarai et al . , 2016; Mariotti et al . , 2007; Muglan et al . , 2016; Nakagaki et al . , 2002; Sethi and Jankovic , 2002 ) .", "In some cases , lesioning or stimulation of the cerebellum has been documented to improve the symptoms , corroborating the notion that in some patients , dystonia can arise from cerebellar dysfunction ( Koch et al . , 2014; Panov et al . , 2013; Teixeira et al . , 2015 ) .", "Our dystonic mouse model of DYT1 allowed us to explore how cerebellar dysfunction causes dystonia .", "We found , similar to what was noted some twenty years ago in the dystonic rat and more recently in dystonic mouse models of RDP , that cerebellar-induced dystonia is associated with bursting cerebellar output ( Fremont et al . , 2014 , 2015; LeDoux and Lorden , 1998 ) .", "Our data along with a number of other mouse models of dystonia suggest that a bursting cerebellar output may be a hallmark of cerebellar-induced dystonia ( Fremont et al . , 2014; LeDoux et al . , 1998; LeDoux and Lorden , 1998; Lorden et al . , 1992 ) .", "Notably , similar bursting cerebellar output has also been documented in a dystonic patient undergoing surgery ( Slaughter et al . , 1970 ) .", "It is important to note that in the case of cerebellar-induced dystonia animal studies suggest that silencing , lesioning or otherwise normalizing cerebellar output would be an effective therapeutic approach ( LeDoux et al . , 1993; Neychev et al . , 2008 ) , and recent reports document that cerebellar deep brain stimulation and transcranial magnetic stimulation might also be effective in lessening dystonia in patients ( Koch et al . , 2014; Teixeira et al . , 2015 ) .", "It is intriguing that cerebellar-induced dystonia is seemingly indistinguishable from dystonia caused by the dysfunction of the basal ganglia .", "A plausible explanation might be that the aberrant cerebellar output ultimately disrupts basal ganglia function to the extent that the disorder manifests as if the dysfunction had originally arisen from the basal ganglia .", "Scrutiny of the dystonic mouse model of RDP suggests that the disynaptic projection from the cerebellum to the dorsolateral basal ganglia via the thalamus , which is present both in rodents and nonhuman primates , might be the conduit by which the abnormal cerebellar output disrupts basal ganglia function to cause dystonia ( Chen et al . , 2014 ) .", "Indeed , severing the link between the cerebellum and basal ganglia by either silencing the relevant thalamic neurons , or lesioning the appropriate region can effectively alleviate cerebellar-induced dystonia in mice ( Calderon et al . , 2011; Chen et al . , 2014 ) .", "With regards to DYT1 , what remains to be established is how loss of torsinA function disrupts cerebellar activity .", "Recently , PNKD , the protein disrupted in paroxysmal nonkinesigenic dyskinesia was found to play a crucial role in neurotransmitter release by regulating exocytosis ( Shen et al . , 2015 ) .", "Several roles for torsinA have been suggested by a number of studies including potential changes in synaptic transmission , endoplasmic reticulum ( ER ) stress , and abnormal levels of eukaryotic initiation factor 2α ( eIF2α ) in the dystonic phenotype in DYT1 ( Beauvais et al . , 2016; Rittiner et al . , 2016 ) .", "Our data provide little mechanistic insight as to the cause of cerebellar dysfunction , although it is clear that at the very least the intrinsic activity of both Purkinje cells and DCN neurons are altered by loss of torsinA .", "The change in intrinsic activity could be due to a direct effect of loss of torsinA , or indirectly through alterations in synaptic transmission .", "The animal model described here provides a valuable platform for future mechanistic studies aimed at elucidating the cellular and molecular pathways that contribute to dystonia in DYT1 .", "To date , the neural substrates of dystonia have been unclear with studies in patients implicating abnormal activity in multiple motor-related areas and circuits ( Neychev et al . , 2011; Zoons et al . , 2011 ) .", "While our findings are not the first to suggest a role for the cerebellum in dystonia ( LeDoux et al . , 1993; Pizoli et al . , 2002 ) , we provide compelling evidence that abnormal cerebellar output may play a role in DYT1 , the most common inherited dystonia .", "Further , the model we have generated faithfully recapitulates the most common and debilitating symptom in DYT1 , dystonia and therefore provides a platform for gaining further understanding into this disorder and developing novel therapeutics ." ], [ "Two different shRNAs against unique aspects of the sequence of each protein examined were identified .", "The sequences were originally generated by the RNAi consortium .", "For torsinA , the identified sequences correspond to TRCN0000008485 ( 5’-CCGGCCGGAACCTCATAGATTATTTCTCGAGAAATAATCTATGAGGTTCCGGTTTTT-3’ ) and TRCN0000008487 ( 5’-CCGGGCTGCAGAAAGATCTGGATAACTCGAGTTATCCAGATCTTTCTGCAGCTTTTT-3’ ) .", "Lentiviruses containing these shRNAs were generated by the Albert Einstein College of Medicine Lentiviral Core , and the efficacy of knockdown was tested in vitro in mouse cortical cultures ( data not shown , see below and above ) .", "Albert Einstein College of Medicine Lentiviral Core generated plasmids containing the effective shRNA sequences .", "AAV9 compatible plasmids containing each shRNA were produced commercially ( Virovek , AAV9-U6-shRNA-CMV-GFP , AAV9-U6-shRNA2-CMV-GFP , AAV9-U6-shRNA119308-CMV-GFP ( Lot# 12–183 ) ) .", "The same company also generated AAV9 virus with an average titer of 2 × 1013 vg/ml .", "A 0 . 22 µm filter sterilized solution containing the virus in DPBS buffer with 0 . 001% pluronic F-68 was directly injected into different brain regions of adult mice .", "Control AAV9 containing non-targeted ( NT ) shRNAs under the same promoter containing the same CMV driven GFP and at an equivalent titer were purchased from Virovek as was AAV9-CMV-GFP .", "The NT-shRNA used were: AAV9-U6-Non-targeted-Control-CMV-GFP ( ( 5'-GAGGATCAAATTGATAGTAAACCGTTTTGGCCACTGACTGACGGTTTACTATCAATTTGATCCTCTTTTT-3' ) Lot# 13–234 , Virovek ) and AAV9-U6-Non-targeted-Control-CMV-GFP ( ( 5'-CCAACTACCCGAACTATTATTCAAGAGATAATAGTTCGGGTAGTTGGCATTTTTT-3' ) Lot# 13–037 , Virovek ) Before injection into live animals , all shRNAs were tested in vitro to determine a relative knockdown efficiency .", "Cortical neuron cultures were prepared from embryonic day 16 ( E16 ) C57BL/6 mice ( Charles Rivers ) .", "The pregnant mouse was anesthetized using isoflurane ( 5% ) and the embryos were dissected out and transferred to ice-cold HEPES-Glucose buffer ( HEPES 10 mM ( Fisher ) , Glucose 33 mM ( Fisher ) , in PBS ( Corning ) ) .", "Embryos were decapitated and cortices were dissected from the rest of the brain and incubated in HEPES-Glucose buffer containing Trypsin at 37°C for 15 min .", "Trypsin was washed with HEPES-Glucose buffer then replaced by DMEM ( Dulbecco's Modified Eagle Medium , GIBCO ) fortified with 10% FBS ( Fetal Bovine Serum , GIBCO ) .", "Cortical tissues were fragmented by pipetting with a Pasteur pipette heated to obtain a narrow bore .", "The cells were plated in 24-well culture plates at a density of 20 , 000 per cm2 .", "Cultures were incubated at 37°C for 2 hr .", "The culture media was replaced with Neurobasal medium ( GIBCO ) fortified with B27 supplement ( 2% , GIBCO ) and Glutamax ( 0 . 25% , GIBCO ) .", "After 5 days , the cultures were transfected with adeno-associated viruses ( AAV ) carrying shRNA against the target protein’s mRNA .", "Cultures were then incubated for 10 days at 37°C .", "Neuronal lysates were subsequently collected using lysis buffer ( SDS 2% , Tris 50 mM , EDTA 2 mM ) .", "Protein lysates were prepared and homogenized in an SDS-based lysis buffer containing 2% SDS , 50 mM Tris and 2 mM EDTA ( pH = 8 ) .", "Brain lysates were prepared on ice .", "Lysates from cultures were prepared at room temperature .", "All lysates were incubated at room temperature for 30 min for protein digestion , and then sonicated and frozen at −20°C .", "The protein concentration was determined by BCA assay ( Pierce ) .", "Samples were run on SDS page gel ( BioRad ) and transferred to PVDF membrane ( BioRad ) .", "Membranes were blocked with milk and subsequently incubated with primary TorsinA antibody for 2 hr or less at room temperature on a rocker followed by the secondary antibody for 30 min .", "PVDF membranes were developed using chemiluminescence and signals were quantified using ImageJ to perform densitometry .", "The following primary antibodies were used: torsinA ( Abcam 34540 , 1:1000 ) , beta-tubulin ( Sigma T5201 , 1:10 , 000 ) , GAPDH ( Cell Signaling 2118 1:5000 ) , B-actin ( Sigma A1978 1:20 , 000 ) .", "Beta-tubulin was used as a loading control in all studies because it is an abundant housekeeping protein .", "All injections were performed in a single surgery .", "During the same surgery , EMG wires and/or a bracket for in vivo recordings were also implanted .", "AAV9-shRNA-GFP was injected in mice at 4 sites of the cerebellum .", "At a rate between 0 . 1 and 0 . 15 μl/min , a total volume of two microliters was injected at each site .", "After the injection , the syringe was left in place for a minimum of 10 min before it was removed .", "The coordinates used were: ( AP:−6 mm , ML:0 mm , DV:−1 . 5 mm ) , ( AP:−6 . 96 mm , ML:0 mm , DV: 1 . 5 mm ) , ( AP: −6 mm , ML: 1 . 8 mm , DV: 2 . 3 mm ) , ( AP: −6 mm , −1 . 8 mm , DV: 2 . 3 mm ) .", "Striatum and globus pallidus: Injections were performed at four sites with two microliters injected at each site at rates identical to cerebellar injections .", "The coordinates used were: ( AP: 0 . 5 mm , ML: 2 mm , DV: 2 . 5 mm ) , ( AP: 0 . 5 mm , ML: −2 mm , DV: 2 . 5 mm ) , ( AP: −0 . 5 mm , ML: 2 . 5 mm , DV: 3 . 5 mm ) , ( AP: −0 . 5 mm , ML: −2 . 5 mm , DV: 3 . 5 mm ) .", "Injections into the substantia nigra were performed at 4 sites with 0 . 5–1 µl infused at each site at a rate of 0 . 05 µl per minute .", "The injections coordinates were: ( AP: −3 mm , ML: 1 . 5 mm , DV: 4 mm ) , ( AP: −3 mm , ML: −1 . 5 mm , DV: 4 mm ) , ( AP: −3 . 6 mm , ML: 1 mm , DV: 4 mm ) , ( AP: −3 . 6 mm , ML: −1 mm , DV: 4 mm ) .", "Mice were trans-cardially perfused with 1X PBS followed by 4% paraformaldehyde .", "Brains were then extracted , kept at four degrees overnight in 4% paraformaldehyde and switched into a 30% sucrose solution before embedding them in Tissue-Tek OCT compound .", "Brains were sectioned at a 50 µm thickness .", "Sections were either Nissl stained or stained with an anti-GFP antibody ( primary: 1:500 , A11122 , molecular probes; secondary: 1:1000 , A11008 , molecular probes ) and Hoechst 3342 ( 1:1000 , H3570 , molecular probes ) .", "Images were taken on a Zeiss Axioskop two plus .", "The presence of dystonia and its severity were quantified using a previously published scale ( Calderon et al . , 2011 ) .", "Briefly , 0 = normal behavior; 1 = abnormal motor behavior , no dystonic postures; 2 = mild motor impairment , dystonic-like postures when disturbed; 3 = moderate impairment , frequent spontaneous dystonic postures; 4 = severe impairment , sustained dystonic postures .", "The videos were assessed independently by four observers who were blinded to the animal’s condition .", "The observers were trained with a video set containing representative examples for each score in which key characteristics were highlighted .", "Electrodes were inserted into the gastrocnemius muscle ( extensor ) and anterior tibialis muscle ( flexor ) of the same leg and wires were routed under the skin to a connector fixed to the skull on animals with a dystonia score greater than 2 .", "Immediately prior to recordings , a head-stage for the Pinnacle EEG/EMG recording system 4100 was secured to the connector .", "For 1–10 min , recordings of muscle activity were performed in the open field while mice were simultaneously videotaped .", "Afterward , the connector was removed and the animal was placed back into the home cage .", "Adult mice were anesthetized with isoflurane and decapitated .", "Sagittal slices of 300 µm thickness were cut from the cerebellum at a temperature of 35°C and then transferred to room temperature after 1 hr .", "Extracellular recordings were obtained from single Purkinje cells using a custom-built amplifier and glass pipette electrodes filled with extracellular solution ( in mM NaCl 125 , KCl 2 . 5 , NaHCO3 26 , NaH2PO4 1 . 25 , MgCl2 1 , CaCl2 2 , glucose 10 , pH 7 . 4 when gassed with 5%CO2:95%O2 ) .", "Slices were recorded on an upright microscope ( Zeiss ) and perfused with 35°C extracellular solution at a rate of 1 . 5 ml/min during recording sessions .", "The perfusion solution contained picrotoxin ( 10 uM ) and CGP55845 ( 1 µM ) to block inhibitory synaptic transmission and kynurenic acid ( 5 mM ) to block excitatory transmission .", "Data was sampled at 10 kHz using an analog to digital converter ( National Instruments SCB-68 ) and analyzed using custom-written LabView software ( available upon request ) .", "Mice were implanted with an L-shaped bracket that was fixed onto the skull with bone screws and dental cement .", "A recording chamber was drilled in the skull on top of the cerebellum , surrounded with dental cement and covered with surgi-foam and bone wax .", "Single-unit neural activity was recorded extracellularly using a tungsten electrode ( Thomas Recording , 2–3 MΩ ) , which was advanced into the cerebellum until either the Purkinje cell layer or the deep cerebellar nuclei were reached .", "Purkinje cells were identified by location , characteristic firing rate , the presence of complex spikes , and post-hoc histology .", "DCN neurons were identified by location , firing rate , and post-hoc histology .", "Signals were filtered ( 200 Hz–20 kHz ) and amplified ( 20000x ) on a custom built amplifier and then digitized ( 20 kHz ) using a National Instruments BNC-2110 .", "Waveforms were sorted offline using characteristics of the spikes such as amplitude and energy , as well as those determined by principal component analysis ( Plexon ) .", "TorsinA injected animals began exhibiting dystonic postures ~13 weeks after injection .", "At 15 weeks post-injection AAV9-TorsinA shRNA-GFP and AAV9-NT shRNA-GFP animals were trans-cardially perfused with 1X PBS followed by 4% paraformaldehyde ( PFA ) .", "Brains were removed , incubated at 4°C overnight in 4% PFA and then transferred to a 30% sucrose solution .", "Brains were then cryopreserved in an OCT compound ( Tissue-Tek ) .", "The cerebellum was cryosectioned coronally in 30 μm slices using a cryostat maintained at −20°C .", "Slides were imaged using a Zeiss Axioskop two microscope to identify the injection sites .", "Slides containing sections up to 1 mm lateral to the injection site were stained using the TUNEL protocol ( Roche ) to label cells undergoing apoptosis .", "Slides were also co-stained with Hoechst to quantify the total number of cells .", "The number of TUNEL positive ( red ) and Hoechst positive cells ( blue ) were quantified in 300 × 300 µm sections imaged at different distances from the injection sites .", "The ratio of apoptotic cells/total number of cells was determined and then used to compare cell loss in the cerebellar cortex and DCN .", "Images were acquired from up to 1 mm lateral , anterior , posterior , and ventral from all four injection sites in each animal .", "For comparing characteristics of firing a student’s t-test was used unless otherwise stated .", "For analysis of behavioral data on the dystonia scale , a Friedman test with post-hoc Dunn’s multiple comparison’s test or a Mann-Whitney test were used to determine significance .", "In all cases data was considered statistically different from control when p<0 . 05 .", "Sample size was not determined using a priori power analysis , but was based on the statistical criteria for significance in observations .", "Experiments were repeated in at least five mice , and the number of samples are noted in text and the appropriate figure legend ." ] ]

[ "DYT1 is a debilitating movement disorder caused by loss-of-function mutations in torsinA .", "How these mutations cause dystonia remains unknown .", "Mouse models which have embryonically targeted torsinA have failed to recapitulate the dystonia seen in patients , possibly due to differential developmental compensation between rodents and humans .", "To address this issue , torsinA was acutely knocked down in select brain regions of adult mice using shRNAs .", "TorsinA knockdown in the cerebellum , but not in the basal ganglia , was sufficient to induce dystonia .", "In agreement with a potential developmental compensation for loss of torsinA in rodents , torsinA knockdown in the immature cerebellum failed to produce dystonia .", "Abnormal motor symptoms in knockdown animals were associated with irregular cerebellar output caused by changes in the intrinsic activity of both Purkinje cells and neurons of the deep cerebellar nuclei .", "These data identify the cerebellum as the main site of dysfunction in DYT1 , and offer new therapeutic targets ." ]

[ "Dystonia is the third most common type of movement disorder after Parkinson’s disease and tremor .", "Patients with dystonia experience prolonged involuntary contractions of their muscles , often causing uncontrollable postures or repetitive movements .", "Almost thirty years ago , genetic studies revealed that a mutation in the gene that encodes a protein called torsinA causes the most common type of dystonia , called DYT1 .", "Exactly how mutations that affect the torsinA protein give rise to DYT1 remains unclear , and there are still no effective treatments for the disorder .", "Part of the problem is that we do not fully understand how torsinA works , or which of its many proposed functions is relevant to dystonia .", "Moreover , attempts to study DYT1 using genetically modified mice have proved largely unsuccessful .", "This is because mice that simply express the same genetic mutations that cause dystonia in humans do not show the overt symptoms of dystonia .", "Fremont , Tewari et al . have now generated a mouse ‘model’ that does show symptoms of dystonia , and used these model mice to investigate the role of torsinA in the disorder .", "Acutely reducing the amount of torsinA protein in a region of the brain called the cerebellum induced the symptoms of dystonia in the mice .", "Conversely , reducing the amount of torsinA in a different brain area known as the basal ganglia had no such effect , even though both the cerebellum and the basal ganglia contribute to movement .", "Furthermore , neither manipulation had any effect in juvenile mice , which suggests that , in contrast to humans , young mice can compensate for the loss of torsinA .", "Fremont , Tewari et al . also found that the loss of torsinA causes the cerebellum to generate incorrect output signals , which in turn trigger the abnormal movements seen in dystonia .", "In the future , further studies of the model mice could identify the exact changes that occur in neurons following the loss of torsinA from the cerebellum .", "Understanding these changes could potentially pave the way for developing effective treatments for DYT1 and other dystonias ." ]

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

[ "neuroscience" ]

[ [ "Learning is an integral part of our everyday life and necessary for survival in a dynamic environment .", "The behavioral changes arising from learning have quite successfully been described by the reinforcement learning principle ( Sutton and Barto , 1998 ) , according to which biological agents continuously adapt their behavior based on the consequences of their actions .", "Thus , reinforcement learning models and most other learning models depend on feedback from the environment .", "Yet , there are important instances of learning where no such external feedback is provided , challenging the generality of these learning models in shaping our behavior .", "A well-studied case of learning is the improvement of performance in perceptually demanding tasks through training or repeated exposure ( Gibson , 1963 ) .", "Such perceptual learning has repeatedly been demonstrated to occur without feedback ( Herzog and Fahle , 1997; Gibson and Gibson , 1955; McKee and Westheimer , 1978; Karni and Sagi , 1991 ) and is therefore ideally suited as a test case to study learning in the absence of external feedback .", "Previous work has emphasized the role of reinforcement learning in perceptual learning ( Kahnt et al . , 2011; Law and Gold , 2009 ) .", "However , these accounts were based on perceptual learning with external feedback and therefore cannot account for instances in which learning occurs without external feedback .", "Here , we pursued the idea that , in the absence of external feedback , learning is guided by internal feedback processes that evaluate current perceptual information in relation to prior knowledge about the sensory world .", "We reasoned that introspective reports of perceptual confidence could serve as a window into such internal feedback processes .", "In this scenario , low or high confidence would correspond to a negative or positive self-evaluation of one’s own perceptual performance , respectively .", "Accordingly , confidence could act as a teaching signal in the same way as external feedback in normative theories of reinforcement learning ( Daniel and Pollmann , 2012; Hebart et al . , 2014 ) .", "Applied to the case of perceptual learning , a confidence-based reinforcement signal could serve to strengthen neural circuitry that gave rise to high-confidence percepts and weaken circuitry that led to low-confidence percepts , thereby enhancing the quality of future percepts .", "We tested this idea in a challenging perceptual learning task , in which participants continuously reported their confidence in perceptual choices while undergoing functional magnetic resonance imaging ( fMRI ) .", "No external feedback was provided; instead , confidence ratings were used as a proxy of internal monitoring processes .", "To account for perceptual learning in the absence of feedback , we devised a confidence-based associative reinforcement learning model .", "In the model , confidence prediction errors ( Daniel and Pollmann , 2012 ) serve as teaching signals that indicate the mismatch between the current level of confidence and a running average of previous confidence experiences ( expected confidence ) .", "Based on recent evidence of confidence signals in the mesolimbic dopamine system ( Daniel and Pollmann , 2012; Hebart et al . , 2014; Schwarze et al . , 2013 ) , we hypothesized to find neural correlates of confidence prediction errors in mesolimbic brain areas such as the ventral striatum and the ventral tegmental area .", "Since confidence prediction errors act as a teaching signal in our model , we hypothesized that the strength of these mesolimbic confidence signals should be linked to individual perceptual learning success ." ], [ "To establish stimulus-specific perceptual learning , we compared perceptual thresholds in pre- and post-experimental sessions between the trained and untrained reference axis ( Figure 2A ) .", "The contrast thresholds improved for the trained ( t28 = 6 . 73 , p < 0 . 001 , two-tailed ) , but not for the untrained reference axis ( t28 = 0 . 41 , p = 0 . 68; interaction of training × time: F1 , 28 = 14 . 2 , p < 0 . 001 ) , demonstrating clear and specific effects of perceptual learning .", "These stimulus-specific training effects could still be detected 10 weeks later ( F1 , 28 = 4 . 3 , p = 0 . 047 ) , indicating long-term stability and thus demonstrating a key characteristic of perceptual learning ( Karni and Sagi , 1993 ) .", "To test whether the effects of learning could already be detected during the training session , we linearly fitted the contrast thresholds across trials in the critical constant performance condition .", "The analysis showed that contrast threshold consistently decreased across runs ( linear slope: −0 . 006 ± 0 . 002 , t28 = −2 . 38 , p = 0 . 024 ) , from 8 . 64% ± 0 . 47 ( mean ± SEM ) in the first training run to 7 . 68% ± 0 . 52 in the last training run . 10 . 7554/eLife . 13388 . 004Figure 2 . Behavioral results .", "( A ) Contrast thresholds across the runs of the training session and in the three test-sessions ( pre/post/long-term ) .", "( B ) Relationship between confidence ratings and performance during the training session .", "Percent correct responses were computed by means of a sliding window across sorted confidence values ( window size: 5% of all trials ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 13388 . 00410 . 7554/eLife . 13388 . 005Figure 2—figure supplement 1 . Eyetracking . Heatmap indicating the percentage of eye gaze position at every pixel of the screen .", "The red circle indicates the area that contained 98% of all eye gaze positions and the white circle depicts the area covered by the Gabor patch .", "On average , 98 . 5 ± 0 . 6% of recorded eye gaze positions during the training session were within the fixation area ( radius r = 2 . 5° of visual angle ) , demonstrating that the participants maintained fixation throughout the fMRI experiment .", "Please note , that one participant was excluded due to fixation failure ( <95% ) and iDOI: http://dx . doi . org/10 . 7554/eLife . 13388 . 00510 . 7554/eLife . 13388 . 006Figure 2—figure supplement 2 . Confidence ratings .", "( A ) Distribution of confidence ratings at the single-subject level .", "( B ) Distribution of the pooled response times of all participants .", "The median response time was 2 . 47 s .", "There was a modest negative relationship between reaction time and confidence ( mean ± SE of individual z-transformed correlation coefficients: rPearson = −0 . 06 ± 0 . 02; one-sample t-test against Fisher z’ = 0: t28 = −3 . 3 , p0 . 002 ) .", "The correlation with choice accuracy was not significant ( rPearson = −0 . 02 ± 0 . 01 , t28 = −1 . 5 , p=0 . 14 ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 13388 . 006 To assess whether participants adequately used the continuous confidence rating scale during training , the relationship between reported confidence and performance ( proportion correct ) was analyzed by means of a sliding window across sorted confidence values .", "The performance increased monotonically with confidence ( main effect of percentile: F22 , 2442 = 5 . 76 , p < 0 . 001 , one-way ANOVA with repeated measures ) , approaching chance at low levels of confidence , without showing ceiling effects at high levels of confidence ( Figure 2B ) .", "This pattern indicates a close link between the decisional certainty of the choice and the reported confidence and shows that confidence represents valuable self-generated feedback .", "To account for perceptual learning without external feedback , we devised an associative reinforcement learning model with confidence as internal feedback ( Figure 3 ) .", "Learning in this model was guided by the combination of a confidence-based reinforcement signal and Hebbian plasticity , inspired by the previously proposed three-factor learning rule ( dopaminergic reinforcement signal , pre-synaptic activity , post-synaptic activity ) for neural plasticity in the mesolimbic system ( Reynolds et al . , 2001; Schultz , 2002 ) .", "Our model assumes that observers improve perceptual performance by optimizing a filter on incoming sensory evidence .", "The filter is represented by two components: signal weights for clockwise ( cw ) and counterclockwise ( ccw ) stimulus orientation ( wccw , ccw; wcw , cw ) , connecting orientation energy detectors Eccw/cw to decision units Accw/cw with same orientations; and noise weights ( wccw , cw; wcw , ccw ) , connecting detectors Eccw/cw to decision units Acw/ccw with opposing orientations .", "The clockwise and counterclockwise orientation energy contained in the stimuli is computed by a simple model of primary visual cortex ( Petrov et al . , 2005 ) .", "The weighted sums of Eccw/cw determine the activities of decision units Acw/ccw which , in a next step , are integrated in a decision value DV = Acw − Accw .", "DV translates into probabilities for clockwise or counterclockwise choices via a softmax action selection rule , and to the model’s equivalent of confidence—decisional certainty—through its absolute value .", "Finally , perceptual learning is based on an associative reinforcement learning rule with two separate components: a reinforcement component utilizes a confidence prediction error ( CPE; denoted as δ ) as internal feedback , representing the mismatch between current confidence and a long-term estimate of expected confidence ( via a learning rate αc ) ; and a Hebbian component ensures that the weights are updated in proportion to how strongly orientation detectors and decision units co-activate .", "In addition , a learning rate αw accounts for inter-individual differences in learning speed .", "Figure 3—figure supplement 1 provides an exemplary time slice of one participant's behavioral reports and accompanying model variables . 10 . 7554/eLife . 13388 . 007Figure 3 . Confidence-based model of perceptual learning . Counterclockwise ( Ecw ) and clockwise ( Eccw ) orientation energy detectors of a dedicated representational subsystem are connected via signal weights ( horizontal ) and noise weights ( diagonal ) to decision units ( Accw , Acw ) .", "Reported choices ( decisions ) d are probabilistically modeled by a decision value DV = Accw− Acw and the reported confidence c is modeled through the absolute value of x .", "Weights are updated through an associative reinforcement learning update rule .", "The reinforcement component is based on a confidence prediction error δ , reflecting the difference between reported confidence and a weighted running average of previous confidence experiences ( expected confidence c¯ ) .", "The Hebbian component ( Ei× Aj ) ensures that the update more strongly affects those connections that contribute more to the final choice .", "Grey-shaded boxes indicate observed variables . DOI: http://dx . doi . org/10 . 7554/eLife . 13388 . 00710 . 7554/eLife . 13388 . 008Figure 3—figure supplement 1 . Exemplary time course of model variables and behavioral reports .", "( A ) Energy .", "Stimulus energy for clockwise ( cw ) and counterclockwise ( ccw ) orientation as computed by the representational subsystem .", "( B ) Signal weights .", "Strength of weights connecting orientation detectors to decisional units of the same orientation .", "( C ) Noise weights .", "Strength of weights connecting orientation detectors to decisional units of the opposing orientation .", "( D ) Choices .", "Depicted are the model’s choice probability for clockwise choices and the subject’s actual choices ( cw = 1 , ccw = 0 ) .", "Correct subject choices are marked by a circle .", "( E ) Confidence .", "Confidence ratings predicted by the model ( corresponding to λ∙|DV| ) and subject’s actual confidence ratings .", "( F ) Confidence prediction error and expected confidence .", "Depicted are the hidden model variables for the confidence prediction error ( CPE ) and expected confidence ( EC ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 13388 . 008 In a first step , we validated the representational subsystem by computing the average energy content for a range of spatial frequencies ( one octave above and below the actual frequency of 1 . 25 cycles/degree ) and for a range of orientations ( −40° to +40° relative to the reference axes ) .", "As expected , the energy content was higher for the spatial frequency and orientations used to generate the Gabor patches relative to other frequencies and orientations ( Figure 4—figure supplement 1 ) .", "Further , when orientation energy was computed separately for correct and incorrect responses , the energy for designated orientations ( i . e . , the orientations used to generate the Gabor patches ) was higher for correct than for incorrect responses ( t28 = 8 . 1 , p < 0 . 001 ) , whereas the energy for opposite orientations ( i . e . , ∓20° if ±20° was presented ) was lower for correct than for incorrect responses ( t28 = −3 . 6 , p = 0 . 001 ) ( Figure 4A ) .", "This pattern demonstrated that the varying orientation energy was directly associated with behavior and adds to the validation of the model .", "This pattern did also hold when the analysis was restricted to trials of the constant contrast condition ( designated orientation: t28 = 6 . 64 , p < 0 . 001; opposite orientation: t28 = −2 . 38 , p = 0 . 024 ) , thereby showing that the representational subsystem accounted for additional variance due to the random noise field over and above the variance due to changes in stimulus contrast . 10 . 7554/eLife . 13388 . 009Figure 4 . Modeling results .", "( A ) Orientation energy computed by the model’s representational subsystem .", "The energy is depicted separately for correct and incorrect responses as well as for designated and opposing orientations .", "( B ) Binned choice probabilities ( clockwise ) for observed data ( black ) and model predictions ( red ) as a function of the model-derived DV ( gry: logistic fit to data ) .", "( C ) Correspondence between participants’ binned confidence ratings and model-based decisional certainty ( grey: linear fit ) .", "( D ) Change of signal and noise weights across training runs .", "All error bars denote SEM corrected for between-subject variance ( Cousineau , 2005 ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 13388 . 00910 . 7554/eLife . 13388 . 010Figure 4—figure supplement 1 . Validation of the representational subsystem . Depicted is the stimulus energy for spatial frequencies ( Gabor frequency , ± 1 octave ) and orientations around the spatial frequency ( 1 . 25 cycles/degree ) and orientations ( −20°/20° and 70°/110° ) of the experimental Gabor stimuli , respectively .", "As expected , the energy content is higher for the spatial frequency and orientations used to generate the Gabor patches relative to other frequencies and orientations , thereby validating the computed orientation energies .", "Error bars represent SEM . DOI: http://dx . doi . org/10 . 7554/eLife . 13388 . 01010 . 7554/eLife . 13388 . 011Figure 4—figure supplement 2 . Choice probabilities and the corresponding model prediction for individual participants . DOI: http://dx . doi . org/10 . 7554/eLife . 13388 . 01110 . 7554/eLife . 13388 . 012Figure 4—figure supplement 3 . Confidence ratings and the corresponding model prediction for individual participants . DOI: http://dx . doi . org/10 . 7554/eLife . 13388 . 012 We then went on to fit the model parameters to participants’ orientation and confidence reports in the training session using maximum likelihood approximation ( median ± SE of the median: αw = 0 . 0018 ± 0 . 0007 , αc = 0 . 533 ± 0 . 077; see Supplementary file 1 for other parameters ) .", "To assess the model fit , we correlated model-based choice probabilities with participants’ actual choices , separately for each participant .", "This analysis showed that the model accounted well for participants’ choices ( mean ± SE of individual z-transformed correlation coefficients: rPearson = 0 . 64 ± 0 . 03; one-sample t-test against Fisher z’ = 0: t28 = 26 . 2 , p < 0 . 001 ) .", "This correspondence is reflected in the fact that participants’ and model-based choice probabilities show a nearly identical ( sigmoidal ) dependency on DV ( Figure 4A; see Figure 4—figure supplement 2 for single-subject fits ) .", "We next assessed whether the model could predict participants’ trial-wise confidence reports .", "A correlation between the model-based decisional certainty and participants’ confidence reports confirmed that confidence , too , was captured by the model ( mean ± SE of rPearson = 0 . 32 ± 0 . 02 , t28 = 15 . 4 , p < 0 . 001; Figure 4B; see Figure 4—figure supplement 3 for single-subject data ) .", "Finally , we evaluated how perceptual learning was reflected in the update of the model’s sensory filter by computing the change of signal and noise weights across runs .", "We expected that an increase of signal weights and a decrease of noise weights over the course of the training session was responsible for perceptual improvements .", "As depicted in Figure 4C , we found a linear increase for signal weights across runs ( mean ± SEM of slope = 0 . 0147 ± 0 . 0036 , t28 = 4 . 1 , p < 0 . 001 ) , and a linear decrease for noise weights ( slope = −0 . 0036 ± 0 . 0010 , t28 = −3 . 5 , p = 0 . 001 ) .", "Furthermore , the individual contrast threshold learning slopes correlated negatively with the slopes of signal weights ( rPearson = −0 . 45 , p = 0 . 013 ) and positively with the slopes of noise weights ( rPearson = 0 . 46 , p = 0 . 011 ) .", "Thus , individual learning was well captured by the signal and noise weights of the model .", "We reasoned that if confidence-based internal feedback and reward-based external feedback share a common neural basis , neural responses in the ventral striatum to high- , average- and low-confidence events should exhibit a qualitatively similar pattern as reported for rewarding , neutral and punishing outcomes in reward-based learning ( Delgado et al . , 2000; Knutson et al . , 2001 ) .", "The results of these previous studies suggest that striatal activation reflects a positive anticipatory response at the beginning of a trial as well as a subsequent prediction error response related to the outcome .", "To simulate the BOLD response that arises from such a scheme , we convolved vectors coding the neural activation for an initial anticipatory response and three different outcome scenarios ( positive , absent and negative prediction error ) with a canonical double-gamma hemodynamic response function ( Figure 5A ) .", "In accordance with the fMRI results of these previous studies , the simulation shows", "( i ) an increase of striatal BOLD responses related to trial onset reflecting the anticipatory signal , and", "( ii ) a subsequent positive , absent , or negative deflection of the BOLD response reflecting prediction errors . 10 . 7554/eLife . 13388 . 013Figure 5 . Confidence signals in the mesolimbic system and their relation to perceptual learning .", "( A ) Neural activation time courses consisting of an anticipatory peak at trial onset and a positive , absent , or negative reward prediction error ( PE ) during outcome ( stimulus onset ) .", "To simulate the associated BOLD response , the time courses were convolved with the standard canonical hemodynamic response function provided by SPM .", "( B ) Event-related BOLD time courses in the ventral striatum for three tertiles of the behavioral confidence reports ( representing 'low' , 'middle' and 'high' confidence trials ) .", "The shaded areas denote SEM .", "( C , D )", "Whole-brain t-maps showing brain regions with a positive relationship between BOLD signal and expected confidence at trial onset ( C ) , and between BOLD signal and CPE at stimulus onset ( D ) .", "The t-maps were thresholded at p<0 . 005 ( C ) and p<0 . 001 ( D ) , uncorrected , for illustration purposes .", "( E ) Scatter plot for the relation between the strength of striatal modulation by confidence prediction errors ( peak values , after age correction ) and individual perceptual learning success . DOI: http://dx . doi . org/10 . 7554/eLife . 13388 . 01310 . 7554/eLife . 13388 . 014Figure 5—figure supplement 1 . Control analyses accounting for effects of absolute orientation energy . The GLM of the model-based analysis was extended with a second parametric regressor for absolute orientation energy ( i . e . , energy for the presented orientation ) in a way that any variance shared between the energy and the CPE regressor would be accounted for by the energy regressor .", "( A ) Whole-brain t-map for a positive relationship between BOLD signal and confidence prediction error ( CPE ) , after accounting for absolute orientation energy ( threshold: p < 0 . 001 , uncorrected ) .", "Even after this correction for stimulus energy , a strong positive relationship in bilateral ventral striatum ( left: peak at [−16 8 −10] , t28 = 7 . 34 , prFWE < 0 . 001; right: peak at [14 14 −6] , t28 = 7 . 53 , prFWE < 0 . 001 ) and in the ventral tegmental area ( peak at [−6 −22 −16] , t28 = 2 . 98 , prFWE = 0 . 028 ) was present .", "( B ) The converse model , in which variance was first accounted for by the CPE regressor and second by the energy regressor , showed no residual activation in the mesolimbic ROIs ( even at a liberal threshold of p < 0 . 05 , uncorrected ) .", "The strongest trends for a modulation by stimulus energy on top of CPEs was present in voxels located within our stimulus localizer ROI ( left occipital cortex: peak at [−42 −74 −8] , t28 = 2 . 85 , p = 0 . 004 , uncorrected; left posterior fusiform gyrus: peak at [−32 −56 −12] , t28 = 2 . 60 , p = 0 . 007 , uncorrected ) .", "Interestingly , the modulation of activity in putative V1 by CPEs ( cf . Supplementary file 2 ) appears to be entirely accounted for by CPEs , as no significant modulation by energy was detectable in this analysis ( p > 0 . 05 , uncorrected ) .", "( C ) Whole-brain t-map for a positive relationship between BOLD signal and energy , without correcting for CPE .", "No cluster survived correction for multiple comparisons at the whole-brain level .", "The strongest activation was found in right dorsolateral prefrontal cortex ( peak at [32 , 38 , 18] , t28 = 5 . 24 , p = 0 . 000007 , uncorrected ) .", "A second notable activation was found in our stimulus localizer ROI ( left fusiform gyrus: peak at [−32 , −56 , −12] , t28 = 3 . 89 , p = 0 . 0003 , uncorrected ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 13388 . 014 To relate the neural signature of confidence in the present study to the simulation and to the results of previous reward-based studies , we binned the data into tertiles of the behavioral confidence rating ( low , middle , and high confidence ) and extracted the average BOLD time course in an anatomical mask of the ventral striatum using the SPM toolbox rfxplot ( Gläscher , 2009 ) .", "As shown in Figure 5B , the obtained event-related BOLD time courses are in remarkable agreement with the predictions of the simulation of Figure 5A and previous empirical findings of reward studies ( Delgado et al . , 2000 ) .", "Specifically , 4–6 s after trial onset ( reflecting the hemodynamic delay ) , the BOLD time courses exhibited a first peak , consistent with an anticipatory confidence signal at the start of a trial; 4–6 s after stimulus onset , the BOLD time courses displayed a positive deflection for high-confidence trials and a negative deflection for low-confidence trials .", "A statistical analysis confirmed above-baseline striatal activation at trial onset , indicative of an anticipatory signal ( left peak at [−10 14 −6] , t28 = 6 . 42 , prFWE < 0 . 001; right peak at [12 14 −8] , t28 = 7 . 78 , prFWE < 0 . 001 ) , as well as a main effect of confidence at stimulus onset in the bilateral ventral striatum ( left peak at [−10 14 −4] , t28 = 10 . 56 , prFWE < 0 . 001; right peak at [16 12 −8] , t28 = 11 . 46 , prFWE < 0 . 001 ) .", "This model-free assessment provides initial support for the idea that reinforcement based on reward and based on confidence share a common neural substrate both in the anticipation and the outcome period .", "In addition , the results lend plausibility to a model utilizing confidence as a reinforcement signal .", "Finally , we investigated whether the strength of the striatal confidence prediction error modulation translated into improvements in perceptual performance .", "For that purpose , we correlated the parameter estimates at the peaks of the bilateral striatal CPE contrast ( coordinates [−16 8 −10] and [16 14 −8] , see above ) with an index for the perceptual learning success that quantified the threshold change for the trained reference axis while accounting for baseline thresholds .", "Age was included as an additional factor in the regression model to preclude confounding effects of age-related variation in striatal BOLD signal ( Duijvenvoorde et al . , 2014 ) .", "As hypothesized , we found a significant relationship between the striatal CPE signal and individual perceptual learning success in the left ventral striatum ( rPearson = 0 . 400 , p = 0 . 016 , one-tailed; without age correction: rPearson = 0 . 37 , p = 0 . 026 ) and a trend in the right ventral striatum ( rPearson = 0 . 268 , p = 0 . 080; without age correction: rPearson = 0 . 24 , p = 0 . 11 ) ( see Figure 5E ) .", "This result is congruent with a viable role of CPE-based feedback signals for perceptual learning in the absence of external feedback ." ], [ "In this study , we used perceptual learning to address the question of how humans can improve performance in the absence of external feedback .", "Previous reinforcement learning accounts of perceptual learning were based on external cognitive and rewarding feedback ( Law and Gold , 2009; Kahnt et al . , 2011 ) and could not explain the established phenomenon of perceptual learning without such feedback ( Herzog and Fahle , 1997; Gibson and Gibson , 1955; McKee and Westheimer , 1978; Karni and Sagi , 1991 ) .", "Here , we suggest that observers are capable of generating internal feedback by utilizing confidence signals that provide a graded evaluation of the correctness of a perceptual decision .", "In this way , confidence may serve as a reinforcement signal similar to reward and guide perceptual learning in cases where no external feedback is provided .", "In support of this view , our model-free fMRI analyses revealed that mesolimbic confidence signals mirror those typically found for reward , both in the anticipation period ( Preuschoff et al . , 2006; Delgado et al . , 2000; Knutson et al . , 2001 ) and for prediction errors ( Schultz et al . , 1997; O’Doherty et al . , 2004; Berns et al . , 2001 ) .", "To establish a mechanistic ground for this suggested parallel , we devised an associate reinforcement learning model , which links behavior to computational variables that each account for a different aspect of the learning process .", "CPEs served as feedback in the model , defined as the difference between the current level of confidence and a long-term estimate of expected confidence .", "The model successfully described the learning process as a continuous adjustment of a perceptual filter linking sensory and decision units .", "Our model-based fMRI analyses confirmed and extended the results of the model-free analyses by demonstrating a parametric modulation in mesolimbic brain areas both by expected confidence and confidence prediction .", "Importantly , the strength of the striatal modulation by CPEs predicted participants’ perceptual improvements , further corroborating the behavioral relevance of these internally-generated feedback signals .", "The observed pattern of confidence-related activity in the mesolimbic system , including the co-modulation of the ventral tegmental area , fit well with the prediction error hypothesis of dopamine , which posits that dopaminergic midbrain neurons and their targets respond at two time points during a learning trial ( Schultz et al . , 1997 ) .", "In this framework , the first response is triggered by an outcome-predictive cue and reflects an anticipatory signal .", "In the case of classical reinforcement learning , such a cue may be probabilistically coupled with rewards of possibly varying magnitudes .", "The anticipated value of the cue is then assumed to be computed as the average reward magnitude—contingent on the cue—in previous trials ( Schultz , 2006 ) .", "Here , we argue that the same principle could hold for confidence: participants learn to anticipate a certain level of confidence for the upcoming trial based on past confidence experiences , and this anticipatory state is activated when the beginning of a new trial is indicated ( equivalent to a cue ) .", "In congruence with this postulation , we indeed found a modulation of striatal activity by expected confidence at trial onsets—as previously reported for expected reward ( Preuschoff et al . , 2006; Delgado et al . , 2000; Knutson et al . , 2001 ) .", "The second response is triggered by the actual outcome and corresponds to a prediction error signal .", "In classical reinforcement learning , the reward prediction error represents the difference between expected value and actual outcome .", "In the confidence domain , the outcome would correspond to the level of confidence calculated from the stimulus and the prediction error would be computed as the difference between expected confidence and actual confidence .", "Overall , our results may therefore indicate that self-generated confidence assumes the role of external reward in dopaminergic prediction-error-based reinforcement learning when no external feedback is available .", "A number of previous studies have used reinforcement learning models to capture the neural underpinnings of perceptual learning ( Law and Gold , 2009; Kahnt et al . , 2011 ) and category learning ( Daniel and Pollmann , 2012 ) .", "In particular , an fMRI study by Kahnt and colleagues ( Kahnt et al . , 2011 ) investigated perceptual learning with external reward and found that behavioral improvements were well explained by a reinforcement learning model .", "Their results exhibit a notable parallel to the present findings: the authors reported stimulus information encoded in visual cortex and model-derived decision value in frontal cortices , in agreement with the findings of the present study .", "In addition , this previous study identified a perceptual learning-related reward prediction error in the ventral striatum , dovetailing with our finding of a perceptual learning-related confidence prediction error in the same brain region .", "Importantly , our combined Hebbian and reinforcement learning model extends and improves previous models in several ways .", "First and foremost , by implementing confidence prediction errors in replacement of reward prediction errors , it extends previous reward reinforcement learning models of perceptual learning ( Law and Gold , 2009; Kahnt et al . , 2011 ) to cases without feedback .", "Second , these previous models were based on the assumption that perceptual performance is determined by a single 'readout weight' , representing the amplification of stimulus information in sensory areas .", "While the simplicity of these models is appealing , they are limited in the sense that negative prediction errors have an unreasonable influence on behavior: according to these models , worse-than-expected feedback reduces the readout weight , which leads to an additional reduction in performance .", "This property runs counter to the idea that reinforcement learning agents improve their behavior through both positive and negative prediction errors .", "By contrast , the associative reinforcement learning rule of the present model entails a behaviorally advantageous and plausible function of negative prediction errors: inhibition of sensory noise .", "Third , a conceptually related reinforcement learning model for perceptual categorization ( Daniel and Pollmann , 2012 ) implies that stimuli exclusively activate the correct stimulus category , an assumption that disregards the fact that the ambiguity of incoming stimulus information is an essential property of perceptually demanding tasks .", "In contrast , the present model utilizes a dedicated representational subsystem ( Petrov et al . , 2005 ) to estimate the activation of all implemented input units , and it is their differential activity that determines perceptual choices .", "The present model and results are biologically plausible and fit well with theoretical accounts of the neural basis of learning .", "The associative reinforcement learning rule in the model was inspired by the three-factor learning rule ( Schultz , 2002; Reynolds et al . , 2001 ) , which has been proposed to underlie the potentiation of synapses in the striatum .", "It proposes that changes in neural transmission in cortico-striatal synapses not only depend on coincident presynaptic and postsynaptic activity ( Hebbian learning ) , but also on the presence of dopamine error signals .", "Indeed , Ashby and colleagues ( Ashby et al . , 2007; Hélie et al . , 2015 ) have previously suggested that the basal ganglia , which represent the predominant site of dopaminergic synaptic plasticity , are themselves a key region for learning in perceptual tasks .", "They proposed that", "( i ) the basal ganglia serve to activate the appropriate target regions in executive frontal cortices shortly after sensory cortex activation; and", "( ii ) such basal ganglia learning is superseded by cortico-cortical Hebbian learning , once the correct cortico-cortical synapses are built .", "This account fits well with the present model , in which perceptual learning corresponds to the process of reweighting connections between sensory and decisional units .", "These considerations in combination with the present results thus lend support to the hypothesis that the optimization of perceptual read-out ( as implicated by our model ) could be mediated via reinforcement learning in the basal ganglia .", "While our study represents a first but important step towards understanding the role of confidence signals in perceptual learning , future studies are needed to investigate in more detail the characteristics of these signals which were not addressed in the current study .", "First , are these signals triggered independent of whether participants have to report their level of confidence after the percept or independent of whether they receive external feedback ?", "Investigating these questions could clarify whether the observed activity in the reward network is an automatic response or depends on the task of the observer .", "Second , are these learning signals independent of making a perceptual decision ?", "In other words , are they triggered only when participants have to engage in a subsequent perceptual choice ?", "Similarly , can these confidence signals be disentangled from choice accuracy , for instance by manipulating stimulus luminance ( Busey et al . , 2000 ) ?", "An answer to this latter question would shed light on the nature of the confidence signals , i . e . whether they can also be affected by metacognitive biases .", "In summary , our study devised and tested a novel model of perceptual learning in the absence of external feedback , utilizing confidence prediction errors to guide the learning process .", "Our analyses revealed a compelling analogy between confidence-based and reward-based feedback , suggesting a similar neural mechanism for learning with and without external feedback .", "Future work could investigate whether a learning mechanism based on such self-generated feedback is also applicable outside the realm of perception , where learning without feedback has likewise been a long-standing puzzle ( Köhler , 1925 ) ." ], [ "Thirty healthy , right-handed female participants took part in the experiment in return for payment after giving written informed consent .", "Participants of only one gender were selected in view of the planned between-subject analysis , because male and female brains on average have different brain volumes ( Ruigrok et al . , 2014 ) , introducing between-subject noise in the spatial normalization procedure , and slightly different hemodynamic response profiles ( Jaušovec and Jaušovec , 2010 ) , which could impact the detectable BOLD signal .", "One participant was excluded due to fixation failure , leaving 29 valid participants ( 24 . 1 ± 2 . 5 years , range 19–31 years ) .", "The relatively large total sample size of 30 size was chosen in view of the planned between-subject correlation between striatal modulation and learning success .", "As the best available study for comparison , we determined Schlagenhauf et al . ( 2013 ) ( N = 28 ) , which investigated the association between striatal prediction error signaling and fluid intelligence .", "The sample size for the present study was estimated through a method recommended by Hulley et al . ( 2013 ) ( N≈zα+zβ0 . 5ln1+r/1-r2+3 ) , thereby applying the observed associative strength in this previous study ( r = 0 . 47 ) , a Type I error rate α = 0 . 05 ( Zα = 1 . 960 ) and a Type II error rate β = 0 . 2 ( Zβ = 0 . 842 ) .", "The present study was conducted according to the declaration of Helsinki , and approved by the ethics committee of the Charité Universitätsmedizin Berlin .", "Each participant came in for four sessions .", "The training session took place in an fMRI scanner with a back-projection screen setup ( Sanyo PLC-XT21L , 60 Hz , resolution 1024 x 768 ) .", "Participants responded with their right hand using an MR-compatible trackball ( Current Designs Inc . , Philadelphia , PA ) by pressing the left button with the thumb , the right button with the middle finger and navigating the trackball with the index finger .", "Around one week ( 7 . 1 ± 0 . 2 days ) before and one day after the training session , the behavioral pre- and post-test took place in a darkened room , in which the participant sat in front of a 17'' LCD monitor ( LG Flatron L1750S , 60 Hz , resolution 1024 x 768 ) and operated with an equivalent trackball device .", "Around 10 weeks ( 70 . 6 ± 2 . 1 days ) after the training session , a long-term test was conducted with a setup identical to the pre- and post-test sessions .", "Each trial started with a fixation screen for 2000 ms ± 1000 ms , followed by the presentation of the stimulus for 100 ms . After 2000 ms ± 1000 ms the white fixation cross turned orange or blue ( depending on the response mapping; see section 'Training' ) for 750 ms to signalize the appearance of the confidence rating scale ( see subsection confidence below ) .", "After adjusting the confidence rating bar , participants made a binary judgment about the stimulus orientation using the two buttons of the trackball device .", "After the button press , the response screen remained up for 1 s .", "To avoid a potential bias by the choice itself ( Kvam et al . , 2015; Sniezek et al . , 1990; Sieck , 2003; Tafarodi et al . , 1999 ) , participants reported their confidence prior to reporting their choice .", "The confidence rating scale was visualized as a half-open circle ( radius r = 4° of visual angle ) that linearly increased in width ( 0 . 1° to 1° visual angle ) and color ( black to green ) , whereby the orientation and angular direction of the circle changed randomly from trial to trial .", "Participants received the following instruction: “After the presentation of the stimulus , a rating scale appears , on which you should indicate how confident you are that your perceived orientation matches the correct orientation of the stimulus . Placing the slider of the rating scale on the thin black end would mean that you have absolutely no confidence in your perceived orientation . Placing the slider at the thick green end would mean , that you are entirely confident about your perceived orientation . Try to rate all intermediate levels of confidence proportionally in between both ends of the scale” To select a confidence rating , participants adjusted a sliding white bar on the rating scale by means of a trackball device .", "No time pressure was imposed .", "The aim of the pre- , post- and long-term test ( henceforth test sessions ) was to determine individual contrast thresholds in the orientation discrimination task .", "The test sessions were divided into blocks of 16 trials with alternating reference axes and continued until the termination criterion of a staircase procedure was reached .", "Each block began with a start screen that indicated the reference axis of the upcoming block .", "The staircase procedure started with a one-up-one-down staircase with a relative stepsize of 0 . 05 log units to rapidly approximate the rough threshold range ( start contrast: cp = 20%; cf . Eq . 1 ) .", "After three reversals , the algorithm switched to a weighted one-up-two-down staircase ( Kaernbach , 1991 ) for fine-tuning .", "The ratio of stepsize down / stepsize up was set to 0 . 5488 ( García-Pérez , 1998 ) with stepsize down set to 0 . 33% , leading to convergence at a performance of 80 . 35 percent correct .", "The termination criterion was the 9th reversal .", "Thresholds for the horizontal and vertical references axes were independently adjusted .", "In order to familiarize with the stimulus materials , all participants performed an additional 8 blocks at maximal contrast ( cp = 100% ) prior to the pre-test .", "The training session in the fMRI scanner comprised an initial adjustment run and nine training runs , each with 48 trials .", "During the adjustment run and all training runs , the participants viewed only one reference axis ( 'trained reference axis’ ) , which was assigned to participants based on the parity of their consecutively numbered participant IDs .", "Participants performed the adjustment run in the scanner prior to the first training run in order to accommodate with the scanner environment and to fine-tune the initial contrast level for the training runs .", "The adjustment run started at the determined contrast threshold of the pre-test and was subsequently adapted with the same weighted one-up-one-down staircase procedure used in the test sessions , targeting a performance of 80 . 35 percent correct .", "In the critical condition during the training runs , performance was kept constant at 80 . 35 percent correct by continuously adapting stimulus contrast through the above-described one-up-one-down staircase procedure .", "The training runs included an additional control condition with constant stimulus contrast in an interleaved half of the trials to permit an assessment of orientation information encoded in activation patterns of visual cortex without the confound of a changing stimulus contrast .", "The instruction for the response mapping between stimulus orientation ( counterclockwise/clockwise ) and response button ( left/right ) was alternated between runs .", "The response mapping was indicated at the beginning of a run and additionally in each trial through the color ( blue/orange ) of the fixation cross ( the assignment of color and orientation being counterbalanced across participants ) .", "The stimuli were based on an additive mixture of a Gabor patch at four possible orientations ( +20° or −20° from the vertical or horizontal reference axis ) and phase-randomized spectrally filtered noise ( Petrov et al . , 2006 ) .", "Each stimulus consisted of a greyscale Gabor patch G ( x , y ) embedded in a larger field of filtered greyscale noise N ( x , y ) ( Petrov et al . , 2006 ) .", "The luminance L ( x , y ) of each pixel was an additive mixture of a Gabor term G ( x , y ) and noise N ( x , y ) , where L0 was the background luminance of the screen ( 51 . 9 Cd/m² ) : ( 1 ) L ( x , y ) =1+Cp100G ( x , y ) +Cn100N ( x , y ) L0 ( 2 ) G ( x , y ) =e-x2+y22σ2sin2πfxcosθ+ysinθ+φ The peak target contrast cp , which could take values between 0% and 100% , was continuously adapted by a staircase procedure .", "The Gabor patches had a fixed phase ψ of 0 . 25 , a spatial frequency f of 1 . 25 cycles per degree visual angle , and the standard deviation σ of their Gaussian envelope was set to 0 . 6 .", "The orientation θ was set to +20° or −20° from the vertical ( 0° ) or horizontal ( 90° ) reference axis .", "The Gabor patch was trimmed at values smaller than 0 . 005 , resulting in a radius of 1 . 87° visual angle .", "The noise field N ( x , y ) was constructed from a random phase and a bandpass-filtered power spectrum .", "The power spectrum was generated by subtracting two Butterworth low-pass filters ( two-dimensional , resolution 300 x 300 pixel , corresponding to 5 . 3° x 5 . 3°; order 3 ) with cut-off frequencies one octave below and one octave above the spatial frequency of the Gabor patch .", "The phase spectrum was sampled as a 300 x 300 matrix of uniformly distributed random numbers .", "The noise contrast cn was fixed at 15% .", "Inverse Fourier transformation of the power and phase spectrum resulted in a noise field that effectively interfered with the spatial frequency of the Gabor patch .", "The additive mixture of the Gabor patch and the noise field was multiplied with a circular filter and cropped at a radius of 2 . 5° visual angle , such that the stimulus became circular and smoothly faded out to background luminance .", "The filter was constructed as the inverse of a two-dimensional 300 x 300 pixel Butterworth low-pass filter of order 7 with cut-off frequency 0 . 275 cycles / degree visual angle .", "The value of the cut-off frequency ensured that the fading zone overlapped with the outer border of the Gabor patch and the high order of the filter ensured that the fading zone was relatively steep .", "The luminance of the monitor in test sessions and the projection setup in the training session was equalized through pre-measured color look-up tables .", "To independently identify stimulus-responsive regions for a multivariate analysis , we conducted a localizer run with 18 stimulus blocks and 18 baseline blocks of 12 s duration in pseudo-randomized order .", "In the stimulus blocks the Gabor patch was shown with maximal contrast ( cp = 100% ) at an eccentricity of 5° visual angle and alternated every 250 ms between phase ψ and counterphase 1-ψ ( phase and eccentricity were identical to the test and training sessions ) .", "The orientation of the Gabor patches alternated block-wise between the two orientations shown in the training session of the respective participant ( ± 20° with respect to the trained reference axis ) .", "The baseline blocks consisted of the fixation cross only .", "To hold participants’ attention , they performed an independent color change detection task on the central fixation cross .", "The task was to press one of the buttons of the trackball device as soon as the fixation cross turned from white to red .", "They were instructed that , while fixating and performing the task , they should still note and make themselves aware of the Gabor stimuli .", "To quantify participants’ perceptual learning success for the trained reference axis , the respective pre-test contrast thresholds were subtracted from post-test thresholds ( threshold improvement ) .", "However , an analysis of the relationship between pre-test thresholds and threshold improvements showed a strong positive correlation ( rPearson = 0 . 73 , p < 0 . 001 ) , suggesting that participants starting at higher thresholds had more room for performance to improve .", "Thus , to correct our perceptual learning index for this substantial learning-unrelated dependency , pre-test thresholds were regressed out from threshold improvements across participants .", "The perceptual learning index then corresponds to the resulting residuals and has mean zero .", "Eyetracking data were successfully collected in 24 participants during the fMRI training session using an infrared video eyetracking system ( iView XTM MRI 50 Hz , SensoMotoric Instruments , Teltow , Germany ) .", "For six other participants , eye tracker calibration failed .", "As a measure of fixation reliability , we computed the percentage of recorded eye gaze positions during stimulus presentation within a circle of 2 . 5° visual angle in radius around the center of the fixation cross .", "This radius corresponded to the eccentricity of the first stimulus pixel .", "The cut-off for exclusion was a percentage of below 95% ." ] ]

[ "It is well established that learning can occur without external feedback , yet normative reinforcement learning theories have difficulties explaining such instances of learning .", "Here , we propose that human observers are capable of generating their own feedback signals by monitoring internal decision variables .", "We investigated this hypothesis in a visual perceptual learning task using fMRI and confidence reports as a measure for this monitoring process .", "Employing a novel computational model in which learning is guided by confidence-based reinforcement signals , we found that mesolimbic brain areas encoded both anticipation and prediction error of confidence—in remarkable similarity to previous findings for external reward-based feedback .", "We demonstrate that the model accounts for choice and confidence reports and show that the mesolimbic confidence prediction error modulation derived through the model predicts individual learning success .", "These results provide a mechanistic neurobiological explanation for learning without external feedback by augmenting reinforcement models with confidence-based feedback ." ]

[ "Much of our behavior is shaped by feedback from the environment .", "We repeat behaviors that previously led to rewards and avoid those with negative outcomes .", "At the same time , we can learn in many situations without such feedback .", "Our ability to perceive sensory stimuli , for example , improves with training even in the absence of external feedback .", "Guggenmos et al . hypothesized that this form of perceptual learning may be guided by self-generated feedback that is based on the confidence in our performance .", "The general idea is that the brain reinforces behaviors associated with states of high confidence , and weakens behaviors that lead to low confidence .", "To test this idea , Guggenmos et al . used a technique called functional magnetic resonance imaging to record the brain activity of healthy volunteers as they performed a visual learning task .", "In this task , the participants had to judge the orientation of barely visible line gratings and then state how confident they were in their decisions .", "Feedback signals derived from the participants’ confidence reports activated the same brain areas typically engaged for external feedback or reward .", "Moreover , just as these regions were previously found to signal the difference between actual and expected rewards , so did they signal the difference between actual confidence levels and those expected on the basis of previous confidence levels .", "This parallel suggests that confidence may take over the role of external feedback in cases where no such feedback is available .", "Finally , the extent to which an individual exhibited these signals predicted overall learning success .", "Future studies could investigate whether these confidence signals are automatically generated , or whether they only emerge when participants are required to report their confidence levels .", "Another open question is whether such self-generated feedback applies in non-perceptual forms of learning , where learning without feedback has likewise been a long-standing puzzle ." ]

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

[ "chromosomes and gene expression", "biochemistry and chemical biology" ]

[ [ "Important features of cellular regulatory programs depend on interactions between the transcriptional machinery and DNA packaged in nucleoprotein complexes in vivo .", "The impact of nucleoprotein on transcriptional regulation has been elucidated in greatest detail in eukaryotes , where nucleosome structure and dynamics affect efficient initiation complex assembly ( Knezetic and Luse , 1986; Lorch et al . , 1987; Li et al . , 2007 ) , affect transcript elongation by RNA polymerase II ( RNAPII ) ( Studitsky et al . , 1997; Kireeva et al . , 2005; Bintu et al . , 2012; Kulaeva et al . , 2013 ) , and , conversely , are modulated by factors associated with elongating RNAPII ( Kristjuhan and Svejstrup , 2004; Workman , 2006 ) .", "However , our understanding of the impact of nucleoprotein on transcription in bacteria is more primitive , principally because the structures of nucleoprotein complexes formed from DNA and nucleoid-associated proteins ( NAPs ) are more heterogeneous in structure , more dynamic , and less stable than nucleosomes .", "The histone-like nucleoid structuring protein ( H-NS ) is the principal NAP in Escherichia coli .", "H-NS binds DNA at high-affinity sites , spreads to form filaments on AT-rich DNA , bridges between filaments on different DNA segments , and inhibits transcription ( reviewed in Ali et al . , 2012; Dorman , 2007 , 2009; Navarre , 2010; Navarre et al . , 2007 ) .", "H-NS is present at ∼2 × 104 copies per cell ( Ali Azam et al . , 1999 ) , enough to cover ∼14% of a single-copy genome , as currently modeled in bridged filaments ( Arold et al . , 2010 ) .", "ChIP experiments in E . coli and Salmonella reveal sequestration in H-NS filaments of ∼350 DNA segments 0 . 5–50 kb in length ( ∼2 kb on average ) that change little in different growth or environmental conditions and correlate with higher AT-content and reduced gene expression ( Oshima et al . , 2006; Noom et al . , 2007; Vora et al . , 2009; Kahramanoglou et al . , 2011; Peters et al . , 2012; Myers et al . , 2013 ) .", "A large fraction of these filaments co-localize in clusters ( ∼2 clusters per chromosome in E . coli ) that likely depend on H-NS bridging ( Wang et al . , 2011 ) , although the extent and time-scale of bridging rearrangements is unknown .", "The 15 . 5-kDa H-NS monomer consists of an N-terminal oligomerization domain with two oligomerization sites ( head and tail; Figure 1—figure supplement 1 ) separated by a 45-aa α-helical linker; a 46-aa C-terminal DNA-minor-groove-binding domain connects to the oligomerization domain through a 10-aa flexible linker ( Shindo et al . , 1999; Arold et al . , 2010; Cordeiro et al . , 2011; Gordon et al . , 2011 ) .", "H-NS lacking DNA-binding domains forms helical proteinaceous filaments with head–head and tail–tail interfaces ( Arold et al . , 2010 ) .", "H-NS binds DNA at discrete high-affinity sites with site-specific regulatory function ( Bouffartigues et al . , 2007; Lang et al . , 2007 ) .", "Depending on surrounding sequence , available H-NS , and other factors ( temperature , solute composition , other NAPs ) , filaments form by spreading ( Amit et al . , 2003; Bouffartigues et al . , 2007; Cordeiro et al . , 2011 ) .", "Current models suggest that the DNA-binding domains of one tail–tail module bind adjacently to a single DNA segment in linear filaments , which form at <5 mM Mg2+ , or contact separate DNAs or DNA segments in bridged filaments favored at >5 mM Mg2+ ( Liu et al . , 2010 ) .", "H-NS filaments ( and associated NAPs ) silence transcription of horizontally transferred DNA ( Navarre et al . , 2007 ) and suppress pervasive noncoding and antisense transcription ( Peters et al . , 2012; Singh et al . , 2014; Wade and Grainger , 2014 ) by controlling RNAP initiation at promoters ( Dame et al . , 2002; Fang and Rimsky , 2008; Dorman , 2009; Singh et al . , 2014 ) , by inhibiting transcript elongation by RNAP ( Dole et al . , 2002 , 2004; Saxena and Gowrishankar , 2011; Peters et al . , 2012 ) , or both .", "For both silencing and suppression of antisense and noncoding transcription , in vivo experiments indicate that H-NS filaments slow or block elongating RNAP , although direct biochemical tests of elongating RNAP–H-NS interactions or insights into the underlying mechanisms have not been reported .", "In vivo assays also establish that the H-NS block to transcription is greater in enterobacteria growing at 20–30°C outside hosts than at 37°C typical for symbiotic or pathogenic growth , and implicate H-NS in switching gene expression upon host invasion ( Goransson et al . , 1990; Trachman and Yasmin , 2004; Ono et al . , 2005; Yang et al . , 2005 ) .", "Rho-dependent termination also plays a key role in suppressing both horizontally transferred genes and pervasive noncoding transcription ( Saxena and Gowrishankar , 2011; Nicolas et al . , 2012; Peters et al . , 2012; Singh et al . , 2014 ) .", "In both cases , a strong association between sites of Rho-dependent termination and sites of H-NS filament formation implicates H-NS in delaying transcript elongation to aid Rho in dissociating elongating RNAP from DNA and preventing synthesis of RNAs potentially deleterious to the cell ( Ali et al . , 2012; Peters et al . , 2012; Singh et al . , 2014 ) .", "To investigate whether H-NS filaments pose direct barriers to transcript elongation and to characterize underlying mechanisms , we focused on an antisense transcription unit in the well-characterized bgl operon of E . coli K-12 .", "bglGFB encodes cryptic genes for ß-glucoside catabolism and is ordinarily silenced by H-NS filaments that emanate from high-affinity sites flanking the promoter ( upstream regulatory element , URE , and downstream regulatory element , DRE , respectively; Figure 1A ) .", "H-NS filaments nucleating on the DRE block RNAP in both the sense and antisense directions ( Dole et al . , 2004; Peters et al . , 2012 ) .", "Using in vitro transcription and direct visualization of H-NS filaments and elongating RNAP by atomic force microscopy ( AFM ) , we found that H-NS filaments directly inhibit elongating RNAP and promote Rho-dependent termination , but surprisingly only when H-NS forms bridging interactions . 10 . 7554/eLife . 04970 . 003Figure 1 . H-NS formed two different filaments depending on concentration .", "( A ) The H-NS-silenced E . coli bgl operon , encoding genes for ß-glucoside catabolism , contains an antisense promoter within bglF ( PAS ) ( Peters et al . , 2012 ) .", "The 1 . 56-kb linear λPR-bgl DNA template contains the λPR promoter followed by a 26-nucleotide C-less cassette ( to allow formation of halted A26 ECs ) and two high affinity H-NS binding sites ( DRE and URE ) ( Dole et al . , 2004 ) .", "Native PAGE of H-NS filaments on 10 pM or 10 nM λPR-bgl template in 8 mM Mg2+ .", "Graphics depicting bridged and linear H-NS filaments are shown left of the gel and related to H-NS molecular structures in Figure 1—figure supplement", "1 . ( B ) Native PAGE of H-NS filaments formed on free DNA or halted A26 complexes ( at 10 nM ) at 2 or 8 mM Mg2+ and 66 H-NS/kb or 200 H-NS/kb ( 1 or 3 μM H-NS respectively ) .", "32P-labeled DNA ( 10 nM ) was used for lanes denoted −RNAP; unlabeled DNA and 32P-labeled RNA formed by incorporation of [a-32P]GTP were used for lanes denoted +RNAP .", "( C ) Representative AFM images of H-NS filaments on DNA or ECs matching the EMSA assays shown in ( B ) .", "DNA or ECs with either 66 H-NS/kb or 200 H-NS/kb were diluted from 10 nM to 2 nM , immediately absorbed on APS-mica , and imaged in air .", "RNAP bound to DNA is indicated by white arrows .", "Cyan arrows indicate linear H-NS complexes ( L ) , interwound H-NS complexes ( I ) , circular H-NS complexes ( C ) , or hairpin H-NS complexes ( H ) .", "Graphics depicting the observed DNA topologies are shown in insets , where gray or black lines are each equivalent to one dsDNA molecule .", "AFM images from which these panels were cropped and additional examples are shown in Figure 1—figure supplement", "2 . ( D ) Pseudo-3D images of complexes similar to those in panel C , but lacking ECs to avoid scaling distortion .", "( E ) Complexes were binned based on their DNA topology defined in ( C ) .", "Interwound , circular , and hairpin H-NS complexes were grouped together as various forms of bridged complexes .", "H-NS complexes formed on template DNA are denoted −RNAP , and +RNAP denotes complexes formed on ECs .", "Only complexes with RNAP bound were counted in the +RNAP samples . DOI: http://dx . doi . org/10 . 7554/eLife . 04970 . 00310 . 7554/eLife . 04970 . 004Figure 1—figure supplement 1 . Model of H-NS filaments . H-NS is a minor groove DNA binding protein that first binds to A/T rich DNA as a dimer through a C-terminal DNA binding domain ( right inset; PDB 2LEV [Cordeiro et al . , 2011] ) , and can form filaments by head-to-head and tail-to-tail contacts of an N-terminal oligomerization domain ( left inset and bottom inset; PDB 3NR7 [Arold et al . , 2010] ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 04970 . 00410 . 7554/eLife . 04970 . 005Figure 1—figure supplement 2 . Interwound filaments formed preferentially in samples of ECs at 8 mM Mg2+ and 66 H-NS /kb .", "( A ) Lower magnification images of AFM as described and shown in Figure 1C , with the additional representative image of linear filaments formed in 8 mM Mg2+ and 200 H-NS/kb ( high concentration H-NS ) .", "Linear filaments formed at high concentrations of H-NS have high background from the additional H-NS .", "Green boxes represent the area shown at higher magnification in Figure 1C .", "Blue box depicts the area under higher magnification in the image to the right .", "( B ) The average contour length of H-NS filaments .", "Error bars represent the standard deviation of at least 25 molecules .", "( C ) The average persistence length of H-NS filaments .", "Error bars represent the standard deviation of at least 25 molecules .", "( D ) Distribution of complexes formed in bridging conditions ( 8 mM Mg2+ and 66 H-NS/kb ) as determined by AFM . DOI: http://dx . doi . org/10 . 7554/eLife . 04970 . 00510 . 7554/eLife . 04970 . 006Figure 1—figure supplement 3 . Temperature affected H-NS bridging interactions . Native PAGE of H-NS complexes assembled at either 20°C ( A ) or 37°C ( B ) on 10 nM λPR-bgl template in 8 mM Mg2+ .", "H-NS-DNA complexes were electrophoresed at 4°C ( left panels ) , 20°C ( middle panels ) , or 37°C ( right panels ) .", "Rf ( retardation factor ) values were calculated as the distance of the H-NS-DNA complexes migrated divided by the distance the DNA template alone migrated .", "The loss of the ∼0 . 45 Rf band in gels run at 37°C but not 20°C is consistent with a loss of bridging interactions at 37°C that occurred after the samples were loaded on the gels ( which were pre-equilibrated to the running temperature ) .", "Weakened H-NS interactions at 37°C also were apparent in the loss of smearing at lower H-NS concentrations ( 0 . 1–0 . 4 µM ) .", "The appearance at 37°C of the ∼0 . 55–0 . 58 Rf in place of the ∼0 . 45 Rf band evident at 20°C or 4°C suggests that linear filaments may persist at elevated temperatures .", "The slower migrating bands at high H-NS concentrations ( ≥10 µM ) that appear prominently at 37°C and to some extent at lower temperatures may reflect either aggregation or restoration of bridging interactions . DOI: http://dx . doi . org/10 . 7554/eLife . 04970 . 006" ], [ "To investigate how H-NS filaments affect transcript elongation by RNAP , we engineered a transcription template that could form λPR promoter-initiated , halted A26 elongation complexes ( ECs ) on a 26 nucleotide C-less cassette placed upstream from the bgl DRE and URE in the antisense direction ( Figure 1A ) .", "On this template , λPR drives synthesis of the bgl antisense transcript from a position ∼1500 bp closer to the DRE than the bglF antisense promoter .", "Initial generation of halted ECs allowed us to uncouple transcription initiation from subsequent H-NS filament formation and transcript elongation , a key experimental feature impossible for in vivo studies .", "To understand filament formation on our bgl template , we examined H-NS–DNA interactions by native PAGE at 8 mM Mg2+ , a condition previously found to favor bridging interactions ( Figure 1A ) .", "At low DNA concentration ( 10 pM ) , we observed half-maximal retardation of radiolabeled DNA electrophoresis at ∼2 nM H-NS , which we infer corresponds to either the Kd of H-NS nucleation on the high-affinity URE and DRE sites or the Kd for filament extension .", "This affinity of H-NS is tighter than previously reported ( Azam and Ishihama , 1999; Dole et al . , 2004 ) , perhaps because prior measurements required >2 nM H-NS to form stable filaments at the DNA concentrations used or because , to mimic in vivo conditions , we substituted glutamate for chloride often used previously .", "As the H-NS concentration was increased , we observed two distinct complexes at both 10 pM and 10 nM DNA .", "A slower migrating band was observed at 66 H-NS/kb ( for 10 nM DNA ) .", "At higher concentrations of H-NS ( 80–200 H-NS/kb ) , the complexes migrated more rapidly , producing a visible gel downshift .", "Both the faster and slower migrating species were detected at both low and high DNA concentrations; however , it took higher H-NS:DNA ratios to achieve the comparable protein–DNA complex shifts at 10 pM DNA ( 333 H-NS/kb for the slower and 3 . 3 × 104 H-NS/kb for the faster migrating species ) .", "With 10 nM DNA , we found that the shift in gel mobility occurred at both low ( 2 mM ) and high ( 8 mM ) Mg2+ when H-NS concentration was increased from 66 H-NS/kb to 200 H-NS/kb either using radiolabeled DNA alone or using DNA containing halted A26 ECs with radiolabeled RNA ( Figure 1B ) .", "We hypothesized that the differences in the filament gel mobility resulted from a switch from bridged to linear filaments as the concentration of H-NS was increased .", "To test this idea , we examined the filaments using AFM .", "We observed four different H-NS-induced topologies , which we classified linear , interwound , circular , and hairpin based on their appearances ( Figure 1C and Figure 1—figure supplement 2D ) .", "On linear filaments , H-NS spread over the entire DNA molecule but interactions between DNA segments or with another DNA molecule were not visible .", "Interwound complexes contained two DNAs , which were sometimes discernible as distinct filaments but generally exhibited increased height above the mica surface compared to linear filaments ( Figure 1D ) .", "Occasionally , linear and bridged filaments were observable in the same AFM field ( Figure 1C , third panel ) .", "Circular and hairpin complexes appeared to have similar bridging to the interwound filaments , but involved only one DNA .", "We categorized the interwound , hairpin , and circular topologies as different forms of bridged filaments , whereas the remaining bound species were characterized as linear filaments .", "These different filament topologies were generally consistent with previously reported AFM images of H-NS–DNA complexes ( Dame et al . , 2000; Maurer et al . , 2009; Liu et al . , 2010 ) , except that RNAP was apparent in images prepared from samples containing A26 ECs ( e . g . , Figure 1C , panel 1 , 2 , 3; Figure 1—figure supplement 2A ) .", "To quantify the types of filaments formed in different conditions , we binned images based on their shape , width , contour length , and height ( ‘Materials and methods’ ) .", "In our transcription conditions ( 10 nM DNA diluted to 0 . 5–1 nM at 4°C for AFM imaging ) , bridged filaments predominated at 8 mM Mg2+ and 66 H-NS/kb ( ∼80% of filaments without RNAP and ∼70% of filaments with RNAP were interwound , hairpin , or circular; Figure 1D ) , whereas linear filaments predominated at lower Mg2+ ( 2 mM ) and higher H-NS:DNA ratio ( 200 H-NS/kb ) at both 2 and 8 mM Mg2+ .", "H-NS filaments also exhibited decreased contour lengths ( were compacted ) and increased persistence lengths ( were stiffer ) , especially in the bridging configuration ( Figure 1—figure supplement 2B , C ) .", "The AFM images were mostly consistent with our hypothesis that the slower migrating band at 8 mM Mg2+ in the EMSA assays contained bridged filaments ( either with or without RNAP ) , although the concentration requirements of AFM imaging precluded direct observations at 10 nM DNA .", "Additionally , the presence of RNAP in halted A26 ECs caused a shift in the conformation of bridged filaments from a near equal distribution of interwound , hairpin , and circular forms without ECs to predominantly interwound when ECs were present ( Figure 1—figure supplement 2E ) .", "Consistent with predictions that H-NS bridged interactions and transcriptional silencing may be disrupted at higher temperatures ( Arold et al . , 2010 ) , we found that the more slowly migrating band in EMSA assays , which we attributed to bridged filaments , disappeared when gels were run at 37°C ( Figure 1—figure supplement 3 ) .", "We conclude that H-NS forms bridged complexes principally at high divalent cation concentrations when H-NS is present at 50–66 H-NS/kb and forms linear filaments when H-NS is bound at 2 mM Mg2+ or at high concentrations of H-NS ( see ‘Discussion’ ) .", "The presence of both slower and faster migrating complexes in EMSA assays of samples formed at 2 mM Mg2+ , where AFM predicts mostly linear filaments , is an inconsistency in our results but could reflect formation of bridged filaments during electrophoresis in the altered environment of the gel .", "In general , the detection of H-NS–DNA nucleoprotein filaments by EMSA in buffers lacking Mg2+ ( see ‘Materials and methods’ ) may reflect the caging effects of PA gels that could stabilize both the bridged and linear filaments .", "We next assessed how the linear or bridged filaments affected transcript elongation during single-round in vitro transcription by adding NTPs ( 30 µM each ) to halted A26 ECs after H-NS filament formation ( Figure 2A ) .", "Strikingly , transcript elongation at 8 mM Mg2+ was dramatically slower on filaments formed at 66 H-NS/kb ( conditions favoring bridged filaments ) than on DNA alone , but returned to nearly the rate observed on DNA alone on filaments formed under conditions that favor linear filaments ( 200 H-NS/kb ) .", "We converted the gel images to plots of transcript length by densitometry and comparison to size standards , which allowed calculation of mean transcript lengths for each time point ( Figure 2B–D ) .", "To assign precise pause positions , we also compared pause bands to 3′-deoxyNTP-generated ladders using shorter templates and high-resolution gels ( Figure 2—figure supplement 1; Table 1 ) .", "These reaction profiles revealed that under bridging conditions H-NS dramatically slowed escape from some pause sites , whereas other pauses remained largely unaffected .", "Some H-NS pauses were long-lived , with dwell times of more than 12 min ( e . g . , starred C347 pause; Figure 2B ) . 10 . 7554/eLife . 04970 . 007Figure 2 . H-NS dramatically decreased transcript elongation in vitro .", "( A ) In vitro transcription in the presence of 66 H-NS/kb or 200 H-NS/kb filaments at 20°C , 8 mM Mg2+ , and 30 µM each NTP .", "ECs ( 10 nM ) were formed at the end of the C-less cassette on the λPR-bgl template ( A26 ECs ) and then incubated with H-NS .", "Samples were removed at 2 , 3 , 4 , 8 , 16 , and 32 min after addition of NTPs and separated by 6% PAGE .", "M , 5′ end-labeled , MspI-digested pBR322 marker .", "RO , run-off RNA .", "Pauses mapped to single-nt resolution in Figure 2—figure supplement 1 and Table 1 are indicated on the right side of the gel in red for H-NS-stimulated pauses and black for H-NS independent pauses .", "( B , C )", "Densitometry profiles of transcripts produced at 8 mM Mg2+ and 20°C from the λPR-bgl template in 66 H-NS/kb or 200 H-NS/kb filaments ( B and C , respectively ) or without H-NS ( see ‘Materials and methods’ ) .", "In ( B ) , the 4-min time point from the gel shown in ( A ) is displayed horizontally to allow alignment with the densitometry profile ( larger transcripts are to the right ) .", "Key pauses are marked in the profiles .", "Insets , mean transcript lengths and standard deviations were calculated from at least four independent experiments .", "( D ) Densitometry profiles of transcripts produced at 2 mM Mg2+ and 20°C from the λPR-bgl template in 66 H-NS monomer/kb compared to without H-NS .", "Inset , mean transcript lengths and standard deviations were calculated from at least four independent experiments . DOI: http://dx . doi . org/10 . 7554/eLife . 04970 . 00710 . 7554/eLife . 04970 . 008Figure 2—figure supplement 1 . Mapping of 3′ ends of pauses on λPR-bgl template .", "( A–K )", "Pauses on the λPR-bgl template were could be mapped to nucleotide resolution up to ∼200 nucleotides downstream from the transcription start site using ladders generated by 3′-deoxy NTP incorporation ( see ‘Materials and methods’ ) .", "To map pauses further downstream , we prepared truncated templates that deleted 5′ portions of the bgl transcribed region ( pMK122 , pMK110-520 , pMK124 , and pMK126; ‘Materials and methods’ ) .", "Halted A26 ECs ( 10 nM ) were formed on pMK110 template ( A–B ) , pMK122 template ( C–E ) , pMK110-520 template ( F–G ) , pMK124 template ( H ) , or pMK126 template ( I–K ) .", "Transcription was then restarted with 30 μM ATP , UTP , GTP , and CTP at 37°C with or without 10–50 μM 3′-deoxy GTP , ATP , UTP , or CTP ( concentrations were adjusted depending on the segment to be examined ) .", "Samples were collected at times indicated on the panels and then separated by 8% PAGE .", "Lanes are marked with sample times or the 3′-deoxyNTP used .", "Contrast in each image was adjusted to increase visibility .", "The mapped sequence is indicated next to each panel with the pause 3′ nucleotide highlighted in red .", "H-NS-stimulated pause sequences are starred .", "In one case ( G ) , transcription was restarted in the presence or absence of 66 H-NS/kb at 20°C to determine which pause was H-NS dependent . DOI: http://dx . doi . org/10 . 7554/eLife . 04970 . 00810 . 7554/eLife . 04970 . 009Figure 2—figure supplement 2 . Linear H-NS filaments had minimal effects on elongation .", "( A ) 10 nM halted ECs formed on the λPR-bgl template were incubated with H-NS in either 2 or 8 mM Mg2+ to reach equilibrium .", "30 μM NTPs were added , time points were taken at 10 , 20 , 40 , 60 , 120 , and 180 s at 20°C , then resolved by 12% PAGE .", "M denotes labeled MspI-digested pBR322 marker .", "02 and 08 refers to the time point taken prior to the addition of NTPs in 2 or 8 mM Mg2+ respectively .", "( B ) PAGE ( 6% PA ) of the 2 mM Mg2+ reactions described in ( A ) .", "Time points were taken at 2 , 3 , 4 , 8 , 16 , and 32 min at 20°C .", "M denotes labeled MspI-digested pBR322 marker , and RO indicates template run-off products .", "( C ) Mean transcript lengths at various time points plotted with error bars depicting standard deviations of at least four independent experiments assembled in 2 mM Mg2+ buffer . DOI: http://dx . doi . org/10 . 7554/eLife . 04970 . 00910 . 7554/eLife . 04970 . 010Figure 2—figure supplement 3 . H-NS effects on transcript elongation also occurred on a different template .", "( A ) The 1 . 27-kb linear pMK121 DNA template ( λPR-bglF template ) similarly contains the λPR promoter followed by a 26-nucleotide C-less cassette and includes a different portion of the bgl operon , the region downstream of the bglF antisense promoter ( PAS ) ( Peters et al . , 2012 ) .", "( B ) Native PAGE of filaments formed on 10 nM labeled λPR-bglF template at increasing H-NS concentrations and 8 mM Mg2+ ( −RNAP lanes ) .", "( +RNAP lanes ) , 10 nM halted A26 ECs at increasing H-NS concentrations and 8 mM Mg2+ .", "( C ) A26 ECs ( 10 nM; λPR-bglF template ) were incubated with H-NS in 8 mM Mg2+ .", "NTPs ( 30 µM ) were added , samples were removed at 10 , 20 , 40 , 60 , 120 , and 180 s and 20°C , and the samples were resolved by denaturing PAGE ( 12% PA ) .", "M , labeled MspI-digested pBR322 marker .", "0 , time point taken prior to the addition of NTPs .", "( D ) PAGE in 6% PA of the reactions described in ( C ) .", "Samples were taken at 2 , 3 , 4 , 8 , 16 , and 32 min , M , labeled MspI-digested pBR322 marker , RO , indicates template run-off products .", "( E ) Densitometry profiles of various time points of reactions with 66 H-NS/kb compared to the absence of H-NS .", "Mean transcript lengths at various time points from two independent experiments are shown in the chart . DOI: http://dx . doi . org/10 . 7554/eLife . 04970 . 01010 . 7554/eLife . 04970 . 011Table 1 . Pause sites and their responses to H-NS and transcription factorsDOI: http://dx . doi . org/10 . 7554/eLife . 04970 . 011Pause positionPausingTerminationSequenceH-NSRfaHGreBGreARhoRho + H-NSRho + NusGPause↓C134↑ ? ?", "? ? ? ?", "CGCUGAUAACUCAAGCUUUCUUCCUGG162↑↓↓ ?", "–––AAUUAAGGCUGAACUGAAAUUUUAUUU169↑–↓↓––↑GCUGAACUGAAAUUUUAUUAAUUGCAC213↑–↓↓–––GCGUGACACCUGCAACAUCCUCCAUAC220↑↓–↓–––ACCUGCAACAUCCUCCAUAUUUCCGCU226↑↓–↓–––AACAUCCUCCAUAUUUCCGCUCAUUUC346↑↑↑–↓––↑–UAGCUGGAACUCUUUCGGGUAAAGCCC370–↓–↓↑––CCGCUGGAUAUCCCACAGCAACGGGUC393 , G394↑↓↓↓–↑↑GGUUGGGCAGCAACACGUUUUGCUGAU588 , U589↑↑↑–↓↓↑––UCAAGGCAUACUCUUUUUCUAUUCCAA593–––––––GCAUACUCUUUUUCUAUUCCACUUGAG624↑↓↓↓–––UUCUUUCGCCAGCGCGUUUUUGAAAGG643↑–↓↓–––UUGAAAGCCAAUUCCGCGCCCCAUGAA746 , U747––↓–↑–↑GCAAGGACCUUUUUUAUAAACAAAAAG926↑–––– ? ?", "AAUAUGACCAUGCUCGCAGUUAUUAAU996↑–↓↓– ? ?", "CCAAUAAUUAAGUUAUUGGGAUUUGUU1011↑↓↓↓– ? ?", "UUGGGAUUUGUCUGGUGAAUUAUUUGU1022 , U1024↑↓↓↓– ? ?", "GUCUGGUGAAUUAUUUGUCGCUAUCUU1079 ( ops ) ––↓↓↑ ? ?", "CUAGUGGCGGUAGCGUGCUUUUUUCAPause positions are given as 3′ RNA nucleotide identity and distance from the transcription start site as mapped by high-resolution PAGE ( Figure 2—figure supplement 1 ) .", "↑ , increased pause or termination .", "↓ , decreased pause or termination .", "In the sequences shown , pause 3′ ends are bold ( under arrow ) and the position corresponding to the incoming NTP is underlined .", "In contrast , ECs elongating through linear H-NS filaments were only modestly slowed relative to transcription of DNA alone ( filaments formed at 200 H-NS/kb in 2 or 8 mM Mg2+; Figure 2C , D , Figure 2—figure supplement 2 ) .", "Filaments formed at 66 H-NS/kb monomer in 2 mM Mg2+ did impede elongation , but to a lesser extent than did filaments formed in bridging conditions ( Figure 2D , Figure 2—Figure supplement 2 ) .", "Although it is possible that pure linear filaments inhibited transcript elongation to some extent , their effects were clearly less than those of bridged filaments .", "The inhibitory effect seen at 2 mM Mg2+ and 66 H-NS/kb monomer could reflect low or transient levels of bridging in these conditions that were not captured by AFM imaging .", "We conclude that bridged H-NS filaments dramatically slow RNAP by increasing pausing at a subset of sites and that linear filaments have lesser or no effects on elongating RNAP .", "Importantly , the large effects of bridged vs linear H-NS filaments were also observed at physiological NTP concentrations ( Figure 3 , Figure 3—figure supplement 1 ) and at higher temperatures ( Figure 4 , Figure 4—figure supplement 1 ) .", "The effects of bridged filaments persisted to 28°C , but the effects of H-NS largely disappeared at temperatures over 30°C .", "Thus , H-NS alone inhibits transcript elongation in conditions found during free-living but not inter-host growth of enteric bacteria , consistent with observations that H-NS helps mediate the bacterial temperature response upon infection ( Trachman and Yasmin , 2004; Ono et al . , 2005 ) .", "We also verified that the preferential effects of bridged vs linear filaments on ECs also occurred on a different template on which RNAP initiated transcription at the start site of the bglF antisense promoter ( Figure 2—figure supplement 3 ) .", "We conclude that H-NS filaments can drastically slow transcript elongation by ECs under physiological conditions that would prevail when E . coli grows at ambient temperatures outside a mammalian host . 10 . 7554/eLife . 04970 . 012Figure 3 . H-NS inhibited transcript elongation at physiological NTP concentrations ( 1 mM each NTP ) .", "Densitometry profiles of transcripts produced at 20°C , 12 mM Mg2+ and 1 mM each NTP from the λPR-bgl template in bridged H-NS filaments ( 66 H-NS/kb ) or in linear H-NS filaments ( 200 H-NS/kb ) .", "Samples were removed at 0 . 66 , 1 , 2 , 3 , 4 , 8 , and 16 min after addition of NTPs and separated by denaturing PAGE .", "Inset , mean transcript lengths at various times were averaged from two independent experiments . DOI: http://dx . doi . org/10 . 7554/eLife . 04970 . 01210 . 7554/eLife . 04970 . 013Figure 3—figure supplement 1 . Electrophoretic gel image showing H-NS inhibited transcription elongation at physiological NTP concentrations ( 1 mM each NTP ) .", "ECs ( 10 nM ) formed on the λPR-bgl template were incubated with bridged H-NS ( 66 H-NS/kb ) or linear filaments ( 200 H-NS/kb ) in 12 mM Mg2+ .", "1 mM NTPs were added , and samples were removed at 0 . 66 , 1 , 2 , 3 , 4 , 8 , and 16 min and resolved by denaturing PAGE .", "M denotes labeled MspI-digested pBR322 plasmid marker , and RO indicates template run-off products . DOI: http://dx . doi . org/10 . 7554/eLife . 04970 . 01310 . 7554/eLife . 04970 . 014Figure 4 . H-NS effects on transcript elongation were reduced at ≥30°C . Densitometry profiles of transcripts produced at 25°C , 30°C , or 37°C , 8 mM Mg2+ and 30 μM each NTP from the λPR-bgl template in the presence of 66 H-NS/kb ( bridged filaments ) .", "Samples were removed at 2 , 4 , 8 , 16 , and 32 min after addition of NTPs and separated by denaturing PAGE .", "Insets , mean transcript lengths at various time points plotted were averaged from two independent experiments . DOI: http://dx . doi . org/10 . 7554/eLife . 04970 . 01410 . 7554/eLife . 04970 . 015Figure 4—figure supplement 1 . Electrophoretic gel image showing reduced H-NS effects on transcription elongation at ≥30°C . 10 nM-halted ECs formed on the λPR-bgl template were incubated with 66 H-NS/kb in 8 mM Mg2+ for 20 min at 25°C , 30°C , or 37°C .", "30 μM NTPs were added , time points were taken at 2 , 3 , 4 , 8 , 16 , and 32 min and resolved by PAGE .", "M denotes labeled MspI-digested pBR322 marker .", "RO indicates template run-off . DOI: http://dx . doi . org/10 . 7554/eLife . 04970 . 015 The preferential inhibition of transcript elongation by bridged vs linear H-NS might reflect either tighter binding of H-NS in the bridging mode or a topological effect of sequestering ECs in a DNA segment flanked by bridged filaments on both sides ( see ‘Discussion’ ) .", "To determine whether bridged H-NS filaments readily re-formed upstream of ECs on recently transcribed DNA segments , we used AFM to examine the configuration of ECs and filaments after ECs had transcribed about half the DNA template ( 8 and 16 min at 30 µM NTPs; Figure 5A ) .", "H-NS was clearly able to bridge around ECs located in the middle of the DNA template ( ∼80% of observed complexes , n = 93; Figure 5A complexes I–X , Figure 5B ) .", "A minority of complexes ( ∼20% ) did not exhibit H-NS bridging on one side of ECs , either because the shorter bridged segments unravel during deposition on AFM slides or because bridged filaments failed to form on newly exposed DNA upstream from elongating ECs ( Figure 5A , complexes XI and XII ) . 10 . 7554/eLife . 04970 . 016Figure 5 . Bridged H-NS filaments reformed upstream of ECs during transcription .", "( A ) Representative AFM images of ECs elongating through bridged filaments ( 66 H-NS/kb; 8 mM Mg2+; 20°C ) sampled at either 8 or 16 min after addition of NTPs ( 30 µM each ) .", "ECs ( 10 nM ) and H-NS filaments were absorbed onto APS-mica and imaged in air .", "ECs are indicated by white arrows .", "Roman numerals and arrows depict two classes of filaments formed during transcription ( cyan , bridged on both sides of the EC; green , unbridged on one side of the EC ) .", "Depictions of ECs and filaments are shown in insets for a subset of panels ( black , gray different DNA duplexes; blue , RNAP ) .", "( B ) Quantification of H-NS filament disposition during EC elongation from AFM images like those shown in ( A ) .", "Cyan bar , bridged H-NS filaments both upstream and downstream of ECs .", "Green bar , bridged H-NS filaments on only one side of ECs . DOI: http://dx . doi . org/10 . 7554/eLife . 04970 . 016 These observations suggest bridged H-NS may constrain ECs within a topologically closed domain that could promote pausing ( see ‘Discussion’ ) but do not rule out the possibility that bridged H-NS binds DNA tighter than in linear H-NS filaments to create a stronger roadblock to transcript elongation .", "To investigate the mechanistic basis of H-NS stimulation of transcription pausing , we examined the precise 3′ ends of paused transcripts stimulated by H-NS ( Table 1 , Figure 2—figure supplement 1 ) .", "In general , the sites of pausing corresponded to the recently mapped consensus pause sequence for E . coli RNAP ( Larson et al . , 2014 ) , with pausing occurring just after pyrimidine addition and just prior to purine addition and with Gs at positions −10 or −11 .", "H-NS increased pausing strongly at a subset of sites ( e . g . , C346 , U588 , and some others; Table 1 ) but had less effect at other pause sites recognized in the absence of H-NS ( e . g . , at C370 and A746 , and at U1079 , the ops site present on the template ) .", "Interestingly , both H-NS-sensitive pauses occurred with Us at positions −2 and −3 , whereas 2 of 3 H-NS resistant pauses lacked this feature ( C370 and A593 ) .", "The H-NS-resistant A746 pause containing a U-tract at these positions and the A593 pause were atypical , as they occurred with a 3′ A . 3′ A is known to be highly sensitive to a 1-nt backtrack conformation , which can be readily cleaved by intrinsic hydrolysis and re-extended ( Sosunova et al . , 2013 ) .", "The 3′-proximal U-tracts observed in the H-NS-stimulated pause sequences should make paused ECs especially sensitive to >1-nt backtracking ( Komissarova and Kashlev , 1997; Nudler et al . , 1997 ) , suggesting that pauses sensitive to multiple-nt backtracking may be most strongly affected by H-NS .", "To investigate further the nature of H-NS-sensitive pauses , we next examined the effects of RfaH and GreB ( Figure 6 ) .", "RfaH and its paralog NusG are thought to suppress entry into pauses by favoring forward translocation and can additionally inhibit hairpin-stabilized pauses by inhibiting RNAP clamp opening through an interaction of their NTDs with the clamp ( Herbert et al . , 2010; Hein et al . , 2014 ) ( Figure 6A ) .", "Although NusG can inhibit backtracking to some extent , GreB is a far more effective anti-backtracking factor because it rescues ≥2-nt backtracked ECs by promoting intrinsic transcript cleavage ( Borukhov et al . , 1993; Laptenko et al . , 2003 ) .", "However , GreB and its paralog GreA ( which promotes cleavage of 1-nt backtracked RNAs ) have little effect on the frequency of pausing overall or on hairpin-stabilized pausing ( Feng et al . , 1994; Artsimovitch and Landick , 2000 ) . 10 . 7554/eLife . 04970 . 017Figure 6 . Bridged H-NS filaments induced RNAP backtracking , which was rescued by GreB .", "( A ) Steps in pausing affected by the NusG-like N-terminal domain ( NGN ) of RfaH ( RfaH-NTD ) or Gre factors ( e . g . , GreB ) .", "Binding of the NGN RfaH-NTD ( cyan ) to the clamp domain ( pink ) of RNAP inhibits clamp motion and suppresses entry into pause states ( Sevostyanova et al . , 2011 ) .", "The duration of pausing once paused ECs form can be increased by backtracking of DNA and RNA through RNAP , during which the 3′ RNA enters the RNAP secondary channel .", "GreB promotes endonucleolytic cleavage of the backtracked RNA in the RNAP active site to convert an offline paused EC back to an active EC ( Laptenko et al . , 2003 ) .", "( B ) Mean transcript lengths were averaged from two independent experiments .", "( C , D )", "Densitometry profiles of transcripts produced at 20°C , 12 mM Mg2+ , and 1 mM each NTP from the λPR-bgl template in bridged filaments ( 66 H-NS/kb ) with or without 300 nM RfaH-NTD ( C ) or 50 nM GreB ( D ) .", "Samples were removed at 0 . 33 , 0 . 66 , 1 , 1 . 5 , 2 , 3 , 4 , 8 , and 16 min after addition of NTPs and separated by denaturing PAGE . DOI: http://dx . doi . org/10 . 7554/eLife . 04970 . 01710 . 7554/eLife . 04970 . 018Figure 6—figure supplement 1 . At 1 mM NTPs , NusG partially suppressed H-NS effects on pausing and GreA more significantly suppressed H-NS effects .", "( A ) A26 ECs ( 10 nM ) formed on λPR-bgl were incubated with H-NS to form bridged filaments at 66 H-NS/kb and 12 mM Mg2+ .", "GreA ( 500 nM ) or NusG ( 75 nM ) was added followed by NTPs ( 1 mM each ) , time points were taken at 0 . 5 , 1 , 1 . 5 , 2 , 4 , and 8 min at 20°C , and separated by PAGE .", "M , labeled MspI-digested pBR322 marker .", "RO , run-off RNAs .", "( B ) Mean transcript lengths were calculated from two independent experiments .", "( C , D )", "Densitometry profiles of transcripts produced from the λPR-bgl templates with or without bridged H-NS and with or without GreA ( C ) or NusG ( D ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 04970 . 01810 . 7554/eLife . 04970 . 019Figure 6—figure supplement 2 . Electrophoretic gel image showing that H-NS-induced RNAP backtracking was rescued by GreB . PAGE corresponding to Figure 6 , where reactions were assembled with 66 H-NS/kb and 50 nM GreB or 300 nM RfaH-NTD . DOI: http://dx . doi . org/10 . 7554/eLife . 04970 . 01910 . 7554/eLife . 04970 . 020Figure 6—figure supplement 3 . The H-NS stimulated C346 pause readily backtracked , whereas H-NS-resistant pausing at C370 occurred without obligate backtracking .", "( A , B )", "Nucleic-acid scaffolds that enable EC reconstitution just upstream of sequences equivalent to the C346 H-NS-stimulated pause ( A ) and the C370 pause not stimulated by H-NS ( B ) .", "Lowercase RNA , nt added by RNAP after reconstitution .", "* , position of [32P]CMP incorporation ( red ) .", "Green , 3′ nt at the pause .", "( C , D )", "Pause assay reaction schemes ( see ‘Materials and methods’ ) .", "For assays on the fly , 10 μM CTP , UTP , and GTP ( for ECC346 ) or CTP , ATP , and GTP ( for ECC370 ) were added to ECs upstream from the pause at either 37°C or 20°C .", "For C346 delay assay ( C ) , 10 μM CTP and UTP extended ECs to the pause and 10 μM GTP was added after 5 min to extend the RNA .", "For C370 delay assay ( D ) , ECs were immobilized on Co2+ magnetic beads and extended to the pause by stepwise incubation with [α-32P] CTP , ATP , and CTP , incubated for 5 min , and then extended from the pause with CTP , ATP , and GTP ( all at 10 μM NTP ) .", "ECs were washed five times with 1 ml EB between steps .", "( E , F )", "Denaturing RNA gels of products of assays depicted in ( C ) and ( D ) .", "C , chase sample incubated with 1 mM all 4 NTPs .", "( G , H )", "C346 or C370 paused ECs formed as shown in ( C ) and ( D ) were resuspended in cleavage buffer ( pH 9 . 0 and 20 mM Mg2+ ) to induce intrinsic cleavage ( G ) or in EB with or without 50 nM GreB to induce GreB-mediated hydrolysis ( H ) .", "Possible 5′ and 3′ cleavage products determined by position of label are illustrated for C346 and C370 between the gel panels . DOI: http://dx . doi . org/10 . 7554/eLife . 04970 . 020 We tested the effects of RfaH-NTD ( which is sufficient for full pause–suppression activity of RfaH ) , NusG , GreB , and GreA .", "Transcription through bridged H-NS filaments in the presence of 300 nM RfaH-NTD and 1 mM NTPs resulted in partial suppression of H-NS-stimulated pausing ( Figure 6B , C ) .", "RfaH-NTD ameliorated the effect of H-NS on the C393 , G624 , A746 , G926 , U996 , U1011 , and U1022 pauses but not on the strong H-NS-stimulated C346 and U588 pauses .", "The effects of RfaH-NTD on individual pauses were more evident than the effect on mean transcript length because RfaH-NTD also caused a strong , H-NS independent pause at the U1079 ops site ( Artsimovitch and Landick , 2002 ) that dominated the mean transcript calculations .", "NusG at 100 nM had similar partial effects on overcoming the H-NS-stimulated pauses ( Figure 6—figure supplement 1A , B , C ) .", "In contrast to these partial effects on H-NS-stimulated pausing , GreA at 500 nM or GreB at 50 nM dramatically reduced the effects of H-NS on elongation ( Figure 6B , D , Figure 6—figure supplement 2A , B , D ) .", "All but one pause at least moderately H-NS-stimulated ( G926 ) and both pauses strongly stimulated by H-NS ( C346 and U588 ) were reduced by GreB , whereas the two pauses not affected by GreB ( C370 and G926 ) were suppressed by RfaH NTD ( Table 1 ) .", "One pause ( C346 ) was affected by GreB less so by GreA , suggesting was backtracked by multiple nt .", "To investigate the difference between pauses strongly affected by H-NS and GreB vs pauses affected by RfaH-NTD but less by H-NS and GreB , we chose the C346 and C370 pauses and engineered scaffolds from the sequences that allowed examination of RNAP behavior with single-nucleotide resolution ( Figure 6—figure supplement 3A , B ) .", "Although scaffolds lack sufficient lengths of duplex DNA to form H-NS filaments , they proved highly informative about the nature of these pauses .", "The C346 was not detectable at 37°C or 20°C during active elongation ( on the fly ) but became a strong pause when RNAP was delayed at the C346 position by transiently withholding GTP ( delay; Figure 6—figure supplement 3C , E ) .", "In contrast , the C370 pause was readily detectable on the fly and increased mostly in the fraction paused rather dwell time when delayed at the C370 position ( Figure 6—figure supplement 3D , F ) .", "To understand whether pausing at C346 or C370 involved backtracking , we tested intrinsic cleavage of the nascent RNA in ECs halted at the pause sites .", "Intrinsic cleavage occurs at internal positions of backtracked transcripts that occupy the RNAP active site and can map the extent of backtracking .", "The C346 but not the C370 RNA was highly sensitive to intrinsic cleavage in halted ECs; the positions of cleavage suggested that halted C346 ECs spontaneously backtracked by 4–6 nt ( Figure 6—figure supplement 3G ) .", "We confirmed this result by testing susceptibility to GreB cleavage .", "Again the C346 halted EC was more sensitive than the C370 halted EC ( Figure 6—figure supplement 3H ) .", "Thus , pausing at a sequence significantly enhanced by H-NS ( C346 ) occurred significantly only when delayed and then readily backtracked to create a long-lived pause , whereas pausing at a sequence much less affected by H-NS ( C370 ) exhibited much less potential for backtracking .", "Taken together , these data suggest that H-NS promotes pausing by stimulating backtracking .", "Both the sequences of strongly H-NS-stimulated pauses and the stronger effects of GreB than RfaH-NTD are consistent with this hypothesis .", "The C370 pause that showed greater response to RfaH-NTD than either GreB or H-NS may reside mostly in the elemental pause state , entry to which may be suppressed by RfaH-NTD-induced forward translocation .", "This pause occurs at a relatively C-rich RNA:DNA hybrid , with both −10 G and −11 G present in the consensus pause sequence and is thus a strong candidate for a non-backtrack , elemental pause .", "Examination of the potential for backtracking at C346 and C370 sequences confirmed this interpretation .", "Genomic locations of H-NS filaments and transcription termination caused by the Rho termination factor are highly correlated , raising the possibility that H-NS-stimulated pausing by aiding Rho-dependent termination ( Peters et al . , 2012 ) .", "Deletion of hns is synthetically lethal with either chemical inhibition of Rho or mutations in rho , suggesting a functional role for H-NS in Rho-dependent termination ( Tran et al . , 2011; Peters et al . , 2012 ) .", "Rho is a homohexameric ATP-dependent RNA helicase that binds to ∼80 nt of unstructured C-rich RNA ( Figure 7A , inset ) ( reviewed in Peters et al . ( 2011 ) ) .", "Once bound to a nascent RNA transcript , Rho translocates 5′–3′ along the RNA until it reaches the EC , where it terminates transcription .", "Thus , the elongation rate of the EC determines a kinetic window during which it can be acted upon by Rho; increases in the frequency and strength of pausing can assist in termination by extending this kinetic window ( Jin et al . , 1992 ) . 10 . 7554/eLife . 04970 . 021Figure 7 . Stimulation of pausing by bridged H-NS filaments aided Rho-dependent termination .", "( A , B )", "Densitometry profiles of transcripts produced at 28°C , 8 mM Mg2+ , and 30 µM each NTP from the λPR-bgl template in bridged filaments ( 66 H-NS/kb ) with or without 5 nM Rho ( A ) and with or without 75 nM NusG ( B ) .", "Samples were removed at 2 , 4 , 8 , 16 , and 32 min after NTPs were added and separated by denaturing PAGE .", "To detect release of Rho-terminated transcripts , a 32-min sample was separated into released and EC-bound fractions using paramagnetic Co2+ beads that bind the His10-tagged RNAP .", "The released supernatant fraction was separated by denaturing PAGE and converted to densitometric profiles shown as darker colors .", "( C , D )", "Mean transcript lengths and standard deviations were calculated from at least two ( C ) or four ( D ) independent experiments .", "( E ) Rho termination efficiencies were calculated as the fraction of released transcripts divided by the total transcripts , with averages and standard deviations from at least three independent experiments . DOI: http://dx . doi . org/10 . 7554/eLife . 04970 . 02110 . 7554/eLife . 04970 . 022Figure 7—figure supplement 1 . Stimulation of pausing by bridged H-NS filaments aided Rho-dependent termination .", "( A ) Halted A26 ECs ( 10 nM ) formed on the λPR-bgl template were incubated with bridged H-NS , 75 nM NusG , or 5 nM Rho at 8 mM Mg2+ at 28°C .", "Samples were then collected at 2 , 4 , 8 , 16 , and 32 min after addition of NTPs ( 30 µM each ) and resolved by PAGE .", "Rho-terminated , released transcripts for the 32-min sample were determined as described in the legend to Figure 7 .", "R , released transcripts .", "M , labeled MspI-digested pBR322 marker .", "RO , run-off RNAs .", "( B ) Densitometry profiles were determined as described in legend of Figure 2 .", "Mean transcript lengths were averaged from at least two independent experiments .", "( C ) Mean transcript lengths and standard deviations with or without bridged H-NS and with or without 5 nM Rho were determined using at least four independent experiments .", "( D ) Mean transcript lengths with or without bridged H-NS , 5 nM Rho , or 75 nM NusG were averaged from two independent experiments . DOI: http://dx . doi . org/10 . 7554/eLife . 04970 . 022 To test directly whether H-NS-stimulated pauses increased the kinetic window for Rho-dependent termination , we added Rho and 30 μM NTPs to halted ECs on DNAs with or without bridged H-NS filaments at 28°C , a temperature at which Rho terminated transcription and H-NS stimulated pausing in control experiments ( not shown ) .", "To distinguish possible Rho stimulation of pausing from true Rho-dependent termination , we separated the RNAs after 32 min into an EC-bound fraction retained on beads via a His10 tag on RNAP and a supernatant fraction containing the terminated RNAs ( Figure 7A , C , Figure 7—figure supplement 1A ) .", "We observed significant termination only when Rho was present; bridged H-NS filaments alone did not cause RNA release ( compare red and purple traces , Figure 7A ) .", "However , Rho terminated ECs at earlier positions on DNA in bridged filaments than DNA lacking H-NS ( compare blue and purple traces , Figure 7A , E ) .", "Strikingly , bridged H-NS facilitated Rho termination at sites poorly utilized by Rho alone and at which H-NS strongly stimulated pausing ( e . g . , C346 and C393 , Figure 7—figure supplement 1A , Table 1 ) .", "The C346 and C393 pauses are strong candidates for backtrack pauses that were affected by both H-NS and GreB , whereas a site of Rho termination observed in both the presence and absence of H-NS ( C370 ) is a candidate for a non-backtracked , elemental pause ( Table 1 , Figure 7E; see above ) .", "This result suggests that non-backtracked pauses may be better natural substrates for Rho than backtracked pauses ( see ‘Discussion’ ) .", "However , strong H-NS stimulation of pausing may allow Rho termination at backtrack sites by increasing the kinetic window for Rho action at the sites as backtracked ECs are known to be poor substrates for Rho ( Dutta et al . , 2008 ) .", "An alternative hypothesis is that direct H-NS–Rho interaction aids termination .", "However , H-NS in linear filaments did not synergize with Rho; the patterns of Rho termination on linear filaments vs DNA alone were almost identical ( Figure 8 , Figure 8—figure supplement 1 ) .", "We conclude that H-NS-stimulated pausing on bridged filaments increases the kinetic window for Rho action ( Jin et al . , 1992 ) , and the apparent preferential effect of Rho on backtrack pauses may allow Rho to terminate at otherwise suboptimal sites . 10 . 7554/eLife . 04970 . 023Figure 8 . Linear H-NS filaments did not aid in Rho-dependent termination .", "( A ) Densitometry profiles of transcripts produced at 28°C , 8 mM Mg2+ , and 30 µM each NTP from the λPR-bgl template in linear filaments ( 200 H-NS/kb ) with or without 5 nM Rho .", "Samples were removed at 2 , 4 , 8 , 16 , and 32 min after addition of NTPs and separated by denaturing PAGE .", "Rho-terminated , released transcripts for the 32-min sample were determined as described in the legend to Figure 7 .", "( B ) Mean transcript lengths were averaged from two independent experiments . DOI: http://dx . doi . org/10 . 7554/eLife . 04970 . 02310 . 7554/eLife . 04970 . 024Figure 8—figure supplement 1 . Linear H-NS Filaments did not aid in Rho termination .", "( A ) Halted A26 ECs ( 10 nM ) formed on λPR-bgl template were incubated with either 66 H-NS/kb or 200 H-NS/kb with or without 5 nM Rho at 8 mM Mg2+ and 28°C .", "Samples were then collected at 2 , 4 , 8 , 16 , and 32 min after addition of NTPs ( 30 µM each ) and resolved by PAGE .", "Rho-terminated , released transcripts for the 32-min sample were determined as described in the legend to Figure 7 .", "R , released RNAs , M , labeled MspI-digested pBR322 marker .", "RO , run-off RNAs . DOI: http://dx . doi . org/10 . 7554/eLife . 04970 . 024 In addition to its pause-suppressing activity , NusG enhances Rho-dependent termination through direct interactions between Rho and NusG ( Figure 7A , inset ) ( Li et al . , 1993; Pasman and von Hippel , 2000; Mooney et al . , 2009; Chalissery et al . , 2011 ) .", "In vivo , NusG improves the efficiency of Rho termination at a fraction ( ∼20% ) of terminators with suboptimal Rho binding sites ( Sullivan and Gottesman , 1992; Peters et al . , 2012 ) .", "Consistent with in vivo effects of H-NS on Rho-dependent termination , genetic studies have shown that gain-of-function mutations in hns suppress loss of function mutations in rho and nusG , suggesting that all three function in the same pathway ( Saxena and Gowrishankar , 2011 ) .", "Further , H-NS filaments are found near NusG-dependent Rho terminators in vivo ( Peters et al . , 2012 ) .", "To test if NusG affects the synergy between Rho and H-NS , we compared the elongation and termination profiles of NusG and Rho to reactions containing NusG , Rho , and bridged H-NS ( Figure 7B , D ) .", "We found that NusG-enhanced Rho termination at H-NS-dependent pause sites that are strong candidates for backtracking .", "The addition of NusG robustly aided in Rho-dependent termination , consistent with previous reports .", "During transcription of bridged H-NS filaments , NusG stimulated termination of the C346 and C393 pauses more than the C370 pause and allowed termination at the upstream U169 pause ( Figure 7B , E , Figure 7—figure supplement 1A ) .", "NusG also altered the preferred positions of termination at the U588 pause .", "These effects were exacerbated when bridged H-NS was present , inhibiting overall elongation and increasing termination relative to samples containing only Rho or both NusG and Rho .", "These results suggest that NusG , like H-NS , may aid Rho in terminating backtracked ECs .", "Synergy between the actions of NusG and H-NS at Rho terminators could play an important role in transcriptional silencing by H-NS in vivo ( see ‘Discussion’ ) ." ], [ "Bridged H-NS filaments slow RNAP at a subset of pause sites at which RNAP appears susceptible to backtracking , as evidenced by the large effect of GreB in suppressing H-NS stimulation of pausing at these sites , by the presence of short U-tracts just upstream from the pause sites at which H-NS has the largest effects , and by direct detection of backtracking for the C346 H-NS stimulated pause .", "The existence of both H-NS-sensitive and largely H-NS resistant pause sites , both of which occur at sequences conforming to the recently described consensus pause sequence ( Larson et al . , 2014 ) , is consistent with the view that RNAP remains in a non-backtracked , elemental pause state at some sites and that such paused ECs are less susceptible to inhibition by H-NS .", "At least two hypotheses might explain the effects of H-NS at the class of sites more susceptible to backtracking: ( 1 ) bridged H-NS might create a physical barrier that blocks forward translocation and thus favors reverse translocation of RNAP ( road-blocking model ) or ( 2 ) the structure of the bridged filament entraps elongating RNAP in small , topologically fixed domains by binding both upstream and downstream of the EC ( topological model; Figure 9A ) .", "Although we lack sufficient information to exclude either mechanism unambiguously and both could be contributory , our results favor the topological model . 10 . 7554/eLife . 04970 . 025Figure 9 . Models for H-NS effects on pausing , Rho termination , and DNA bridging .", "( A ) As RNAP elongates through bridged filaments , pause durations increase for one or both of two reasons:", "( i ) the off-rate of bridged H-NS is slower than the elongation rate of RNAP , leading to a roadblock ( physical barrier; black bar ) ; or", "( ii ) H-NS bridging creates a closed topological domain that accumulates positive and negative supercoiling ( torsional stress ) in front and behind the EC , respectively , because free rotation of the DNA is blocked by bridged H-NS contacts and free rotation of the EC is blocked by steric clash between the bridged H-NS–DNA filament and the nascent RNA , including macromolecules like Rho or ribosomes bound to the nascent RNA ( Liu and Wang , 1987 ) .", "Blue arrows depict the rotation of DNA required to avoid torsional strain when the DNA is unconstrained .", "Both the under-winding ( behind EC ) and over-winding ( in front of EC ) torsional stresses will increase the propensity for RNAP to backtrack , thus increasing the duration of pauses that involve backtracking and increasing the kinetic window for Rho-dependent termination at backtrack pauses .", "( B ) At 66 H-NS/kb , H-NS-free DNA segments allow DNA-binding domains from initially formed linear filaments to interact and form bridged filaments ( top ) .", "At 200 H-NS/kb , all DNA segments become occupied by H-NS , leaving no available unbound DNA for formation of bridged filaments ( bottom ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 04970 . 025 If the road-blocking mechanism is correct , then H-NS must bind DNA significantly more tightly in the bridging mode than in the linear mode .", "The off-rate from DNA of H-NS in bridged filaments has been estimated to be 1 . 5 s−1 at 20°C ( Dame et al . , 2006 ) .", "However , we observed strong H-NS effects at RNAP pauses with much longer dwell times under similar conditions ( pauses of minutes or more at 20°C and 30 µM NTPs ) and where the average elongation rate of RNAP is ∼1 . 3 s−1 .", "A priori , H-NS dissociates too fast to slow pause escape so dramatically .", "Although direct measures of H-NS exchange rates under our conditions will be needed to draw firm conclusions , the available data appear inconsistent with a road-blocking mechanism .", "Further , prior demonstrations of RNAP road-blocking used lac repressor or non-cleaving EcoRI proteins that bind with Kds of 0 . 3–1 pM ( ∼104 tighter than we observe for H-NS , 2 nM ) , whereas only partial road-blocking was observed with an EcoRI mutant exhibiting a Kd of 30 pM ( still 100× tighter than H-NS ) ( Deuschle et al . , 1986; Pavco and Steege , 1990 ) .", "These data lead us to favor the topological model .", "A topologically constrained domain created by H-NS bridging around elongating RNAP will cause over-winding ( positive supercoiling ) of DNA downstream of an EC and under-winding ( negative supercoiling ) of DNA upstream of an EC by ∼1 turn for every 10 . 3 rounds of nucleotide addition ( Figure 9A ) ( Liu and Wang , 1987 ) .", "These topological forces strongly inhibit translocation and favor backtracking of RNAP ( Ma et al . , 2013 ) .", "In contrast , linear H-NS filaments will allow either the EC or the upstream and downstream DNA to freely rotate as transcript extension occurs .", "Thus , the topological model readily explains why the shift to linear filaments at higher H-NS concentrations reverses the pause-stimulating effects of H-NS , whereas the road-block model must invoke a large change in H-NS affinity that , while possible , lacks obvious physical rationale .", "The preferential effect of H-NS on backtrack pauses also may explain the variable effects of Rho and NusG at different pause sites .", "Rho terminates non-backtracked ECs more readily than backtracked ECs ( Pasman and von Hippel , 2000; Dutta et al . , 2008 ) and NusG enhances the rate at which Rho dissociates ECs ( Burns and Richardson , 1995 ) .", "In the absence of H-NS , Rho terminated apparent non-backtracked pauses more readily than apparent backtracked pauses .", "By increasing the time window for Rho action on backtrack pauses , H-NS may increase the sites at which Rho can dissociate a significant fraction of ECs .", "NusG , which exhibited a preferential effect on the apparent backtracked pauses , may be especially synergistic with H-NS because it both shifts ECs to less backtracked registers and directly aids EC dissociation by Rho , thus overcoming the apparently greater barrier for Rho dissociation at backtrack pause sites .", "Previous studies have established that <5 mM Mg2+ favors linear ( stiffened ) H-NS filaments and >5 mM Mg2+ favors bridging ( Liu et al . , 2010 ) , Mg2+ levels within those estimated for the E . coli cytoplasm ( 1–10 mM free Mg2+; ≥100 mM including bound Mg2+ ) ( Moncany and Kellenberger , 1981; Cayley et al . , 1991 ) .", "Our AFM observations support bridged-linear filament switching in the 1–10 mM Mg2+ range , but it is currently unclear whether Mg2+ binds H-NS , DNA , or both to favor the switch to bridging .", "Biophysical studies of H-NS are needed to elucidate this mechanism .", "Our results add H-NS concentration as a second important parameter governing the bridged-linear switch .", "As H-NS levels saturate all available DNA binding sites , assuming a site size of ∼5 bp/H-NS monomer , bridging becomes disfavored ( Figure 1 ) .", "The simplest explanation for this effect is that bridging requires the interaction of an H-NS-free DNA segment with an H-NS bound segment to nucleate formation of a 2-duplex bridged structure such as that proposed by Arold et al . ( 2010 ) ( Figure 9B , Figure 1—figure supplement 1 ) .", "At high H-NS levels , all DNA segments may become occupied by H-NS even if bound by only one of the two available DNA-binding domains at each CTD–CTD junction in the filament .", "At this point , linear filaments may become favored because no free DNA sites are sterically available , even if the intrinsic stability of the linear filament is less than that of the bridged filament structure .", "Based on prior measurements ( Ali Azam et al . , 1999 ) , H-NS is present in E . coli well below the saturating levels needed to produce linear filaments ( equivalent to 2 H-NS/kb in exponentially growing cells containing ∼2 genome equivalents of DNA ) .", "However , many additional factors including sequestration of DNA by other proteins , low Mg2+ concentration , or elevated expression of H-NS could make the switch to linear filaments possible .", "If the topological model of H-NS action on ECs is correct , then one particularly interesting aspect of bridged-linear switching is that linear filaments might readily inhibit transcription initiation at promoters by interfering with RNAP binding with less effect on elongating RNAP , whereas bridged filaments might inhibit both initiation and elongation .", "The roles of H-NS nucleoprotein filaments in repressing transcription initiation by occluding RNAP or activator binding at promoters are well established , including repressing horizontally transferred genes , allowing expression of genes in pathogenicity islands during bacterial infections and suppression of pervasive antisense and non-coding transcription ( Dorman , 2007; Fang and Rimsky , 2008; Fass and Groisman , 2009; Singh et al . , 2014 ) .", "Effects on elongating RNAP have been strongly indicated by frequent downstream locations of H-NS binding sites that affect gene expression and by H-NS synergy with Rho in vivo ( Dole et al . , 2004; Saxena and Gowrishankar , 2011; Peters et al . , 2012 ) .", "However , direct biochemical evidence has been lacking , and some results have questioned whether H-NS can affect elongating RNAP ( Lucht et al . , 1994; Dame et al . , 2006 ) .", "Our finding that bridged filaments enhance pausing and Rho-dependent termination in vitro provides a mechanistic basis for the in vivo effects of H-NS on transcript elongation and suggests several ways that changes in the effects of H-NS filaments can contribute to bacterial gene regulation .", "Several factors known to affect the formation of bridged filaments or elongating RNAP are also known to vary among growth environments or conditions .", "Increased temperature upon bacterial invasion of a mammalian host is thought to play a key role in H-NS-mediated up-regulation of genes involved in pathogenesis or symbiotic growth ( Goransson et al . , 1990; Amit et al . , 2003; Trachman and Yasmin , 2004; Ono et al . , 2005; Yang et al . , 2005; Bouffartigues et al . , 2007 ) .", "Our results suggest that H-NS bridging is inhibited at 37°C and that effects of H-NS on elongating RNAP mediated by bridged H-NS decrease above 30°C .", "Thus , an important component of H-NS-mediated temperature regulation of laterally transferred genes , pathogenesis genes , and transcriptionally silenced genes may be aided by decreased H-NS stimulation of Rho-dependent termination at temperatures encountered in mammalian hosts .", "The concentration of H-NS is highest in exponential phase , and decreases by about a factor of four in stationary phase ( Ali Azam et al . , 1999 ) ; conversely , H-NS is induced by cold–shock ( La Teana et al . , 1991 ) .", "Although the complexity of proteins interacting with DNA in cells precludes simple inferences , these changes in H-NS concentrations might influence levels of bridging and thus the magnitude of effects on elongating RNAP similar to the H-NS concentration effects we observed in vitro .", "Because small changes in effects on transcript termination are cumulative , unlike the switch-like effects that operate during initiation , modest changes in the amounts of bridging could be magnified ( e . g . , increasing termination from 1% to 10% at 20 sites in an operon will reduce expression by a factor of 7 ) .", "Changes in Mg2+ levels , which strongly influence the propensity for bridging and the magnitude of effects on RNAP over relatively narrow changes in concentration ( 2 mM to 8 mM; Figure 2 ) are thought to decrease in response to external Mg2+ levels during some bacterial infections ( Groisman , 1998 ) .", "Combined with increases in temperature , which decrease effects of H-NS on elongating RNAP and increase during infection , reduced Mg2+ levels could increase gene expression by decreasing H-NS bridging .", "Finally , multiple additional proteins that interact with H-NS could strongly influence the effects of H-NS on transcript elongation .", "For example , the H-NS paralog StpA may be present in filaments with H-NS ( Leonard et al . , 2009; Uyar et al . , 2009 ) and could alter bridging potential ( Lim et al . , 2012 ) .", "The E . coli Hha and YdgT proteins , although lacking DNA-binding domains , share the oligomerization fold of H-NS , may interact with H-NS filaments , and may modify their properties ( Saxena and Gowrishankar , 2011; Ali et al . , 2013; Ueda et al . , 2013 ) .", "Indeed our preliminary results suggest that both StpA and Hha-modified H-NS filaments more strongly inhibit transcript elongation by RNAP than bridged H-NS filaments alone ( MK , BB , DH and RL , unpublished observations ) .", "Hha is thought to govern both initiation and elongation of the hly operon in uropathogenic E . coli ( Juarez et al . , 2000; Nieto et al . , 2000; Madrid et al . , 2002; Ueda et al . , 2013 ) and intriguingly is regulated by RfaH , which we found modestly aided elongation through H-NS filaments .", "Taken together , these results suggest multiple ways that inhibition of transcript elongation by bridged H-NS filaments may play crucial roles in bacterial gene regulation .", "Like the growing appreciation of the interplay between chromatin structure , promoter-proximal pausing , transcriptional regulation , and RNA processing in eukaryotes , these effects of H-NS on elongating bacterial RNAP are variable and may be modulated by cellular and environmental conditions .", "Much work remains to establish the mechanistic bases of the links between these effects and the repression of horizontally transferred genes , pathogenicity islands , and pervasive antisense and noncoding transcription .", "Our study provides a first step toward this mechanistic understanding ." ], [ "DNA oligonucleotides were obtained from Integrated DNA Technologies ( Coralville , IA ) , [α-32P]NTPs and [γ32P]ATP were from PerkinElmer Life Sciences ( Waltham , MA ) , NTPs were from GE Healthcare Life Sciences ( Piscataway , NJ ) , and 3′–deoxyNTPs were from Trilink ( San Diego , CA ) .", "The C-terminally His6-tagged H-NS expression plasmid pET21-HNS-cHis6 was the kind gift of Sohail Akhtar and Aseem Ansari .", "Plasmid pMK110 encoding the bgl transcription template ( Figure 1 ) was described previously ( Haft et al . , 2014 ) .", "Plasmid pMK121 ( Figure 2—figure supplement 3 ) was constructed by inserting a 1091-bp PCR product containing bglF from the E . coli bgl operon between the SpeI and PstI sites of pIA267 ( Artsimovitch and Landick , 2002 ) .", "PCR was performed using E . coli chromosomal DNA , forward primer 5′-TCGAGCACTAGTCAGGCGATAACAAAGGGGTA , and reverse primer 5′-TATGCTCTGCAGGAATTCTGCGCAACGCGATTACGTT .", "After purification , the PCR product was digested with SpeI and PstI and ligated into similarly cut pIA267 .", "Plasmids pMK122 , pMK124 , and pMK126 ( Figure 2—figure supplement 1 ) were constructed by inserting regions from 312 , 707 , and 912 nt downstream of the transcription start site of pMK110 , respectively , into the SpeI site of pIA267 using forward primers 5′-GCATACTAGTTTAACGCTTCTTCCCCTAGC ( pMK122 ) , 5′-GCATACTAGTTTTTGGTGATTTGCATGTTCA ( pMK124 ) , and 5′-GCATACTAGTGACCATGCTCGCAGTTATT ( pMK126 ) along with the reverse primer 5′-GCATACTAGTGGCGATGAGCTGGATAAACT .", "Transcription templates were generated by PCR amplification from pMK110 , pMK121 , pMK122 , pMK124 , and pMK126 using forward primer 5′-CGTTAAATCTATCACCGCAAGGG and reverse primer 5′-CAGTTCCCTACTCTCGCATG .", "pMK110-520 template was generated from pMK110 with the same reverse primer used above and forward primers 5′-CACTAATTTATTCCATGTCACACTTTTCGCATCTTTTTTATGCTATAATTATTTCATGTAGTAAAGAGGAATATGACTTAAGAGTTCGC or 5′-CACTAATTTATTCCATGTCACACTTTT .", "PCR products were electroeluted from a 1% agarose gel , phenol extracted , and ethanol precipitated .", "RNAP ( Nayak et al . , 2013 ) , σ70 ( Gribskov and Burgess , 1983 ) , NusG ( Mooney et al . , 2009 ) , RfaH-NTD ( Kolb et al . , 2014 ) , GreA ( Feng et al . , 1994 ) , GreB ( Feng et al . , 1994 ) , and Rho ( Mooney et al . , 2009 ) were purified as described previously .", "H-NS was purified by overexpression in E . coli strain BL21 ( λDE3 ) containing pET21-HNS-cHis6 , and grown at 37°C in Luria broth supplemented with 0 . 1 mg Ampicillin/mL to an apparent OD600 of 0 . 4 .", "The temperature was lowered to 30°C , 500 μM isopropyl-1-thio-β-D-galactopyranoside was added , and the culture was grown for 4 hr with shaking .", "Cells were pelleted at 3000×g for 15 min at 4°C , resuspended in H-NS lysis buffer ( 20 mM Tris–HCl pH 7 . 5 , 100 mM NaCl , 5% glycerol , 2 mM EDTA , 1 mM dithiothreitol [DTT] , and 1 mM β-mercaptoethanol ) supplemented with 0 . 1 mg PMSF/ml and a protease inhibitor mix ( 0 . 0125 mg of benzamide/ml , 2 × 10−4 mg of chymostatin/ml , 2 × 10−4 mg of leupeptin/ml , 4 × 10−5 mg of pepstatin/ml , 4 × 10−4 mg of aprotonin/ml , and 4 × 10−4 mg of antipain/ml ) , and lysed by sonication .", "All the subsequent steps were performed at 4°C .", "The H-NS containing lysate was then enriched by mixing with polyethylenimine ( PEI; avg . MW 60 K; Acros Organics ) to 0 . 6% , incubating for 5 min , and then collecting the H-NS-containing precipitate at 11 , 000×g for 15 min .", "The PEI pellet was washed by gently resuspending in PEI wash buffer ( 10 mM Tris–HCl pH 7 . 5 , 150 mM NaCl , 0 . 1 mM EDTA , 5% glycerol , and 1 mM DTT ) , followed by centrifugation at 11 , 000×g for 15 min .", "H-NS was eluted from the nucleic acid pellet by gently resuspending in PEI elution buffer ( 10 mM Tris–HCl pH 7 . 5 , 600 mM NaCl , 0 . 1 mM EDTA , 5% glycerol , and 1 mM DTT ) followed by centrifuging at 11 , 000×g for 15 min .", "The eluted supernatant was then precipitated by slow addition of ammonium sulfate with gentle stirring to 37% .", "The solution was stirred overnight , and the precipitate was collected by centrifugation at 27 , 000×g for 15 min .", "The H-NS precipitate was resuspended in 35 ml of buffer A ( 20 mM Tris–HCl pH 7 . 5 , 500 mM NaCl , and 5 mM β-mercaptoethanol ) containing 5 mM imidazole , loaded onto a 5 ml HisTrap column ( GE Healthcare ) , washed with 30 ml of buffer A containing 5 mM imidazole , and eluted over a gradient of 5–500 mM imidazole over 10 min at a flow rate of 2 ml/min .", "H-NS containing fractions were combined and dialyzed against buffer B ( 10 mM Tris–HCl pH 7 . 5 , 0 . 1 mM EDTA , 5% glycerol , 100 mM NaCl , and 1 mM DTT ) overnight .", "The dialyzed H-NS was loaded onto a 5 ml HiTrap heparin column ( GE healthcare ) , washed with 30 ml of buffer B , and eluted over a gradient of 0 . 1–0 . 9 M NaCl over 12 min .", "Fractions containing H-NS were pooled and dialyzed into H-NS storage buffer ( 20 mM Tris–HCl pH 7 . 5 , 300 mM KCl , and 10% glycerol ) .", "RNAP holoenzyme ( core ββ′α2ω plus σ70 ) was prepared by incubating twofold molar excess of σ70 with core for 30 min at 30°C in RNAP storage buffer ( 20 mM Tris–HCl pH 7 . 9 , 100 mM NaCl , 10 mM MgCl2 , 0 . 1 mM EDTA , 1 mM DTT , and 40% glycerol ) .", "Halted ECs were formed by incubating 10 nM λPR-bgl template ( linear pMK110 template ) and 15 nM RNAP holoenzyme in EMSA buffer ( Stonehouse et al . , 2011 ) ( 40 mM HEPES-KOH pH 8 . 0 , 100 mM potassium glutamate , 0 . 022% NP-40 , 100 μg bovine serum albumin/ml , and 10% glycerol supplemented with 8 or 2 mM magnesium aspartate ) and combined with 150 μM ApU , 10 μM ATP and UTP , 2 . 5 μM GTP , and 0 . 37 μM ( 10 μCi ) [α-32P]GTP for 10 min at 37°C to trap A26 ECs .", "H-NS ( 1 or 3 μM ; 66 H-NS/kb or 200 H-NS/kb . respectively ) was added to the A26 ECs and incubated for 20 min at 20°C .", "When specified in the figure legends , 75 nM NusG , 5 nM Rho , 50 nM GreB , 500 nM GreA , or 300 nM RfaH-NTD was added .", "Transcription was then allowed to resume by adding ATP , UTP , CTP , and GTP ( 30 μM each ) , 100 μg rifampicin/ml , and 0 . 1 U RNasin/μl ( Promega , Madison , WI ) .", "Samples ( 10 μl ) were taken at indicated time points by mixing with EDTA ( 20 mM final ) , immediately phenol:cholorfom extracted , and ethanol precipitated .", "Reaction pellets were resuspended in Formamide Running Dye ( 95% formamide , 15 mM EDTA pH 8 , 0 . 05% bromophenol blue , and 0 . 05% xylene cyanol ) .", "Samples were heated for 2 min at 90°C and separated by electrophoresis in a denaturing 6% or 12% polyacrylamide gel ( 19:1 acrylamide:bisacrylamide ) in 1 . 25 mM Na2EDTA , and 44 mM Tris borate , pH 8 . 3 ( 0 . 5× TBE ) plus 7 M urea .", "Gels were exposed to a PhosphorImager screen , scanned using a Typhoon PhosphorImager , and quantified using ImageQuant software ( GE Healthcare , Waukesha , WI ) .", "Densitometry profiles and mean transcript lengths were generated as previously described ( Haft et al . PNAS 2014 ) .", "ECs in reactions chased with 1 mM NTPs were formed in EMSA buffer supplemented with 12 mM magnesium aspartate .", "All transcription experiments were repeated at least twice .", "Halted A26 complexes were formed on the λPR-bgl template with 10 μM ATP and UTP and 2 . 5 μM GTP supplemented with 0 . 37 μM ( 10 μCi ) [α-32P] GTP .", "Transcription was restarted with 30 μM ATP , UTP , GTP , and CTP in the presence of 100 μg rifampacin/ml .", "Samples were collected and stopped as above ( where H-NS was present ) or by addition to 2× urea stop dye ( 10 M urea , 30 mM Na2EDTA , 0 . 05% each of bromophenol blue , and xylene cyanol ) and then separated by 8% denaturing PAGE ( 0 . 5× TBE plus 7 M urea ) .", "RNA ladders were generated by adding 10–50 μM of one 3′-deoxyNTP ( G , A , U , or C ) and 4 NTPs ( 30 µM each ) to 10 nM halted ECs and incubating for 10 min before stopping by addition to 2× urea stop dye .", "RNA ladders were run alongside time points .", "Gels were visualized as described above .", "Nucleic-acid scaffolds to reconstitute elongation complexes were assembled as described previously ( Kyzer et al . , 2007 ) using sequences shown in Figure 2—figure supplement 1 .", "Briefly , ( 1 μM RNA ) and template DNA ( 2 μM ) were annealed in reconstitution buffer ( 10 mM Tris–HCl , pH 7 . 9 , 40 mM KCl , and 5 mM MgCl2 ) .", "Scaffolds were diluted 10-fold with elongation buffer ( EB; 25 mM HEPES-KOH , pH 8 . 0 , 50 mM KCl , 5 mM MgCl2 , 1 mM DTT , 5% glycerol , and 25 μg acetylated bovine serum albumin/ml ) and incubated with 0 . 5 μM RNAP for 10 min at 37°C .", "300 nM non-template DNA ( 300 nM ) was then added and incubation continues for 10 min at 37°C to form halted ECs .", "The RNA was extended one nucleotide with [α-32P]CTP to form C346 scaffold ( ECC342 ) or C370 scaffold ( ECC368 ) .", "On-the-fly transcription complexes were diluted 20-fold , and 10 μM CTP , UTP , and GTP ( ECC346 ) or CTP , ATP , and GTP ( ECC370 ) were added at either 37°C or 20°C .", "Samples were quenched by addition of an equal volume of 2× stop dye ( 10 M urea , 50 mM EDTA , 90 mM Tris-borate buffer , pH 8 . 3 , 0 . 02% bromophenol blue , and 0 . 02% xylene cyanol ) at 10 , 20 , 40 , 60 , 90 , 120 , 150 , and 180 s at 37°C , or 20 , 40 , 60 , 90 , 120 , 150 , 180 , and 360 s at 20°C .", "RNA was resolved by electrophoresis through a 15% denaturing polyacrylamide gel ( 0 . 5× TBE plus 7 M urea ) .", "For delayed transcription from ECC342 , CTP and UTP ( 10 µM each ) were added at 37°C for 5 min to halt complexes at ECC346 .", "GTP ( 10 µM ) was added , and samples were collected as described above .", "Delayed ECC370s were walked to the pause by binding the His10-tagged RNAP to Co2+ magnetic beads during the last 5 min of incorporation labeling with [α-32P]CTP .", "The bead-bound ECs were washed five times with 1 ml of EB and extended one nucleotide with 10 μM ATP .", "The complexes were washed again and extended to the pause site by addition of 10 μM CTP .", "Subsequently , CTP , ATP , and GTP ( 10 μM each ) were added , and samples were collected as described above .", "For intrinsic or GreB-mediated cleavage assays , ECC346 or ECC370 complexes were elongated to the pause as described above .", "ECs were washed with 5× 1 ml of EB .", "Complexes were resuspended in cleavage buffer ( 25 mM Tris–HCl , pH 9 . 0 , 50 mM KCl , 20 mM MgCl2 , 1 mM DTT , 5% glycerol , and 25 μg acetylated bovine serum albumin/ml ) to induce intrinsic cleavage or were suspended in EB with or without 50 nM GreB to assay GreB-mediated hydrolysis .", "Samples were collected as described above and separated by 20% denaturing PAGE ( 0 . 5× TBE plus 7 M urea ) .", "Gels were exposed to a PhosphorImager screen , scanned using a Typhoon PhosphorImager , and quantified using ImageQuant software ( GE Healthcare ) .", "5′-32P-labeled linear pMK110 fragment ( 10 nM or 10 pM; generated by PCR as described above ) was incubated in EMSA buffer supplemented with 2 or 8 mM magnesium aspartate with various amounts of H-NS and incubated at 20°C for 20 min .", "Reactions were then resolved by electrophoresis though a 3% native PA gel ( 19:1 acrylamide:bisacrylamide ) cast and run in 0 . 5× TBE plus 2 . 5% glycerol for 5 hr at 4°C , 20°C , or 37°C .", "The resulting polyacryamide gel was dried , exposed to PhosphorImager screen , and scanned using a Typhoon PhosphorImager .", "H-NS-DNA protein complexes were adsorbed onto APS-mica .", "Mica was functionalized with APS as described previously ( Shlyakhtenko et al . , 2013 ) .", "166 . 7 μM APS solution was incubated with mica for 30 min at room temperature .", "APS-mica was washed with water , dried under a stream of argon and then cured overnight and stored under argon at reduced pressure .", "10 nM or 2 nM pMK110 template only or A26 ECs ( described above ) were incubated with various amounts of H-NS in AFM buffer ( 40 mM HEPES-KOH pH 8 . 0 and 100 mM potassium glutamate ) supplemented with either 2 or 8 mM magnesium aspartate for 20 min at 20°C .", "A fraction of the H-NS complexes was loaded onto 3% native PAGE ( described above ) .", "A fraction of complexes was directly applied to the APS-mica surface , incubated for 2 min at room temperature , washed with 600 μl water , dried under a stream of argon , and cured overnight under vacuum .", "Another fraction of the complexes was incubated on ice for 2 min and diluted 1:4 in ice-cold AFM buffer .", "The diluted complexes were deposited onto APS-mica at 4°C , incubated for 2 min at 4°C , washed with 600 μl ice-cold water , dried under a stream of argon , and cured overnight under vacuum .", "Dilution of the H-NS-DNA complexes was performed to lower H-NS background and aid identification of complexes in the higher undiluted samples .", "The remaining H-NS-ECs were used in transcription assays conducted in parallel ( as described above ) .", "Images of samples were obtained with a MultiMode AFM ( Digital Instruments NanoScope IV; now Bruker , Santa Barbara , CA ) in tapping mode in air using TESPA-V2 cantilevers ( Bruker ) .", "Images were analyzed for changes in topology , changes in complex height and width with Gwyddion software .", "Additional contour length and persistence length measurements were made with Femtoscan software which uses previously established algorithms ( Rivetti et al . , 1996 ) ." ] ]

[ "Bacterial H-NS forms nucleoprotein filaments that spread on DNA and bridge distant DNA sites .", "H-NS filaments co-localize with sites of Rho-dependent termination in Escherichia coli , but their direct effects on transcriptional pausing and termination are untested .", "In this study , we report that bridged H-NS filaments strongly increase pausing by E . coli RNA polymerase at a subset of pause sites with high potential for backtracking .", "Bridged but not linear H-NS filaments promoted Rho-dependent termination by increasing pause dwell times and the kinetic window for Rho action .", "By observing single H-NS filaments and elongating RNA polymerase molecules using atomic force microscopy , we established that bridged filaments surround paused complexes .", "Our results favor a model in which H-NS-constrained changes in DNA supercoiling driven by transcription promote pausing at backtracking-susceptible sites .", "Our findings provide a mechanistic rationale for H-NS stimulation of Rho-dependent termination in horizontally transferred genes and during pervasive antisense and noncoding transcription in bacteria ." ]

[ "Genes—which are made of DNA—contain the genetic blueprint of an organism .", "Different genes are switched on ( expressed ) at various points in an organism's life when they are needed .", "When a gene is switched on or ‘expressed’ , the DNA is copied using molecules of ribonucleic acid ( RNA ) that can then be used as templates to make proteins .", "One way that the expression of genes can be controlled is by the way that DNA is packaged in cells .", "In humans and other eukaryotic organisms , DNA is packaged within groups or ‘complexes’ of proteins called histones .", "The histones can interact with the cellular machinery that moves along the gene and makes the RNA copies .", "In doing so , the histones can alter the length of the RNA copies , and if the RNAs are too short , they will not be used to make proteins so the gene is effectively switched off .", "We know much less about how DNA packaging in bacteria cells affects gene expression .", "There are many different DNA packaging complexes in bacteria and they are less stable than the histones in eukaryotes , which makes them harder to study .", "In the bacteria Escherichia coli , the main DNA packaging protein is the histone-like structuring protein ( H-NS ) .", "This protein binds to the DNA at select sites and forms filaments that can link to each other to form bridges between different stretches of DNA .", "In this study , Kotlajich et al . found that the bridged filaments formed by the H-NS protein interfere with the production of RNA copies .", "It is normal for the enzyme that makes RNA copies from DNA—called RNA polymerase—to make short pauses while it moves along the DNA .", "However , the bridged filaments made by H-NS cause RNA polymerase to pause for longer periods of time .", "These delays provide more time for another protein that halts gene copying to bind to the site where the RNA polymerase has paused , leading to RNA molecules that are too short to make proteins .", "It has previously been shown that there is less bridging between H-NS filaments in some bacteria when they are grown in warmer temperatures—around 37°C—than when they are grown in cooler temperatures of around 20–30°C .", "This may allow bacteria that cause diseases in animals to increase the expression of genes that help them outwit the host's defenses and to resist antibiotic treatments when they enter a warm animal body from a colder environment ." ]

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

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

[ [ "α-Synuclein ( α-Syn ) is an abundant presynaptic protein that is expressed throughout the central nervous system ( CNS ) and associated with synaptic vesicles ( Maroteaux et al . , 1988; Jensen et al . , 1998 ) .", "Pathologically , missense mutations ( A30P , E46K , A53T ) of α-Syn ( Polymeropoulos et al . , 1997; Kruger et al . , 1998; Zarranz et al . , 2004 ) and duplications and triplications of the gene that encodes α-Syn ( Singleton et al . , 2003; Chartier-Harlin et al . , 2004 ) are linked to early onset Parkinson's disease ( PD ) .", "Moreover , Lewy bodies observed in Parkinson's disease , dementia with Lewy bodies , and other neurodegenerative diseases contain high concentrations of α-Syn aggregates ( Spillantini et al . , 1997; Hardy and Gwinn-Hardy , 1998; Spillantini et al . , 1998 ) .", "In aqueous solution , α-Syn is a largely unfolded protein and has a propensity to aggregate in a nucleation-dependent manner , forming β-sheet rich amyloid-like fibrils ( Serpell et al . , 2000; Zhao et al . , 2011; Burré et al . , In press ) .", "Fibrils , protofibrils and/or large oligomers are believed to be cytotoxic ( Conway et al . , 2000; Goldberg and Lansbury , 2000; Bucciantini et al . , 2002 ) , and may be responsible , at least in part , for the neurodegeneration observed in diseases featuring Lewy bodies .", "Physiologically , α-Syn increases the rate of SNARE-complex formation at the synapse via binding to the SNARE protein synaptobrevin-2 ( also called VAMP2 , vesicle associated membrane protein 2 ) and to phospholipids ( Burré et al . , 2010 ) .", "In adult canaries and zebra finches , α-Syn expression correlates with plasticity in the developing song control system ( Clayton and George , 1998 ) .", "However , deletion of α-Syn in mice has been reported to show either no or very small and opposing effects on neurotransmitter release ( Abeliovich et al . , 2000; Cabin et al . , 2002; Chandra et al . , 2004; Liu et al . , 2004; Yavich et al . , 2004; Unger et al . , 2006; Senior et al . , 2008; Burré et al . , 2010; Garcia-Reitbock et al . , 2010; Scott et al . , 2010; Anwar et al . , 2011 ) .", "Similarly , overexpression of α-Syn either in transgenic mice or using stereotactic injections of adeno-associated virus have resulted in conflicting effects on neurotransmitter release , although chronic overexpression nearly always leads to neurodegeneration ( Liu et al . , 2004; Larsen et al . , 2006; Burré et al . , 2010; Nemani et al . , 2010; Gaugler et al . , 2012 ) .", "Structurally , α-Syn is a 14-kDa protein composed of an amphipathic , positively charged 100 residue N-terminal domain with a lysine-rich N-terminus that binds reversibly to anionic membranes ( Rhoades et al . , 2006 ) , and a 40-residue highly acidic C-terminal domain that interacts with the N-terminal sequence of synaptobrevin-2 ( Burré et al . , 2010 ) .", "Recombinant , erythrocyte , and brain α-Syn is predominantly monomeric in solution with a smaller fraction of multimeric species , and is largely unstructured with a small ( approximately 21–24% ) helical contribution ( Fauvet et al . , 2012; Burré et al . , 2013 ) .", "At high concentrations ( ∼500 μM ) and in the presence of β-octylglucoside , nuclear magnetic resonance spectra showed weak intramolecular NOEs in regions of α-helical propensity but no tertiary interactions , reminiscent of a molten globule ( Wang et al . , 2011 ) .", "The interaction with anionic lipids induces α-helicity in α-Syn , as revealed by CD spectroscopy ( Davidson et al . , 1998; Jo et al . , 2000 ) .", "Moreover , solution NMR studies , EPR analyses , calorimetry , and single molecule fluorescence resonance transfer experiments in the presence of SDS micelles and small unilamellar vesicles revealed that the N-terminal 100 residues form a broken amphiphatic α-helix upon binding to anionic lipids ( Bussell and Eliezer , 2003; Chandra et al . , 2003; Ulmer et al . , 2005; Jao et al . , 2008; Ferreon et al . , 2009; Lokappa and Ulmer , 2011; Maltsev et al . , 2013 ) .", "Binding of α-Syn to the membrane is transient , and the C-terminal 40 residues of α-Syn are unstructured and not bound to the membrane ( Bodner et al . , 2009 ) .", "N-terminal acetylation of α-Syn does not induce significant structure in recombinant α-Syn in solution ( i . e . , absence of detergents or lipids ) , but further enhances its binding to anionic membranes and induced α-helicity ( Maltsev et al . , 2012 ) .", "Although initially largely monomeric , purified brain α-Syn associates into partially folded multimers and aggregates in a time-dependent manner ( Burré et al . , 2013 ) .", "It is likely that these different monomeric and oligomeric states exist in an equilibrium in vivo , which is modulated by a variety of factors and interactions , such as membrane binding .", "This labile mixture of conformations provides a potential explanation for why α-Syn is so susceptible to pathological aggregation and fibril formation as observed in multiple neurodegenerative disorders ( Devine et al . , 2011; Martin et al . , 2011 ) .", "To investigate the effect of native α-Syn on synaptic vesicle fusion , we employed a recently developed in vitro system ( Kyoung et al . , 2011; Diao et al . , 2012a ) that allowed us to measure the effect of α-Syn on Ca2+-triggered vesicle fusion with reconstituted neuronal SNAREs , synaptotagmin-1 , and complexin-1 on a sub-sec timescale .", "We found that native α-Syn does not significantly affect the efficiency and kinetics of Ca2+-triggered synaptic protein-mediated fusion itself .", "Rather , α-Syn induces clustering of vesicles with reconstituted synaptobrevin-2 or with both reconstituted synaptobrevin-2 and synaptotagmin-1 .", "The clustering effect is dependent on specific binding to both anionic lipid membranes and synaptobrevin-2 as revealed by deletion and mutagenesis studies , including the Parkinson's disease mutant A30P that disrupts lipid binding .", "Furthermore , clustering is induced by both recombinant and brain-purified native α-Syn , both of which are initially in a largely monomeric state in the absence of membranes .", "This clustering or lack thereof may account for the increase in SNARE-complex levels observed upon overexpression of native α-Syn ( Burré et al . , 2010 ) , and , conversely , the decrease in SNARE-complex assembly in αβγ-Syn triple knockout mice ( Burré et al . , 2010 ) . 10 . 7554/eLife . 00592 . 003Box 1 . Definitionsv-vesicle: proteoliposome with reconstituted synaptobrevin-2 in all experiments except Figures 1 , 2 , and 4D where both synaptobrevin-2 and synaptotagmin-1 were reconstituted .", "We also refer to these vesicles as “synaptic-vesicle mimics” .", "t-vesicle: proteoliposome with reconstituted syntaxin-1A and SNAP-25A .", "We also refer to these vesicles as “plasma-membrane mimics” .", "v-/t-vesicle association: fluorescent spot arising from labeled v-vesicles ( or small clusters of v-vesicles ) that bind to a surface with immobilized t-vesicles , using the protocol described in the ‘Single vesicle-vesicle Ca2+-triggered fusion experiments’ section in ‘Materials and Methods’ .", "v-/v-vesicle cluster: fluorescent spot arising from labeled v-vesicles that bind to a surface with immobilized v-vesicles , using the protocol described in the ‘Single-vesicle clustering experiments’ section in ‘Materials and Methods’ .", "DOI: http://dx . doi . org/10 . 7554/eLife . 00592 . 003" ], [ "To address the question of whether native α-Syn influences Ca2+-triggered synaptic-protein mediated membrane fusion , we employed our recently developed in vitro single vesicle–vesicle fusion system using vesicles with reconstituted synaptic proteins ( Kyoung et al . , 2011; Diao et al . , 2012a; Kyoung et al . , 2012 ) .", "Free-floating ‘v-vesicles’ ( with reconstituted synaptobrevin-2 and synaptotagmin-1 ) mimicked synaptic vesicles , while surface-immobilized ‘t-vesicles’ ( with reconstituted syntaxin-1 and SNAP-25 ) mimicked the presynaptic plasma membrane ( Figure 1A ) .", "A defined volume of v-vesicle solution was incubated with t-vesicles together with complexin-1 , establishing a metastable state at zero Ca2+ .", "We tested the effect of recombinant α-Syn with our system by adding it to the v-vesicle incubation stage .", "For the v-vesicles that bound , native α-Syn had little effect on the efficiency ( defined as the number of fusion events per docked v-vesicles ) and the kinetics of Ca2+-triggered fusion upon injection of 500 μM Ca2+ ( Figure 1B ) , and it did not alter the corresponding cumulative fusion distribution plot ( Figure 1C ) . 10 . 7554/eLife . 00592 . 004Figure 1 . Native α-Syn has no effect on Ca2+ -triggered v-/t-vesicle fusion .", "( A ) Experimental scheme of our single v-/t-vesicle content mixing system ( Kyoung et al . , 2011; Kyoung et al . , 2012 ) , with improvements described in ( Diao et al . , 2012a ) , and further modifications described in ‘Materials and methods’ .", "( B ) and ( C ) α-Syn has little effect on the probability of triggered vesicle fusion upon injection of 500 μM Ca2+ .", "Panels show histograms of the occurrence ( B ) and the corresponding cumulative distribution ( C ) of Ca2+-triggered complete fusion ( content mixing ) in presence or absence of 2 μM α-Syn .", "In the absence of α-Syn , we observed 166 fusion events out of ∼2000 docked v-vesicles ( identified as fluorescent spots that were present prior to Ca2+ injection ) , whereas in the presence of α-Syn , we observed 84 fusion events out of ∼1300 docked v-vesicles within the observation period of 50 s .", "The time-binning was 1 s .", "Black lines are fits to bi-exponential decay functions over the entire observation period of 50 s .", "In the absence of α-Syn , the fitted function is f ( t ) = −0 . 0046 + 0 . 048 e-t/27 . 1 + 0 . 26 e-t/0 . 58 , whereas in the presence of α-Syn , the fitted function is f ( t ) = −0 . 001 + 0 . 064 e-t/15 . 4 + 0 . 22 e-t/0 . 51 with t in sec . DOI: http://dx . doi . org/10 . 7554/eLife . 00592 . 004 Although native α-Syn showed no effect on Ca2+-triggered fusion activity , we noticed that α-Syn reduced the number of fluorescent spots that appeared when v-vesicles bound to immobilized t-vesicles during the incubation stage at zero Ca2+ ( Figure 2A ) .", "However , the addition of native α-Syn produced the emergence of brighter spots between 1 . 5 and 6 a . u . in the fluorescence emission histogram ( insert , Figure 2B ) , along with occasional very large fluorescent spots ( black arrow , lower right image in Figure 2A ) .", "The major peak ( <1 . 5 a . u . ) at low fluorescence intensity corresponds to single donor v-vesicles docked to the imaging surface , while the high intensity tail ( >2 a . u . ) corresponds to small clusters of v-vesicles docked to the surface .", "In the presence of α-Syn , we thus observed a reduction of the single vesicle population and a concomitant increase of a population of vesicle clusters .", "We note that the same finite volume and concentration of individual v-vesicles was used during the incubation stage with and without native α-Syn .", "Thus , the observed decrease in the total number of fluorescent spots ( referred to as ‘v-/t-vesicle associations’ ) in the presence of native α-Syn ( Figure 2A ) can be explained by a reduction of the number of free-floating particles by clustering of individual v-vesicles in the finite volume of the sample chamber ( Figure 2C ) . 10 . 7554/eLife . 00592 . 005Figure 2 . Native α-Syn decreases the number of v-/t-vesicle associations .", "( A ) Plotted is the number of content dye fluorescent spots in presence of 2 μM complexin-1 and with or without 2 μM α-Syn ( tag-free construct ) ; example images are shown in the lower two panels .", "Scoring ( ‘counts’ ) excluded the few very large fluorescent spots that appeared in the presence of α-Syn; an example is shown in the lower right panel , marked by a black arrow .", "Error bars are standard deviations from 10 random imaging locations within the same sample channel .", "*** indicates p<0 . 001 by the Student's t-test .", "( B ) Distribution of the fluorescence intensity of all fluorescent spots as shown in panel ( A ) .", "In the presence of α-Syn , the number of low intensity fluorescent spots ( up to 1 . 5 a . u . ) decreased , which was accompanied by an increase in higher intensity fluorescent spots ( see inset ) due to the formation of v-vesicle clusters .", "The very large fluorescent spot ( black arrow in the lower right image in panel ( A ) ) was excluded in this distribution .", "( C ) Illustration that clustering of v-vesicles induced by α-Syn reduces the effective number of fluorescent spots ( corresponding to docked single or multiples of v-vesicles ) in a finite volume since the starting concentration of individual v-vesicles was identical with and without α-Syn . DOI: http://dx . doi . org/10 . 7554/eLife . 00592 . 005 We next confirmed the clustering activity of native α-Syn on v-vesicles with a different assay that was designed to directly probe the interaction between v-vesicles ( Figure 3A ) .", "One population of v-vesicles was anchored to an imaging surface , while the other population was free-floating .", "Both vesicle populations were labeled with spectrally distinct fluorescent lipid analogues allowing tracking of the location of the free-floating vesicles and assessment of the surface coverage of the immobilized vesicles , respectively .", "Interactions between both populations of v-vesicles were measured by counting the number of initially free-floating v-vesicles that bound to the immobilized v-vesicles .", "We refer to this quantity as ‘clustering’ between vesicles of the same type ( v-/v-vesicle clustering ) .", "The protocol of this clustering experiment included an incubation step with native α-Syn followed by buffer exchange , in order to measure the interaction between immobilized v-vesicles with bound α-Syn and free-floating v-vesicles without α-Syn ( Figure 3B ) .", "This procedure prevented formation of the large fluorescent spots that we observed in the v-/t-vesicle fusion experiments , and it also reduced the likelihood of non-specific vesicle clustering by α-Syn oligomerization . 10 . 7554/eLife . 00592 . 006Figure 3 . Native α-Syn induces clustering of synaptobrevin-2 v-vesicles .", "( A ) Experimental scheme of our single-vesicle assay for monitoring clustering of synaptobrevin-2 v-vesicles .", "A saturated layer of DiD-labeled v-vesicles with reconstituted synaptobrevin-2 was immobilized on an imaging surface via biotin/neutravidin interactions .", "Free-floating DiI-labeled vesicles with reconstituted synaptobrevin-2 were injected into the sample chamber ( ‘Materials and methods’ ) .", "The number of v-/v-vesicle interactions ( clustering ) in presence or absence of α-Syn was determined by counting the number of spots arising from fluorescence emission of DiI upon excitation at 532 nm .", "( B ) Experimental flow:", "1 . Immobilization of DiD-labeled v-vesicles on the imaging surface through biotin/neutravidin interactions .", "2 . Buffer exchange .", "3 . α-Syn incubation of the surface with immobilized DiD v-vesicles .", "4 . Buffer exchange , removing unbound or weakly bound α-Syn molecules .", "5 . Injection of DiI-labeled v-vesicles without α-Syn .", "Following another buffer exchange , the channels were imaged on the microscope . DOI: http://dx . doi . org/10 . 7554/eLife . 00592 . 006 Using this clustering assay , we confirmed that native α-Syn increases the clustering of v-vesicles in a concentration-dependent manner ( Figure 4A ) .", "We note that the specified α-Syn concentration refers to the incubation stage with immobilized v-vesicles ( Figure 3B , middle panel ) .", "Subsequent to the incubation stage , a buffer exchange with α-Syn-free buffer was performed for 6 s which removed unbound α-Syn .", "Moreover , it is likely that the buffer exchange also removed some of the v-vesicle-bound α-Syn molecules since kinetic studies by NMR spectroscopy revealed that α-Syn binding to membranes is transient with exchanges occurring in the range of 1–10 s-1 ( Bodner et al . , 2009 ) .", "Thus , the effective α-Syn concentration during the final clustering step ( Figure 3B , right panel ) is lower than 2–20 μM , as only bound α-Syn is present at this stage .", "Indeed , as Figure 4A shows , v-vesicle clustering has not reached saturation at 2 μM α-Syn incubation concentration , so v-vesicles were not fully covered with α-Syn molecules at that particular α-Syn incubation concentration . 10 . 7554/eLife . 00592 . 007Figure 4 . Native α-Syn ( tag-free construct ) promotes v-vesicle clustering by binding to both synaptobrevin-2 and anionic membranes .", "( A ) α-Syn increases the number of interacting DiI-labeled v-vesicles on the imaging surface in a concentration-dependent manner .", "Bar graph: quantitation of interacting vesicles .", "Bottom panel: representative fluorescence images of interacting vesicles on the imaging surface .", "Error bars are standard deviations from 15 random imaging locations in the sample channel obtained .", "( B ) Number of interactions between free-floating and immobilized v-vesicles in presence of wildtype α-Syn and its mutant that does not bind to synaptobrevin-2 ( α-Syn[1-95] with myc-tag ) at indicated concentrations .", "( C ) Purified α-Syn wildtype and α-Syn[1-95] ( with myc-tag ) expressed in bacteria were analyzed by SDS-PAGE and Coomassie staining .", "( D ) Number of interactions in the presence of 20 μM α-Syn ( myc-tag construct ) .", "At variance to panels ( A , B , and C ) , both free-floating and immobilized v-vesicles were reconstituted with synaptobrevin-2 ( Syb2 ) , synaptotagmin-1 ( Syt1 ) , or both ( Syb2/Syt1 ) .", "Note , that the slight variation in the ‘absolute’ number of observed fluorescent spots between comparable experiments in panels ( A , B , and D ) arises mainly from tagged vs untagged versions of α-Syn , as well as from different liposome and imaging surface preparations .", "Yet , the ‘relative’ differences were statistically similar for different protein preparations .", "In all panels , error bars are standard deviations from 10 random imaging locations in the same sample channel , and *** indicates p<0 . 001 by the Student's t-test . DOI: http://dx . doi . org/10 . 7554/eLife . 00592 . 007 As another independent confirmation of v-vesicle clustering , a cryo-electron microscopy experiment revealed large v-vesicle clusters in the presence of native α-Syn ( Figure 5 ) .", "Note , that the vesicle concentration used for the cryo-EM experiment was 100-fold higher and the effective α-Syn concentration was also higher than that used for the single-vesicle clustering experiments , explaining the formation of the fairly large clusters in the cryo-EM experiment at that concentration .", "V-vesicles clustering does not occur in the absence of α-Syn ( Kyoung et al . , 2011 ) and is therefore specific to native α-Syn .", "It is also entirely dependent on the presence of synaptobrevin-2 , as lack of synaptobrevin-2 ( Figure 4A ) , or truncation of C-terminal residues of α-Syn that bind to synaptobrevin-2 ( Burré et al . , 2010 ) but do not participate in lipid binding of α-Syn ( Burré et al . , 2010 , 2012 ) significantly reduced clustering of v-vesicles ( Figure 4B , C ) .", "Furthermore , clustering of v-vesicles depended on the presence of negatively charged ( anionic ) lipids ( Figure 4A , right most condition ) , which is known to induce α-helix formation in native α-Syn and lipid binding of α-Syn .", "As a control , and in agreement with previous studies ( Davidson et al . , 1998; Jo et al . , 2000; Eliezer et al . , 2001 ) , we observed α-helicity in the CD spectrum of native α-Syn in the presence of protein-free vesicles with the same lipid composition used for our fusion and v-vesicle clustering experiments ( Figure 6 ) .", "Taken together , our clustering experiments suggest that native α-Syn has to bind to both , synaptobrevin-2 and anionic lipids , in order to cluster v-vesicles , confirming the protein- and anionic-lipid specificity of this phenomenon . 10 . 7554/eLife . 00592 . 008Figure 5 . Cryo-electron microscopy image of clusters of synaptobrevin-2 v-vesicles as induced by native α-Syn .", "Scale bar is 200 nm . The dark feature is the holey carbon grid .", "In contrast , reconstituted v-vesicles in the absence of α-Syn do not form clusters ( Kyoung et al . , 2011 ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 00592 . 00810 . 7554/eLife . 00592 . 009Figure 6 . Native α-Syn undergoes a conformational transition from a predominantly unstructured state in solution to an α-helical state upon binding to protein-free vesicles as measured by CD spectroscopy . The protein-free vesicles had the same lipid composition as the v-vesicles used throughout this work .", "The molar protein-to-lipid ratio was 1:530 , and tag-free wildtype α-Syn was used . DOI: http://dx . doi . org/10 . 7554/eLife . 00592 . 009 In the next experiment , we asked if the inclusion of synaptotagmin-1 in the synaptobrevin-2 vesicles would alter v-vesicle clustering induced by native α-Syn ( Figure 4D ) .", "We found that v-vesicle clustering induced by native α-Syn was similar with and without synaptotagmin-1 within experimental error .", "Furthermore , v-vesicles with only reconstituted synaptotagmin-1 did not cluster , consistent with the requirement of synaptobrevin-2 for native α-Syn-induced v-vesicle clustering ( Figure 4A ) .", "For the remaining experiments we therefore used v-vesicles that only contained synaptobrevin-2 .", "To further investigate the membrane-binding role of native α-Syn on v-vesicle clustering , and to extend our findings to PD , we performed experiments with the three PD-related mutants A30P , E46K , and A53T ( Figure 7A ) .", "While all these mutants retain the ability to bind synaptobrevin-2 , only A30P shows reduced membrane binding ( Burré et al . , 2012 ) .", "We measured the effect of these mutations on membrane-binding using a lipid flotation assay ( Figure 7B ) , and on v-vesicle clustering using single vesicle microscopy ( Figure 7C ) .", "Out of the mutants tested , only A30P showed ∼ 50% decrease in membrane binding and v-vesicle clustering compared to native wildtype α-Syn .", "Thus , the effect of the three mutations in v-vesicle clustering correlated well with their effect on native α-Syn binding to PS-containing vesicles .", "This suggests that membrane-binding by native α-Syn is essential for its clustering activity . 10 . 7554/eLife . 00592 . 010Figure 7 . V-vesicle clustering correlates with lipid binding of native α-Syn .", "( A ) Purification of α-Syn expressed in bacteria .", "Purified α-Syn wildtype and the three Parkinson's disease ( PD ) -related mutants A30P , E46K , and A53T ( all tag-free constructs ) were analyzed by SDS-PAGE and Coomassie staining .", "( B ) Lipid binding of recombinant α-Syn wildtype , and of the PD mutants A30P , E46K , and A53T .", "α-Syn was incubated with negatively charged liposomes ( 30% phosphatidylserine , 70% phosphatidylcholine , or 100% phosphatidylcholine as control ) , and subjected to a flotation assay .", "Eight fractions were collected from top to bottom of the flotation gradient , and equal volumes of each fraction were separated by SDS-PAGE and immunoblotted for α-Syn .", "Top two fractions were defined as lipid-bound , and quantitated as percent of total α-Syn .", "Data shown are means ± SEM; n = 3 . ( C ) Impaired lipid-binding of α-Syn decreases v-vesicle clustering .", "In order to measure vesicle clustering , the number of DiI-labeled v-vesicles that were docked to immobilized v-vesicles was counted , as illustrated in Figure", "3 . ( D ) Impaired lipid-binding of α-Syn increases the number of v-/t-vesicle associations .", "The number of fluorescent spots arising from labeled v-vesicles were counted that were docked to t-vesicles ( immobilized to the imaging-surface ) , as illustrated in Figure 2 , but in the presence of the specified α-Syn mutants without complexin-1 .", "In all panels , error bars are standard deviations from 10 random imaging locations in the same sample channel , and *** indicates p<0 . 001 by the Student's t-test . DOI: http://dx . doi . org/10 . 7554/eLife . 00592 . 010 To confirm that v-vesicle clustering induced by native α-Syn depends on lipid binding , we also measured the effect of the PD mutants on the number of v-/t-vesicle associations ( Figure 7D ) .", "Only the A30P mutant increased the number of v-/t-vesicle associations .", "As mentioned above , the reduction of the number of v-/t-vesicle associations by native wildtype α-Syn is a consequence of the finite volume used during the incubation stage and the reduction of the number of free particles in the sample chamber by v-vesicle clustering .", "Thus , the numbers of v-vesicle clusters ( Figure 7C ) and v-/t-vesicle associations ( Figure 7D ) are anti-correlated .", "In order to test whether native brain-purified α-Syn has the same activity as native recombinant α-Syn in clustering v-vesicles , we purified native α-Syn from mouse brain without detergents or denaturants using multiple chromatography steps to a purity of >90% ( Burré et al . , In press ) .", "We found that the induction of v-vesicle clustering by native brain purified α-Syn was similar to that of native recombinant α-Syn ( compare Figures 4A and 8 ) , confirming that the observed clustering activity is not sensitive to the origin of α-Syn . 10 . 7554/eLife . 00592 . 011Figure 8 . Native α-Syn purified from mouse brain induces v-vesicle clustering . Error bars are standard deviations from 20 random imaging locations in the same sample channel .", "*** indicates p<0 . 001 by the Student's t-test . DOI: http://dx . doi . org/10 . 7554/eLife . 00592 . 011" ], [ "The mechanism by which native α-Syn promotes SNARE-complex assembly in vitro and in vivo remains unknown ( Chandra et al . , 2005; Burré et al . , 2010 ) .", "Moreover , young αβγ-synuclein triple knockout mice showed no obvious phenotype , but developed neurological impairments during aging and revealed age-dependent impairments in SNARE-complex assembly ( Burré et al . , 2010 ) .", "Effects on neurotransmission were observed only upon prolonged trains of high-frequency stimulation ( Abeliovich et al . , 2000; Cabin et al . , 2002 ) ( however , see Chandra et al . , 2004 for different results ) .", "Transgenic mice with overexpressed human α-Syn invariably exhibit signs of neurodegeneration , but impairment in synaptic vesicle exocytosis and a reduction in neurotransmitter release were observed only in some studies ( Nemani et al . , 2010; Scott et al . , 2010 ) but not in another study ( Burré et al . , 2010 ) .", "Overexpression of α-Syn in the substantia nigra pars compacta of rodents using viral vectors also invariably leads to neurodegeneration ( Burré et al . , 2012 ) , and may cause an associated decrease in dopamine release ( Gaugler et al . , 2012; Lundblad et al . , 2012 ) .", "We tested a possible function of native α-Syn using a reconstituted system consisting of v-vesicles that mimic synaptic vesicles , and t-vesicles that mimic the presynaptic plasma membrane .", "We found that native α-Syn did not affect the Ca2+-triggered fusion efficiency or fusion kinetics on a sub-second timescale ( Figure 1 ) .", "The lack of a noticeable effect of native α-Syn on Ca2+-triggered fusion in our synthetic system agrees well with extensive in vivo studies with synuclein double and triple knockout mice that showed little effect on synaptic strength ( Chandra et al . , 2004; Burré et al . , 2010 ) .", "Although there was no effect on Ca2+-triggered fusion , we observed clustering of v-vesicles in the presence of native α-Syn ( Figures 2 , 4 , and 5 ) .", "Native α-Syn clustered both synaptobrevin-2 and synaptobrevin-2/synaptotagmin-1 v-vesicles ( Figure 4D ) , and the clustering ability was dependent on the synaptobrevin-2 binding domain of α-Syn and the ability of α-Syn to bind to anionic lipids ( Figures 4A , B and 7C ) .", "Furthermore , both recombinant and brain purified native α-Syn induced v-vesicle clustering ( Figures 4 and 8 ) .", "The observed reduction in the number of associations between v-vesicles ( single or multiple ) and t-vesicles ( Figure 2A ) was correlated with an increase in the clustering of v-vesicles in the presence of α-Syn ( Figures 4 and 5 ) .", "This seemingly paradoxical result can be readily explained since the observed reduction in the number of v-/t-vesicle associations by native α-Syn is due to clustering of v-vesicles into larger particles , which effectively reduces the concentration of individual particles in the sample chamber ( Figure 2C ) , and thus reduces the number of fluorescent spots .", "The clustering activity of native α-Syn that we uncovered is very different from the effect of dopamine-induced large oligomers/aggregates of α-Syn ( Choi et al . , 2013 ) .", "We note that although both our v-/t-vesicle assay and the single-vesicle lipid mixing assay by ( Choi et al . , 2013 ) revealed a reduction of the number of fluorescent ( ‘docked’ ) spots in v-/t-vesicle experiments , the underlying mechanism is different: while native α-Syn clusters v-vesicles and reduces the number of free particles in our v-/t-vesicle assay without interfering with SNARE interactions , large α-Syn oligomers interfere with SNARE complex formation and reduce docking in the lipid mixing assay of ( Choi et al . , 2013 ) .", "It has been suggested that clusters of synaptic vesicles could act as a protein buffer ( Denker et al . , 2011 ) to prevent the loss of accessory proteins involved in vesicle recycling .", "How could this be accomplished , and also result in an increase of SNARE complex assembly rate as observed by ( Chandra et al . , 2005; Burré et al . , 2010 ) ?", "We propose the following model ( Figure 9 ) : Under normal conditions , native α-Syn would contribute to clustering of synaptic vesicles at the active zone , and thereby assist with the formation of neuronal SNARE complexes by constraining additional synaptic vesicles to be close to the active zone .", "We found that the Parkinson's disease related A30P point mutant reduced the clustering activity of native α-Syn ( Figure 7C ) , suggesting a possible loss-of-function phenotype for this mutant .", "Under certain other diseased conditions , excess amount of α-Syn could induce severe synaptic vesicle aggregation and thereby result in a reduced readily releasable synaptic vesicle pool , thus affecting neurotransmission . 10 . 7554/eLife . 00592 . 012Figure 9 . Proposed model of native α-Syn function . Native α-Syn binds at the same time to the synaptic vesicle membrane and synaptobrevin-2 .", "Synaptic vesicle fusion with the presynaptic plasma membrane is mediated by the three neuronal SNARE proteins synaptobrevin-2 ( on the synaptic vesicle membrane ) , SNAP-25 and syntaxin-1 ( on the presynaptic plasma membrane ) , which form the synaptic SNARE-complex .", "Native α-Syn clusters synaptic vesicles depending on binding to synaptobrevin-2 and the vesicles themselves , which may result in a local increase of synaptic vesicles at the presynaptic plasma membrane , and a subsequent increase in SNARE-complex formation . DOI: http://dx . doi . org/10 . 7554/eLife . 00592 . 012" ], [ "Full-length human α-Syn cDNA was cloned into modified pGEX-KG vectors ( GE Healthcare , Uppsala , Sweden ) , containing an N-terminal TEV protease recognition site .", "Tag-free and myc-tagged constructs were prepared: The TEV protease cleavage site introduced one extra glycine residue at the N-terminus of the tag-free α-Syn sequence whereas the myc epitope-tagged α-Syn construct encoded the following extra N-terminal residues: GLEEQKLISEEDLGSGS ( the myc epitope is underlined ) .", "All experiments except those shown in Figure 4B , D employed the tag-free α-Syn construct; we used the myc-tag construct for the experiments in Figure 4B since the C-terminal truncation mutant of α-Syn[1-95] could not be immuno-detected with our antibody .", "Mutant α-Syn constructs were generated by site-specific mutagenesis , according to the manufacturer's protocol ( Stratagene; Agilent Technologies , Santa Clara , CA ) .", "All proteins were expressed as GST fusion proteins in bacteria ( BL21 strain ) , essentially as described ( Burré et al . , 2010 ) .", "Bacteria were grown to OD 0 . 6 ( measured at 600 nm ) , and protein expression was then induced with 0 . 05 mM isopropyl β-D-thiogalactoside ( IPTG ) for 6 hr at ambient temperature .", "Bacteria were harvested by centrifugation for 20 min at 4500 g , and pellets were resuspended in solubilization buffer ( PBS , 0 . 5 mg/ml lysozyme , 1 mM PMSF , DNase , and an EDTA-free protease inhibitor cocktail; Roche Diagnostics Corporation , Indianapolis , IN ) .", "Cells were broken by sonication , and insoluble material was removed by centrifugation for 30 min at 20 , 000×gav and 4°C .", "Proteins were affinity-purified using glutathione sepharose bead ( GE Healthcare ) incubation overnight at 4°C , followed by TEV protease ( Invitrogen , Grand Island , NY ) cleavage overnight at ambient temperature .", "His-tagged TEV protease was removed by incubation with Ni-NTA overnight at 4°C .", "The protein concentration was assessed using BCA method according to the manufacturer's protocol ( Thermo Scientific , Rockford , IL ) .", "The protein sample was flash-frozen and stored at −80°C .", "All DNAs encode rat proteins , and were expressed and purified as described by ( Kyoung et al . , 2011 ) with modifications ( Diao et al . , 2012a ) .", "Briefly , his-tagged syntaxin-1A , synaptobrevin-2 , SNAP-25A , and synaptotagmin-1 were expressed in E . coli and purified using a combination of Ni-NTA affinity ( Qiagen , Hilden , Germany ) and size exclusion chromatography on a Superdex 200 column ( GE Healthcare ) .", "Synaptotagmin-1 was further purified using cation exchange chromatography on a Mono-S column ( GE Healthcare ) .", "His-tags were removed from syntaxin-1A , synaptobrevin-2 , and SNAP-25A with TEV protease , or from synaptotagmin-1 with PreScission protease ( GE Healthcare ) .", "Wildtype complexin-1 was purified essentially as described ( Diao et al . , 2012a ) with modifications .", "Briefly , it was expressed as an N-terminal hexa-his tagged protein from pET28a ( Novagen , EMD Chemicals , Gibbstown , NJ ) in BL21 ( DE3 ) at 30°C using an auto-induction system ( Studier , 2005 ) .", "After binding to Ni-NTA beads , the protein was eluted by overnight cleavage with thrombin .", "Thrombin was then inactivated with 1 mM PMSF .", "The cleaved protein was subjected to size exclusion chromatography using a Superdex 200 10/300 column ( GE Healthcare ) and concentrated to 200 µM .", "10% glycerol was added to all purified protein solutions .", "Complexin-1 and SNAP-25A were flash-frozen and stored as aliquots at −80°C , whereas synaptobrevin-2 , synaptotagmin-1 , and syntaxin-1A were used from freshly-made preparations .", "Mouse brains homogenized in phosphate-buffered saline ( PBS ) and protease inhibitors ( Roche Diagnostics Corporation , Indianapolis , IN ) were fractionated by ultracentrifugation ( 3 × 280 , 000×gav ) .", "α-Syn was purified at 4°C by Q-Sepharose ( GE Healthcare ) anion exchange chromatography where α-Syn eluted at 0 . 3–0 . 5 M NaCl , 20 mM Tris–HCl pH 7 . 4 , by phenyl-Sepharose ( Sigma-Aldrich , St . Louis , MO ) hydrophobic interaction chromatography where α-Syn was recovered in the flow-through in 1 M ( NH4 ) 2SO4 , and by gel filtration on a Superdex 200 10/300 GL column ( AKTA; GE Healthcare ) in PBS .", "Details can be found in Burré et al . ( 2013 ) .", "Preparation of vesicles and liposome flotation assay were performed as previously described ( Burré et al . , 2010 ) .", "Briefly , 1 mg brain phosphatidylcholine ( PC; Avanti Polar Lipids , Alabaster , AL ) or 0 . 7 mg PC and 0 . 3 mg brain phosphatidylserine ( PCPS; Avanti Polar Lipids ) in chloroform were dried in a glass vial under a nitrogen stream .", "Residual chloroform was removed by lyophilization for 2 hr .", "To obtain a solution of 1 mg/ml lipids , 1 ml PBS was added to each vial and vortexed well .", "Small unilamellar vesicles were formed by sonicating lipid solutions on ice ( Barenholz et al . , 1977 ) .", "For α-Syn binding to vesicles , 5 μg proteins were incubated with 100 µg vesicles for 2 hr at ambient temperature .", "Samples were then subjected to a liposome flotation assay as previously described ( Burré et al . , 2010 ) .", "Quantitative immunoblotting experiments were performed with iodinated secondary antibodies as described previously ( Rosahl et al . , 1995 ) .", "Protein samples were separated by SDS-PAGE , and transferred onto nitrocellulose membranes .", "Blots were blocked in Tris-buffered saline ( TBS ) containing 0 . 1% Tween-20 ( Sigma-Aldrich ) and 3% fat-free milk for 30 min at ambient temperature .", "The blocked membrane was incubated overnight in blocking buffer containing primary antibody , followed by five washes .", "The washed membrane was incubated in blocking buffer containing either horseradish peroxidase ( HRP ) -conjugated secondary antibody ( 1:5000; MP Biomedicals , Santa Ana , CA ) for 1 hr at ambient temperature , or 125I-labeled secondary antibodies ( 1:1000; PerkinElmer , Santa Clara , CA ) .", "HRP immunoblots were developed using enhanced chemiluminescence ( GE Healthcare ) .", "125I blots were exposed to a PhosphorImager screen ( GE Healthcare ) overnight and scanned using a Typhoon scanner ( GE Healthcare ) , followed by quantitation with ImageQuant software ( GE Healthcare ) .", "α-Syn ( cl . 610786; BD Transduction , San Jose , CA ) , c-myc ( cl . 9E10; Developmental Studies Hybridoma Bank , Iowa City , IA ) .", "CD spectra were collected with an Aviv model 202-01 spectrometer ( Aviv Biomedical , Lakewood , NJ ) .", "Spectra are averages of eight replicates collected at 1-nm steps from 195 to 240 nm with a spectral bandwidth of 1 nm and a 1-s averaging time .", "The spectra of 1 . 4 μM α-Syn were acquired in pH 7 . 4 , 18 mM NaCl , 4 mM HEPES , 4 μM EGTA , 0 . 2% β-mercaptoethanol with and without 960 µM lipids in protein-free vesicles that were formed with the same protocol ( Kyoung et al . , 2012 ) used for the fusion and clustering experiments .", "Our experimental setup was similar to that previously described ( Diao et al . , 2012a; Kyoung et al . , 2012 ) ( Figure 1A ) , except that lipid dye was not used , and the time stamp of Ca2+ arrival in the evanescent field was determined by the instance of the first content-mixing event ( i . e . , stepwise increase of content dye fluorescence intensity ) among all docked vesicles .", "T-vesicles ( with reconstituted syntaxin-1A and SNAP-25A ) and v-vesicles ( with reconstituted synaptobrevin-2 and synaptotagmin-1 ) were prepared as previously described ( Diao et al . , 2012a; Kyoung et al . , 2012 ) .", "We showed previously that our particular vesicle preparation and protein reconstitution protocols ( Kyoung et al . , 2012 ) produce homogeneous populations of vesicles with an average diameter of approximately 80 nm , and physiological protein concentrations ( Kyoung et al . , 2011 ) .", "The resulting t-vesicle solution was diluted 10× and immobilized on an imaging surface via biotin/neutravidin interactions under saturating conditions .", "Specifically , 100 µl of the diluted t-vesicle solution was injected into the sample chamber and incubated for 30 min , followed by a 200 μl vesicle buffer exchange ( vesicle buffer is defined as 90 mM NaCl , 20 mM HEPES pH 7 . 4 , 20 μM EGTA , 1% β-mercaptoethanol ) .", "V-vesicles ( with reconstituted synaptobrevin-2 and synaptotagmin-1 ) containing self-quenched content dye ( sulforhodamine B; Invitrogen ) were prepared as previously described ( Diao et al . , 2012a; Kyoung et al . , 2012 ) .", "The v-vesicle solution was diluted 150× and added to the immobilized t-vesicle surface with 2 μM complexin-1 in presence or absence of 2 μM α-Syn .", "The presence of 2 μM α-Syn corresponds to a v-vesicle-lipid to α-Syn-protein ratio of 11:1 , with the actual lipid to protein ratio being larger due to the presence of the saturated t-vesicle surface; at this condition , we did not observe destabilization of the v-SNARE vesicles by α-Syn since no leaking of content dyes from docked v-vesicles was observed .", "The system was incubated for 30 min , followed by buffer exchange ( 1 × 200 μl vesicle buffer for 6 s ) , removing unbound or weakly bound complexin and α-Syn .", "Then , 500 μM Ca2+ buffer was injected into the sample chamber , and the fluorescence intensity of the content dye for each docked v-vesicle ( but excluding few large fluorescent spots , see arrow in the lower right image of Figure 2A ) was monitored with total internal reflection ( TIR ) wide-field microscopy ( Nikon Instruments , Melville , NY ) using an electron multiplying charge-coupled device ( CCD ) camera ( iXon+ DV 897E; Andor Technology USA , South Windsor , CT ) .", "The smCamera program ( written in C++ ) from Taekjip Ha's laboratory was used for data acquisition and analysis as described previously ( Diao et al . , 2012a; Kyoung et al . , 2012 ) .", "The number of v-/t-vesicle associations was calculated by counting the number of fluorescent spots prior to Ca2+ injection , averaged over the specified number of images in the same sample channel .", "This simple averaging procedure was possible since the surface coverage was homogenous within the sample channel .", "An instance of complete fusion was characterized by a stepwise increase in the content dye fluorescence intensity; this increase resulted from the approximately twofold dilution of the content dye upon fusion between a v- and a t-vesicle , and subsequent dequenching of the dye .", "For the histograms shown Figure 1B , the time stamp of Ca2+ injection was defined as the instance of the first content-mixing event among all docked vesicles within a particular field of view .", "Histograms were combined from several fields of views by using these time stamps for post-synchronization .", "Histograms were self-normalized with respect to total number of fusion events , and cumulative distributions calculated .", "Histograms were fitted to bi-exponential decay functions using OriginPro 8 . 6 ( OriginLab , Inc . Northampton , MA ) .", "For the clustering experiments the same v-vesicle preparation and protein reconstitution , and vesicle immobilization protocols were used as for the single vesicle-vesicle fusion experiments described in the previous section , except that lipid dyes were added .", "Specifically , a saturated layer of 1 , 1′-dioctadecyl-3 , 3 , 3′ , 3′-tetramethylindodicarbocyanine perchlorate , DiIC18 ( 5 ) ( DiD ) labeled synaptobrevin-2 containing v-vesicles was immobilized on an imaging surface via biotin/neutravidin interactions .", "The DiD-labeled synaptobrevin-2 v-vesicle solution ( obtained by our reconstitution protocol; Diao et al . , 2012a; Kyoung et al . , 2012 ) was diluted 20× .", "100 µl of the diluted vesicle solution was injected into the sample chamber and incubated for 30 min ( Figure 3B , left panel ) , followed by buffer exchange ( 1 × 200 μl vesicle buffer for 6 s ) .", "We confirmed that the vesicle-covered surfaces were saturated and produced a homogeneous distribution for each surface preparation with red laser excitation ( 633 nm ) of the DiD-labeled immobilized vesicles ( Figure 3B , right image ) .", "As previously reported , more than 1000 vesicles could be immobilized with this method ( Yoon et al . , 2008 ) .", "The immobilized v-vesicle surface was incubated for 30 min at the specified α-Syn concentration ( Figure 3B , middle panel ) ; note that this is an additional step compared to the v-/t-vesicle fusion experiments described above .", "This step was followed by buffer exchange ( 1 × 200 μl vesicle buffer for 6 s ) , removing unbound or weakly bound α-Syn .", "Next , 1 , 1′-dioctadecyl-3 , 3 , 3′ , 3′-tetramethylindodicarbocyanine perchlorate , DiIC18 ( 5 ) ( DiI ) -labeled synaptobrevin-2 vesicles were also prepared with our reconstitution protocol ( Diao et al . , 2012a; Kyoung et al . , 2012 ) and diluted 500× .", "100 µl of diluted free-floating DiD-labeled v-vesicle solution was injected into the sample chamber ( Figure 3B , right panel ) ; note that this v-vesicle solution did not contain α-Syn in order to prevent pre-clustering of this vesicle solution .", "After an incubation period of 120 min , unbound v-vesicles were removed by buffer exchange ( 2 × 200 μl vesicle buffer for ∼20 s ) .", "Sample slides with multiple channels were monitored in a wide-field TIR fluorescence microscope , data were acquired with a CCD camera , and analyzed with the smCamera program , similar to the single vesicle-vesicle fusion experiments described above .", "A specified number of images were taken at random locations within each channel on the quartz slide .", "Details regarding software , slide assembly , and imaging protocols are described in reference ( Diao et al . , 2012b ) .", "The number of v-/v-vesicle interactions ( clustering ) was determined by counting the number of fluorescent spots from emission of DiI upon excitation at 532 nm ( Figure 3A ) .", "The counts were averaged over the specified number of images at random locations in each sample channel .", "This simple averaging procedure was possible since the surface coverage was homogeneous .", "For each set of comparisons between different conditions and/or mutants ( Figures 4A , B , D , 7C , D , and 8 ) , the same protein preparations and surface preparations ( quartz slide with immobilized vesicles ) were used , and the conditions were run in separate channels on the same slide; although there was some variation in absolute numbers of counts ( e . g . , due to the use of tag-free vs tagged a-Syn constructs , Figure 4A vs Figure 4B , D ) , the relative differences were statistically similar for different protein preparations .", "Frozen-hydrated samples of synaptobrevin-2 v-vesicles in the presence of 20 μM α-Syn were prepared using the procedures for observation in cryo-EM as described previously ( Kyoung et al . , 2011 ) .", "Briefly , the synaptobrevin-2 v-vesicle solution obtained by our reconstitution protocol ( Diao et al . , 2012a; Kyoung et al . , 2012 ) was diluted 5× in the presence of 20 μM α-Syn ( resulting in v-vesicle-lipid to α-Syn-protein ratio of 33:1 ) and then incubated on a glow-discharged lacey Form var/carbon 300 mesh copper grid ( Ted Palla , Redding , CA , USA ) , followed by blotting and plunging into liquid ethane .", "Thus , the concentration of the vesicles used in the cryo-EM experiments was 100-fold higher than in the single vesicle-vesicle clustering experiments .", "The frozen-hydrated specimen was subsequently observed at liquid nitrogen temperature using a CM200F electron microscope ( FEI , Hillsboro , OR , USA ) operating at 200 kV , under low dose conditions .", "Images were collected at a 50 , 000× magnification and 1 . 5 µm under-focus on a 2k × 2k UltraScan 1000 camera ( Gatan , Inc . , Pleasanton , CA ) ." ] ]

[ "α-Synuclein is a presynaptic protein that is implicated in Parkinson's and other neurodegenerative diseases .", "Physiologically , native α-synuclein promotes presynaptic SNARE-complex assembly , but its molecular mechanism of action remains unknown .", "Here , we found that native α-synuclein promotes clustering of synaptic-vesicle mimics , using a single-vesicle optical microscopy system .", "This vesicle-clustering activity was observed for both recombinant and native α-synuclein purified from mouse brain .", "Clustering was dependent on specific interactions of native α-synuclein with both synaptobrevin-2/VAMP2 and anionic lipids .", "Out of the three familial Parkinson's disease-related point mutants of α-synuclein , only the lipid-binding deficient mutation A30P disrupted clustering , hinting at a possible loss of function phenotype for this mutant .", "α-Synuclein had little effect on Ca2+-triggered fusion in our reconstituted single-vesicle system , consistent with in vivo data .", "α-Synuclein may therefore lead to accumulation of synaptic vesicles at the active zone , providing a ‘buffer’ of synaptic vesicles , without affecting neurotransmitter release itself ." ]

[ "The central nervous system coordinates many different activities by sending instructions to large numbers of cells and , simultaneously , processing all the signals that are sent back to the brain .", "All these messages are carried by electrical pulses that travel along chains of neurons , with neurotransmitter molecules enclosed inside synaptic vesicles conveying the messages across the synapses between neurons .", "A protein called α-synuclein is thought to have a role in the transport of neurotransmitter molecules across synapses , but the details of its involvement are not fully understood .", "Mutations in the gene that codes for α-synuclein , and also duplications and triplications of this gene , are known to lead to an increased risk of early onset Parkinson's disease , a condition where the central nervous system degenerates .", "Moreover , the Lewy bodies found in the neurons of patients with Parkinson's disease contain high concentrations of α-synuclein .", "Again , however , none of this is fully understood .", "Diao et al . have shed new light on these questions by creating synthetic vesicles to mimic what happens in real synapses , and using optical microscopy to observe the behaviour of these vesicles .", "They found that native α-synuclein ( and another set of membrane proteins ) increases the availability of synthetic vesicles at the synapse by causing them to cluster together .", "In a second experiment , Diao et al . showed that native α-synuclein does not decrease calcium-triggered fusion between membranes , the process that releases neurotransmitter into the synaptic cleft .", "In contrast , it is known that pathogenic α-synuclein aggregates directly interfere with the release of the neurotransmitter molecules .", "Moreover , when Diao et al . used a particular mutant form of α-synuclein that is associated with Parkinson's disease , the vesicles did not form clusters .", "If these results are confirmed in vivo , the role played by native α-synuclein in the central nervous system , and the connection between α-synuclein and Parkinson's disease , will be much clearer ." ]

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

[ "developmental biology" ]

[ [ "Coronary artery disease is the leading cause of death worldwide , but there is currently no effective method to regenerate new coronary arteries ( CA ) in diseased or injured hearts ( Rubanyi , 2013 ) .", "This is likely due to our limited understanding of CA progenitor cells and the signaling pathways that activate their differentiation .", "CAs are the vessels that supply blood to ventricular heart muscle and are composed of an inner endothelial cell lining wrapped by a smooth muscle covering .", "Coronary artery smooth muscle cells ( caSMC ) are particularly important due to their role in the pathogenesis of coronary artery disease .", "However , caSMC development , both normally during embryogenesis and ectopically during coronary artery disease , is a poorly understood process .", "CaSMC development occurs during arterialization of immature coronary plexus vessels .", "In the murine heart , coronary endothelial cells derived primarily from the sinus venosus and endocardial cells that sprout onto the heart to form an early vascular plexus ( Kattan et al . , 2004; Chen et al . , 2014b; Red-Horse et al . , 2010; Tian et al . , 2013; Wu et al . , 2012 ) .", "A subset of the plexus vessels differentiates into arteries once the network attaches to the aorta and begins receiving blood flow ( Chen et al , 2014a; Hood et al . , 1992; Peeters et al . , 1997 ) .", "Although ultimately adjacent to CA endothelial cells , the source of caSMCs is different .", "These cells arise from the mesothelial covering of the heart called the epicardium ( Cai et al . , 2008b; Mikawa and Gourdie , 1996; Wilm et al . , 2005; Zhou et al . , 2008 ) .", "In rodents , caSMCs reside deep within the myocardium while epicardial cells are located on the outermost layer of the heart .", "During embryonic development , many epicardial cells undergo an epithelial-to-mesenchymal transition ( EMT ) and migrate into the deeper layers of the myocardium to form the stromal cells of the heart , including cardiac fibroblasts and caSMCs .", "Inhibition of epicardial migration from the surface by deletion of Platelet Derived Growth Factor Receptor β ( PDGFRβ ) diminishes caSMC development ( Mellgren , et al . , 2008; Smith et al . , 2011 ) .", "However , the migrating epicardial-derived cell type fated to become caSMCs has not been discovered , and the mechanisms that trigger this intermediate progenitor to differentiate into caSMCs are unknown .", "Smooth muscle within other internal organs , including vascular smooth muscle , also arises from an outer mesothelial covering .", "Lineage tracing Mesothelin ( Msln ) -positive mesothelial cells and their prospective isolation and transplantation has shown that many of the abdominal and thoracic organs derive their smooth muscle and fibroblasts from the surface serosa layer ( Rinkevich et al . , 2012 ) .", ".", "Mesothelial to vascular smooth muscle differentiation occurred almost exclusively during developmental and early postnatal stages , but , similar to the heart , the cellular pathway bridging the surface to internal arteries has not been identified .", "Epicardial mesothelium of the heart also has a developmental restriction .", "Most migrating caSMC progenitors leave the heart surface before embryonic day 12 . 5 and are no longer able to migrate into the adult heart , either normally or following myocardial infarction ( Wei et al . , 2015; Zhou et al . , 2011 ) .", "However , since this time point is long before caSMCs appear , it is unclear what cell type resides in the heart to eventually receive signals to become new smooth muscle around forming arteries and whether this cell type persists in the adult .", "The discovery of such an intermediate progenitor could identify a cell type that could aid collateral artery formation during disease .", "Here , we find that cells resembling vascular pericytes ( PDGFRβ+Notch3+NG2+SM-MHC−SMα−PDGFRα− , wrapping microvessels , embedded within a basement membrane ) are intermediate progenitors for smooth muscle in the developing heart .", "Pericytes are mural cells that wrap microvascular blood vessels and regulate their development and function ( Armulik et al . , 2011; Cappellari et al . , 2013 ) .", "Pericytes share features with smooth muscle cells including close apposition to the vessel and some molecular markers , but differ in their contractile protein expression , cell shape , location on capillaries instead of large vessels , and discontinuous covering of the endothelium .", "Since their identification in 1873 ( Rouget , 1873 ) , biologists have wondered whether pericytes and smooth muscle differentiate into each other , but direct evidence has been lacking ( Armulik et al . , 2011; Cappellari et al . , 2013; Majesky , 2011 ) .", "We provide evidence that pericytes lining the coronary vascular plexus respond to Notch3 signaling at arterial remodeling zones to become mature caSMCs during embryonic development .", "We also observed caSMC related pericytes in the adult heart presenting the possibility that the Notch3 pathway could be manipulated in these cells to participate in CA regeneration ." ], [ "To identify where an epicardial-derived caSMC progenitor would be found , we determined where caSMCs first appear .", "CA development was followed using confocal imaging of intact mouse hearts immunostained for vascular endothelial-cadherin ( VE-cadherin ) and smooth muscle-myosin heavy chain ( SM-MHC ) , one of the most specific markers for mature smooth muscle ( Miano et al . , 1994; Seidelmann et al . , 2013 ) .", "Hearts are shown on the right lateral side to most effectively display developing CAs .", "Development of CAs begins with the invasion of VE-cadherin+ endothelial cells that form an immature coronary vascular plexus ( Kattan et al . , 2004; Chen et al . , 2014b; Red-Horse et al . , 2010 ) , which is initially devoid of blood flow ( Figure 1A–A’” ) ( Chen et al . , 2014a ) .", "At embryonic day ( e ) 13 . 5 , the plexus vessels attach to the aorta and begin to receive blood flow ( Chen et al . , 2014a ) .", "Subsequently , vascular remodeling , or fusion and enlargement of plexus vessels , is observed directly downstream of the aortic attachment site where future arteries will form ( Chen et al . , 2014a ) .", "SM-MHC protein expression was first observed at e14 . 5 in plexus vessels that had begun to remodel into arteries ( Figure 1B–B’” ) .", "At e14 . 5 smooth muscle coverage was patchy and consisted of cells with both low and high SM-MHC ( SM-MHClow or SM-MHChigh ) protein expression ( fluorescence measured from single confocal z-planes ) ( Figure 1B”’ , Figure 1—figure supplement 1 ) .", "As this initial remodeling zone transitioned into recognizable arterial vessels at e15 . 5 , caSMC coverage increased and most cells were SM-MHChigh ( Figure 1C–C”’ ) .", "Remodeling zones with patchy SM-MHClow cells were now just distal to the more mature vessels ( Figure 1C”’ ) .", "These distal remodeling zones eventually transformed into mature vessels in the next developmental stage at e16 . 5 ( Figure 1D–D”’ ) .", "These data identify when and where SM-MHC+ caSMCs differentiate within the context of the whole heart at single-cell resolution ( Figure 1E ) . 10 . 7554/eLife . 10036 . 003Figure 1 . Coronary artery smooth muscle differentiation is initiated early during vascular remodeling .", "( A–D )", "Whole mount confocal images of the developing right coronary artery at embryonic ( e ) days 13 ( A–A’’’ ) , 14 . 5 ( B–B’’’ ) , 15 . 5 ( C–C’’’ ) , and 16 . 5 ( D–D’’’ ) immunostained with VE-cadherin ( blue ) and SM-MHC ( red ) .", "Higher magnification views ( z-stack subsets ) from boxed regions ( A’–D’ ) and separated channels ( A”–D”’ ) show that smooth muscle first appears at early remodeling zones ( arrowheads ) and further accumulates as these transform into coronary arteries ( arrows ) .", "( E ) Schematic representation showing coronary artery ( CA ) smooth muscle cell ( red ) development coincident with aortic attachment and initiation of blood flow at the coronary artery remodeling zone ( CA rz ) .", "( F ) Boxed region in C”’ highlighting SM-MHClow cells ( arrowheads ) at the remodeling zone in comparison to the higher expression in cells surrounding a more mature coronary artery ( arrow ) .", "( G ) Histogram plotting SM-MHC expression shows that mural cells of the remodeling zone are SM-MHClow while those around mature arteries are SM-MHChigh ( n = 16 cells/region from 4 embryos ) .", "( H ) Histogram plotting distance of SM-MHClow cells from the epicardium ( n = 22 cells from 5 hearts ) .", "( I ) Proposed model where an intermediate progenitor ( IP ) bridges epicardial cells ( Epi ) and coronary artery smooth muscle .", "Ao , aorta; pt , pulmonary trunk .", "Scale bars , 100 µm . DOI: http://dx . doi . org/10 . 7554/eLife . 10036 . 00310 . 7554/eLife . 10036 . 004Figure 1—figure supplement 1 . Morphology of SM-MHClow cells .", "( A ) SM-MHClow cells ( lower intensity green ) develop among PDGFRβ+ perivascular cells ( red ) that coat coronary vessels ( blue ) .", "( B ) Boxed region with SM-MHClow cells ( arrowheads ) show their cellular morphology .", "Scale bar , 100 µm . DOI: http://dx . doi . org/10 . 7554/eLife . 10036 . 004 The temporal appearance and location , i . e . at the remodeling zone early in arterial development , of SM-MHClow cells suggested that they have recently initiated caSMC differentiation while cells expressing higher SM-MHC levels were more mature ( Figure 1F , G ) .", "Measuring the distance between SM-MHClow cells and the epicardium revealed that they were not on the surface of the heart , but on average 50 µm deep ( Figure 1H ) .", "Thus , a putative intermediate progenitor would need to migrate through approximately 7 cell layers between the epicardium and nascent CAs where they first express the mature smooth muscle cell marker ( Figure 1I ) .", "Because most caSMCs derive from the epicardium ( Cai et al . , 2008b; Mikawa and Gourdie , 1996; Pérez-Pomares et al . , 2002; Wilm et al . , 2005; Zhou et al . , 2008 ) , we aimed to find the epicardial-derived progenitor that eventually differentiates into caSMCs .", "Epicardial-driven Cre-expressing mice for lineage tracing exist; however , these constructs label all the other epicardial-derived stromal cells in the heart in addition to caSMCs .", "We needed to study the smooth muscle lineage in isolation , which is done most accurately using clonal level labeling where single cells and all their progeny are genetically marked with a fluorescent tag ( Buckingham and Meilhac , 2011 ) .", "Our approach was to identify caSMC progenitors by analyzing fluorescently labeled sister cells within clonal clusters that also contain caSMCs .", "To produce clones , epicardial cells were sparsely labeled using T-box 18 ( Tbx18 ) -Cre ( Cai et al . , 2008b ) coupled with the Mosaic Analysis with Double Markers ( MADM ) Cre reporter system ( Zong et al . , 2005 ) .", "Tbx18-Cre was selected because , in control experiments with other Cre reporters , recombination was induced in the large majority of epicardial cells ( Figure 2—figure supplement 1A ) .", "The MADM system fluorescently labels Cre-expressing cells through a rare interchromosomal recombination event such that , when coupled with Tbx18-Cre , hearts contained isolated clusters of fluorescently labeled cells ( Figure 2—figure supplement 1B ) .", "We analyzed tightly associated clusters ( at least 100 µm from any other labeled cell ) that contained epicardium , caSMCs , and other clonally related cells , the latter of which could be the progenitor population .", "Initial experiments analyzing Tbx18-Cre , MADM clones based on cellular morphology and location revealed that clones containing epicardial cells and caSMCs always contained sister cells with long , thread-like processes that wrapped around coronary vessels ( Figure 2A ) .", "This was in contrast to sister cells from clones that were adjacent to , but did not incorporate into , the caSMC layer , which were mostly located in the space between vessels instead of wrapping around them ( Figure 2B ) .", "The morphology of the caSMC-associated sister cells was characteristic of vascular pericytes , which are mural cells that tightly associate with small blood vessels and regulate their development and function ( Armulik et al . , 2011 ) .", "To investigate the presence of pericytes in caSMC clones , we validated the use of a set of markers that would allow us to identify pericytes and caSMCs in the developing heart .", "These were then used to analyze an additional set of clones .", "The following describes our marker selection criteria . 10 . 7554/eLife . 10036 . 005Figure 2 . Characterization of epicardial-derived pericytes in the developing heart .", "( A–G )", "Whole mount confocal images of hearts immunostained with the indicated antibodies and/or fluorescent labels .", "( A and B )", "Images from Tbx18-Cre , MADM hearts show that coronary artery smooth muscle cell ( caSMC ) containing clones ( blue ) always include pericyte-like sister cells with long extended processes ( arrows ) that travel along VE-cadherin+ blood vessels ( red ) ( A ) .", "In contrast , cells within clones not containing caSMCs ( other cell type clone ) are generally located in between vessels ( B ) .", "High magnifications are shown on the right .", "( C–J )", "Mural cell characterization in hearts from mice of the indicated genotypes .", "( C and D )", "Smooth muscle and pericytes can be distinguished by immunolabeling for smooth muscle cell contractile proteins ( SM-MHC and SMα ) and PDGFRβ .", "( C ) Smooth muscle surrounding coronary arteries ( CA ) is positive for SM-MHC and PDGFRβ ( arrows ) while pericytes only stain for PDGFRβ ( arrowheads ) .", "( D ) PDGFRβ+ pericytes are not labeled with SMα-specific antibodies ( arrowheads ) .", "caSMCs around large arteries are positive for both markers ( arrows ) .", "Some cardiomyocytes expression low levels of SMα ( outlines ) .", "( E ) SM-MHC is expressed in small and large arteries ( arrowheads ) , while SMα only marks the caSMC coating around larger , more mature vessels ( arrows ) .", "( F ) PDGFRβ+ cells display a pericyte-like morphology with long processes that wrap around microvessels ( arrowheads ) .", "( G ) PDGFRβ immunostaining of PDGFRα-GFP hearts demonstrate that the two markers do not significantly overlap .", "PDGFRβ+ cells ( red ) wrap around the vessel ( arrowhead ) , while PDGFRα+ cells usually exist in-between vessels ( asterisk ) .", "( H ) PDGFRβ+ cells ( arrow ) are embedded within a Collagen IV+ basement membrane ( arrowhead ) .", "( I and J )", "PDGFRβ overlaps with NG2-DsRed labeling ( I ) and Notch3 immunostaining ( J ) .", "( K ) Quantification of marker expression and lineage labeling in PDGFRβ+ cells in the free walls of the developing heart ventricles .", "The number of cells analyzed are indicated .", "( L ) PDGFRβ+ pericytes are the most numerous epicardial-derived cell type at the e14 . 5 coronary artery remodeling zone .", "72% of Tbx18-Cre , Rosatdtomato lineage traced cells are pericytes ( PDGFRβ+ ) ( n = 14 hearts from 6 litters ) .", "The epicardial derived PDGFRβ- fraction contains mostly PDGFRα+ fibroblasts .", "( M ) Tbx18-Cre , Rosatdtomato lineage tracing shows that the majority of caSMCs are epicardial derived .", "Scale bars , A and B , 20 µm; C–E , 50 µm; F and H , 10 µm; G , 50 µm; I and J , 100 µm; M , 200 µm . DOI: http://dx . doi . org/10 . 7554/eLife . 10036 . 00510 . 7554/eLife . 10036 . 006Figure 2—figure supplement 1 . Tbx18-Cre lineage tracing and clonal analysis .", "( A ) Widespread epicardial labeling ( green ) in Tbx18-Cre hearts containing the RosamTmG Cre reporter allele .", "( B ) Examples of labeled clonal clusters ( teal , arrowheads ) in Tbx18-Cre hearts containing the MADM Cre reporter alleles .", "Ao , aorta; pt , pulmonary trunk .", "Scale bars: 100 µm . DOI: http://dx . doi . org/10 . 7554/eLife . 10036 . 00610 . 7554/eLife . 10036 . 007Figure 2—figure supplement 2 . Calponin is not expressed in PDGFRβ+ perivascular cells that wrap microvessels .", "( A ) PDGFRβ+ perivascular cells that surround capillary plexus vessels ( arrows ) are not labeled with a Calponin 1-specific antibody , while surrounding cardiomyocytes are intensely labeled ( red ) .", "( B ) Calponin1 labels a subset of smooth muscle cells around large coronary arteries ( arrowheads ) .", "Many smooth muscle cells are negative ( arrow ) .", "Scale bars: A , 20 µm; B , 50 µm . DOI: http://dx . doi . org/10 . 7554/eLife . 10036 . 00710 . 7554/eLife . 10036 . 008Figure 2—figure supplement 3 . PDGFRβ+ perivascular cells are adjacent to vessels and within the basement membrane .", "( A ) PDGFRβ immunostaining of PDGFRα-GFP hearts demonstrates that PDGFRβ+ cells ( red ) are adjacent to vessels while PDGFRα+ cells ( green ) are interspersed .", "( B ) Fluorescent intensity measurements along the white line in A show that PDGFRβ+ cells ( red peaks and arrows ) are closely associated with vessels ( blue peaks and arrows ) while PDGFRα+ cells ( green peaks and arrows ) frequently reside in between vessels .", "( C and D )", "Whole mount confocal microscope images of e13 . 5 hearts showing that PDGFRβ+ ( red ) perivascular cells ( arrows ) are embedded within a collagen IV+ basement membrane ( arrowheads ) .", "Images in C are z-stack projections , and D represents a single Z plane .", "Scale bars: A , 50 µm; C and D , 10 µm . DOI: http://dx . doi . org/10 . 7554/eLife . 10036 . 00810 . 7554/eLife . 10036 . 009Figure 2—figure supplement 4 . Characterization of PDGFRβ perivascular cells in the developing heart .", "( A and B )", "PDGFRβ pericytes co-develop with coronary vessels ( CV ) .", "Dorsal view of the e11 . 5 heart ( A ) and right lateral view at e13 . 5 ( B ) showing CVs with associated pericytes ( arrowheads ) .", "Right panels are boxed regions .", "Solid lines outline the heart .", "( C ) Dorsal view of the e13 . 5 heart .", "PDGFRβ cells coat the developing coronary plexus .", "Ao , aorta; epi , epicardium; endo , endocardium; sv , sinus venosus .", "Scale bars , 100 µm . DOI: http://dx . doi . org/10 . 7554/eLife . 10036 . 00910 . 7554/eLife . 10036 . 010Figure 2—figure supplement 5 . Epicardial-derived cells at the arterial remodeling zone are largely pericytes . Confocal images of an arterial remodeling zone from a Tbx-18-Cre , RosatdTomato lineage traced heart immunostained for VE-cadherin , PDGFRβ+ ( A ) , and PDGFRα ( B ) .", "The majority of epicardial-derived cells are PDGFRβ+ and tightly associate with the vessel ( arrowheads ) ( A ) while fewer are PDGFRα+ and localize in between vessels ( asterisks ) ( B ) .", "Note in ( A ) that PDGFRβ has a punctate distribution on perivascular cells and , unlike the lineage marker , does not uniformly label the entire cell and all of its cell processes .", "Quantification is shown in ( Figure 2K ) .", "Scale bars: 20 µm . DOI: http://dx . doi . org/10 . 7554/eLife . 10036 . 010 We found that whole mount immunostaining with PDGFRβ and SM-MHC appeared to distinguish between pericytes and caSMCs .", "CaSMCs were positive for SM-MHC and PDGFRβ and were around mature and developing arteries ( Figure 2C ) .", "In contrast , PDGFRβ perivascular cells around microvessels were SM-MHC negative ( Figure 2C ) .", "We also analyzed the expression of two additional smooth muscle markers , Smooth Muscle Alpha Actin ( SMα and Calponin 1 ( CNN1 ) ( Majesky , 2011 ) .", "Aside from a low level in some cardiomyocytes , SMα immunolabeling was only in larger , more proximal CAs , and not in PDGFRβ+ cells around plexus capillaries ( Figure 2D ) or in SM-MHC+ cells in smaller vessels and remodeling zones ( Figure 2E ) .", "Thus , in the coronary system , SMα appeared to be induced later than SM-MHC since its expression domain was less extensive and only in more mature arteries .", "CNN1 was highly expressed in cardiomyocytes , but did not appear in intervening PDGFRβ+ cells ( Figure 2—figure supplement 2A ) .", "Around large coronary arteries a subset of caSMCs expressed CNN1 ( Figure 2—figure supplement 2B ) .", "Cells that surrounded plexus capillaries and were positive for PDGFRβ , but negative for SM-MHC , SMα , and CNN1 ( Figure 2C , D , Figure 2—figure supplement 2A ) , displayed a morphology similar to vascular pericytes with long processes that wrap the endothelium ( Figure 2F ) .", "To investigate whether PDGFRβ+ perivascular cells around microvessels could be cardiac pericytes ( Armulik et al . , 2011 ) , we further characterized this population .", "Whole mount immunohistochemistry showed that PDGFRβ+ cells surrounding plexus capillaries in the free walls of the developing heart ventricles were always closely apposed to endothelial cells in contrast to PDGFRα+ fibroblasts , which were dispersed between the endothelium ( Figure 2G and Figure 2—figure supplement 3A , B ) .", "PDGFRβ+ cells were also embedded within a Collagen IV-containing basement membrane ( Figure 2H and Figure 2—figure supplement 3C , D ) .", "These are critical attributes for pericyte identification ( Armulik et al . , 2011 ) .", "They also all expressed the pericyte markers NG2 ( Figure 2I ) and Notch3 ( Figure 2J ) ( Liu et al . , 2010 ) ( quantification shown in Figure 2K ) .", "PDGFRβ+ cells were observed on the earliest coronary sprouts when the vessels first migrate directly beneath the epicardium and surrounded the entire plexus endothelium as it developed ( Figure 2—figure supplement 4 ) .", "Later , at the arterial remodeling zone where caSMCs first differentiate , 98 ± % of PDGFRβ+ cells were Tbx18-Cre lineage labeled consistent with most arising from the epicardium ( Figure 2K ) .", "In addition , PDGFRβ+ cells were the most numerous Tbx18-Cre traced cell type in this location ( Figure 2L and Figure 2—figure supplement 5A ) .", "The remaining 28% were predominately fibroblasts as defined by their expression of PDGFRα and variable proximity to the vessels ( Figure 2L and Figure 2—figure supplement 5B ) .", "At time points after arteries are formed , much of the coronary smooth muscle was also lineage labeled with Tbx18-Cre ( Figure 2M ) in line with other studies showing an epicardial origin for these cells ( Cai et al . , 2008b; Wilm et al . , 2005; Zhou et al . , 2008 ) .", "The epicardium is not thought to give rise to cells of the hematopoietic lineage , and , accordingly , PDGFRβ cells did not overlap with CD45 staining ( data not shown ) .", "It must be noted that although eight criteria ( PDGFRβ+Notch3+NG2+SM-MHC−SMα−PDGFRα− , wrapping microvessels , embedded within basement membrane ) suggested that PDGFRβ+ perivascular cells are pericytes , we cannot exclude the possibility that the discovery of different markers will reveal this population to be a mix of pericytes and pericyte-like cells with different functions .", "However , based on current knowledge , the above described cell type will be referred to as pericytes below .", "In summary , given their marker expression , localization within the tissue , and cellular morphology , we defined PDGFRβ+ cells to be cardiac pericytes of the developing coronary vasculature .", "We also show that they are the most common epicardially-derived cell type at the arterial remodeling zone making them a prime candidate for caSMC progenitors .", "To evaluate the likely clonality of the observed fluorescently labeled cell clusters , we mathematically modeled the process of clone formation using realistic parameters estimated from the imaging data of the e13 . 5 developing heart ( see details in Materials and methods ) .", "In brief , we simulated the process of clone formation as a three dimensional Poisson process and estimated the underlying rate of clone generation that best fit the observed experimental data of ~∼3 . 3 fluorescently labeled clusters per half heart ( Figure 3A ) .", "For the estimated clone generation rate , we then calculated the fraction of simulated clusters that were truly clonal ( not comprised of more than one overlapping clones ) to evaluate the likelihood that the observed clusters are indeed clonal .", "This analysis revealed that ∼82% of the observed clusters were likely to derive from a single labeling event ( Figure 3B ) .", "This same clonality rate was maintained when considered for hearts having a range of 1–6 clusters each ( Figure 3C ) , which was the range observed in our e13 . 5 data set ( mean value of 3 . 3 ± 1 . 6 ) .", "We repeated the entire modeling process with different settings for the discrimination distance that defines clonal clusters ( see details in Materials and methods ) confirming the robustness of the estimated clonality rate ( Figure 3—figure supplement 1 ) .", "These observations provide evidence that greater than 80% of the observed clusters were truly clonal . 10 . 7554/eLife . 10036 . 011Figure 3 . Coronary artery smooth muscle cells and pericytes are clonally related .", "( A–C )", "Simulation-Based Analysis of Clonality as outlined in Materials and methods .", "Mathematical modeling was performed to estimate the underlying clone generation rate that best fit the experimental data ( denoted by the horizontal red line ) ( A ) , and to evaluate the corresponding overall average rate of clonality ( denoted by the vertical red line ) ( B ) as well as the clonality rates for simulated half heart regions with the designated numbers of observed clusters ( C ) .", "( D ) Confocal images of clones from indicated ages ( e13 . 5 , e15 . 5 , and adult ) .", "Left panels are low magnification views of entire clones and middle panels are internal views of circled clones , which are near coronary arteries in e15 . 5 and adult .", "Boxed regions are separated channels as examples of marker expression with white showing clone label for morphology .", "Note that PDGFRβ staining is punctate while the clone label is uniform throughout the cell .", "Asterisks indicate long cellular processes in adult pericytes .", "Schematics of each are on the far right .", "( E ) Graph showing cell types within individual clones .", "( F ) Quantification of the percentage of pericytes and smooth muscle cells in clones and their ratios among total mural cells in adult hearts .", "caSMC , coronary artery smooth muscle cell; epi , epicardial cell; LV , left ventricle; peri , pericyte; RV , right ventricle; Sep , septum .", "Scale bars: left panels , 100 µm; middle panels , 20 µm . DOI: http://dx . doi . org/10 . 7554/eLife . 10036 . 01110 . 7554/eLife . 10036 . 012Figure 3—figure supplement 1 . Influence of discrimination distance parameter on estimated clonality rate . The results of our mathematical simulations to evaluate the clonality of the observed fluorescently labeled cell clusters is presented as in Figure 3A–C .", "Simulations were performed as described ( Materials and methods ) , with the value of the discrimination distance parameter set to be 25 µm ( A ) , 75 µm ( B ) , or 100 µm ( C ) .", "Left panels display the mean number of observed clusters per simulation plotted against the underlying clone generation rate ( representing the average number of clones produced per half heart region ) .", "The horizontal red line indicates the mean number of clusters recorded in the experimental dataset ( 3 . 3 clusters per half heart ) .", "Right panels show the resulting clonality rate ( defined as the fraction of observed clusters consisting of a single clone ) plotted as a function of the underlying clone generation rate .", "The vertical red line indicates the estimated clone generation rate that best fit the experimental data , and its associated clonality rate .", "The estimated clonality rates were 88% for a discrimination distance of 25 µm ( A ) , 77% for a value of 75 µm ( B ) , and 69% for a value of 100 µm ( C ) .", "Each data point in both panels represents the results of 10 , 000 simulated half heart regions containing at least one clone . DOI: http://dx . doi . org/10 . 7554/eLife . 10036 . 01210 . 7554/eLife . 10036 . 013Figure 3—figure supplement 2 . Additional examples of pericyte-coronary artery smooth muscle clones and quantification of cell location and number .", "( A ) Box-and-whisker plot depicting the location of cell types in e15 . 5 clones with respect to the epicardium [n = 91 , 249 , 37 and 150 for epicardial cells ( epi ) , pericytes ( peri ) , coronary artery smooth muscle cells ( caSMCs ) , and other cell types , respectively] .", "( B ) A pericyte-caSMC clone ( green ) located near a coronary artery ( CA ) ( red ) .", "Endothelial cells shown in blue .", "Top left: low magnification .", "Top middle: A deeper slice from the boxed region shows continuous sister cells with pericyte and caSMC identity .", "Top right: higher magnification of middle panel .", "Lower panels show PDGFRβ staining in pericytes from top middle panel .", "Schematic is on the far right .", "( C ) Quantification of the number of cells contained in e15 . 5 Tbx18-Cre , MADM clones .", "( D ) An adult pericyte-caSMC clone ( green ) located near a CA ( red ) .", "Pericytes display long extended processes ( arrowheads ) along capillaries ( blue ) .", "Arrows point to caSMCs .", "Low magnification is left , higher magnification is middle , and separated channels of boxed regions are right .", "Scale bars: 100 µm . DOI: http://dx . doi . org/10 . 7554/eLife . 10036 . 013 We next used PDGFRβ and SM-MHC to analyze Tbx18-Cre-derived clonal cell clusters isolated at e13 . 5 and 15 . 5 , before and after caSMCs develop , respectively .", "This analysis revealed a clonal and spatial relationship between caSMCs and pericytes .", "At e13 . 5 before caSMCs are present , we obtained clones consisting of just epicardial cells and pericytes , the latter of which were always located directly beneath epicardial sister cells ( Figure 3D-e13 . 5 , E ) .", "At e15 . 5 , clones with caSMCs always contained pericytes that filled the region of the myocardium between the epicardium and smooth muscle and were continuous with , and often touching , related caSMCs ( Figure 3D-e15 . 5 , E , Figure 3—figure supplement 2A , B , and Videos 1 , 2 ) .", "As expected from the Tbx18-Cre lineage trace data ( Figure 2L ) , pericytes were the most numerous cell type in these clones ( Figure 3—figure supplement 2C ) .", "Tbx18-Cre labels cardiomycytes ( Cai et al . , 2008b ) , but these cells never appeared in clonal clusters with pericytes or smooth muscle .", "Together , the temporal sequence and spatial arrangement of Tbx18-Cre-derived clones suggested that pericytes are the intermediate differentiation step between epicardium and caSMCs . 10 . 7554/eLife . 10036 . 014Figure 4 . Pericytes differentiate into coronary artery smooth muscle .", "( A and B )", "Schematics describing the experimental design for cardiac pericyte lineage tracing ( A ) and part of the differentiation pathway being interrogated ( B ) .", "( C ) No recombination occurs in NG2-CreER , Rosatdtomato animals in the absence of tamoxifen ( tam ) .", "( D ) Quantification of Cre labeling ( i . e . recombination efficiency ) in NG2+Notch3+ pericytes alongside levels of smooth muscle lineage labeling .", "( E ) E11 . 5 dosing of NG2-CreER induces lineage labeling ( green ) in pericytes ( arrowhead ) , smooth muscle , and some cardiomyocytes ( arrow ) .", "( F ) Boxed region in E showing lineage labeled pericytes ( green , arrowheads ) and coronary artery smooth muscle ( yellow , arrows ) ( n = 10 hearts from 3 litters ) .", "Endothelial cells are in blue ( VE-cadherin+ ) .", "Right panel is boxed region in far left panel .", "( G ) Labeled pericytes ( arrowhead ) , smooth muscle , and rare cardiomyocytes ( arrow ) in Notch3-CreER lineage trace .", "( H ) Boxed region in F showing lineage labeled pericytes ( green , arrowheads ) and coronary artery smooth muscle ( yellow , arrows ) ( n = 11 hearts from 2 litters ) .", "Endothelial cells are in blue ( VEGFR2+ ) .", "Right panel is boxed region in far left panel .", "Ao , aorta; caSMC , coronary artery smooth muscle cell; epi , epicardium; r ven , right ventricle , Scale bars: C , E and G , 100 µm; F and H 50 µm . DOI: http://dx . doi . org/10 . 7554/eLife . 10036 . 01410 . 7554/eLife . 10036 . 015Video 1 . Following an epicardial/pericyte/smooth muscle cell clone from the surface of the heart to a coronary artery deep in the myocardium . Movie showing single confocal Z-planes of the clone in Figure 3A-e15 . 5 going from the surface of the heart to deeper regions where a coronary artery is located ( μms from the epicardium are indicated ) .", "A single epicardial cell is present at the surface while tightly clustered pericytes span the region between the surface and coronary artery where clonal cells are incorporated into the smooth muscle cell layer .", "VE-Cadherin ( blue ) labels the vasculature , SM-MHC ( red ) labels coronary artery smooth muscle , and the Tbx18-Cre , MADM lineage label is green . DOI: http://dx . doi . org/10 . 7554/eLife . 10036 . 01510 . 7554/eLife . 10036 . 016Video 2 . Pericytes and smooth muscle clone cells express PDGFRβ .", "Clone in Video 1 ( Figure 3A-e15 . 5 ) showing the fluorescent channels for PDGFRβ in red and the Tbx18-Cre , MADM lineage label in green .", "Note that PDGFRβ is localized in a punctate pattern while the lineage label fills the entire cell so that only portions of the cell appear as double positive ( yellow ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 10036 . 016 We next tested whether the pericytes that were clonally related to caSMCs during embryogenesis persisted into adulthood or were depleted after development .", "Strikingly , adult Tbx18-Cre , MADM hearts frequently contained tightly packed clusters of lineage labeled cells consisting of pericytes and caSMCs ( Figure 3D-adult ) ( n = 20 ) .", "Pericytes at this stage exhibited even longer , thinner processes that tracked along the vessels for greater than 5–10 times the length of their cell bodies ( Figure 3D-adult and Figure 3—figure supplement 2D ) .", "The fact that these cells were not dispersed , even five weeks after labeling , shows that pericytes and caSMCs do not migrate significantly once their positions are established during development .", "In addition , our analysis indicates that pericytes around capillaries that are clonally related to caSMCs remain and function as cardiac pericytes in the adult heart ( Figure 3E ) .", "Within established adult clones , the ratio between the number of pericytes and caSMCs was comparable to ratios calculated for total mural cells from three different regions of the heart ( Figure 3F ) .", "This is the expected result if the epicardial to pericyte pathway is a prominent source of caSMCs .", "In total , our clonal analysis suggests that epicardial-derived pericytes surround the entire coronary plexus during embryonic development , but that these cells differentiate into caSMCs if located around vessels that enlarge to become arteries during remodeling .", "These pericytes also surround capillaries in the adult heart and could potentially become new caSMC if arterial remodeling were induced at this stage .", "The above described composition and arrangement of epicardial-derived clones suggests that pericytes differentiate into caSMCs .", "However , direct lineage tracing of pericytes is required to confirm this sequence of events .", "NG2 and Notch3 are well-established pericyte markers in other tissues ( Armulik et al . , 2011; Kofler et al . , 2011 ) .", "In the myocardium of the developing ventricle free walls , NG2- and Notch3-positive cells on the microvasculature always expressed PDGFRβ and wrapped vessels ( Figure 2I–K ) , which identified them as cardiac pericytes ( Figure 2 ) .", "NG2 and Notch3 also completely overlapped ( n = 185 cells counted , data not shown ) .", "To test if pericytes differentiate into smooth muscle , we performed lineage tracing using two independent mouse lines , NG2-CreER ( Zhu et al . , 2011 ) and Notch3-CreER ( Fre et al . , 2011 ) .", "In embryos containing either the NG2-CreER or Notch3-CreER allele coupled with the fluorescent Cre reporter gene Rosatdtomato , pericytes , but not caSMCs , were labeled by injecting tamoxifen at either e10 . 5 or e11 . 5 .", "This restricts Cre activity to a time point after pericytes had formed but before caSMCs appear ( Figure 4A ) .", "If caSMCs arise from pericytes , this strategy should result in lineage labeled caSMCs , i . e . tdtomato ( Figure 4B ) .", "These lineage tracing experiments resulted in labeled caSMCs with both Cre drivers .", "Importantly , no labeling was detected in the absence of tamoxifen ( Figure 4C and data not shown ) .", "Quantifying the proportion of lineage labeled pericytes in the compact myocardium indicated that Cre recombination efficiency in PDGFRβ+SM-MHC- perivascular cells ( pericytes ) was 21% and 38% with NG2-CreER and Notch3-CreER , respectively ( Figure 4D ) .", "SM-MHC+ caSMCs also expressed the tdtomato lineage label in both NG2-CreER ( Figure 4E , F ) and Notch3-CreER ( Figure 4G , H ) strains .", "Similar percentages of labeling were seen in pericytes and caSMC ( quantified in the compact myocardium ) consistent with low recombination efficiencies in pericytes accounting for the incomplete caSMC tracing ( Figure 4D ) .", "However , our analysis does not exclude the possibility of an additional source , particularly in regions not analyzed , i . e . the ventricular septum .", "One potential confounding factor is that both transgenes exhibited sporadic labeling in cardiomyocytes ( Ozerdem et al . , 2001 ) , which was more rare in Notch3-CreER ( Figure 4E , G ) .", "However , control experiments using Myh6-CreER showed that cardiomyocyte lineage tracing never labeled caSMCs ( data not shown ) .", "Finally , NG2 and Notch3 were expressed in PDGFRβ perivascular cells that were characterized as cardiac pericytes ( Figure 2 ) , but , as stated above , we cannot exclude the possibility that these experiments traced pericyte-like SMC progenitors sharing attributes with traditional pericytes , specifically PDGFRβ+Notch3+NG2+SM-MHC−SMα−PDGFRα− , wrapping microvessels , and embedded within the basement membrane .", "In summary , lineage tracing data from two independent Cre lines support a model where pericytes differentiate into caSMCs .", "To gain further evidence that pericytes were caSMC progenitors , we analyzed mutants with deficient caSMC layers and asked if this phenotype was correlated with defects in pericytes at the arterial maturation zone .", "PDGFRβ-null mice have reduced caSMCs ( Figure 5A ) ( Hellström et al . , 1999; Smith et al . , 2011 ) ; however , they undergo vascular remodeling exhibiting a recognizable arterial remodeling zone and CA ( Figure 5B ) .", "Analyzing the total number of Notch3+ pericytes at the remodeling zone revealed that they were significantly decreased ( Figure 5B , C ) .", "Pericytes and caSMCs were not completely gone ( Figure 5B , C ) suggesting the presence of a very inefficient compensatory mechanism .", "The coincident decrease in pericytes and caSMC support a model where pericytes are progenitors that are required for smooth muscle formation , and that PDGFRβ functions at the surface to induce the first transition of the pathway from epicardial cell to pericyte ( Figure 5D ) . 10 . 7554/eLife . 10036 . 017Figure 5 . Coronary artery smooth muscle and pericytes are decreased in PDGFRβ-null mice .", "( A ) Absence of SM-MHC+ smooth muscle around coronary arteries ( CA ) in PDGFRβ knockout hearts .", "( B ) Notch3+ mural cells ( green ) are decreased at the coronary artery remodeling zone ( CA rz ) in PDGFRβ-deficient hearts ( n = 8 from 4 litters ) .", "( C ) Quantification of pericyte numbers per field of view ( FOV ) ( wild type , n = 7 hearts; mutant , n = 5 ) .", "Error bars are s . d . ; *p≤0 . 05 .", "( D ) Schematic demonstrating the hypothesized epicardial to smooth muscle differentiation pathway and how it is affected in PDGFRβ-null mice .", "Greyed cells are reduced or absent .", "Ao , aorta; CA rz , coronary artery remodeling zone; caSMC , coronary artery smooth muscle cell; epi , epicardium; r ven , right ventricle .", "Scale bars: A , 100 µm; B , 50 µm . DOI: http://dx . doi . org/10 . 7554/eLife . 10036 . 017 To identify molecular regulators of the pericyte to caSMC differentiation step , we characterized the expression of mural cell markers at the arterial remodeling zone .", "Among those investigated , Notch3 was upregulated in pericytes at the region where SM-MHC protein expression was initiated ( Figure 6A , B ) .", "In fact , the arterial remodeling zone can be identified based on Notch3 staining intensity without visualizing vessel structure ( Figure 6A ) .", "This pattern was in contrast to PDGFRβ , which was uniformly expressed in mural cells throughout the vasculature ( Figure 6A , B ) .", "Jagged-1 has been shown to be a major ligand for Notch3 in other organs , where it is expressed in arterial endothelium and stimulates smooth muscle differentiation including inducing the expression of SM-MHC ( Briot et al . , 2014; Doi et al . , 2006; High et al . , 2008; Hofmann et al . , 2012; Jin et al . , 2008; Kofler et al . , 2011; Manderfield et al . , 2012; Yang and Proweller , 2011 ) .", "We found high levels of Jagged-1 to be specifically expressed at arterial remodeling zones where SM-MHC+ cells were developing and in CAs ( Figure 6C and Figure 6—figure supplement 1 ) .", "Given that Jagged-1 has previously been identified as a shear stress-induced molecule in vitro ( McCormick et al . , 2001 ) , we investigated whether it could be regulated by blood flow in the heart .", "Coronary vessels are initially unperfused , but begin to receive blood flow after they attach to the aorta at e13 . 5 ( Chen et al . , 2014a ) .", "This time point correlated with the onset of robust Jagged-1 expression in endothelial cells of vessels directly downstream of the attachment site ( Figure 6D–WT and Figure 6—figure supplement 1 ) .", "We analyzed a model of delayed CA stem attachment to the aorta , Isl1 heterozygosity ( Cai et al . , 2008a ) , which postpones the initiation of blood flow ( Chen et al . , 2014a ) .", "Immunostaining Isl1 mutant hearts revealed that Jagged-1 was only upregulated in arterial remodeling zones of e13 . 5 hearts that had formed CA stems on the aorta to receive blood flow ( Figure 6D , E ) .", "Isl1 mutant hearts also failed to upregulate Notch3 in pericytes suggesting that this change also requires signals downstream of blood flow ( n = 9 , data not shown ) .", "In summary , Notch3 is upregulated in pericytes at the arterial remodeling zone while its ligand Jagged-1 is induced in endothelial cells in the same region following the initiation of blood flow suggesting that this receptor-ligand pair could trigger caSMC differentiation in coronary pericytes . 10 . 7554/eLife . 10036 . 018Figure 6 . Notch3 and Jagged-1 expression at the arterial remodeling zone .", "( A and B )", "Mural cells around coronary vessels increase Notch3 protein expression at the coronary artery remodeling zone ( CA rz ) while PDGFRβ levels remain the same .", "( A ) Confocal image of a representative remodeling zone .", "( B ) Quantification of marker expression .", "Error bars are s . d . ( C and D ) Confocal images immunostained for VE-cadherin ( blue ) and Jagged-1 ( green ) .", "( C ) Jagged-1 is specifically expressed in coronary arteries ( CA ) and the CA rz after attachment to the aorta ( ao ) and induction of blood flow .", "( D ) Jagged-1 is expressed in coronary vessels at e13 . 5 soon after aortic attachment and CA stem formation , but not in Isl1 mutant littermates with delays in attachment and arterial blood flow .", "( E ) Table of Jagged-1 protein expression in wild type ( Wt ) and Isl1 mutants .", "Scale bars: 100 µmDOI: http://dx . doi . org/10 . 7554/eLife . 10036 . 01810 . 7554/eLife . 10036 . 019Figure 6—figure supplement 1 . Characterization of Jagged-1 expression during coronary artery development . Confocal images of VE-cadherin and Jagged-1 immunostaining in hearts from the indicated ages .", "Jagged-1 expression is initiated right after coronary vessels ( VE-cadherin+ ) connect to the aorta in the vessels directly downstream of the attachment site ( arrowheads ) .", "Ao , aorta; ca , coronary artery; cv , coronary vessels .", "Scale bars: 50 µm . DOI: http://dx . doi . org/10 . 7554/eLife . 10036 . 019 We next analyzed Notch3-deficient hearts to investigate whether this signaling pathway could be involved in the pericyte to caSMC transition .", "CAs in Notch3-null mice displayed significantly reduced levels of SM-MHC when compared to controls at two time points examined , e15 . 5 and e17 . 5 ( Figure 7A , B and data not shown ) .", "SMα was also reduced ( Figure 7C ) .", "In contrast , CA coverage by PDGFRβ- cells was similar to wild type counterparts ( Figure 7D ) , and PDGFRβ staining revealed that pericytes were still present in normal numbers in the absence of Notch3 ( Figure 7D , E ) .", "We next used EdU labeling to mark cycling cells in wildtype and knockout hearts .", "Quantification of EdU+ mural cells ( PDGFRβ+ ) showed no difference between the two genotypes ( Figure 7F ) .", "Thus , epicardial to pericyte differentiation and arterial coverage is not severely affected , but caSMC maturation , specifically the induction of contractile proteins ( SM-MHC and SMα ) , is disrupted ( Figure 7G ) .", "These data show that coronary vessels upregulate Jagged-1 after attaching to the aorta to receive arterial blood flow , and that Notch3 is required for pericyte to caSMCs differentiation , possibly in response to Jagged-1 expression at the arterial remodeling zone . 10 . 7554/eLife . 10036 . 020Figure 7 . Notch3 is required for coronary artery smooth muscle development .", "( A ) SM-MHC+ coronary artery smooth muscle cell ( caSMCs ) are significantly reduced in Notch3-null hearts although coronary artery ( CA ) caliber ( dotted lines ) is comparable .", "( B ) Quantification of caSMC coverage in Notch3-deficient hearts where dots are individual samples and error bars are s . d . *p≤0 . 05 .", "( C ) SMα protein expression is reduced on arteries from Notch3-deficient hearts .", "( D ) PDGFRβ+ cells cover CAs in both wild type and knockout , and pericytes ( peri ) are not significantly reduced .", "( E ) Quantification of pericyte numbers in Notch3-deficient hearts .", "( F ) Quantification of mural cell proliferation in Notch3-deficient hearts at the capillary plexus and CA remodeling zone ( CA rz ) .", "( G ) Schematic demonstrating the hypothesized epicardial to smooth muscle differentiation pathway and how it is affected in the absence of Notch3 .", "Greyed cells are reduced .", "Scale bars: A , 100 µm; C , 50 µm; D , 25 µm . DOI: http://dx . doi . org/10 . 7554/eLife . 10036 . 020 We next investigated whether the pericyte to smooth muscle transition occurs in the kidney , another organ who’s internal arteries receive smooth muscle from the surface mesothelium ( Rinkevich et al . , 2012 ) .", "Characterization of smooth muscle development in the kidney using whole mount confocal microscopy showed that SM-MHC+ cells first appear at e14 on developing intralobular arteries ( Figure 8A ) .", "As in the heart , NG2 also labels pericytes in the developing kidney ( Lin et al . , 2008 ) .", "We therefore lineage traced either NG2- or Notch3-positive cells by inducing labeling of the Rosatdtomato Cre reporter before smooth muscle appears ( Figure 8B ) .", "Both approaches resulted in robust lineage labeling of arterial smooth muscle cells in the kidney at e15 . 5 as well as other perivascular cells including those within the glomerulus ( Figure 8C and data not shown ) .", "Although understanding the precise cellular pathway from mesothelium to smooth muscle requires further study , these data suggest that , similar to the heart , pericytes form an intermediate stage during this differentiation process in the kidney . 10 . 7554/eLife . 10036 . 021Figure 8 . NG2+ and Notch3+ cells differentiate into smooth muscle cells in the kidney .", "( A ) Whole mount confocal imaging of embryonic kidneys ( outlined with dotted lines ) from the indicated ages immunostained for SM-MHC and VE-cadherin .", "Mature smooth muscle differentiation is detected at e14 .", "( B ) Schematic describing lineage tracing experimental design .", "( C ) e11 . 5 dosing of NG2-CreER , Rosatdtomato animals induces labeling ( green ) in smooth muscle ( red , arrows ) ( n = 11 kidneys from 2 litters ) .", "Cells within the glomerulus are also labeled ( arrowheads ) .", "Scale bars: 100 µm . DOI: http://dx . doi . org/10 . 7554/eLife . 10036 . 021" ], [ "Due to the cells’ proximity and similar marker expression , vascular biologists have long wondered if pericytes and smooth muscle interconvert ( Armulik et al . , 2011; Cappellari et al . , 2013; Majesky , 2011 ) .", "Here , we show evidence , for the first time , that pericytes differentiate into smooth muscle cells during coronary artery development in the mouse heart and developing kidney .", "Previous identification of the epicardial-derived caSMC progenitor had been hampered by the fact that multiple developmental pathways occur downstream of epicardial differentiation .", "We have overcome this hurdle by using clonal analysis to label differentiation steps downstream of a single epicardial cell .", "This single cell tracing analyzes whether cells are clonally related eliminating one caveat of population level Cre labeling experiments where a mistake in the cell types expressing Cre can produce misleading results .", "Our clonal analysis of epicardial-derived cells identified cells with the molecular marker and morphological profile of vascular pericytes as being lineage related to caSMCs .", "The temporal presence and position of pericytes in epicardial-derived clones suggested that pericytes were the intermediate differentiation step between the epicardium and smooth muscle .", "This sequence of events was confirmed by direct pericyte lineage tracing using two independent pericyte Cre lines .", "In addition , pericytes were the most numerous epicardial-derived cell type at the CA remodeling zone where they wrapped developing arteries , a location that would allow them to respond to arterial maturation signals .", "Thus , we used complimentary lineage analysis experiments consisting of clonal and population-based tracing to show that pericytes ( or pericyte-like cells ) travel along blood vessels as they enter the myocardium and function as progenitors for caSMCs .", "Since pericytes wrap all small coronary vessels , we explored the signals that trigger their differentiation into caSMCs specifically at CA remodeling zones .", "We found that upon attachment to the aorta and establishment of blood flow , coronary endothelial cells downstream of the attachment site , which presumably receive the strongest blood flow , induce Jagged-1 .", "Jagged-1 was not robustly expressed in a model of delayed aortic attachment and delayed initiation of blood flow .", "The Jagged-1 ligand Notch3 was upregulated in pericytes at the arterial remodeling zone , and caSMCs failed to differentiate in Notch3-null hearts .", "A delay in smooth muscle differentiation in the absence of Notch3 and Jagged-1 has been observed in other organ systems ( Briot et al . , 2014; Doi et al . , 2006; High et al . , 2008; Hofmann et al . , 2012; Jin et al . , 2008; Manderfield et al . , 2012; Yang and Proweller , 2011 ) , however nothing is known about their roles in caSMC differentiation .", "Furthermore , due to the previous lack of knowledge of the lineage relationship between pericytes and smooth muscle cells , we are the first to show the importance of Notch3 signaling in stimulating the pericyte to smooth muscle transition .", "Jagged-1 and Notch signaling has been reported to be upregulated by sheer stress in vitro ( McCormick et al . , 2001; Theodoris et al . , 2015 ) , and we provide evidence that this occurs in vivo following coronary plexus attachment to the aorta .", "Together , our data support a working model ( Figure 9 ) where epicardial cells differentiate into Notch3low pericytes that cover the entire coronary plexus as it populates the myocardium .", "Then , the pericytes located on plexus vessels that receive blood flow-induced Jagged-1 upregulate Notch3 and differentiate into caSMCs .", "Thus , blood flow initiation is correlated with molecular changes that mark the site of arterial development in the coronary vascular plexus and may stimulate vessel remodeling and pericyte differentiation into smooth muscle . 10 . 7554/eLife . 10036 . 022Figure 9 . Model and summary .", "( A ) Different parts of the hypothesized epicardial to caSMC pathway were dissected using the indicated experiments .", "( B ) Working model for caSMC differentiation .", "CA , coronary artery; caSMC , coronary artery smooth muscle cell; Epi , epicardium; Peri , pericytes . DOI: http://dx . doi . org/10 . 7554/eLife . 10036 . 022 Our observations on the pericyte to smooth muscle transition are consistent with studies analyzing the timing of epicardial differentiation and its emergence into the myocardium .", "Studies dissecting the role of PDGFRs , TCF21 , and Myocardin-Related Transcription Factors have provided evidence that the decision to differentiate into either the cardiac fibroblast or caSMC lineage occurs in epicardial cells at the heart’s surface ( Acharya et al . , 2012; Braitsch et al . , 2012; Mellgren , et al . , 2008; Smith et al . , 2011; Trembley et al . , 2015 ) .", "In addition , progression down the caSMC lineage occurs early in development , mostly between e10 . 5 and 12 . 5 ( Wei et al . , 2015 ) .", "We show that endothelial cells migrating directly beneath the epicardium at this time point acquire pericytes and pericytes within caSMC containing clones are usually located near the heart’s surface as well as deeper layers .", "Thus , migrating coronary vessels could trigger adjacent epicardial cells to differentiate into pericytes at the surface and support their movement into the myocardium along with invading endothelial cells .", "Then , the subset of these pericytes that traveled to arterializing vessels receives signals to become caSMCs .", "Clones also generally segregated between those containing pericytes/caSMCs and those with other cell types not positive for our cell type specific markers , likely fibroblasts , the latter of which did not integrate into the smooth muscle layer even when directly adjacent to the artery .", "Together , these findings provide strong evidence that two different epicardial derived pathways diverge at the surface of the heart where the pericyte/caSMC lineage travels along vessels as they invade the myocardium to provide the different mural cells of the coronary vasculature .", "Analyzing pericyte-caSMC clones in adults showed that caSMC related pericytes persist after development is complete .", "Labeled cells in the adult heart were found in surprisingly tight clusters .", "This suggests that embryonic pericytes within caSMC clones were not merely an intermediate cell type that is depleted during development , but that cells not differentiating remain and function as cardiac pericytes throughout life .", "The tight clustering in adult hearts also indicate that pericytes travel very little along a vessel once they establish their location during development , at least in this organ .", "Thus , the embryonic heart appears to have developed an efficient method of distributing smooth muscle progenitors , cells that our data suggests maintain their cell-type specific function as pericytes at vessels that do not receive arterial blood flow and differentiation signals .", "Studies in the adult heart and other adult tissues report that perivascular cells are a heterogeneous population with mesenchymal stem cell properties that can contribute to tissue fibrosis ( Chen et al . , 2015; Ding et al . , 2012; Kramann et al . , 2014; Dulauroy et al . , 2012; Stallcup , 2013 ) .", "Because a large number of caSMCs arise from the epicardium , we characterized epicardial-derived cells at the coronary artery remodeling zone .", "These were found to be almost exclusively either PDGFRβ+Notch3+NG2+PDGFRα- ( pericytes ) or PDGFRβ-Notch3-PDGFRα+ ( fibroblasts ) suggesting less heterogeneity in the epicardial-derived cellular compartment at this age .", "This could be due to development being an early and protected stage in the animal’s life or that the cellular milieu is more complex in the adult when immunity is active .", "Additional complexity is likely induced in tissue injury models .", "Although lineage tracing shows that pericytes are a large source , our data does not exclude other pathways to caSMC development .", "In fact , developmental studies have show that an unidentified compensatory progenitor can provide caSMC if the epicardium is inhibited , although this source predisposes the arteries to disease ( Smith et al . , 2011; Wei et al . , 2015 ) .", "Regardless , the description of pericytes as progenitors and their presence in the adult identifies the possibility that they could be utilized to produce new caSMCs during CA regeneration .", "Identifying the cellular pathway between the epicardium and smooth muscle is therefore important , particularly since it is known that epicardial cells do not enter the adult heart , even after myocardial infarction ( Zhou et al . , 2011 ) .", "Our data showing that pericytes are coronary artery smooth muscle progenitors and that they persist in the adult present the possibility that pericytes could be targeted without relying on the original progenitors ( epicardial cells ) being transported to form new collateral arteries .", "Future experiments will investigate whether diverted arterial blood flow following coronary artery stenosis and/or blockage induces Notch signaling and differentiation of pericytes into caSMCs as part of the process that forms collateral vessels .", "If so , it will be important to ascertain whether collateral formation can be made more efficient by stimulating Notch activity .", "Our data suggest a similar pericyte to smooth muscle pathway in the kidney .", "However , it will be important to investigate whether smooth muscle in all mesothelial covered abdominal and thoracic organs and other tissues pass through a similar pericyte intermediate as it differentiates into organ-specific smooth muscle ( Rinkevich et al . , 2012 ) ." ], [ "All animal experiments were performed according to protocols approved by the Stanford University Institutional Animal Care and Use Committee ( IACUC ) .", "Mouse strains used: CD1 mice were used for wild type analysis and obtained from Charles River ( San Diego , California ) .", "NG2-CreER ( Jax strain: B6 . Cg-Tg ( Cspg4-cre/Esr1* ) BAkik/J ) .", "( Zhu et al . , 2011 ) , NG2-DsRed ( Jax strain: ( cspg4-DsRed . T1 ) 1Akik/J ) , PDGFRα ( Jax strain: B6 . 129S4-Pdgfratm11 ( EGFP ) Sor/J ) , α-MHC-Cre ( Jax strain: Tg ( Myh6-cre ) 1Jmk/J ) , MADM TG/TG ( Jax strain: Iis2tm2 ( ACTB-tdTomato , -EGFP ) Luo/J ) , MADM GT/GT ( Jax strain: Iis2tm1 ( ACTB-EGFP , -tdTomato ) Luo/J ) , Rosa-tdTomato ( Jax strain: B6 . Cg-Gt ( ROSA ) 26Sortm9 ( CAG-tdTomato ) Hze/J ) , PDGFRβ flox ( Jax strain: 129S4/SvJae-Pdgfrbtm11Sor/J ) , and Rosa-mTmG ( Jax strain: Gt ( ROSA ) 26Sortm4 ( ACTB-tdTomato , -EGFP ) Luo/J ) were all obtained from Jackson Laboratories .", "Conditional PDGFRβ flox animals were converted to full knockouts by crossing with HPRT-Cre females .", "Isl1 heterozygous ( Cai et al . , 2008a ) , Tbx18-Cre ( Cai et al . , 2008b ) , Notch3-CreER ( Fre et al . , 2011 ) and Notch3 ( Krebs et al . , 2003 ) have been previously described .", "Staged embryonic hearts were obtained by timed pregnancies ( morning plug designated e 0 . 5 ) and were dissected and fixed in 4% paraformaldehyde , washed and stored at 4°C in phosphate buffered saline ( PBS ) .", "Whole mount fluorescence microscopy was performed on intact hearts .", "Staining was performed in 1 . 5 ml tubes subjected to constant rotation .", "Primary antibodies were diluted in blocking solution ( 5% goat serum , 0 . 5% TritonX-100 in PBS ) and incubated with tissues overnight at 4°C .", "Tissues were then washed with PBT ( PBS with 0 . 5% TritonX-100 ) four times for one hour before another overnight incubation with secondary antibodies diluted in blocking solution .", "Specimens were then washed again , placed in Vectashield ( Vector Labs #H-1000 ) , and imaged using an inverted Zeiss LSM-700 confocal microscope .", "Images were digitally captured and processed using Zeiss Zen software ( 2011 ) .", "The following primary antibodies were used: VE-cadherin ( BD Biosciences , 550548 , 1:100 ) , PDGFRβ ( R&D Systems , #BAF1042 1:50; eBioscience 14-1402-81 , 1:100 ) , Notch3 ( Santa Cruz Biotechnology #M-134 1:100 ) , SM-MHC ( Biomedical Technologies , BT-562 , 1:300 ) , VEGFR2 ( R&D Systems #AF644 , 1:125 ) ; Jagged-1 ( R&D Systems AF599 , 1:125 ) ; SMα Quartzy #D00019 1:300 ) ; Calponin ( Sigma-Aldrich #C6047 , 1:250 ) Secondary reagents were Alexa Fluor conjugated antibodies ( 405 , 488 , 555 , 637 ) from Life technologies used at 1:250or streptavidin conjugates ( Life technologies #S21374 ) used at 1:500 .", "In Figure 1H , distance of SM-MHClow cells from the epicardium was measured from Z-plane views of whole mount confocal images using Zeiss Zen software .", "Fluorescence intensity values were calculated from single Z-planes using the same software package .", "For Figure 1G , individual cells in single Z-planes were encircled and intensity values in the SM-MHC channel were recorded for mural cells surrounding the capillary plexus ( n = 16 ) , remodeling zone ( n = 16 ) , and mature arteries ( n = 16 ) from 4 different hearts each .", "For Figure 6B , values in the PDGFRβ n = 9 cells/region from 3 hearts ) , Notch3n = 21 cells/region from 7 hearts ) , and SM-MHC ( n = 15 cells/region from 3 hearts ) channels were recorded .", "For Figure 2—figure supplement 2B , localization of PDGFRα+ and PDGFRβ+ cells was measured using the profile option where fluorescent intensities are graphed along a line drawn across the XY-plane of a confocal image .", "Quantification of PDGFRβ , NG2 , and Notch3 overlap ( mentioned in the text and shown in Figure 2F , G ) was performed by randomly designating a field of view , encircling all the cells positive for either PDGFRβ or NG2 , and counting the number of those circled also positive for the additional markers .", "Number of cells analyzed is stated in Figure 2H counted from 3–4 hearts per marker , each from multiple litters .", "Mice were bred so that embryos receive each of three alleles: Tbx18-Cre , a MADM GT cassette , and a TG cassette ( Zong et al . , 2005 ) .", "Embryonic hearts were isolated at e13 . 5 , e15 . 5 and 3–5 weeks of age and immunostained with antibodies for VE-cadherin , SM-MHC and PDGFRβ , and imaged as described above .", "For adults , 50 µm cryosectioning was performed and the sections were stained with the staining protocol as described above .", "Clusters of labeled cells were considered clonal if they were clearly distinct and at least 100 µm away from other labeled cells .", "For e13 . 5 , e15 . 5 and adult clones a total of 12 , 13 and 7 caSMC clones were quantified , respectively .", "Cell identities were assigned by the immunostaining criteria described in the main text and assessed by two individual researchers in the laboratory .", "Depth below the epicardium was analyzed using measurement tools included in the Zeiss Zen software ( Figure 3—figure supplement 1A ) .", "Quantification of pericytes , caSMCs and other cell types included all e15 . 5 clonal cells from 14 clones where no cells were excluded .", "In total , n values were 91 for epicardial cells , 249 for pericytes , 37 for caSMCs , 150 for other cell types .", "To assess the expected clonality rate for the observed fluorescently labeled cell clusters , we mathematically modeled the process of clone formation in three dimensions based on parameters estimated from the imaging data of the e13 . 5 developing heart .", "The surface area of the heart was approximated as that of a sphere with a radius of 315 µm , yielding a value of 1 , 246 , 898 square µm ( A = 4πr2 ) .", "We then modeled regions representing each half of the heart as square tiles with an equivalent surface area ( height x width = 623 , 449 µm2 ) and a depth of 97 . 5 µm , corresponding to the estimated distance from the epicardium to the endocardium .", "Groups of clonally related labeled cells were modeled as spheres with a radius of 54 µm , based on the average dimensions of the e13 . 5 clones .", "We then defined a discrimination cutoff of 50 µm ( based on a conservative analysis of the maximum intra-clone distance between any labeled cell and its nearest neighbor within the same clone ) as the minimum distance required between any cells from two different groups of clones such that those clones were considered to be distinct entities .", "Using these empirically determined parameters , we then performed simulations to estimate the underlying clone generation rate ( and its corresponding clonality rate ) that best fit the observed data as follows .", "First , we tested a range of suitable values ( from 0 . 5 to 10 . 0 ) for the clone generation rate ( m ) , which specifies the average number of clones that are stochastically generated in each simulated half heart .", "For any given value of the generation rate , we randomly simulated 10 , 000 half hearts , with the number of clones ( X ) assigned to each heart randomly determined according to the Poisson distribution ( Prob ( X = k ) = e-m mk / k !", ") .", "The centroids of each clone were then randomly assigned with uniform probability to locations within the volume of the simulated region , and any clones that were located closer than the allowed discrimination distance of each other were merged together to form a single ‘cluster’ .", "Then , the mean number of observed ‘clusters’ ( clusters may either consist of a single clone or a set of neighboring merged clones ) was calculated as a function of the clone generation rate .", "The underlying clone generation rate that best matched the experimental data with an average of 3 . 3 clusters per region was then chosen as the estimated true generation rate , and its associated clonality rate , defined as the fraction of observed clusters that are comprised of individual clones , was computed from the results of 10 , 000 simulations .", "The calculated clonality rates represent the likelihood that any individual cluster of labeled cells is indeed clonal , based on the results of this simulation analysis .", "Finally , we repeated the entire modeling process with different settings for the discrimination distance , in order to assess the robustness of the estimated clonality rate to different values for this parameter ( Figure 3—figure supplement 1 ) .", "Adult hearts ( n = 3 ) were dissected and fixed in 4% paraformaldehyde ( PFA ) for one hour at 4oC , washed in PBS , and cryoprotected in 30% sucrose for 30 min .", "Hearts were then oriented apex-down within a mold of Tissue-Tek® O . C . T . ™ compound before snap freezing and cryosectioning ( 20 µm ) .", "Sections were immunostained for SM-MHC and PDGFRβ as previously described , placed in Vectashield® with DAPI ( Vector Labs , #H-1200 ) , and frozen at -20oC overnight .", "A section from each heart that contained left ventricle , right ventricle , and septum tissue was chosen and 3 different fields of view ( FOV ) for each region of each heart were collected through 40X oil immersion microscopy .", "The total number of DAPI-labeled , PDGFRβ+ cells signified pericytes in each FOV , while the total number of DAPI-labeled , SM-MHC+ cells denoted smooth muscle cells .", "Both NG2-CreER and Notch3-CreER males were crossed to Rosa-tdTomato reporter mice .", "Cre activity was activated by tamoxifen that was dissolved in corn oil and delivered to pregnant dames by intraperitoneal injection at either e10 . 5or 11 . 5 ( 4 mg ) with identical results .", "Dames impregnated by NG2-CreER and Notch3-CreER males were sacrificed at e15 . 5 .", "Embryonic hearts and kidneys were stained with SM-MHC , PDGFRβ and VE-cadherin as described above before each cell type was assessed for lineage labeling .", "NG2-CreER traced smooth muscle was observed in 10 hearts/kidneys from three different litters .", "Notch3-CreER traced coronary smooth muscle was observed in 11 hearts and kidneys from three litters .", "The percentage of lineage labeled pericytes and caSMCs as shown in Figure 4D was quantified using Zeiss Zen imaging software to count individually labeled pericytes and ImageJ to measure the percentage of the linage label overlapping with SM-MHC immunoreactivity .", "A total of 12 mutant hearts from 5 litters were analyzed all of which showed the same phenotype .", "For Figure 5C , the number of Notch3+ cells was recorded from a total of 7 wild type and 5 mutant hearts from 4 embryo litters from multiple 15 , 000 µm2 fields of view: remodeling zone ( Wt: n = 19 fields; -/-: n = 11 fields ) and mature arteries ( Wt: n = 11 fields; -/-: n = 11 fields ) .", "Embryos were collected from Notch3 heterozygous crosses , and the hearts immunostained for VEGFR2 , SM-MHC , and PDGFRβ as described above .", "A total of 20 mutants hearts from 10 litters from time points e15 . 5 and 17 . 5 were analyzed , all of which showed the same phenotype .", "For quantification in Figure 6F , 9 mutant and 10 wild type hearts from 6 litters were analyzed ( heterozygous hearts were not included in this analysis ) .", "Confocal z-stacks ( with 14 µm intervals between z-planes ) through the right lateral side of each heart were collected and imported into ImageJ ( NIH ) .", "The 'segmented line' tool was used to measure the total length of the right coronary artery ( and its auxiliary branches ) covered either partially or fully by SM-MHC+ cells .", "Discontinuous lengths of smooth muscle coverage were summed for each heart .", "Similar results were obtained when assessing SMα expression ( n = 3 mutant hearts from three different litters ) .", "To assess cell proliferation , mitotically active cells were measured through the incorporation of 5-ethynyl-2’-deoxyuridine ( EdU ) .", "Pregnant females from Notch3 heterozygous crosses received a single intraperitoneal injection of 400 ug of Edu dissolved in 200 u L of dimethyl sulfoxide ( DMSO ) .", "Three hours later , embryonic hearts were dissected , fixed , and immunostained for VEGFR2 and PDGFRβ as described above .", "EdU incorporation into DNA was detected through the Click-iT® EdU Alexa Fluor® 555 Imaging Kit performed at room temperature and using the protocol recommended by the manufacturer ( Invitrogen , #C10338 ) .", "A total of 4 wild type , 11 heterozygous , and 10 mutant hearts from 5 litters at time point e14 . 5 were analyzed .", "Confocal z-stacks at 20X magnification ( with 3 um intervals between z-planes ) through the right lateral side of each heart were collected and imported into Zeiss Zen imaging software .", "A 150 um x 150 um square was drawn at both the vascular plexus and at a coronary artery remodeling zone .", "The percentage of EdU-positive pericytes was determined by quantifying the number of cells in each region that were PDGFRβ+ and EdU+ compared to only PDGFRβ+ .", "Embryos were collected at e13 . 5 from crosses between Isl1 heterozygous and wild type mice .", "The hearts were immunostained for Jagged-1 , SM-MHC , and VE-Cadherin as described above .", "A total of 27 heterozygous and 26 wild type hearts from 5 litters were analyzed .", "Confocal z-stacks ( with 14 µm intervals between z-planes ) through the right lateral side of the each heart were collected and analyzed .", "For each heart , the relative fluorescence intensity of Jagged-1 was recorded as a range from absent or very low to very high expression .", "Statistical analyses were performed using SigmaPlot version 12 . 0 ( Systat Software Inc ) or Prism ( Graphpad ) , where appropriate normality and variation were calculated .", "Data are represented as mean ± standard deviation ( sd ) .", "Mann-Whitney Rank Sum tests were performed as appropriate for two-group comparisons , and one-way ANOVA was performed for multiple-group ( more than 2 groups ) comparisons and post hoc analysis was used with a Holm-Sidak post hoc test .", "A p <0 . 05 was considered statistically significant .", "Samples sizes were chosen so that statistically significant values would be obtained ." ] ]

[ "Epicardial cells on the heart’s surface give rise to coronary artery smooth muscle cells ( caSMCs ) located deep in the myocardium .", "However , the differentiation steps between epicardial cells and caSMCs are unknown as are the final maturation signals at coronary arteries .", "Here , we use clonal analysis and lineage tracing to show that caSMCs derive from pericytes , mural cells associated with microvessels , and that these cells are present in adults .", "During development following the onset of blood flow , pericytes at arterial remodeling sites upregulate Notch3 while endothelial cells express Jagged-1 .", "Deletion of Notch3 disrupts caSMC differentiation .", "Our data support a model wherein epicardial-derived pericytes populate the entire coronary microvasculature , but differentiate into caSMCs at arterial remodeling zones in response to Notch signaling .", "Our data are the first demonstration that pericytes are progenitors for smooth muscle , and their presence in adult hearts reveals a new potential cell type for targeting during cardiovascular disease ." ]

[ "The heart is a complex organ composed of several different cell types .", "Muscle cells of walls of the heart contract to pump blood around the body .", "These muscle cells are themselves supplied with blood from the coronary arteries that penetrate deep into this muscle tissue .", "The lining of the coronary arteries is made of endothelial cells , while smooth muscle cells ( or SMCs for short ) surround the arteries and provide support .", "The SMCs can also contract to increase or decrease blood flow to the heart , depending on the heart rate .", "Endothelial cells and SMCs of the coronary arteries physically interact but develop from different precursor cells .", "The coronary artery SMCs are derived from cells that comprise the outer layer of the heart ( called the epicardium ) and move inwards during embryonic development .", "However , it was not clear exactly what kind of cells these precursor cells are , or which molecular signals trigger their conversion into SMCs .", "Volz et al . have studied cardiac development in mice and used fluorescent labels to observed individual cells of the epicardium as they divided and moved .", "This revealed that when epicardial cells developed into the coronary artery SMCs , there was always an intermediate cell type that wrapped around the developing blood vessels .", "Upon further investigation , Volz et al . found that these cells were so-called pericytes , which otherwise support small blood vessels throughout the body .", "Furthermore , the pericytes that did not develop into SMCs remained near the coronary arteries and were still present in adult hearts .", "Lastly , experiments showed that a protein called Notch-3 is expressed on pericytes and interacts with another protein called Jagged-1 on endothelial cells to prompt the conversion of pericytes into SMCs .", "Since heart development is similar in mice and humans , these findings may have implications for future therapies of coronary artery disease , the most common cause of death worldwide .", "Currently there are no methods to trigger the formation of new coronary arteries after injury or blockage , but knowledge of the pericyte precursors and the signaling pathways that turn them into SMCs could eventually lead to new treatments ." ]

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

[ "immunology and inflammation" ]

[ [ "Our ability to mount adaptive immune responses against skin-invading pathogens depends on the delivery of antigens to lymph nodes ( LNs ) for encounter by naive lymphocytes ( Cyster , 2010; Qi et al . , 2014 ) .", "However , activation , clonal expansion and effector lymphocyte differentiation takes several days , whereas pathogens can undergo marked replication in a matter of hours .", "The skin contains immune effector cells that help keep pathogen replication in check , in a process referred to as ‘barrier immunity’ ( Belkaid and Segre , 2014 ) .", "Despite this , in many cases , intact pathogens travel within minutes via lymph fluid to draining LNs .", "Indeed some pathogens , such as Yersinia pestis , appear to have evolved to undergo marked expansion only after arrival in the LN ( St John et al . , 2014 ) .", "Recently , there has been evidence indicating the existence of barrier immunity within LNs .", "The first LN cells exposed to lymph-borne antigens include the CD169+ macrophages that extend between the subcapsular sinus ( SCS ) or medullary sinuses and the underlying parenchyma ( Barral et al . , 2010; Iannacone et al . , 2010; Phan et al . , 2009 ) .", "Crosstalk between sinus-associated macrophages and IFNγ precommitted CD8 T cells and NK cells is important for mounting rapid Th1-like and NK cell responses against acute infection by Pseudomonas aeroguinosa , Salmonella typhimurium and Toxoplasma gondii ( Coombes et al . , 2012; Kastenmüller et al . , 2012 ) .", "IL17 is a cytokine with roles in anti-bacterial and anti-fungal defense that is made abundantly by effector T cells at epithelial surfaces ( Littman and Rudensky , 2010 ) .", "Whether IL17 is produced rapidly during responses to subcapsular sinus-invaders in LNs is unclear .", "In recent work , our group and others identified populations of innate-like ( pre-formed effector ) lymphocytes that are enriched near the SCS in peripheral LNs and are pre-committed to produce IL17 ( Do et al . , 2010; Doisne et al . , 2009; Gray et al . , 2012; Roark et al . , 2013 ) .", "These cells express high amounts of the chemokine receptors Ccr6 and Cxcr6 as well as the cytokine receptor IL7R , and they include a majority of αβ T cells but also considerable numbers of γδ T cells as well as non-T cells ( Gray et al . , 2012 ) .", "Within the IL17-committed γδ T cell population a major subset expresses a Vγ4-containing TCR ( according to the nomenclature of [Heilig and Tonegawa , 1986] ) , and undergoes expansion in response to challenge with imiquimod or complete Freund’s adjuvant ( Gray et al . , 2013; Ramirez-Valle et al . , 2015; Roark et al . , 2013 ) .", "In previous work , we found that innate-like lymphocytes isolated from peripheral LNs were heavily coated with CD169+ macrophage-derived membrane fragments ( ‘blebs’ ) ( Gray et al . , 2012 ) .", "This observation suggested there may be strong adhesive interactions between these cells and the CD169+ macrophages .", "CD169 is the founding member of the Siglec family of sialic acid-binding lectins ( Crocker et al . , 2007; Macauley et al . , 2014 ) .", "Although CD169 is a defining feature of LN SCS macrophages and targeting antigens to CD169 can promote antibody responses ( Macauley et al . , 2014 ) , the function of CD169 on these cells is not fully understood .", "Pre-enrichment of innate-like lymphocytes near LN sinuses is thought to be important for allowing very rapid responses against lymph-borne invaders ( Gray et al . , 2012; Kastenmüller et al . , 2012 ) .", "Despite this , it is not known whether IL17-committed innate-like lymphocytes in LNs respond rapidly upon pathogen challenge , and little is understood about how these cells localize to or move in the subcapsular region .", "In this study , we found IL7RαhiCcr6+ innate-like lymphocytes were mostly LN resident and they produced IL17 within hours of bacterial or fungal challenge .", "Their proximity to the SCS was mediated by Ccr6 and was important for the rapid induction of IL17 following bacterial challenge .", "Real time intravital two photon microscopy and in vivo labeling procedures revealed that innate-like lymphocytes exchanged between the LN parenchyma and the SCS .", "Movement into the SCS was S1pr1 dependent , whereas return to the parenchyma involved LFA1 and ICAM1 .", "Within the SCS , CD169-mediated adhesive interactions that helped retain the cells , presumably against the shear stresses exerted by lymph flow .", "This requirement was most prominent for the Vγ4+γδ T cell subset of innate-like lymphocytes .", "These observations provide a model for understanding the mechanism by which innate-like lymphocytes survey the pathogen-exposed surface of the LN to protect the organ from infection ." ], [ "IL7RαhiCcr6+ innate-like lymphocytes within peripheral LNs express high amounts of Cxcr6 and they include ~70% αβ T cells , ~20% γδ T cells and 5–10% non-T cells ( Figure 1A , B ) ( Gray et al . , 2012 ) .", "Consistent with previous findings , the IL7RαhiCcr6+gd T cell subset produced IL17 rapidly upon treatment with phorbol 12-myristate 13-acetate ( PMA ) and ionomycin or with the cytokines IL1β and IL23 ( Figure 1C , lower graph ) ( Cai et al . , 2014; Gray et al . , 2012; Gray et al . , 2011 ) .", "These treatments also triggered rapid IL17 production from the IL7RαhiCcr6+αβ T cells ( Figure 1C ) .", "We therefore tested whether both the αβ and γδ subsets of IL7RαhiCcr6+ T cells produce IL17 in skin draining LNs following bacterial or fungal challenge .", "IL17 is known to play a role in host defense against cutaneous Candida albicans and Staphyloccus aureus infection ( Cho et al . , 2010; Conti and Gaffen , 2015 ) and to be induced in rats by Y . pestis ( Comer et al . , 2010 ) .", "Three hours after heat-killed C . albicans , S . aureus bioparticle , or attenuated Y . pestis footpad challenge , IL17 production in draining popliteal LNs was observed ( Figure 1D ) .", "IL7RαhiCcr6+ lymphocytes were the dominant IL17 producers at this early time point after challenge ( Figure 1D ) .", "The induction of IL17 expression in IL7RαhiCcr6+ cells upon bacterial challenge was dependent on CD169+ SCS macrophages as the response was greatly blunted in CD169-DTR mice ( Miyake et al . , 2007 ) pretreated with DT to ablate these cells ( Figure 1E ) .", "Since IL1β and IL23 induced IL17 production from IL7RαhiCcr6+ T cells in vitro , we looked for expression of these cytokines after S . aureus bioparticle challenge .", "Transcripts for both Il1b and Il23a were upregulated ( Figure 1F ) .", "Induction of IL17 following S . aureus bioparticle challenge was compromised in mice lacking the ASC ( Apoptosis-associated speck-like protein containing a CARD ) inflammasome subunit ( Figure 1G ) , consistent with a role for IL1β in activating IL7RαhiCcr6+ cells during the response to these bacteria . 10 . 7554/eLife . 18156 . 003Figure 1 . Rapid induction of IL17 expression by IL7RαhiCcr6+ innate-like lymphocytes in a CD169+ macrophage-dependent manner following bacterial and fungal challenge .", "( A ) Representative FACS plot showing IL7RαhiCcr6+ staining of peripheral LN cells from a Cxcr6GFP/+ mouse , and Cxcr6-GFP intensity on the gated cells .", "( B ) Representative FACS plots showing CD3ε , TCRβ and TCRγδ staining of the IL7RαhiCcr6+ population .", "Bar graph shows summary frequency data ( mean ± sd ) for more than 10 mice .", "( C ) Intracellular FACS showing IL17 production among LN cells after 3 hr in vitro stimulation with IL1β and IL23 or phorbol 12-myristate 13-acetate and ionomycin .", "Graph shows summary data from 3 experiments .", "( D ) Representative FACS plots showing IL17 production among popliteal LN cells 3 hr after footpad challenge with heat inactivated C . albicans , S . aureus coated bioparticles , and attenuated Y . pestis .", "Summary graph shows% IL17+ cells among IL7RαhiCcr6+ cells .", "( E ) IL17 production by IL7RαhiCcr6+ cells in control and CD169-DTR macrophage ablated mice treated with S . aureus bioparticles as in D , Summary graph shows% IL17+ cells among IL7RαhiCcr6+ cells .", "( F ) Il1b and Il23a mRNA level in popliteal LNs of S . aureus bioparticle challenged mice relative to controls , determined by qRT-PCR .", "( G ) Summary graph to show% IL17+ cells among IL7RαhiCcr6+ cells between control and ASC-deficient mice after 3 hr S . aureus bioparticle challenge .", "***p<0 . 001 by student’s t test .", "Data are representative of at least two experiments for panels A–C .", "Data are representative of two or more experiments with at least two mice per group for panels D–G .", "LN , Lymph node .", "DOI: http://dx . doi . org/10 . 7554/eLife . 18156 . 003 IL7RαhiCcr6+ lymphocytes make up ~0 . 5% of peripheral LN cells yet they represent only ~0 . 1% of cells in blood ( Figure 2A ) , suggesting that the cells are largely non-recirculatory under homeostatic conditions .", "To test this more directly , we ‘time stamped’ cells in inguinal LNs of KikGR photoconvertible protein-expressing transgenic mice by brief violet light exposure ( Gray et al . , 2013 ) .", "At 24 hr after photoconversion almost three quarters of the IL7RαhiCcr6+ cells and a similar fraction of the Vγ4+Ccr6+ cells remained resident in the LN , whereas more than 80% of the conventional αβ T cells and Ccr6– Vγ4+ T cells had left the LN and been replaced by newly arriving cells .", "At 48 hr , the innate-like lymphocyte pool showed little further exchange , whereas naive αβ T cells were 90% replaced ( Figure 2B ) .", "In a further approach , we examined the amount of cell exchange that occurred in parabiotic mice .", "Two weeks following surgery the naive αβ T cell compartment and the Ccr6– Vγ4+ T cells had achieved full chimerism , whereas the innate-like lymphocytes showed only limited exchange between the paired mice ( Figure 2C ) .", "Taken together , these findings indicate that most IL7RαhiCcr6+ cells are resident in the LN for multiple days and are not extensively recirculating under homeostatic conditions . 10 . 7554/eLife . 18156 . 004Figure 2 . IL7RαhiCcr6+ innate-like lymphocytes are mostly LN resident .", "( A ) Representative FACS plots showing frequency of IL7RαhiCcr6+ and Vγ4+Ccr6+ cells in LNs and blood .", "Graphs show summary data for more than 30 mice of each type .", "( B ) FACS analysis of LN IL7RαhiCcr6+ cells and naïve αβ T cells in KikGR mice before , immediately after and 24 and 48 hr after photoconversion .", "Summary data are pooled from three experiments and each point indicates an individual mouse .", "( C ) FACS analysis of LN IL7RαhiCcr6+ cells and naive αβ T cells in GFP-host and GFP+ host from parabiotic pairs .", "Summary data are pooled from two experiments and each point indicates an individual mouse .", "**p<0 . 01 , ***p<0 . 001 , by student’s t test .", "Data are representative for at least two experiments .", "LN , Lymph node . DOI: http://dx . doi . org/10 . 7554/eLife . 18156 . 004 Cxcr6-GFP is highly expressed by all IL7RαhiCcr6+ cells ( Figure 1A ) , and in previous work , we observed that Cxcr6GFP/+ cells migrate extensively in outer and inter-follicular regions , often in close association with CD169+ SCS macrophages ( Gray et al . , 2012 ) .", "A closer examination of Cxcr6GFP/+ cell behavior in this region revealed that the cells frequently made contact with CD169+ macrophages , and occasionally , lymphocytes could be observed crossing the layer of macrophages to reach the SCS ( Figure 3A and Videos 1 and 2 ) .", "Reciprocally , cells that were initially detected within the SCS could be observed migrating across the thick CD169+ macrophage layer into the LN parenchyma ( Figure 3A and Videos 1 and 2 ) .", "To quantify the crossing events , cell tracks were generated automatically ( Figure 3—figure supplement 1 ) and tracks crossing the SCS floor were manually enumerated .", "Among all the tracks of Cxcr6GFP/+ cells within 50 µm of the capsule , ~25% were in the SCS ( Figure 3B ) .", "Cell tracking analysis showed that about 3% of the cells in the SCS region traveled from the parenchyma into the sinus , and 3% of the cells traveled in the reverse direction , in the 30 min imaging periods ( Figure 3C ) . 10 . 7554/eLife . 18156 . 005Figure 3 . Migration dynamics , sinus exposure and CD169+ macrophage interaction of LN innate-like lymphocytes .", "( A ) Time series of Cxcr6GFP/+ cell movement with respect to CD169+ SCS macrophages .", "Upper panels: white arrow indicates a Cxcr6GFP/+ lymphocyte in the LN parenchyma that crosses into the SCS .", "300 s time series was taken from a 46 μm z stack .", "Lower panel: white arrow indicates a Cxcr6GFP/+ lymphocyte that begins in the SCS and crosses into the LN parenchyma .", "360 s time series was taken from a 34 μm z stack .", "Green , Cxcr6-GFP+ lymphocytes; Red , CD169+ macrophages; Blue , second harmonic .", "White dashed line indicates boundary between SCS and LN parenchyma .", "( B , C )", "Percent tracks in SCS compartment among the total tracks enumerated ( B ) and frequency of tracks crossing from the parenchyma into the SCS or out of the SCS into the parenchyma ( C ) in Cxcr6GFP/+ control mice .", "Each point represents data from a single movie ( two independent experiments ) .", "( D ) In vivo 5 min Thy1-PE labeling of IL7RαhiCcr6+ cells , Vγ4+Ccr6+ cells and αβ T cells , analyzed by flow cytometry .", "Data are representative of at least 10 mice .", "( E ) In vivo Thy1-PE labeling of cells analyzed in tissue sections .", "Costaining was with CD3-A488 ( green ) and Lyve1-A647 ( blue ) .", "White arrows point out Thy1 and CD3 costained cells .", "( F ) Frequency of IL7RαhiCcr6+ , Vγ4+Ccr6+ and naive αβ T cells positive for CD169 .", "Data are representative of 10 mice .", "( G ) In vivo Thy1-PE labeling and CD169 staining on IL7RαhiCcr6+ , Vγ4+Ccr6+ and naive αβ T cells , analyzed by flow cytometry .", "Data are representative of at least two experiments in each panel . DOI: http://dx . doi . org/10 . 7554/eLife . 18156 . 00510 . 7554/eLife . 18156 . 006Figure 3—figure supplement 1 . Example of automatically generated tracks for Cxcr6-GFP+ cells in a Cxcr6GFP/+ mouse LN . Green , Cxcr6-GFP+ lymphocytes; Red , CD11b+ macrophages .", "5 min~30 min tracks for Cxcr6-GFP+ lymphocytes are shown in colored lines .", "LN , Lymph node . DOI: http://dx . doi . org/10 . 7554/eLife . 18156 . 00610 . 7554/eLife . 18156 . 007Video 1 . Cxcr6-GFP+ cell shuttling between parenchyma and the SCS . Representative intravital time-lapse imaging of the popliteal LNs from two Cxcr6GFP/+ mice .", "Overhead 3D video exemplifies the dynamic movement of Cxcr6-GFP+ cells ( green ) within the LN .", "Two-dimensional video of a 20 µm maximal intensity projection from an orthogonal plane demonstrates the anatomy of the SCS region .", "An afferent lymphatic vessel ( rarely visualized ) drains into the SCS , bounded by the collagenous LN capsule ( blue , second harmonic signal ) and CD11b+ SCS macrophages ( red ) .", "The second example further reveals the motility of Cxcr6-GFP+ cells both within the SCS and the LN parenchyma .", "Cxcr6-GFP+ cells are observed to cross from within the LN parenchyma into the SCS , as well as from within the SCS into the LN parenchyma ( examples highlighted by circles ) .", "LN , Lymph node; SCS , Subcapsular sinus . DOI: http://dx . doi . org/10 . 7554/eLife . 18156 . 00710 . 7554/eLife . 18156 . 008Video 2 . Representative examples of individual Cxcr6-GFP+ cells crossing into and out of SCS . Intravital time-lapse imaging of the popliteal LN from a Cxcr6GFP/+ control mouse , highlighting one cell crossing from the parenchyma into the SCS , and one crossing from the SCS into the parenchyma .", "Cells such as these that clearly crossed from one region were manually identified from automated tracking of all Cxcr6-GFP+ cells in an experiment .", "SCS , Subcapsular sinus . DOI: http://dx . doi . org/10 . 7554/eLife . 18156 . 008 To quantitate the proportion of cells in the LN lymphatic sinuses at a given moment in time , we optimized an in vivo procedure to label lymph-exposed cells based on the established method of antibody pulse-labeling of blood-exposed cells ( Cinamon et al . , 2008 ) .", "We targeted Thy1 since this marker is expressed by all the IL7RαhiCcr6+ cells while not being present on SCS macrophages ( not shown ) .", "PE-conjugated antibody was used as the large size of PE ( 250 kD ) reduces the rate at which the antibody accesses the lymphoid tissue parenchyma ( Pereira et al . , 2009 ) .", "Thy1-PE antibody ( 0 . 2 μg ) was injected into the footpad , the draining popliteal LN was isolated 5 min later and the frequency of labeled cells determined by flow cytometry .", "Approximately 10% of the total IL7RαhiCcr6+ cells were brightly labeled with Thy1-PE antibody compared with about 0 . 3% of naive T cells ( Figure 3D ) .", "Among the innate-like lymphocytes , γδ T cells ( predominantly Vγ4+Ccr6+ cells ) were preferentially labeled , with around 15–20% of these cells being antibody exposed .", "By immunofluorescence microscopy , Thy1-PE-labeled CD3ε+ cells were observed in the SCS and in nearby lymphatic sinuses , and few labeled cells were detected within the LN parenchyma , confirming that footpad injection of PE-conjugated antibody led to preferential labeling of lymph-exposed LN cells ( Figure 3E ) .", "The broader labeling of the sinus by Thy-1 than by CD3 reflects the expression of Thy1 by lymphatic endothelial cells ( Jurisic et al . , 2010 ) .", "We also obtained information about lymphocyte-SCS macrophage proximity by following up on our finding that isolated innate-like lymphocytes are heavily coated with CD169+ macrophage-derived membrane fragments ( ‘blebs’ ) ( Gray et al . , 2012 ) .", "This coating is thought to occur at the time of LN cell dissociation , possibly because the SCS macrophages are tightly bound to the extracellular matrix and become fragmented during mechanical preparation of the tissue .", "Importantly , the blebs only become bound to cells that are associated with the macrophages at the time of isolation since co-preparation of LN cells from congenically distinct animals did not lead to cross acquisition of macrophage-derived blebs by innate-like lymphocytes from the different LNs ( Gray et al . , 2012 ) .", "In accord with previous findings , 30–40% of the IL7RαhiCcr6+ innate-like lymphocytes isolated from control mice were CD169 macrophage-derived membrane bleb positive ( Figure 3F ) .", "A higher frequency ( 40–55% ) of the Vγ4+γδ T cells were CD169 bleb positive , suggesting these cells may be preferentially associated with SCS macrophages ( Figure 3F ) .", "Combining this analysis with Thy1-PE labeling showed that lymph-exposed innate-like lymphocytes , but not total αβ T cells , were enriched for CD169-bleb-positive cells ( Figure 3G ) .", "The Thy1-PE+ CD169– cells amongst total αβ T cells most likely correspond to recirculating cells that are exiting the LN via cortical and medullary sinuses .", "Given the high Ccr6 expression on the innate-like lymphocyte population , we asked whether this CCL20 receptor had a role in guiding innate-like lymphocytes to the subcapsular region .", "Although CCL20 is not abundantly expressed in LNs , it is expressed in LN lymphatic endothelial cells ( LECs ) at levels more than 100-fold higher than other LN lymphoid stromal cells ( Figure 4A ) .", "Immunofluorescence microscopy showed evidence of CCL20 protein in the subcapsular sinus region ( overlying follicular and interfollicular regions ) but not in the medullary sinus region ( Figure 4B , Figure 4—figure supplement 1 ) consistent with findings in primate LNs ( Choi et al . , 2003; Pegu et al . , 2007 ) .", "Innate-like lymphocytes were responsive to CCL20 by in vitro migration assays ( Figure 4C ) .", "When CCL20 was injected subcutaneously into Cxcr6GFP/+ mice , IL7RαhiCcr6+Cxcr6hi lymphocytes became clustered near and within the SCS in the draining LNs ( Figure 4D , Figure 4—figure supplement 2 ) .", "Cells from these LNs showed reduced surface Ccr6 , and increased CD169 staining and Thy1 labeling , consistent with their having been exposed to increased amounts of CCL20 and localizing near and within the SCS ( Figure 4E ) . 10 . 7554/eLife . 18156 . 009Figure 4 . Ccr6 promotes innate-like lymphocyte positioning near the SCS .", "( A ) Ccl20 mRNA abundance in sorted LN lymphatic endothelial cells ( LEC ) , blood endothelial cells ( BEC ) , fibroblastic reticular cells ( FRC ) and double negative stromal cells ( DN ) determined by qRT-PCR , shown relative to Hprt .", "( B ) CCL20 staining of LN section ( red ) .", "The control ( no primary ) section was stained with the secondary anti-goat-Cy3 antibody alone .", "B cells were detected in blue ( B220 ) .", "( C ) Transwell migration of IL7RαhiCcr6+ cells to CCL20 .", "( D ) Distribution of Cxcr6GFP/+ cells in LNs 3 hr after saline or CCL20 s . c . injection .", "Sections were stained to detect Cxcr6-GFP ( green ) and Lyve1 ( blue ) .", "( E ) Representative FACS plots show Ccr6 surface level , in vivo Thy1-PE labeling and CD169 macrophage bleb level on IL7RαhiCcr6+ cells from control ( con ) or CCL20 injected mice .", "Summary graph shows comparison of Thy1-PE labeling and CD169+ staining frequency of IL7RαhiCcr6+ and Vγ4+Ccr6+ cells from control or CCL20 injected mice .", "( F ) LN sections from Ccr6GFP/+ or Ccr6GFP/GFP mice stained for EGFP ( green ) and B220 ( blue ) .", "White arrow indicates subcapsular sinus area; FO: B cell follicle; T: T zone .", "( G ) Comparison of Thy1-PE labeling on IL7RαhiCcr6+ cells from WT and Ccr6GFP/+ or Ccr6GFP/GFP mice .", "Ccr6 in Het and KO mice was detected based on GFP reporter expression .", "( H ) Comparison of CD169 staining on IL7RαhiCcr6+ cells from WT , Ccr6GFP/+ or Ccr6GFP/GFP mice .", "( I ) Representative FACS plots showing Vγ4+Scart2+ cells amongst γδT cells and the fraction that are Ccr6+ ( upper ) , and in vivo Scart2-BV605 labeling and CD169 staining ( lower ) .", "Graph shows summary data .", "( J ) LN sections from Ccr6 Het or KO mice stained for Scart2+ ( green ) and B220 ( blue ) .", "White arrow indicates subcapsular sinus area; FO: B cell follicle; T: T zone .", "( K ) Representative histogram plot and summary mean fluorescence intensity ( MFI ) data of IL17 intracellular staining in IL7RαhiCcr6+ LN cells from Ccr6 Het or KO mice 3 hr after S . aureus bioparticle challenge .", "*p<0 . 05 , **p<0 . 01 , ***p<0 . 001 , by student’s t test .", "Data are representative of at least two experiments for panel A–D , F , J . Data are representative of two or more experiments with at least two mice per group for panels E , G–I , K . LN , Lymph node; SCS , Subcapsular sinus . DOI: http://dx . doi . org/10 . 7554/eLife . 18156 . 00910 . 7554/eLife . 18156 . 010Figure 4—figure supplement 1 . CCL20 distribution in inguinal LN . Serial sections were stained with anti-CCL20 ( A ) or without primary antibody ( B , C ) ( red ) and anti-B220 ( blue ) .", "The arrows in A point to staining in the SCS region adjacent to B cell follicles .", "The controls show that there is non-specific staining in some regions , particularly , in the medulla , but this is minimal in the SCS .", "The weak staining of the T zone in A but not in the control ( panel B ) may reflect non-specific binding by the primary antibody since T zone stromal cells showed minimal CCL20 transcript expression ( Figure 4A ) .", "SCS , Subcapsular sinus . DOI: http://dx . doi . org/10 . 7554/eLife . 18156 . 01010 . 7554/eLife . 18156 . 011Figure 4—figure supplement 2 . Movement of Cxcr6-GFP+ cells to SCS location following CCL20 injection . Cxcr6GFP/+ mice were injected s . c . with saline ( A ) or CCL20 ( B ) and 1 hr later inguinal LN sections were stained to detect Cxcr6-GFP ( green ) , and Lyve1 ( blue ) .", "LN , Lymph node . DOI: http://dx . doi . org/10 . 7554/eLife . 18156 . 01110 . 7554/eLife . 18156 . 012Figure 4—figure supplement 3 . Ccr6 is required for positioning of Ccr6+ cells at the SCS . Inguinal LN sections from Ccr6GFP/+ ( A ) and Ccr6GFP/GFP ( Ccr6-deficient ) ( B ) mice were stained to detect GFP ( green ) and B220 ( blue ) .", "( C ) GFP+B220– cells were counted manually and percentage of cells within 100 µm of the capsule ( indicated by the yellow dashed line in A , B ) was calculated .", "Quantification was done for serial sections of LNs from three Ccr6GFP/+ and 3 Ccr6GFP/GFP mice .", "LNs , Lymph nodes . DOI: http://dx . doi . org/10 . 7554/eLife . 18156 . 01210 . 7554/eLife . 18156 . 013Figure 4—figure supplement 4 . Ccr6 is required for positioning of Scart2+γδT cells at the SCS . Inguinal LN sections from control ( Ccr6+/– ) ( A ) and Ccr6–/– ( Ccr6 deficient ) ( B ) mice were stained to detect Scart2 ( green ) and B220 ( blue ) .", "( C ) Scart2+ cells were counted manually and percentage of cells within 100 µm of the capsule ( indicated by the yellow dashed line in A , B ) was calculated .", "Quantification was done for serial sections of LNs from 3 Ccr6+/– and 3 Ccr6–/– mice .", "LN , Lymph node . DOI: http://dx . doi . org/10 . 7554/eLife . 18156 . 013 We next examined the distribution of Ccr6-deficient ( KO ) cells in Ccr6GFP/GFP mice and found that the cells were reduced in density near the SCS ( Figure 4F , Figure 4—figure supplement 3 ) despite being slightly increased in total frequency in the LN ( not shown ) .", "Instead , the cells were often distributed along the B-T zone interface ( Figure 4F , Figure 4—figure supplement 3 ) .", "In Thy1-PE labeling experiments , Ccr6 KO mice showed reduced frequencies of labeled cells , a result that was most significant for the Vγ4+ population ( Figure 4G ) .", "Consistent with reduced proximity to SCS macrophages , Ccr6-GFP+ IL7Rαhi cells and the Vγ4+ subset from Ccr6 KO mice were associated with less CD169+ blebs compared with Ccr6-sufficient controls ( Figure 4H ) .", "The CD169+ macrophage population in LN sections was unaffected by Ccr6-deficiency ( not shown ) .", "Given that the Vγ4+Ccr6+ cell population was most dependent on Ccr6 for Thy1 labeling and macrophage bleb acquisition ( Figure 4G , H ) , we further studied the properties of these cells .", "Vγ4+Ccr6+ T cells uniquely express the surface marker Scart2 ( Figure 4I ) ( Gray et al . , 2013; Kisielow et al . , 2008 ) .", "In a complementary approach to Thy1 labeling , Scart2 antibody treatment was found to label fewer Vγ4+ cells in LNs from Ccr6-deficient mice than from wild-type mice ( Figure 4I ) .", "By immunofluorescence microscopy , Scart2+ γδ T cells were underrepresented in the SCS region in LN sections from Ccr6-deficient mice compared with those from control mice ( Figure 4J , Figure 4—figure supplement 4 ) .", "Together these data support the conclusion that Ccr6 plays a role in guiding innate-like lymphocytes toward the SCS region .", "Importantly , when Ccr6 KO mice were immunized with S . aureus bioparticles , the IL7RαhiCcr6+ cells mounted a diminished IL17 response ( Figure 4K ) , whereas they responded normally to activation stimuli in vitro ( not shown ) .", "These data provide further evidence that proximity to SCS macrophages is important for innate-like lymphocytes to mount rapid IL17 responses following pathogen exposure .", "S1pr1 is needed in naive lymphocyte for access to cortical and medullary lymphatic sinuses during LN egress ( Cyster and Schwab , 2012 ) .", "S1pr1 is also required for marginal zone ( MZ ) B cell shuttling between the S1P high MZ and the S1P low lymphoid follicle ( Arnon et al . , 2013 ) .", "We therefore tested whether S1pr1 played a role in guiding innate-like lymphocytes into the SCS .", "The innate-like lymphocytes had detectable surface S1pr1 ( Figure 5A ) and they responded to S1P by in vitro migration in a Transwell assay ( Figure 5B ) .", "Pretreatment of mice for 6 hr with FTY720 , a functional antagonist of S1pr1 , greatly diminished in vivo Thy1-PE labeling on IL7RαhiCcr6+ lymphocytes ( Figure 5C ) .", "Similarly , in vivo Scart2 antibody labeling of Vγ4+γδ T cells was decreased after FTY720 treatment ( Figure 5D ) .", "FTY720 also decreased the CD169 membrane bleb positive fraction to 20–25% in both the total IL7RαhiCcr6+ population and the γδ T cell subpopulation ( Figure 5C ) .", "The reductions in Thy1-PE-labeled cells ( from ~10 to ~1% ) and in CD169-bleb+ cells ( from ~35 to~25% ) were similar , representing ~10% of the total IL7RαhiCcr6+ population in both cases ( Figure 5C ) , suggesting that the reduced frequency of CD169-bleb+ cells was due to the loss of cells accessing the sinus .", "Comparable findings were made after treatment with the more selective S1pr1 functional antagonist , AUY954 ( Figure 5E ) .", "These data provided evidence that S1pr1 was required for the cells to have normal access to the SCS .", "Examination of tissue sections by immunofluorescence microscopy showed a loss of Cxcr6GFP/+ cells from the SCS following FTY720 treatment ( Figure 5F ) .", "FTY720 treatment also caused a depletion of Scart2+ cells from the SCS ( Figure 5G , Figure 5—figure supplement 1 ) .", "When FTY720-treated mice were challenged with S . aureus bioparticles , the IL7RαhiCcr6+ population mounted an IL17 response of normal magnitude ( not shown ) .", "We speculate that SCS access is needed for other types of responses . 10 . 7554/eLife . 18156 . 014Figure 5 . S1pr1 is required for innate-like lymphocyte movement into the SCS .", "( A ) S1pr1 surface expression on IL7RαhiCcr6+ and Vγ4+Ccr6+ cells from control or FTY720 treated mice .", "Negative indicates samples stained with no primary antibody .", "( B ) Transwell migration assay showing % of input cells that migrated to the indicated amounts of S1P or SDF .", "( C ) Representative FACS plots and summary data of in vivo Thy1-PE labeling and CD169 staining on IL7RαhiCcr6+ and Vγ4+Ccr6+ LN cells from control or FTY720 treated mice .", "( D ) Summary graph showing in vivo Scart2 labeling on IL7RαhiCcr6+ Vγ4+ cells from control or FTY720-treated mice .", "( E ) Representative histogram plot and summary data of CD169 staining on IL7RαhiCcr6+ and Vγ4+Ccr6+ LN cells from AUY954-treated mice .", "( F ) GFP , Lyve1 and TCRγδ staining of LN sections from control and FTY720-treated Cxcr6-GFP+ mice .", "White , SCS: white arrow indicates subcapsular sinus area; FO: B cell follicle; T: T zone .", "( G ) Scart2 and Lyve1 staining of LN sections from FTY720 treated and control mice .", "White arrow indicates subcapsular sinus area; FO: B cell follicle; T: T zone .", "( H ) Summary data of in vivo Thy1-PE labeling and CD169 staining of the indicated cells in Lyve1-Cre Sphk1fl/-Sphk2-/- ( Sphk DKO ) and control mice .", "( I ) Representative FACS plot showing S1pr1 staining on IL7RαhiCcr6+ cells from a control mouse and histogram plots of CD169 staining on the indicated cells .", "( J ) Representative FACS plot showing S1pr1 staining on IL7RαhiCcr6+ cells from a control and 5-day tamoxifen-treated S1pr1f/f CreERt2 mouse , Graphs show summary data for frequency of Thy1-PE labeled and CD169+ IL7RαhiCcr6+ and Vγ4+Ccr6+ cells in control ( con ) or tamoxifen ( tam ) treated mice .", "( K ) Representative FACS plots of the type in I for cells from a 2-day tamoxifen-treated S1pr1f/f CreERt2 mouse .", "Graph shows CD169+ cell frequency amongst S1pr1-negative IL7RαhiCcr6+ cells .", "*p<0 . 05 , **p<0 . 01 , ***p<0 . 001 , by student’s t test .", "Data are representative of at least two experiments for panels A–B , E–G , I and two or more experiments with at least two mice per group for panels C–D , H . Data are representative of at least three experiments with at least one control and one S1pr1f/f CreERt2 mouse for panels J and K . ( L ) Percent tracks in SCS compartment among the total tracks enumerated in FTY720 treated mice .", "Each point represents data from a single movie ( three independent experiments ) .", "Dashed line is the mean for control mice ( data shown in Figure 3B ) .", "The frequency of cells in the SCS differed significantly from the control ( p<0 . 05 by students t test ) .", "( M ) Frequency of tracks crossing into and out of the SCS of FTY720-treated mice , enumerated as in L . Dashed lines are the means for control mice ( data shown in Figure 3C ) .", "Frequency of tracks crossing into the SCS differed significantly from the control ( p<0 . 05 by students t test ) .", "SCS , Subcapsular sinus . DOI: http://dx . doi . org/10 . 7554/eLife . 18156 . 01410 . 7554/eLife . 18156 . 015Figure 5—figure supplement 1 . FTY720 treatment depletes SCART2+γδT cells from the SCS . Mice were treated i . v . with saline ( A ) or FTY720 ( B ) and 6 hr later inguinal LN sections were stained to detect Scart2 ( green ) and Lyve1 ( blue ) .", "SCS and medulla indicate subcapsular sinus area and medullary area . DOI: http://dx . doi . org/10 . 7554/eLife . 18156 . 01510 . 7554/eLife . 18156 . 016Figure 5—figure supplement 2 . Frequency of Cxcr6GFP/+ cells plotted against their depth from the surface of the LN capsule . Each individual cell’s location was determined using the Spots tool in Imaris .", "The depth , or minimum distance from the cell’s center as a Surface object created in Imaris to the capsule , was calculated using a custom Matlab script and the ImarisXT interface .", "Plots are an aggregate of all experiments for each presented condition ( n = 3–6 mice per condition ) .", "( A ) Representative anatomy for reference against the plotted depth .", "The region adjacent to the LN capsule ( blue in image ) can be divided into the sinus ( depth defined as 0–20 µm below capsule , represented by gray bars ) and the LN parenchyma ( depth defined as > 20 µm below capsule , see Video 1 for dynamics in these regions ) .", "Cxcr6+ cells ( green in image ) at the floor of the sinus ( depth 15–20 µm , dark gray bar ) and close to the sinus but within the parenchyma ( depth 20–28 µM , blue bar ) are both adjacent to CD11b+ subcapsular macrophages ( red in image ) .", "( B ) Sixteen hr after FTY720 treatment , a buildup of Cxcr6GFP/+ cells is seen in the parenchyma adjacent to the sinus ( blue bar ) , while a corresponding depletion of cells within the sinus is observed , compared to control mice . DOI: http://dx . doi . org/10 . 7554/eLife . 18156 . 016 In mice unable to produce lymphatic S1P due to generalized Sphk2 deficiency and ablation of Sphk1 in lymphatic endothelium , innate-like lymphocytes had less CD169+ macrophage-derived membrane blebs and less in vivo Thy1-PE antibody labeling compared with control mice ( Figure 5H ) .", "These observations are consistent with the conclusion that the S1P-S1pr1 axis plays a role in guiding innate-like lymphocytes into the SCS .", "In accord with S1pr1 having an intrinsic role in promoting SCS access of IL7RαhiCcr6+ cells , staining for S1pr1 and CD169 showed the macrophage-derived bleb coating was restricted to the S1pr1+ cells ( Figure 5I ) .", "This analysis required use of an unconjugated rat antibody to detect S1pr1 .", "Since the Thy1-PE is a rat antibody , it was not possible to combine the S1pr1 stain with the in vivo Thy1-PE labeling procedure .", "To further test whether S1pr1 in innate-like lymphocytes was required for SCS access , S1pr1f/f CreERt2+ mice were bred .", "Tamoxifen treatment of adult mice for 5 days caused a loss of S1pr1 in innate-like lymphocytes ( Figure 5J ) .", "Analysis in separate mice showed that the loss of S1pr1 was associated with reduced in vivo Thy1-PE labeling and reduced CD169 on IL7RαhiCcr6+ cells ( Figure 5J ) .", "However , this approach could not exclude a role for S1pr1 in other cell types .", "Attempts to test the intrinsic role of S1pr1 using BM chimeras were unsuccessful due to difficulties in achieving efficient reconstitution of IL7RαhiCcr6+ cells and our finding that many of the cells developing in the chimeric mice appeared activated based on CD69 expression ( not shown ) .", "In another approach , mice were treated with tamoxifen for a short time to cause a ~50% reduction in the fraction of cells that were S1pr1+ ( Figure 5K ) .", "We reasoned that if S1pr1 were acting cell intrinsically in IL7RαhiCcr6+ cells then under conditions of partial ablation the S1pr1-deleted cells should lose their CD169 association whereas this would not occur if the receptor were acting in another cell type .", "Consistent with an intrinsic role , there was little CD169 staining of S1pr1-negative cells in the tamoxifen-treated mice ( Figure 5K ) .", "These data support the conclusion that S1pr1 acts intrinsically in innate-like lymphocytes to promote close associations with SCS macrophages .", "We also examined the effect of S1pr1 antagonism on innate-like lymphocyte migration dynamics using intravital two photon microscopy .", "Visual inspection of the imaging data for FTY720-treated versus control LNs suggested that there were fewer Cxcr6GFP/+ innate-like lymphocytes in the SCS after FTY720 treatment and less examples of cells migrating into the sinus ( Video 3 ) .", "Quantification of the number of tracks present in the SCS and the parenchyma in four imaging experiments confirmed that there were fewer cells in the sinus ( Figure 5L ) , and there was a significant reduction in the number of tracks that crossed from the parenchyma into the sinus ( Figure 5M ) .", "We also plotted the frequency of cells versus distance from the LN capsule for multiple experiments determined using a computational approach ( see Materials and methods ) and this confirmed that FTY720 caused a depletion of Cxcr6GFP/+ cells from the sinus region ( Figure 5—figure supplement 2 ) .", "These data are consistent with the conclusion that S1pr1 antagonism prevents innate-like lymphocyte access to the sinus . 10 . 7554/eLife . 18156 . 017Video 3 . Cxcr6-GFP+ cellular dynamics following FTY720 treatment . One hour time-lapse imaging of a popliteal LN in a Cxcr6GFP/+ mouse 16 hr after treatment with FTY720 .", "Cxcr6-GFP+ cells ( green ) can be seen accumulated on the parenchymal side of the SCS macrophages ( red , CD11b+ ) , and depleted from the SCS .", "LN capsule appears blue ( second harmonic signal ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 18156 . 017 Cell movement from vascular locations into the tissue parenchyma often involves integrin-mediated adhesion .", "Innate-like lymphocytes express high levels of LFA1 ( αLβ2 ) integrin ( Figure 6A ) and ICAM1 is highly expressed by LECs lining the SCS ( Cohen et al . , 2014 ) ( Figure 6B ) .", "ICAM1 is also expressed by CD169+ SCS macrophages ( not shown ) .", "In adhesion assays , IL7RαhiCcr6+ cells bound avidly to ICAM1 ( Figure 6C ) .", "The Vγ4+ subset had slightly lower LFA1 than the total IL7RαhiCcr6+ population and adhered less strongly to ICAM1 ( Figure 6A , C ) .", "Given these findings , we hypothesized that LFA1-ICAM1 interaction may have a role in innate-like lymphocyte movement from the SCS into the LN parenchyma .", "Consistent with this model , 6-hr αL blocking antibody treatment caused increased Thy1-PE labeling on total IL7RαhiCcr6+ lymphocytes and on the Vγ4+γδ T cell subset , without influencing their total number in the LN ( Figure 6D , Figure 6—figure supplement 1A ) .", "A similar increase in the frequency of Thy1-PE labeled innate-like lymphocytes was observed in Icam1-/- mice compared to littermate controls ( Figure 6E ) .", "However , Icam1-/- mice had less IL7RαhiCcr6+ and Vγ4+Ccr6+ cells compared with ICAM1 sufficient control mice , whereas the cells were present at an increased frequency in blood ( Figure 6F , G ) .", "When mice were treated with αL blocking antibody for 3 days , there was a similar reduction in IL7RαhiCcr6+ cell number in LNs and a marked increase in their numbers in blood ( Figure 6—figure supplement 1B ) .", "These data indicate that , unlike short-term integrin blockade , long-term deficiency of ICAM1 or sustained blockade of LFA1 causes a loss of innate-like lymphocytes from the LN . 10 . 7554/eLife . 18156 . 018Figure 6 . LFA1 and ICAM1 control innate-like lymphocyte access to the LN parenchyma from the SCS .", "( A ) Representative FACS histogram showing LFA1 staining of IL7RαhiCcr6+ , Vγ4+Ccr6+ and naïve αβ T cells .", "( B ) ICAM1 staining of WT and ICAM1 KO LN sections .", "( C ) Adhesion of IL7RαhiCcr6+ , Vγ4+Ccr6+ cells and naïve αβ T cells to ICAM1 or BSA .", "( D ) Representative FACS plots and summary graph showing in vivo Thy1-PE labeling of IL7RαhiCcr6+ cells in 6 hr control or αL blocking antibody treated mice .", "( E ) Summary graph showing in vivo Thy1-PE labeling of IL7RαhiCcr6+ cells in ICAM1 Het and KO mice .", "( F , G )", "IL7RαhiCcr6+ and Vγ4+Ccr6+ cell frequencies in LNs ( F ) and blood ( G ) from ICAM1 Het and KO mice .", "( H ) Distribution of IL7Rhi cells in LN sections from control and αL-antibody-treated mice .", "White , SCS: white arrow indicates subcapsular sinus area; FO: B cell follicle; T: T zone .", "( I ) Left panels: Time series of Cxcr6GFP/+ cells in LN of control or anti-αL treated mice .", "Mice were treated with CD11b-PE to label macrophages .", "Right panel: Axis ratio of Cxcr6GFP/+ cells in control and anti-αL treated mice .", "Cxcr6GFP/+ cells in SCS contacting CD11b+ macrophages were measured .", "Data are pooled from two independent experiments .", "*p<0 . 05 , **p<0 . 01 , ***p<0 . 001 , by student’s t test .", "Data are representative of at least two experiments for panels A–C , H–I and two or more experiments with at least two mice per group for panels D–G .", "( J ) Percent of tracks in the SCS compartment among the total tracks enumerated in anti-αL-treated mice .", "Each point represents data from a single movie ( three independent experiments ) .", "Dashed line is the mean for control mice ( data shown in Figure 3B ) .", "The frequency of cells in the SCS differed significantly from the control ( p<0 . 05 by students t test ) .", "( K ) Frequency of tracks crossing into and out of the SCS of anti-αL-treated mice , enumerated as in J . Dashed lines are the means for control mice ( data shown in Figure 3C ) .", "Frequency of tracks crossing out of the SCS differed significantly from the control ( p<0 . 05 by students t test ) .", "SCS , Subcapsular sinus . DOI: http://dx . doi . org/10 . 7554/eLife . 18156 . 01810 . 7554/eLife . 18156 . 019Figure 6—figure supplement 1 . Effects of αL blockade on innate-like lymphocyte distribution .", "( A ) Frequency of IL7RαhiCcr6+ and Vγ4+Ccr6+ LN cells 6 hr after treatment with saline or αL blocking antibody .", "( B ) Number of IL7RαhiCcr6+ cells in peripheral LNs and blood 3 days after treatment with saline or αL blocking antibody .", "( C ) Frequency of Cxcr6GFP/+ cells plotted against their depth from the surface of the LN capsule .", "Four hours after treatment with αL blocking antibody ( alphaL ) , a marked increase in the number of Cxcr6GFP/+ cells at the floor of the sinus ( dark gray bar ) is observed compared to control mice .", "LN , Lymph node . DOI: http://dx . doi . org/10 . 7554/eLife . 18156 . 019 By immunofluorescence microscopy , IL7Rαhi cells were found enriched in the SCS in αL blocked mice ( Figure 6H ) .", "These data suggested that innate-like lymphocytes were trapped in the SCS following αL blockade , leading to their increased exposure to the lymph-borne Thy1-PE antibody .", "To further examine this possibility , intravital two photon microscopy was performed , comparing control and 6 hr αL blocked Cxcr6GFP/+ mice .", "Visual inspection of the movies revealed many Cxcr6GFP/+ cells that appeared to be ‘stuck’ to the SCS floor with their cell bodies ‘fluttering’ in the sinus , possibly being moved by passing lymph ( Figure 6I and Video 4 ) .", "These cells had an elongated morphology compared to the rounded shape of cells in the SCS of control mice ( Figure 6I and Video 4 ) .", "Consistent with the in vivo Thy1-PE labeling and IF microscopy , quantitative analysis of the imaging experiments by enumerating cell tracks and by a computational approach revealed that αL treatment caused an increase of cells in the sinus in association with the macrophage layer ( Figure 6J , Figure 6—figure supplement 1C ) .", "Moreover , the cell tracking analysis showed that while there was not a significant change in the frequency of tracks crossing from the parenchyma into the SCS , there was a significant reduction in cells traveling from the SCS into the parenchyma ( Figure 6K ) .", "These data support the conclusion that LFA1-ICAM1-mediated adhesion is required for innate-like lymphocytes to migrate from the SCS into the LN parenchyma . 10 . 7554/eLife . 18156 . 020Video 4 . Cxcr6-GFP+ cell fluttering in the SCS following αL blockade . In Cxcr6GFP/+ mice , four hours after treatment with αL blocking antibody , Cxcr6-GFP+ cells ( green ) are observed to flutter at the floor of the SCS .", "Cxcr6-GFP+ cells appear attached to CD11b+ SCS macrophages ( red ) while being buffeted by bulk lymph flow in the SCS , yielding a characteristic fluttering dynamic ( video inset and arrowheads ) .", "LN capsule appears blue .", "LN , Lymph node; SCS , Subcapsular sinus . DOI: http://dx . doi . org/10 . 7554/eLife . 18156 . 020 Our finding that innate-like lymphocytes are coated with CD169+ macrophage-derived membrane blebs ( Gray et al . , 2012 ) together with in vitro evidence that CD169 can support adhesion of certain cell types ( Crocker et al . , 1995; Crocker and Gordon , 1989; van den Berg et al . , 2001 ) led us to test whether CD169 contributed to innate-like lymphocyte migration or adhesion in the SCS .", "CD169 binds α2–3 linked sialic acid on surface glycoproteins ( Macauley et al . , 2014 ) .", "By CD169-Fc staining , we found IL7RαhiCcr6+ lymphocytes had a high amount of CD169 ligand on their surface , and neuraminidase treatment of the cells abolished their ability to bind CD169 ( Figure 7A ) .", "The acquisition of abundant macrophage membrane fragments by innate-like lymphocytes was dependent on CD169 , since in mice in which CD169 was blocked with a neutralizing antibody ( Crocker and Gordon , 1989 ) or in mice deficient in CD169 , there were no CD11b+ macrophage membrane fragments on innate-like lymphocytes ( Figure 7B and Figure 7—figure supplement 1A ) . 10 . 7554/eLife . 18156 . 021Figure 7 . CD169 mediates SCS retention of innate-like lymphocytes .", "( A ) CD169-Fc binding of innate-like lymphocytes .", "( B ) Effect of CD169-deficiency or blocking antibody treatment on macrophage bleb acquisition by IL7RαhiCcr6+ cells .", "Cells were stained to detect the macrophage markers CD169 and CD11b .", "( C ) Frequency of tracks crossing into and out of the SCS of anti-CD169-treated mice .", "Each point represents data from a single movie ( three independent experiments ) .", "Frequency of tracks crossing into and out of the SCS differed significantly from the control ( p<0 . 05 by students t test ) .", "( D ) Number of Vγ4+Ccr6+ cells in LN after 30 hr of CD169 blockade .", "( E ) In vivo Thy1-PE labeling on Vγ4+Ccr6+ cells after 6 hr CD169 blockade .", "( F ) Number and in vivo Thy1-PE+-labeled Vγ4+Ccr6+ cell frequency in LNs of control ( con ) or CD169-DTR+ mice after DT treatment .", "( G ) Change in innate-like lymphocyte frequency and Thy1-PE labeling over time after treating mice with αL and CD169 blocking antibodies .", "( H ) Effect of 6 hr combined αL and CD169 blockade on IL7RαhiCcr6+ cell frequency in LN and blood , and fraction of LN cells that are in vivo Thy1-PE labeled .", "( I ) Effect of αL blocking in CD169 KO mice on IL7RαhiCcr6+ cell number and in vivo Thy1-PE labeling .", "( J ) IL7RαhiCcr6+ cell adhesion to CD169-Fc and R97A-Fc-coated plates .", "*p<0 . 05 , **p<0 . 01 , ***p<0 . 001 , by student’s t test .", "Data are representative of at least two experiments for panels A–B , F , I . Data are representative of two or more experiments with at least two mice per group for panels C–E , G–H .", "LN , Lymph node; SCS , Subcapsular sinus . DOI: http://dx . doi . org/10 . 7554/eLife . 18156 . 02110 . 7554/eLife . 18156 . 022Figure 7—figure supplement 1 . Effects of CD169 blockade on innate-like lymphocyte properties and distribution .", "( A ) CD169 CD11b costaining of IL7RαhiCcr6+ cells from control and CD169-deficient LNs .", "( B ) Frequency and Thy1-PE labeling of IL7RαhiCcr6+ and Vγ4+Ccr6+ LN cells in control and CD169-deficient mice .", "( C ) Frequency of IL7RαhiCcr6+ and Vγ4+Ccr6+ LN cells 6 hr after treatment with saline or CD169 blocking antibody .", "( D ) Frequency of Cxcr6GFP/+ cells plotted against their depth from the surface of the LN capsule .", "Only slight changes in distribution of Cxcr6GFP/+ cells are observed 4 hr following treatment with CD169 blocking antibody ( CD169 ) , or in CD169-/- Cxcr6GFP/+ mice ( CD169 KO ) , compared to control mice .", "( E ) Frequency of Cxcr6GFP/+ cells plotted against their depth from the surface of the LN capsule .", "Mice treated for 4 hr with both αL blocking antibody and CD169 blocking antibody ( alphaL+CD169 ) show a marked decrease in the frequency of Cxcr6+ cells at the sinus floor compared to control mice or mice treated with 4 hr aL blocking antibody ( alphaL ) alone . DOI: http://dx . doi . org/10 . 7554/eLife . 18156 . 022 By real-time two photon microscopy , CD169 blockade in Cxcr6GFP/+ mice appeared to increase the amount of innate-like lymphocyte migration within the SCS and between parenchyma and sinus ( Video 5 ) .", "Quantitation of the cell tracks crossing between compartments confirmed that there was an increase in bidirectional exchange ( Figure 7C ) .", "We also examined Cxcr6-GFP+ cells in CD169 KO mice and here too the cells seemed to move extensively in the SCS region ( Video 5 ) .", "These data are consistent with the idea that CD169 blockade or deficiency disrupts stable interactions between innate-like lymphocytes and SCS macrophages . 10 . 7554/eLife . 18156 . 023Video 5 . Increased movement of SCS Cxcr6-GFP+ cells after CD169 blockade and in CD169–/– mice . Representative time-lapse images of Cxcr6GFP/+ mice 4 hr after treatment with CD169 blocking antibody , as well as Cxcr6GFP/+ CD169–/– mice .", "In both conditions , there appeared to be an increased frequency of Cxcr6-GFP+ cell ( green ) crossing events , both from the SCS into the LN parenchyma and from the parenchyma into the SCS .", "LN capsule appears blue and SCS macrophages ( CD11b ) red .", "LN , Lymph node; SCS , Subcapsular sinus . DOI: http://dx . doi . org/10 . 7554/eLife . 18156 . 023 We therefore asked whether CD169 plays a role in mediating lymphatic sinus retention of innate-like lymphocytes .", "After 30 hr of treatment with CD169-blocking antibody , there was no change in total IL7RαhiCcr6+ cell frequency , but there was a two-fold loss of Vγ4+Ccr6+ cells in the LN ( Figure 7D ) .", "We speculated that if this loss was occurring due to inhibited adherence of cells in the SCS then the lymph exposed cells might be lost rapidly following CD169-blockade .", "Indeed , in 6-hr blockade experiments , there was a significant reduction in the fraction of Vγ4+Ccr6+ cells that were Thy1-PE labeled ( Figure 7E ) .", "There was only a slight reduction in the total numbers of Vγ4+Ccr6+ cells after this short period ( Figure 7—figure supplement 1B ) consistent with only a low fraction of the total population being in the sinus at a given time .", "These observations suggest that CD169 blockade caused a loss of cells in lymphatic sinuses and this in turn led to a loss of cells from the LN over time .", "A loss of Vγ4+Ccr6+ cells was also observed following CD169+ macrophage ablation using CD169-DTR mice ( Figure 7F ) .", "In considering explanations for why these effects were mostly selective to the Vγ4+Ccr6+ subpopulation , we found it notable that in ICAM1 adhesion assays , Vγ4+Ccr6+ cells were less capable of binding ICAM1 compared with other innate-like lymphocytes ( Figure 6C ) .", "Taking this observation together with the finding that a higher fraction of Vγ4+Ccr6+ cells were lymph-exposed in the steady state ( Figure 3D ) , we speculated that the non-γδT innate-like lymphocytes – but not the Vγ4+Ccr6+ cells – in the SCS were able to travel back into the LN parenchyma even in the absence of CD169 .", "In an effort to reveal a role of CD169 in sinus retention of the total innate-like lymphocyte population , we blocked αL prior to blocking CD169 .", "In the first 4 hr following αL treatment , we observed a gradual increase in the fraction of cells that were Thy1-PE+ ( Figure 7G ) , consistent with the accumulation of cells in the sinus ( Figure 6 ) .", "Within 2 hr of anti-CD169 treatment , this enhanced Thy1 labeling was lost , indicating loss of innate-like lymphocytes from the sinus , and there was a reduction in IL7RαhiCcr6+ cell frequency ( Figure 7G ) .", "IL7RαhiCcr6+ cell frequency declined further after 4 and 6 hr of double blockade ( Figure 7G ) .", "When αL and CD169 were both blocked continually for 6 hr , there was a 50% loss of total innate-like lymphocytes in LNs , and this was accompanied by an increase of the cells in blood ( Figure 7H ) .", "In CD169 Het and KO mice , there were comparable starting frequencies of IL7RαhiCcr6+ cells ( Figure 7—figure supplement 1C ) , but after αL blocking antibody treatment there was a loss of Thy1-PE labeling and in total IL7RαhiCcr6+ cells in the CD169 KO mice compared with the Het controls ( Figure 7I ) .", "The decrease in innate-like lymphocytes in LNs 6 hr after αL and CD169 double blockade was also evident in intact LNs visualized by real-time two photon microscopy ( Video 6 ) .", "Importantly , unlike αL blockade , which caused many innate-like lymphocytes to become non-migratory and apparently stuck to the SCS floor ( Video 4 ) , applying anti-CD169 in addition to anti-αL led to innate-like lymphocyte detachment from CD169+ macrophages and loss in the lymph flow ( Video 7 ) .", "In some regions , it was also possible to observe cells moving from the parenchyma into the sinus , but then failing to attach and being carried away in the lymph flow ( Video 7 ) .", "By quantitative analysis of three imaging experiments , CD169-blockade or deficiency was found to have little effect on the density of Cxcr6GFP/+ cells in the SCS , but combined αL and anti-CD169 treatment caused a decrease of cells from the sinus region ( Figure 7—figure supplement 1D , E ) .", "These data provide evidence that CD169 has a role in mediating lymphatic sinus retention of most innate-like lymphocytes . 10 . 7554/eLife . 18156 . 024Video 6 . Decreased Cxcr6-GFP+ cell frequency in SCS following αL and CD169 double blockade . Decreased numbers of Cxcr6-GFP+ cells ( green ) are observed in the SCS and LN parenchyma following dual antibody blockade of αL and CD169 .", "Time-lapse imaging in a Cxcr6GFP/+ mouse beginning 4 hr post treatment .", "LN capsule appears blue and SCS macrophages ( CD11b ) red .", "LN , Lymph node; SCS , Subcapsular sinus . DOI: http://dx . doi . org/10 . 7554/eLife . 18156 . 02410 . 7554/eLife . 18156 . 025Video 7 . Addition of CD169 blockade causes release of Cxcr6-GFP+ cells from floor of SCS when pre-treated with αL blocking antibody . Representative time-lapse imaging beginning 5 min after treatment with CD169 blocking antibody in Cxcr6GFP/+ mice pretreated 4 hr before with αL blocking antibody .", "Upon addition of CD169 blockade , Cxcr6-GFP+ cells ( green ) detached from the floor of the SCS and entered the bulk lymph flow , rapidly moving away from the field of view ( inset and arrowheads ) .", "LN capsule appears blue and SCS macrophages ( CD11b ) red .", "LN , Lymph node; SCS , Subcapsular sinus . DOI: http://dx . doi . org/10 . 7554/eLife . 18156 . 025 Finally , we examined the sufficiency of CD169 to support adhesive interactions of innate-like lymphocytes .", "In adhesion assays , IL7RαhiCcr6+ lymphocytes showed binding to plates coated with recombinant WT but not a binding site mutant of CD169 ( Figure 7J ) .", "By contrast , naïve αβ T cells showed minimal binding to CD169 ( Figure 7J ) ." ], [ "The above findings show that the Ccr6-dependent positioning of IL7RαhiCcr6+ innate-like lymphocytes near the LN SCS is important for their rapid cytokine production following bacterial or fungal challenge .", "The data support a model ( Figure 8 ) where lymphatic endothelial-derived CCL20 acts on Ccr6 to attract the innate-like lymphocytes into proximity with SCS macrophages .", "This enhances their exposure to macrophage-derived cytokines that promote IL17-production and likely other effector functions of the lymphocytes10 . 7554/eLife . 18156 . 026Figure 8 . Model of requirements for innate-like lymphocyte surveillance of the LN SCS .", "( A ) Diagram of skin draining LN .", "( B ) Model showing effect of Ccr6-deficiency on innate-like lymphocyte positioning and associated defect in ability to upregulate IL17 in response to IL1-family cytokines produced by SCS macrophages in an ASC-dependent manner .", "( C ) Model showing role of Ccr6-CCL20 in guiding innate-like lymphocyte to lymphatic sinus , S1pr1-S1P in promoting trans-cellular migration into sinus , CD169 on macrophage ( MØ ) in mediating retention of CD169-ligandhi lymphocyte ( green ) against lymph flow , and LFA1-ICAM1 in promoting adhesion and transmigration .", "Green arrows show cell migration and orange arrows show lymph flow .", "LN , Lymph node; SCS , Subcapsular sinus . DOI: http://dx . doi . org/10 . 7554/eLife . 18156 . 026 Our findings also reveal an unusual migratory behavior of innate-like lymphocytes in the SCS region that involves exchange of cells between the parenchyma and SCS .", "Movement across the CD169+ macrophage layer into the sinus is promoted by S1pr1 and lymphatic endothelial cell-derived S1P .", "Within the sinus CD169 on macrophages binds to sialylated ligands on the innate-like lymphocytes and helps prevent loss of the cells in lymph flow , with the Vγ4+ subset of innate-like lymphocytes being most dependent on this adhesive system .", "LFA1 binding to ICAM1 also contributes to retaining innate-like lymphocytes in the sinus .", "Finally , return of cells to the parenchyma involves LFA1 and ICAM1 , presumably to support transmigration across the lymphatic endothelium .", "Although this local migratory behavior was not essential for mounting IL17 responses against the pathogens tested in this study ( data not shown ) , we propose that this surveillance program allows innate like lymphocytes to interrogate the CD169+ macrophages and SCS for pathogen- or commensal-derived molecules for which they have appropriate receptors .", "The cues required for homeostatic positioning of cells in LN T zone and follicles have been well studied , with CCL21/CCL19 and CXCL13 having dominant roles ( Cyster , 2005 ) .", "Our findings add CCL20 as an additional homeostatic organizer , acting to recruit Ccr6+ IL17-committed cells to the LN SCS region .", "As well as expression by innate-like lymphocytes , Ccr6 is abundant on Th17 cells ( Littman and Rudensky , 2010 ) and we speculate that Th17 effector cells that remain in the LN following immunization or infection may position in proximity with the SCS .", "Although Cxcr6 is also abundantly expressed by innate-like lymphocytes , Cxcr6-deficiency did not appear to affect their distribution in the LN ( unpubl . obs ) .", "The expression of CCL20 in a subcapsular region in primate LNs ( Choi et al . , 2003; Pegu et al . , 2007 ) makes it likely that our findings with Ccr6 in mice will extend to humans .", "Ccr6 antagonism was suggested to reduce the egress of Ccr6+ effector CD4 T cells from LNs in a mouse EAE model ( Liston et al . , 2009 ) .", "CCL20 can be strongly induced in inflamed tissue ( Mabuchi et al . , 2013 ) , and we speculate that under the inflammatory condition associated with EAE , CCL20 travels to LNs from the inflamed site and attracts Ccr6+ cells into the sinus lumen as we observed following CCL20 injection .", "In the case of Ccr6+ CD4 effector T cells , this might then favor their exit from the LN .", "S1P and S1pr1 have a critical role in promoting egress of T and B cells from LNs , acting at the step of transmigration into cortical and medullary sinuses ( Grigorova et al . , 2009; Sinha et al . , 2009 ) .", "We describe here a further function for this ligand-receptor pair in promoting cell migration into the SCS .", "As for movement into cortical and medullary sinuses , this response involves S1P production by lymphatic endothelial cells .", "S1pr1 on naive lymphocytes is required during the egress commitment step and did not appear to have a role in promoting chemotaxis to the sinus ( Grigorova et al . , 2009 ) .", "Consistent with those findings , we did not observe an obvious effect of S1pr1 antagonism on innate-like lymphocyte density near the SCS , only a loss of cells from within the sinus .", "We therefore favor the model that the cells approach the sinus-lining lymphatic endothelial cells in a CCL20-dependent manner and S1pr1 commits some of the cells to cross the lymphatic endothelium ( and associated CD169+ macrophage layer ) and enter the sinus .", "Within the sinus , we anticipate that the cells are exposed to high amounts of S1P that cause rapid , GRK2-dependent ( Arnon et al . , 2011 ) , internalization and desensitization of S1pr1 , allowing the cells to respond to cues ( possibly including CCL20 ) that can promote their return to the parenchyma .", "This type of shuttling behavior has been described in the spleen for another population of innate-like lymphocytes , the MZ B cells , with movement into the blood-rich MZ being S1pr1 dependent and return to the parenchyma being CXCL13 dependent ( Arnon et al . , 2013 ) .", "One function of MZ B cell shuttling is to deliver immune complexes from blood to B cell follicles ( Cinamon et al . , 2008 ) .", "It will be interesting to see if cell shuttling in LNs contributes to cargo delivery from lymph to the LN parenchyma .", "CD169 is abundantly expressed on the lymph-exposed heads of SCS macrophages as well as on the tails that extend into the LN parenchyma ( Phan et al . , 2007 ) .", "We show that CD169 supports adhesion of innate-like lymphocytes in the sinus and helps restrain them against lymph flow .", "A number of in vitro studies have shown an ability of CD169 to support cell-cell adhesion ( Crocker et al . , 1995; Crocker and Gordon , 1989; van den Berg et al . , 2001 ) .", "Recent work has also revealed that CD169 on LN SCS macrophages can play a role in the capture of lymph-borne exosomes ( Saunderson et al . , 2014 ) and retroviruses ( Sewald et al . , 2015 ) .", "The present work builds upon those observations to provide in vivo evidence that this Siglec family member can support shear stress-resistant adhesion of cells .", "Although lymph flow rates in the mouse popliteal LN have not been directly measured , a modeling study estimated wall shear stresses of several dyn/cm2 in the SCS close to afferent lymphatic vessels ( Jafarnejad et al . , 2015 ) .", "These shear stresses are similar to those in blood vessels that support leukocyte adhesion ( Finger et al . , 1996 ) .", "A recent genomics study found that high endothelial venules in Peyer’s patches highly express St6gal1 , an enzyme that can generate ligands for CD22 ( Siglec-2 ) ( Lee et al . , 2014 ) .", "In transfer experiments , CD22-deficient B cells showed reduced homing to Peyer’s patches ( Lee et al . , 2014 ) .", "CD22 also contributes to B cell homing to the bone marrow ( Nitschke et al . , 1999 ) .", "These studies together with our work provide evidence that Siglecs are a second class of lectin , after the selectins , that functions in mediating shear-resistant adhesion of cells .", "A key feature that facilitates selectin function is the high density of glycosylated ligands on the target cell and the very rapid on- and off-rates of lectin-ligand interactions ( Rosen , 2004 ) .", "We suggest that similar features contribute to the function of CD169 as a shear-resistant adhesion receptor .", "The long ectodomain of CD169 ( with 17 Ig-domains ) also seems likely to contribute to an ability to ‘capture’ cells that have become dislodged by lymph flow and to allow their re-adhesion and subsequent transmigration .", "A similar activity may be involved in capturing CD169-ligand high cells arriving in the SCS from the afferent lymphatic .", "Our study provides evidence that LFA1 and ICAM1 are required for retaining IL7RαhiCcr6+ innate-like lymphocytes in the SCS and supporting their return from the sinus to the parenchyma .", "These findings are in accord with the well-established roles of LFA1 and ICAM1 in supporting adhesion to and transmigration of lymphocytes across blood vessel endothelium into tissues ( Rot and von Andrian , 2004 ) .", "However , they are in discord with a study showing that migration of BM-derived DCs from the SCS into the LN parenchyma was integrin independent ( Lämmermann et al . , 2008 ) .", "The basis for this discrepancy is not yet clear but might reflect general differences in the properties of lymphocytes and BM-derived DCs .", "Moreover , integrins may contribute to DC trafficking into LNs under conditions of inflammation ( Teijeira et al . , 2013 ) .", "Our conclusion that LFA1 and ICAM1 function during innate-like lymphocyte movement from the SCS into the LN parenchyma is based on four sets of observations in mice treated with αL blocking antibody: ( 1 ) innate-like lymphocytes transiently accumulate in the SCS; ( 2 ) the accumulated cells exhibit an unusual elongated morphology ( suggesting a less adhesive state ) ; ( 3 ) tracking analysis shows reduced numbers of cells migrating from the SCS into the parenchyma; ( 4 ) the cells are more sensitive to dislodgement by CD169 blocking antibody .", "We feel that the most parsimonious explanation for these data is that LFA1 supports adhesive interactions between innate-like lymphocytes and ICAM1+ sinus-associated cells ( lymphatic endothelial cells , macrophages ) that are needed for movement into the parenchyma .", "However , given the heterogeneity of the innate-like lymphocyte population , we cannot exclude the possibility that LFA1 blockade also decreases retention of some cells within the LN parenchyma , thereby increasing their movement into the SCS .", "A study of naive lymphocyte egress from LNs showed that LFA1 and ICAM1 can contribute to promoting retention of cells in the LN and this was suggested to reflect a role for LFA1 in limiting the rate of cell movement across the endothelium into the sinus lumen ( Reichardt et al . , 2013 ) .", "More studies will be needed to fully address all the functions of LFA1 and ICAM1 in innate-like lymphocyte migration dynamics .", "The migration of innate-like lymphocytes in close association with SCS macrophages seems likely to ensure that factors made by the macrophages , such as IL1-family cytokines , can rapidly engage the lymphocytes .", "It presumably also ensures that signaling in the reverse direction , from the innate-like lymphocyte to the CD169+ cells , can take place efficiently .", "Such signaling may serve to augment the antibacterial , antifungal or antiviral activities of the macrophages .", "The importance of cell movement into the lumen of the SCS is not yet clear but might allow prompt surveillance of pathogens and endogenous cells ( e . g . cancer cells ) arriving via the lymph , prior to their accessing the LN parenchyma .", "The greater CD169 coating of Vγ4+γδT cells and their stronger dependence on CD169 for retention in the SCS than the other innate-like lymphocytes suggests these cells interact more strongly or in a more selective way with CD169+ macrophages .", "This likely reflects requirements for recognizing and responding to unique ligands beyond IL1β and IL23 .", "Following Toxoplasma infection or after vaccinia virus injection , NK cell interaction with CD169+ macrophages is induced ( Coombes et al . , 2012; Garcia et al . , 2012 ) .", "These studies observed NK cell movement on collagen fibers and of NK cells slowing or stopping in contact with CD169+ cells .", "Whether collagen fibers guide the movement of IL7RαhiCcr6+ cells needs further study , although in contrast to NK cells , the IL7RαhiCcr6+ cells had minimal expression of the collagen binding α2 integrin ( not shown and [Gray et al . , 2012] ) .", "Another distinction between these cell types is that LN NK cells lack Ccr6 ( unpubl . obs ) .", "A previous study showed that NK1 . 1+ cells in the LN are concentrated in medullary regions ( Kastenmüller et al . , 2012 ) , consistent with their homeostatic positioning being controlled by cues other than CCL20 .", "NK cell movement to the SCS might occur in response to inflammation-induced chemoattractants such as CXCR3 ligands that can be upregulated in this region and function in recruiting activated CD4 T cells and CD8 central memory cells ( Garcia et al . , 2012; Groom et al . , 2012; Sung et al . , 2012 ) .", "NKT cell interaction with SCS macrophages was observed following immunization with α-galactosylceramide ( Barral et al . , 2010 ) .", "Since 15–20% of the IL7RαhiCcr6+ innate-like lymphocytes are NKT cells ( Gray et al . , 2012 ) , it is likely that these cells are continually surveying the SCS macrophages in a manner similar to the bulk Cxcr6+ population studied here .", "As well as macrophages , there are DCs in the SCS region ( Gerner et al . , 2015 ) and the migration behavior we describe may help ensure efficient surveillance of SCS-associated DCs .", "Following exposure to strong inflammatory signals , SCS macrophages move into the follicle ( Gaya et al . , 2015 ) .", "It will be interesting to examine how this reorganization modifies the innate-like lymphocyte trafficking behavior .", "Previous work has shown that the IL7RαhiCcr6+γδT cells in peripheral LNs and related cells in the dermis are precommitted to IL17 production , with stimulation by IL1β and IL23 being sufficient to strongly promote IL17 production by these cells ( Cai et al . , 2011; Gray et al . , 2011; Haas et al . , 2009; O'Brien and Born , 2015; Ramirez-Valle et al . , 2015 ) .", "Our studies here show that the IL7RαhiCcr6+αβT cell population also readily produces IL17 upon IL1β and IL23 stimulation .", "These cells are double negative for CD4 and CD8 ( Gray et al . , 2012 ) .", "LN DN T cells highly express IL23R and respond to this cytokine ( Mizui et al . , 2014; Riol-Blanco et al . , 2010 ) .", "Our findings in ASC-deficient mice provide in vivo evidence that IL1-family cytokines are involved in activating the cells .", "These observations are reminiscent of findings for innate-like CD8 T cells in LNs that rapidly make IFNγ upon cytokine ( IL18 and IL12 or IL18 and IFNα ) stimulation , and for various types of memory T cells that make IFNγ upon IL12 and IL18 exposure ( Jameson et al . , 2015; Kastenmüller et al . , 2012 ) .", "While our data suggest cytokines may be sufficient to activate the innate-like T cells under some conditions , this does not exclude a role for TCR stimulation in promoting activation or augmenting responses under other conditions .", "In summary , we demonstrate that IL17-committed innate-like lymphocytes survey the pathogen-exposed surface of peripheral LNs and respond rapidly upon challenge with bacterial and fungal pathogens .", "Ccr6-guided proximity to the SCS is important for these cytokine-driven responses .", "S1pr1 , CD169 and LFA1 function to promote migration between parenchyma and SCS in close association with CD169+ macrophages in a program that we suggest allows innate-like lymphocytes to survey for a range of pathogen- and commensal-derived antigens and mount appropriately tailored responses ." ], [ "Wild-type C57BL/6NCr mice of 7–9 weeks of age were purchased from the National Cancer Institute ( Frederick , MD ) .", "Cxcr6GFP/+ ( RRID:MGI:3616633 ) , Ccr6GFP/+ ( RRID:MGI:3852186 ) , Ccr6+/– ( RRID:MGI:4359785 ) , Pycard+/– , CAG-KiKGR+ , S1pr1f/f , CreERt2+ , Lyve1-Cre+Sphk1f/f Sphk2 –/– mice ( RRID:MGI:4421697 ) , and Icam1+/– mice were previously described and were from JAX or from an internal colony .", "CD169-DTR mice were provided by Masato Tanaka ( Miyake et al . , 2007 ) .", "Littermate mice were evenly distributed into control or treatment groups and mice of both groups were co-caged whenever possible .", "All mice were adult and were studied between 7 and 20 weeks of age .", "Animals were housed in a specific-pathogen-free environment in the Laboratory Animal Research Center at the University of California , San Francisco , and all experiments conformed to ethical principles and guidelines approved by the Institutional Animal Care and Use Committee , protocol approval number: AN107975-02 .", "Mice were treated with FTY720 in saline at a dose of ~1 ug/g i . p . Mice were analyzed for Thy1-PE labeling or CD169 bleb association at 6 hr or O/N post treatment .", "Control mice were treated with saline i . p . AUY954 in saline was given i . p . in 300 μl at 300 μM .", "Mice were analyzed for CD169 bleb association O/N post treatment .", "Control mice were treated with saline i . p . 100 μg αL blocking antibody ( clone M17/4 ) or anti-CD169 blocking antibody ( Ser4 ) or both antibodies was injected i . v . Mice were analyzed 6 hr or 30 hr post treatment .", "For 3-day experiments , mice were injected with 100 μg αL blocking antibody at day-3 and day-1 , and experiments were done at day 0 .", "Mice were challenged with 2 × 107 CFU of heat inactivated C . albicans , attenuated Yersinia pestis , or 150 μg S . aureus bioparticle ( Invitrogen , Cat S2859 ) through the footpad .", "Control mice were treated with 25 μl saline .", "Draining popliteal LNs were dissected and analyzed 3 hr post challenge .", "For IL17 staining , popliteal LN cells were incubated in Golgi plug for 2 hr at 37°C , stained for surface antigens , treated with BD Cytofix Buffer and Perm/Wash reagent ( BD Biosciences ) , and stained with anti-IL-17A .", "0 . 2 μg Thy1 . 2-PE antibody ( 30-H12 ) was injected through the footpad in 25 μl saline .", "Labeling was done for 5 min .", "Draining popliteal LNs were harvested for flow cytometric or immunofluorescence analysis .", "WT and CD169-DTR/+ were treated with 0 . 75 μg DT on Day-5 and Day-2 .", "Experiments were performed and the mice analyzed on Day 0 .", "For full deletion , WT and S1pr1f/- ERcre+ mice were treated with tamoxifen at Day -5 to Day -1 and analyzed on Day 0 .", "For transient deletion , mice were treated with one dose on Day -2 and analyzed on Day 0 .", "Tamoxifen was dosed at 5 mg/mouse/day orally .", "Cells were stained in 'FACS buffer' ( PBS with 0 . 1% sodium azide , 2% FBS and 1 μM EDTA ) with antibodies to TCRγδ ( GL3 ) , TCRβ ( H57-597 ) , IL17A ( eBio17B7 ) , Ccr6 ( 140706 ) , IL7Rα ( A7R34 ) , CD3ε ( clone 145-2C11 ) , CD11b ( clone Mac-1 ) , Vγ4 ( clone UC3-10A6 ) , CD90 . 2 ( 30-H12 ) , S1pr1 ( R&D , MAB7089 , clone 713412 ) , anti-CD169 ( clone MOMA-1 and clone Ser4 ) ; anti-scart2 antibody was kindly provided by Dr . Klaus Karjalainen .", "Molecular Probes Monoclonal Antibody Labeling Kits ( Invitrogen ) were used to directly conjugate antibody to Alexafluor647 or Pacific Blue dyes .", "During analysis , singlets were gated based on peak FSC-H/FSC-W and SSC-H/SSC-W .", "These gates encompassed more than 90% of total events and were set sufficiently wide to include singlet events of variable size while avoiding the main doublet peak .", "To detect IL-17A , cells were stimulated for 3 hr with 50 ng/ml phorbol 12-myristate 13-acetate ( PMA , Sigma ) and 1 µg/ml Ionomycin ( I , EMD Biosciences ) or 3 hr with 10 ng/ml IL1β and 10 ng/ml IL23 in Golgi plug ( BD Biosciences ) at 37°C , stained for surface antigens , treated with BD Cytofix Buffer and Perm/Wash reagent ( BD Biosciences ) , and stained with anti-IL-17A .", "1 μg/ml CD169-Fc or R97A CD169-Fc and 3 μg/ml anti-human IgG-PE antibody ( Jackson Immunoresearch , Cat 109-116-098 ) was preincubated at 4°C for 1 hr .", "After pre-binding , the mixture was added to lymphocytes from LNs and staining was done for 1 hr on ice .", "Cells were then stained as normal for FACS analysis .", "5 μg Scart2 antibody in 100 μl volume of saline was injected s . c , draining inguinal lymph node was analyzed by flow cytometry 1 hr post antibody injection .", "The mouse was anesthetized with ketamine , shaved and antiseptically prepared with 0 . 02% chlorhexidine gluconate .", "The mouse was then draped and a ~1 . 5 cm incision was made in the abdominal skin to expose the left inguinal LN .", "A silver LED 415 ( Prizmatix ) , set to maximum intensity , with a high numerical aperture polymer optical fiber ( core diameter , 1 . 5 mm ) light guide and fiber collimater , was used as a 415 nm violet light source .", "During the 15 min exposure period , the tissue was kept moist with saline .", "After photoconversion , the skin was closed with two autoclips ( Thermo Fisher Scientific ) .", "~0 . 1 mg/kg buprenorphine in saline was given i . p immediately before and after surgery , and every 4–12 hr as needed thereafter .", "The mice were closely monitored for signs of pain .", "Mice were analyzed immediately before and after photoconversion , and 24 hr and 48 hr post photoconversion .", "Flow cytometry was used to analyze left and right inguinal LN cells as previously described ( Gray et al . , 2013 ) .", "The staining was done with antibodies against Vγ4 , IL7Rα , Ccr6 and TCRβ .", "Parabiosis surgery followed previously described procedures ( Smith et al . , 2015 ) .", "Mirror-image incisions at the left and right flanks were made through the skin and shorter incisions were made through the abdominal wall .", "The peritoneal openings of the adjacent parabionts were sutured together .", "Elbow and knee joints from each parabiont were sutured together and the skin of each mouse was stapled ( 9 mm Autoclip , Clay Adams ) to the skin of the adjacent parabiont .", "Each mouse was injected subcutaneously with Baytril antibiotic and Buprenex as directed for pain and monitored during recovery .", "For overall health and maintenance behavior , several recovery characteristics were analyzed at various times after surgery , including paired weights and grooming behavior .", "Mice were sacrificed for flow cytometric analysis two weeks post parabiotic surgery .", "Mice were anaesthetized by intraperitoneal injection of 10 ml/kg saline containing xylazine ( 1 mg/ml ) and ketamine ( 5 mg/ml ) .", "Maintenance doses of intramuscular injections of 4 ml/kg of xylazine ( 1 mg/ml ) and ketamine ( 5 mg/ml ) were given approximately every 30 min .", "To image the popliteal LN , the mouse’s hind leg was immobilized using thermal putty to a Biotherm stage warmer at 37°C ( Biogenics ) for the duration of the surgery and subsequent imaging .", "A small incision was made directly behind the knee , and a ~0 . 5 cm square region of underlying tissue was exposed .", "The fat pad surrounding the popliteal LN was carefully dissected away without damaging surrounding vasculature and the afferent and efferent lymphatic vessels .", "After visualization of the popliteal LN , a 3D-printed plastic tissue mount was attached to the surrounding tissue using Vetbond .", "The tissue mount was immobilized with additional thermal putty , and the area above the LN was submerged in PBS for imaging .", "Images were acquired with ZEN2009 ( Carl Zeiss , Germany ) using a 7 MP two-photon microscope ( Carl Zeiss ) equipped with a Chameleon laser ( Coherent ) .", "For video acquisition , a series of planes of 3 μm z-spacing spanning a depth of 90 μm were collected every 30 s .", "Excitation wavelengths were 905 nm .", "Emission filters were 500–550 nm for GFP , 570–640 nm for PE , and 450–490 for second harmonic signal .", "Videos were made and analysed with Imaris 7 . 4 . 2 × 64 ( Bitplane ) .", "Two hours prior to all imaging experiments , 2 μg CD11b-PE or 3 μg MOMA1-TxRed antibody was injected through footpad .", "Cell tracking was performed using Imaris Bitplane software .", "Tracks generated using the software were manually confirmed and tracks that were a minimum of 5 min in duration were grouped according to whether they were exclusively in the parenchyma , exclusively in the SCS or crossed between compartments .", "Each movie contained a total of between 100–300 tracks in the region of interest .", "Approximately 15% of the Imaris generated tracks could not be confirmed as representing the migration path of a single cell and these were excluded .", "To quantify the depth of cells from the LN capsule in the imaging data , we used a computational procedure .", "First , a surface object was created in Imaris over the LN capsule .", "The positions of both Cxcr6+ cells and CD11b+ subcapsular macrophages were determined using spots objects in Imaris .", "The minimum distance between each spot and the capsule was determined using a custom MATLAB ( MathWorks ) script and the ImarisXT interface .", "These data were exported into the R programming environment for analysis and plotting ( ggplot2 package ) .", "The depth of the subcapsular sinus in individual LNs varied , and for each experiment was computationally determined as the peak frequency on a plot of CD11b+ subcapsular macrophage’s depth below the LN capsule , minus 2 µm , which agreed across experiments with visual estimates of the subcapsular sinus site .", "As the sinus size varied across experiments from 15 to 40 µm , for the graphs of cell frequency against depth below the capsule the size of the sinus was normalized across experiments to 20 µm .", "Only the region within the sinus was normalized .", "Axis ratio was calculated as the ratio of ellipticity ( prolate ) /ellipticity ( oblate ) .", "These dimensions were obtained by first creating a surface object for each cell using Imaris Bitplane software .", "Final videos were annotated and exported in Premiere ( Adobe ) .", "Lymphocytes from LN were allowed to transmigrate for 4 hr across 5 μm transwell filters ( Corning Costar , Corning , NY ) toward medium or SDF , CCL20 , or S1P and enumerated by flow cytometry as described ( Ramirez-Valle et al . , 2015 ) .", "From paraformaldehyde-fixed tissue , 7 μm sections were prepared , as previously described ( Gray et al . , 2012 ) .", "In some cases , sections were fixed by acetone ( Gray et al . , 2012 ) .", "Sections were stained with the flowing antibodies: anti-Lyve1-A647 , anti-CD3ε-bio ( clone 145-2C11 ) , Goat anti-CCL20 ( AF760 ) , Polyclonal Rabbit anti-GFP ( Thermo Fisher Scientific ) , anti-B220-A647 ( RA3-6B2 ) , anti-ICAM1-bio ( 3E2 ) , anti-IL7Rα-A647 ( A7R34 ) , rat anti-scart2 , Hamster anti-TCRγδ ( GL3 ) , Donkey anti-Goat-biotin Jackson immunoresearch ) , Goat anti-Armenian Hamster-bio ( Jackson immunoresearch ) , Donkey anti-Rat-bio ( Jackson immunoresearch ) , Anti-biotin-A488 ( Jackson immunoresearch ) , Anti-biotin-Cy3 ( Jackson immunoresearch ) .", "Images were captured with a Zeiss AxioOberver Z1 inverted microscope .", "Isolated LN cells from 2–4 Cxcr6GFP/+ mice in complete RPMI media ( with 2% FBS ) were washed twice and resuspended in 200~400 μl PBS ( with 2% FBS ) .", "Cells were stained with anti CD62L-bio ( clone MEL-14 ) , anti CD19-bio ( clone MB19-1 ) , anti CD11c-bio ( clone N418 ) , anti NK1 . 1-bio ( clone PK136 ) for 30 min on ice , washed twice , resuspended in 300 μl PBS ( 2% FBS ) with 10~20 μl anti-Biotin MACS beads ( 5 μl per mouse ) and incubated on ice for 30 min .", "Cells were then washed with and resuspended in 2 ml MACS buffer ( PBS , 2% FBS , 2 mM EDTA , pH 7 . 2 ) , filtered and loaded onto a MACS column , following manufacturer instructions to negatively select Cxcr6GFP/+ cells .", "FACS was used to check enrichment and yield .", "ICAM1 adhesion assay: 96-Well Costar Assay Plates ( High binding polystyrene , Corning ) were coated with ICAM1 at 10 μg/ml concentration in 0 . 1 M Na2CO3/NaHCO3 pH 9 . 5 buffer at 4°C O/N .", "Plates were then blocked with 1% BSA in RPMI for 30 min at room temperature .", "Plates were washed twice with RPMI with 0 . 5% BSA .", "One million Lymphocytes were added and adhesion assays performed at 37°C for 30 min .", "A 200-μl pipette was used to wash cells ( 3x 12 o’clock , 3x 6 o’clock , 1x 12 o’clock , 3 o’clock , 6 o’clock and then 9 o’clock ) .", "After washing , cells were eluted with 5 mM EDTA in RPMI with 0 . 5% BSA and incubation on ice for 15 min .", "Cells were stained and analyzed by FACS .", "CD169-Fc/R97A CD169-Fc adhesion assay: Goat Anti-human IgG antibody ( Jackson Immunoresearch ) was coated on 96-Well Costar Assay Plate ( High binding polystyrene , Corning ) at 15 μg/ml in 0 . 1 M Na2CO3/NaHCO3 pH9 . 5 buffer at 4 degree O/N .", "After coating , plates were washed twice with PBS .", "After washing , 0 . 5 μg CD169-Fc or R97A CD169-Fc in 100 μl PBS was added .", "After 30 min binding , plates were washed twice with PBS , and then blocked with RPMI with 1% BSA for 30 min at room temp .", "After blocking , plates were washed twice with RPMI with 0 . 5% BSA .", "Enriched Cxcr6GFP/+ cells or total LN cells ( in RPMI with 0 . 5% BSA ) were added to the plates .", "Adhesion assays were performed at 37°C for 30 min .", "After binding , cells were washed once ( carefully using a 200 μl pipette to remove the medium ) with RPMI with 0 . 5% BSA solution and adherent cells were analyzed by immunofluorescence microscopy or bright field microscopy for quantification .", "Total RNA from sorted LECs or whole LN was isolated and reverse-transcribed , and quantitative PCR was performed for IL1β , IL23a and CCL20 as described .", "Data were analyzed using the comparative CT ( 2−ΔΔCt ) method using Hprt as the reference .", "Prism software ( GraphPad ) was used for all statistical analysis .", "Statistical comparisons were performed using a two-tailed Student’s t-test .", "p values were considered significant when less than 0 . 05 ." ] ]

[ "Lymph nodes ( LNs ) contain innate-like lymphocytes that survey the subcapsular sinus ( SCS ) and associated macrophages for pathogen entry .", "The factors promoting this surveillance behavior have not been defined .", "Here , we report that IL7RhiCcr6+ lymphocytes in mouse LNs rapidly produce IL17 upon bacterial and fungal challenge .", "We show that these innate-like lymphocytes are mostly LN resident .", "Ccr6 is required for their accumulation near the SCS and for efficient IL17 induction .", "Migration into the SCS intrinsically requires S1pr1 , whereas movement from the sinus into the parenchyma involves the integrin LFA1 and its ligand ICAM1 .", "CD169 , a sialic acid-binding lectin , helps retain the cells within the sinus , preventing their loss in lymph flow .", "These findings establish a role for Ccr6 in augmenting innate-like lymphocyte responses to lymph-borne pathogens , and they define requirements for cell movement between parenchyma and SCS in what we speculate is a program of immune surveillance that helps achieve LN barrier immunity ." ]

[ "The lymphatic system is a network of vessels and a vital part of our immune system .", "Amongst other things , the lymphatic system carries microbes that have entered the body – for example via to a cut or mosquito bite – to small , oval-shaped organs called lymph nodes .", "The lymph nodes are packed with immune cells that can be activated to help fight off infections , however certain microbes actually replicate inside the lymph nodes themselves .", "Lymph nodes protect themselves from these infections by having some pre-armed immune cells that are ready to respond rapidly as soon as an invading microbe is detected .", "These cells , referred to as innate-like lymphocytes , position themselves at the exposed surfaces of the lymph node – the locations where microbes are most likely to enter the organ .", "However , it was not known which cues caused these immune cells to assemble and remain at these locations .", "Zhang et al . now reveal that a signaling molecule called CCL20 attracts the innate-like lymphocytes to the lymph node’s exposed surfaces , while a protein known as CD169 helps to securely attach the innate-like lymphocytes in place .", "Further experiments then confirmed that positioning the innate-like lymphocytes at this location made mice more able to fight off the disease-causing bacterium Staphyloccus aureus .", "Unexpectedly , Zhang et al . also found that innate-like lymphocytes can move from the surfaces of lymph node through to the underlying tissue .", "This unusual migratory behavior might allow the lymphocytes to search a larger area for the infectious microbes , though further studies are needed to test this hypothesis .", "Future studies are also likely to focus on elucidating how the innate-like lymphocytes recognize different types of invaders , and how their activity keeps the lymph nodes healthy ." ]

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

[ "cell biology" ]

[ [ "The proteome must continuously adapt to changing environmental conditions and exposure to extrinsic proteotoxic stressors that challenge cellular , tissue , and organismal health .", "A prominent source of proteotoxic stress arises during translation where transcriptional or mRNA processing errors can result in the translation of defective or truncated proteins and lead to the accumulation of toxic nascent protein products ( Brandman and Hegde , 2016; Schuller and Green , 2018 ) .", "Failure to remove these deleterious proteins can lead to aggregation and contribute to human pathologies including a wide range of neurodegenerative disorders ( Gestwicki and Garza , 2012 ) .", "A variety of cellular quality control and stress response pathways have evolved to guard against the accumulation of these aberrant nascent polypeptides and maintain cellular homeostasis ( Dubnikov et al . , 2017; Lykke-Andersen and Bennett , 2014; Sontag et al . , 2017 ) .", "One prominent example is the integrated stress response ( ISR ) which is activated by a variety of protein homeostasis stressors .", "ISR activation results in rapid global protein synthesis attenuation while also stimulating the translation of critical stress response factors , including protein chaperones and ubiquitin ligases , that assist in rebalancing homeostasis ( Guan et al . , 2017; Pakos-Zebrucka et al . , 2016 ) .", "Quality control pathways safeguard against the accumulation of potentially toxic misfolded or otherwise aberrant proteins .", "The ribosome-associated quality control ( RQC ) pathway is one such quality control system that identifies elongating ribosomal complexes whose progression is halted due to a defect in the translating mRNA or emerging nascent chain ( Brandman and Hegde , 2016 ) .", "After the initial recognition event , RQC pathway components catalyze the degradation of both the mRNA and nascent polypeptide , followed by ribosome subunit recycling ( Ikeuchi et al . , 2018 ) .", "Defects within the RQC pathway result in the production of aberrant protein products and an eventual accumulation of protein aggregates ( Choe et al . , 2016; Defenouillère et al . , 2016; Yonashiro et al . , 2016 ) .", "Protein ubiquitylation plays a key role during these stress response and quality control pathways to facilitate the degradation of misfolded or damaged proteins ( Bengtson and Joazeiro , 2010; Brandman et al . , 2012; Joazeiro , 2017; Joazeiro , 2019; Pilla et al . , 2017 ) .", "Monoubiquitylation , which typically does not target proteins for degradation , of distinct ribosomal proteins is also stimulated in response to ISR activation and conditions that stimulate RQC suggesting that ubiquitylation regulates these pathways beyond protein degradation ( Garzia et al . , 2017; Higgins et al . , 2015; Ikeuchi et al . , 2019; Juszkiewicz et al . , 2018; Matsuo et al . , 2017; Simms et al . , 2017; Sugiyama et al . , 2019; Sundaramoorthy et al . , 2017 ) .", "Studies in both S . cerevisiae and mammalian systems have identified a list of RQC factors and have delineated a series of events that occur when ribosome progression is slowed enough to initiate a QC response ( Joazeiro , 2019 ) .", "Regulatory ribosomal ubiquitylation ( RRub ) has emerged as a conserved critical initiating signal during RQC events ( Ikeuchi et al . , 2019; Juszkiewicz and Hegde , 2017; Matsuo et al . , 2017; Simms et al . , 2017; Sundaramoorthy et al . , 2017 ) .", "In mammals , the ubiquitin ligase ZNF598 catalyzes site-specific ubiquitylation of eS10 ( RPS10 ) and uS10 ( RPS20 ) to resolve ribosomes that have stalled during decoding of polyA sequences ( Juszkiewicz and Hegde , 2017; Sundaramoorthy et al . , 2017 ) .", "Ablation of ZNF598 or the ribosomal protein RACK1 , as well as conserved ubiquitylated target lysines in uS10 or eS10 results in RQC failure and subsequent readthrough of stall inducing sequences ( Juszkiewicz and Hegde , 2017; Sundaramoorthy et al . , 2017 ) .", "Similar , yet distinct ubiquitylation events regulate RQC in yeast ( Matsuo et al . , 2017 ) .", "Current models suggest that ribosome collisions are the key initiation signal which recruits critical ubiquitin ligases to facilitate RRub allowing for subsequent nascent chain ubiquitylation , mRNA degradation , and ribosome recycling ( Ikeuchi et al . , 2019; Juszkiewicz et al . , 2018; Simms et al . , 2017 ) .", "The observation that both uS10 and eS10 ubiquitylation are required for mammalian RQC suggest a potential structured order of ubiquitylation events may be needed to specifically mark collided ribosomes .", "While it is clear that RRub is required for downstream RQC events , the precise mechanistic role the 40S ubiquitylation plays during RQC and the consequence of ubiquitylation on target ribosomal proteins remain open questions .", "Activation of the integrated stress response ( ISR ) in mammalian cells triggers an additional set of RRub events on uS3 ( RPS3 ) and uS5 ( RPS2 ) that do not require ZNF598 and do not function within the RQC pathway and whose function remains uncharacterized ( Higgins et al . , 2015 ) .", "The presence of two separate ubiquitylation events on neighboring ribosomal proteins again suggests a possible hierarchical relationship among distinct RRub events that likely impart separate functions .", "Studies in mammalian cells have demonstrated that the extent of ISR-stimulated uS3 and uS5 monoubiquitylation diminished upon removal of ISR agonists ( Higgins et al . , 2015 ) .", "This observation suggests that either RRub events are reversed by the action of deubiquitylating enzymes ( Dubs ) or that ubiquitin-modified ribosomal proteins are degraded after RQC events .", "Here , we establish that regulatory ribosomal ubiquitylation events are reversible and mediated by deubiquitylating enzymes following activation of the ISR or RQC pathways .", "We utilized an overexpression screen to identify two Dubs , USP21 and OTUD3 , whose expression stimulates readthrough of poly ( A ) -mediated ribosome stalls .", "We demonstrate that USP21 and OTUD3 can directly antagonize ZNF598-mediated eS10 and uS10 ubiquitylation events .", "Further , we show that USP21 and OTUD3 expression results in augmented removal of ubiquitin from eS10 and uS10 following UV-induced RQC .", "USP21 expression also represses ISR-stimulated uS3 and uS5 ubiquitylation .", "Importantly , cells lacking USP21 or OTUD3 display reduced levels of poly ( A ) -mediated stall readthrough and a delay in eS10 demodification following UV-induced RQC activation .", "Expression of OTUD3 results in enhanced stall readthrough compared to knock-in cell lines engineered to lack either eS10 or uS10 RRub sites indicating that combinatorial ribosomal ubiquitylation is required for optimal RQC function .", "Interestingly , we demonstrate that uS10 ubiquitylation is dependent upon eS10 ubiquitylation and that uS5 ubiquitylation requires uS3 ubiquitylation further suggesting a hierarchical relationship upon RRub events .", "Taken together , our results establish that RRub events are reversible by deubiquitylating enzymes and that RRub represents a combinational post-translational code that imparts distinct functional outcomes on ribosomes ." ], [ "Previous studies demonstrated that the integrated stress response ( ISR ) -stimulated regulatory ubiquitylation of uS5 ( RPS2 ) and uS3 ( RPS3 ) is diminished upon cessation of the ISR ( Higgins et al . , 2015 ) .", "The reduced levels of ubiquitylated uS5 or uS3 after ISR stimulation could be the result of demodification by a deubiquitylating enzyme ( Dub ) or turnover of ribosomal proteins .", "We also reasoned that the ZNF598-catalyzed ubiquitylation of uS10 ( RPS20 ) and eS10 ( RPS10 ) , a critical initiating signal within the ribosome associated quality control ( RQC ) pathway , may also be reversible .", "To examine the reversibility of RRub events , HCT116 cells were treated with the translation elongation inhibitor anisomycin ( ANS ) to induce both ribosome stalling , as well as inhibit global protein synthesis ( Juszkiewicz et al . , 2018 ) .", "This allowed us to simultaneously observe the timing of RRub demodification relative to total protein turnover in the absence of new protein synthesis .", "Consistent with previous studies , ANS induced ubiquitylation of both eS10 and uS10 ( Figure 1A; Juszkiewicz et al . , 2018; Matsuo et al . , 2017 ) .", "While eS10 ubiquitylation diminished and uS10 ubiquitylation persisted over time , there was no discernable reduction in the relative amount of the unmodified form of each protein ( Figure 1A , B ) .", "Further no accumulation of unmodified or ubiquitylated eS10 or uS10 was observed with combined treatment of proteasome inhibitors and ANS over 12 hr indicating that ribosomal proteins are either not rapidly degraded when ribosome stalling is stimulated , or degradation cannot be detected within the limit of immunoblotting approaches used in these experiments .", "To address whether deubiquitylation is observed with uS5 and uS3 RRub events , we transiently exposed HCT116 cells with the ISR agonist dithiotheritol ( DTT ) alone or in combination with the protein synthesis inhibitor cycloheximide ( CHX ) followed by DTT washout with and without cycloheximide .", "DTT stimulated uS5 and uS3 ubiquitylation which subsequently diminished to pre-treatment levels over time ( Figure 1C ) .", "Further , the relative amount of the unmodified protein remained stable despite global protein synthesis attenuation ( Figure 1C , D ) .", "The varying kinetics of demodification observed with CHX treatment as compared to DTT alone correlates with heightened ISR activation and prolonged phosphorylation of eIF2α which results in sustained uS3 and uS5 ubiquitylation .", "Together these results suggest that the loss in the amount of ubiquitylated ribosomal proteins likely results from demodification by deubiquitylating enzymes .", "To further examine the reversibility and timing of distinct RRub events , we utilized UV-induced ISR activation which stimulates uS5 , uS3 , eS10 and uS10 regulatory ubiquitylation ( Elia et al . , 2015; Higgins et al . , 2015 ) .", "We exposed 293T cells to UV and allowed cells to recover for increasing periods of time .", "All observed RRub events diminished over time further indicating the RRub events are reversible ( Figure 1E , F ) .", "Interestingly , eS10 and uS10 ubiquitylation preceded uS5 and uS3 ubiquitylation after UV exposure ( Figure 1E , F ) .", "Consistent with previous studies demonstrating that ISR-stimulated uS5 and uS3 RRub events require eIF2α phosphorylation , uS5 and uS3 ubiquitylation occurred coincidently with eIF2α phosphorylation ( Higgins et al . , 2015 ) .", "This timing offset between RRub events and the demonstration that uS10 and eS10 ubiquitylation is catalyzed by ZNF598 whereas uS5 and uS3 ubiquitylation does not require ZNF598 suggests that uS5 and uS3 RRub events are functionally distinct from the more immediate eS10 and uS10 ubiquitylation that likely occur as a direct result of UV-induced ribosomal stalls .", "To investigate the importance of individual RRub events , we generated point mutant knock-in HCT116 cell lines in which the endogenous eS10 , uS10 , uS3 or uS5 loci were modified by CRISPR/Cas9 approaches to replace previously identified ubiquitylated lysine residues with arginine .", "We first examined if mutating RRub lysine residues resulted in altered protein stability .", "We observed no appreciable change in total protein abundance in the eS10 K138R/K139R knock-in ( eS10-KI ) and uS10 K4R/K8R knock-in ( uS10-KI ) cells following ANS treatment ( Figure 2A , B and Figure 2—figure supplement 1A , B ) .", "Similarly , the inability to ubiquitylate uS5 K54R/K58R ( uS5-KI ) and uS3 K214R ( uS3-KI ) did not change the steady-state abundance or the turnover of uS5 or uS3 ( Figure 2C , D and Figure 2—figure supplement 1C , D ) .", "Interestingly in the course of this experiment and validation of these cell lines we noticed a hierarchal relationship among the ubiquitylation events ( Figure 2—figure supplement 1E , F ) .", "As expected , eS10-KI cell lines completely lack ANS-induced eS10 ubiquitylation .", "However , loss of eS10 ubiquitylation substantially reduces uS10 ubiquitylation ( Figure 2A and Figure 2—figure supplement 1F ) as compared to control cell lines .", "The inability to ubiquitylate eS10 had a negligible impact on the levels of UV-induced uS5 and uS3 ubiquitylation ( Figure 2—figure supplement 1F ) .", "In contrast , uS10-KI cells maintained their ability to ubiquitylate eS10 , uS5 , and uS3 despite the expected loss of uS10 ubiquitylation ( Figure 2B and Figure 2—figure supplement 1E ) .", "These results indicate that eS10 ubiquitylation may be required for optimal uS10 ubiquitylation upon induction of RQC events .", "Similar to the hierarchy of eS10 and uS10 , the lack of DTT-induced uS3 ubiquitylation in the uS3-KI cells results in complete ablation of uS5 modification while loss of uS5 ubiquitylation did not effect uS3 DTT-stimulated RRub ( Figure 2C , D ) .", "Combined , these results suggest that hierarchical relationships exist within distinct classes of RRub events and imply a specific order of ubiquitylation events .", "Our results implicate the direct involvement of deubiquitylating enzymes in regulating RRub and RQC function .", "To identify and characterize deubiquitylating enzymes ( Dubs ) that operate within the RQC pathway , we utilized a previously established dual-fluorescence RQC reporter assay in which a stall-inducing poly ( A ) sequence placed between GFP and cherry fluorescent protein ( ChFP ) coding sequences results in the repression of downstream ChFP fluorescence as compared to GFP , indicative of an RQC event initiating upon translation of the poly ( A ) sequence .", "( Juszkiewicz and Hegde , 2017; Sundaramoorthy et al . , 2017 ) .", "Previous studies demonstrated that loss of ZNF598 function and the resulting decrease in eS10 and uS10 ubiquitylation results in readthrough of poly ( A ) sequences and a subsequent increase in the ChFP:GFP ratio of the stall reporter ( Juszkiewicz and Hegde , 2017; Sundaramoorthy et al . , 2017 ) .", "Overexpression of a deubiquitylating enzyme that mediates deubiquitylation of eS10 and uS10 RRub events would phenocopy ZNF598 loss-of-function and enhance the amount of poly ( A ) readthrough .", "Based on this rationale , a panel of 60 human Dub expression plasmids were individually co-transfected with the poly ( A ) stall reporter plasmid into 293T cells and the corresponding ChFP:GFP ratio was measured by flow cytometry .", "Immunoblotting confirmed the expression of 58 Dubs albeit to varying expression levels ( Figure 3—figure supplement 1 ) .", "A Z-score analysis of the ChFP:GFP ratio for the stall reporter identified six candidate Dubs whose expression resulted in the largest enhancement of poly ( A ) readthrough above the population mean ( Figure 3A ) .", "We validated that expression of the six candidate Dubs resulted in a reproducible enhancement of poly ( A ) -stall readthrough and a subsequent increase in ChFP:GFP values using the stall-reporter assay ( Figure 3B ) .", "To directly validate that the resulting increase in the ChFP:GFP ratio was specific to the poly ( A ) reporter , each candidate Dub was expressed with a control plasmid lacking the internal poly ( A ) sequence ( Figure 3B ) .", "OTUB2 , OTUD3 , USP10 and UCHL1 expression did not alter the ChFP:GFP ratio of the control reporter while OTUD1 and USP21 only modestly elevated the ChFP:GFP ratio indicating that the identified Dubs specifically alter the ability of ribosomes to progress through a poly ( A ) -induced ribosomal stall ( Figure 3B ) .", "To examine whether the overexpression-induced increase in poly ( A ) -mediated stall readthrough was dependent on the catalytic activity of each of the identified Dubs , catalytically inactive versions of each Dub were generated by site-directed mutagenesis to convert the critical catalytic cysteine residue to serine .", "Each Dub and the respective catalytically inactive mutant ( CS ) were co-expressed with the poly ( A ) -stall reporter ( Figure 3C ) .", "Expression of OTUB2 and OTUD1 inactive mutants resulted in an equivalent degree of poly ( A ) -stall readthrough as compared to the respective wild type enzymes ( Figure 3C ) .", "Additionally , expression of inactive UCHL1 resulted in enhanced readthrough of the poly ( A ) -sequence compared to wild type UCHL1 .", "This result suggests that the observed increase in ChFP fluorescence does not require the catalytic activity of OTUB2 or OTUD1 .", "In contrast , expression of the inactive mutants for the deubiquitylating enzymes USP21 , OTUD3 , and USP10 resulted in a substantial reduction of the ChFP:GFP ratios compared to wild type versions .", "Expression of inactive OTUD3 or USP21 resulted in elevated stall readthrough compared to control transfections indicating a possible alternative role for OTUD3 and USP21 within the RQC pathway that is ZNF598 independent .", "Together , these results indicate that USP21 , OTUD3 , and USP10 expression results in elevated poly ( A ) -mediated stall readthrough in an activity-dependent manner .", "Having demonstrated that exogenous expression of USP21 , OTUD3 , and USP10 enhanced poly ( A ) stall-induced readthrough , we wanted to examine the ability of the Dubs to directly antagonize ZNF598-mediated translational stalling of the poly ( A ) reporter .", "As expected , exogenous expression of wild type ZNF598 resulted in decreased ChFP:GFP ratios as compared to control transfections ( Figure 3D ) .", "Next , the poly ( A ) reporter and candidate Dubs were expressed along with exogenous ZNF598 .", "Both USP21 and OTUD3 , when co-expressed with ZNF598 , resulted in a greater than 2 . 5 fold increase in the ChFP:GFP poly ( A ) reporter ratio relative to what was observed when ZNF598 was expressed in isolation ( Figure 3D ) .", "Antagonism with ZNF598 was not observed for USP10 which suggests its role within the RQC operates independently of ZNF598 .", "Combined expression of OTUD3 and USP21 did not further enhance poly ( A ) -mediated stall readthrough events or result in enhanced antagonism of ZNF598 ( Figure 3—figure supplement 2A ) .", "These results are consistent with the hypothesis that USP21 and OTUD3 directly antagonize ZNF598-mediated RRub events .", "Immunoblot analysis revealed that cells solely overexpressing ZNF598 displayed a 5-fold increase in the abundance of ubiquitylated eS10 compared to untransfected cells ( Figure 3E ) .", "Exogenous expression of USP21 substantially reduced the ZNF598-stimulated eS10 and uS10 ubiquitylation in an activity-dependent manner ( Figure 3E ) .", "The same result was observed upon expression of OTUD3 and ZNF598 ( Figure 3E ) .", "These results demonstrate the ability of USP21 and OTUD3 to remove ubiquitin from eS10 and uS10 following ZNF598-mediated RRub events .", "Because USP21 and OTUD3 were the only Dubs to show activity dependent antagonism of ZNF598 in our stall readthrough assay , these Dubs were selected for subsequent analyses .", "To further examine the antagonism between ZNF598 and OTUD3 and USP21 , parental HCT116 cells and ZNF598 knockout ( ZNF598-KO ) cells were transfected with either USP21 or OTUD3 expressing plasmids and the poly ( A ) stall-inducing reporter .", "We reasoned that expression of these Dubs in the absence of ZNF598 would not impact the amount of poly ( A ) -stall readthrough beyond that observed with loss of ZNF598 expression .", "As expected , expression of either Dub along with the poly ( A ) -reporter in parental HCT116 cells markedly increased the ChFP:GFP ratio of the stall reporter ( Figure 4A , B ) .", "ZNF598-KO cells displayed the expected elevated ChFP:GFP ratio of the stall reporter which was modestly enhanced upon further expression of either Dub ( Figure 4A , B ) .", "This modest enhancement was also observed upon expression of either the wild type or inactive versions of other Dubs , OTUB2 , OTUD1 , and UCHL1 in the ZNF598-KO cell line suggesting that the increased readthrough is possibly non-specific and does not require Dub activity ( Figure 3—figure supplement 2B ) .", "However , the observation that USP21 or OTUD3 expression modestly augments readthrough of poly ( A ) sequences in cells lacking ZNF598 suggests that USP21 and OTUD3 may function within the RQC in a ZNF598 independent manner while also directly antagonizing ZNF598 ribosomal ubiquitylation .", "To investigate the role of individual RRub events during RQC , we utilized the uS10-KI and eS10-KI HCT116 cell lines to examine if the enhanced readthrough of poly ( A ) stall-inducing sequences observed upon USP21 or OTUD3 overexpression required uS10 or eS10 ubiquitylation .", "Consistent with our previous results , eS10-KI and uS10-KI cell lines allowed for enhanced readthrough of poly ( A ) -mediated stall events using our stall reporter FACS assay whereas uS3-KI and uS5-KI cell lines did not appreciably alter reporter levels compared to parental cells ( Figure 2—figure supplement 1G; Sundaramoorthy et al . , 2017 ) .", "We expressed OTUD3 or USP21 in uS10 or eS10 knock-in cell lines along with the poly ( A ) stall reporter .", "USP21 or OTUD3 overexpression in either eS10-KI or uS10-KI cells resulted in a further enhancement of the ChFP:GFP ratio above the respective transfection controls ( Figure 4C , D ) .", "This enhancement was largely activity-dependent as expression of inactive OTUD3 reduced the extent of readthrough compared to wild type in both eS10 and uS10 knock-in cell lines ( Figure 4D ) .", "Expression of inactive USP21 resulted in reduced readthrough compared to wild type in uS10-KI cells but not eS10-KI cells ( Figure 4C ) .", "These results indicate that OTUD3 and USP21 can demodify both uS10 and eS10 , consistent with our immunoblotting data ( Figure 3E ) .", "Further , these results indicate that the combined loss of uS10 and eS10 RRub events results in a stronger RQC defect than loss of either uS10 or eS10 ubiquitylation events alone .", "To validate the poly ( A ) -reporter results , we immunoblotted cell lysates in which we expressed either wild type or inactive USP21 or OTUD3 in parental or eS10-KI or uS10-KI cell lines to visualize eS10 and uS10 ubiquitylation .", "As expected , ZNF598 expression stimulated eS10 and uS10 ubiquitylation in parental HCT116 cells ( Figure 4E ) .", "ZNF598 expression in uS10-KI cells failed to induce uS10 ubiquitylation without impacting the ability of ZNF598 to ubiquitylate eS10 .", "Conversely , ZNF598 expression failed to ubiquitylate eS10 in eS10-KI cells , and uS10 ubiquitylation was substantially reduced compared to ZNF598 expression in parental cells .", "This is consistent with a model in which eS10 ubiquitylation is needed prior to uS10 ubiquitylation .", "While expression of wild type USP21 or OTUD3 reduced the abundance of both monoubiquitylated eS10 and uS10 in parental HCT116 cells , expression of the inactive variants restored ubiquitylation to steady-state levels ( Figure 4E ) .", "Expression of either Dub in the eS10-KI cell line could further demodify the small amount of uS10 ubiquitylation observed upon ZNF598 expression ( Figure 4E ) .", "Similarly , USP21 and OTUD3 antagonized the ZNF598-dependent eS10 ubiquitylation in uS10-KI cells in an activity dependent manner .", "Taken together , these results indicate that USP21 or OTUD3 can deubiquitylate both eS10 and uS10 , resulting in enhanced readthrough of poly ( A ) -mediated stall events .", "Further , these results demonstrate that the combined ubiquitylation of eS10 and uS10 is required for optimal resolution of RQC events .", "Examination of quantitative proteomic datasets from human cell lines revealed that ZNF598 protein levels are 19-fold in excess of OTUD3 while USP21 levels were undetectable indicating that ZNF598 protein levels are in vast excess of either RRub Dub at steady state ( Itzhak et al . , 2016 ) .", "Given the relative excess of ZNF598 compared to its antagonizing Dubs , we set out to examine how varying the levels of the Dubs relative to ZNF598 would impact RQC events .", "We transfected increasing amounts of a ZNF598 expressing plasmid in ZNF598-KO cell lines and examined poly ( A ) -mediated stall readthrough events using the stall reporter assay .", "Expression of the poly ( A ) -reporter with increasing concentrations of exogenous ZNF598 in isolation did not result in a dose-dependent decrease in the ChFP:GFP ratio suggesting that ZNF598 expression at the lowest levels tested were sufficient to fully restore RQC function and that elevated ZNF598 levels do not further enhance ribosome stall resolution ( Figure 4F , G and Figure 3—figure supplement 2C , D ) .", "We then varied the relative ZNF598 expression levels compared to either USP21 or OTUD3 and examined the impact on the ChFP:GFP ratio of the stall reporter .", "When equal amounts of USP21 or OTUD3 and ZNF598 expression plasmids were transfected , a substantial increase in the ChFP:GFP ratio was observed as compared to ZNF598 expressed alone which further verified the direct antagonism observed previously ( Figure 4F , G and Figure 3—figure supplement 2C , D ) .", "The reporter ChFP:GFP ratio declined as ZNF598 plasmid transfections were doubled and tripled with respect to USP21 or OTUD3 plasmid DNA ( Figure 4F , G and Figure 3—figure supplement 2C , D ) .", "Conversely , doubling and tripling the expression of USP21 while holding ZNF598 expression levels constant revealed additional readthrough of the poly ( A ) stall-inducing sequence with increasing ChFP:GFP ratios , suggesting further antagonism of the ligase .", "This result suggests that maintaining ZNF598 expression levels high relative to USP21 and OTUD3 is a feature that may be required to enable rapid 40S ribosomal ubiquitylation upon RQC-triggering events that are not immediately removed by antagonistic Dubs .", "These results also indicate that OTUD3 or USP21 overexpression ( 40-fold and 100-fold above endogenous , respectively ) is required to compete with endogenous ZNF598 activity .", "To examine the ability of USP21 and OTUD3 to catalyze deubiquitylation of RRub events , we generated doxycycline ( Dox ) -inducible 293 cell lines that conditionally express either the wild type or inactive version of each Dub .", "To observe the reversal of RRub events , we induced ribosome stalling and subsequent RRub using UV exposure .", "To test the impact of Dub expression , cells were either treated with or without Dox for 16 hr prior to UV exposure .", "Cells were then UV irradiated and allowed to recover for increasing periods of time .", "Based on our previously established reversibility of RRub events , we suspected that overexpression of wild type USP21 or OTUD3 would induce a more rapid removal of eS10 and uS10 ubiquitylation during recovery from UV-induced stress .", "Control cells without induction of Dub overexpression displayed induced eS10 and uS10 ubiquitylation immediately following UV treatment , followed by rapid demodification 4 hr after UV exposure ( Figure 5A–D and Figure 5—figure supplement 1 ) .", "Interestingly , in cells overexpressing exogenous wild type USP21 , the amount of detectable eS10 ubiquitylation rapidly declined 1 hr post UV exposure , while uS10 ubiquitylation was completely ablated ( Figure 5A and Figure 5—figure supplement 1 ) .", "These observations suggest that USP21 can demodify eS10 and uS10 following UV-induced stress .", "To substantiate these observations , we induced the expression of inactive USP21 and determined the dynamicity of UV-induced RRub events .", "Immunoblots confirmed that the dynamics of eS10 and uS10 ubiquitylation following UV treatment in cells expressing inactive USP21 were unaltered compared to cells without Dox-treatment .", "( Figure 5B and Figure 5—figure supplement 1 ) .", "These findings confirm that USP21 can remove ubiquitin from eS10 and uS10 in an activity-dependent manner .", "Similar to what was observed upon USP21 expression , OTUD3 expression resulted in substantially reduced eS10 ubiquitylation and complete ablation of uS10 ubiquitylation following UV treatment compared to uninduced cells ( Figure 5C and Figure 5—figure supplement 1 ) .", "This reduction in eS10 and uS10 ubiquitylation was activity dependent as induction of inactive OTUD3 did not alter either eS10 or uS10 ubiquitylation upon UV treatment ( Figure 5D and Figure 5—figure supplement 1 ) .", "It was surprising that overexpression of the inactive versions of USP21 or OTUD3 did not result in a dominant negative phenotype with enhanced eS10 or uS10 ubiquitylation at steady state or reduced demodification kinetics following UV exposure .", "This observation suggests that the Dubs do not compete for the same binding surface on the ribosome despite their ability to demodify the same RRub sites .", "To determine if USP21 or OTUD3 loss of function impacted RRub or RQC activity , we generated USP21 and OTUD3 knockout cell lines and USP21/OTUD3 double knockout cell lines using genome engineering approaches ( Figure 6—figure supplement 1A ) .", "Two separate knockout clones for both USP21 and OTUD3 displayed reduced poly ( A ) -mediated stall readthrough which is consistent with our demonstration that overexpression of OTUD3 or USP21 enhances stall readthrough ( Figure 6A ) .", "Combined loss of USP21 and OTUD3 did not further reduce stall readthrough compared to individual knockouts suggesting that the Dubs may be acting at distinct points within the RQC pathway ( Figure 6A ) .", "We then evaluated the kinetics of eS10 demodification following UV-induced RQC activation in parental and knockout cell lines .", "Consistent with previous results , eS10 ubiquitylation was rapidly induced following UV exposure and was fully demodified to pre-exposure levels by 16 hr in parental 293T cells ( Figure 6B and Figure 6—figure supplement 1B ) .", "USP21 or OTUD3 knockout cells displayed reduced demodification kinetics following UV exposure with eS10 ubiquitylation persisting up to 24 hr in OTUD3 knockout cells ( Figure 6B and Figure 6—figure supplement 1B ) .", "OTUD3 and USP21 double knockout cells also displayed sustained eS10 ubiquitylation following UV exposure compared to parental controls ( Figure 6B and Figure 6—figure supplement 1B ) .", "While a delay in eS10 demodification was observed in the knockout cells , the extent of eS10 ubiquitylation is clearly reduced compared to peak levels in all knockout cells indicating that other Dubs can compensate for the loss of USP21 or OTUD3 and that further redundancy exists in the pathway .", "It is notable that we failed to observe an RQC phenotype upon siRNA-mediated knockdown of USP21 , OTUD3 , or combinations of candidate Dubs ( Figure 6—figure supplement 1C–F ) .", "We also could not detect reduced stall readthrough upon knockdown of 24 additional Dubs that were not represented in our overexpression library ( Figure 6—figure supplement 1G ) .", "These results demonstrate that complete genetic ablation of OTUD3 or USP21 expression is required to observe defects with RQC and RRub demodification following RQC activation .", "Stressors that induce the integrated stress response ( ISR ) also induce RRub events on uS3 and uS5 that function in a distinct manner compared to eS10 or uS10 RQC ubiquitylation events ( Higgins et al . , 2015 ) .", "To examine if OTUD3 or USP21 act specifically on eS10 and uS10 RQC RRub events or act more broadly on ubiquitylated ribosomes we utilized the OTUD3 and USP21 inducible cell lines .", "After induced Dub expression , cells were treated with DTT or harringtonine ( HTN ) which stimulates uS5 and uS3 RRub events in a distinct manner ( Higgins et al . , 2015 ) .", "Interestingly , USP21 expression reduced both HTN and DTT-induced uS5 and uS3 ubiquitylation in an activity-dependent manner ( Figure 7A ) .", "However , OTUD3 expression reduced uS5 ubiquitylation , albeit to a lesser extent than UPS21 expression , and had no impact on HTN or DTT-induced uS3 ubiquitylation ( Figure 7B ) .", "These results indicate that USP21 expression has a larger impact on all RRub events examined and that OTUD3 primarily demodifies ZNF598-catalyzed eS10 and uS10 ubiquitylation events .", "Given the ability of USP21 and OTUD3 to remove RRub events , we examined if OTUD3 or USP21 associated with ribosomes .", "We treated OTUD3 or USP21 inducible cells with ANS in the presence or absence of Dox and then pelleted ribosomes by running whole cell extracts through a sucrose cushion .", "As expected , ANS induces eS10 ubiquitylation which is abrogated upon OTUD3 or USP21 expression ( Figure 7C , D ) .", "Importantly , both OTUD3 and USP21 are present in the ribosome pellet fraction suggesting that OTUD3 and USP21 associates with ribosomes .", "To determine which ribosomal subpopulation is associated with OTUD3 upon RQC stimulation , we treated OTUD3 expressing cells with anisomycin and separated whole cell extracts over a linear sucrose gradient .", "Subsequent immunoblotting of gradient fractions revealed OTUD3 to be present in 40S containing fractions and largely absent from 80S and polysome containing fractions ( Figure 7E ) .", "These results suggest that OTUD3 may preferentially demodify ubiquitylated 40S proteins that arise after subunit splitting and position OTUD3-mediated deubiquitylation as a putative late step during RQC events .", "Overall , our observations indicate OTUD3 and USP21 can demodify distinct sets of RRub events and regulate RQC activity ." ], [ "Proteomics studies have revealed that a substantial portion of the ubiquitin-modified proteome may play a role in regulating cellular processes in a non-degradative capacity rather than targeting substrates for degradation ( Kim et al . , 2011 ) .", "Several of these putative regulatory ubiquitylation events appear to be conserved , including many ribosomal monoubiquitylation events .", "Previous studies have established that conserved site-specific regulatory 40S ubiquitylation ( RRub ) is among the first signaling events required for proper RQC execution ( Garzia et al . , 2017; Juszkiewicz and Hegde , 2017; Matsuo et al . , 2017; Sundaramoorthy et al . , 2017 ) .", "While the ubiquitin ligase that catalyzes the RQC-specific RRub events has been characterized in both mammals and yeast , whether RRub demodification was a critical step in ultimate resolution of RQC events and the identity of potential RRub Dubs remained unknown .", "Here we identify two Dubs , USP21 and OTUD3 , that contribute to the removal of ubiquitin from 40S ribosomal proteins .", "Overexpression of USP21 or OTUD3 results in enhanced readthrough of a poly ( A ) stall-inducing sequence .", "USP21 and OTUD3 have overlapping yet distinct ribosomal protein substrate specificities .", "The ubiquitin-specific proteases ( USP ) and the ovarian tumor ( OTU ) family make up the two largest Dub families .", "While USP Dubs are typically more promiscuous with regards to the types of polyubiquitin linkages they demodify ( Faesen et al . , 2011 ) , OTU Dubs have been shown to exhibit ubiquitin-chain linkage specificity ( Mevissen et al . , 2013 ) .", "Consistent with these observations , USP21 is more promiscuous than OTUD3 in that USP21 can hydrolyze all four tested RRub events while OTUD3 preferentially demodifies ZNF598-catazlyed eS10 and uS10 ubiquitylation events .", "These results establish that Dubs can play a regulatory role within the RQC pathway .", "The factors that govern the regulation of these Dubs and when RRub events are removed within the RQC pathway remain unclear .", "We postulate two ways that Dubs may act as regulators of the ISR and RQC pathways .", "First , USP21 and OTUD3 may limit the activity of ZNF598 and other RRub ligases through direct antagonism to prevent spurious RRub .", "Unregulated 40S ubiquitylation could result in premature translational attenuation and destruction of properly processed mRNAs .", "Though plausible , the observed substochiometric ratio of OTUD3 and USP21 relative to ZNF598 suggests that Dubs may not directly limit ZNF598 activity .", "The low expression levels of USP21 and OTUD3 relative to ZNF598 may ensure that when progression of the ribosome is halted to a degree that requires RQC activity , the forward reaction is favored .", "A second possibility is that following 80S splitting , Dubs serve to strip the 40S of its ubiquitin in order to recycle unmodified 40S complexes which can reenter the translation cycle .", "Implicit in this model is that a ubiquitylated 40S may prevent or reshape translation initiation events , a scenario which has not been directly established .", "More in-depth biochemical studies are required to identify the factors and mechanisms that regulate these reversible ribosomal regulatory ubiquitylation events , and the timing by which OTUD3 and USP21 exert their activity .", "The mechanistic role of these regulatory ubiquitylation events remains obscure .", "One possibility is that ubiquitylation serves as a scaffold for targeting endo- or exoribonucleases responsible for downstream degradation of the mRNA .", "Recent work has shown that the endonuclease Cue2 is recruited to collided ribosomal complexes and is responsible for cleaving the mRNA within the A site ( D'Orazio et al . , 2019 ) .", "With the unique interface formed by collided disomes and the positioning of ubiquitylated eS10 and uS10 , it is conceivable that Cue2 uses its two ubiquitin binding domains to latch onto the stalled complex .", "Another possibility is that ribosome ubiquitylation represents a ubiquitin code that distinguishes ribosomes that are simply paused at a specific codon to allow for proper nascent chain folding or to relocalize translation from those that are terminally stalled due to an irreconcilable defect in the mRNA .", "A ribosomal ubiquitin code implies a possible order of operations and suggests that individual RRub events may not be occurring simultaneously , but rather in succession .", "Support for this model comes from the hierarchical relationship we observed among the different sets of RRub events , where the loss of eS10 ubiquitylation results in a reduction in uS10 modification ( Figure 2A ) .", "This observation suggests that eS10 is the first ubiquitylation event required for RQC initiation .", "Taken together , these results suggest that the combined modification of both eS10 and uS10 is required for robust resolution of stalled ribosomes .", "This observation is replicated for ISR stimulated uS3 and uS5 ubiquitylation where loss of the ability to ubiquitylate uS3 results in a complete ablation of uS5 modification which suggest that hierarchical RRub events may regulate translation beyond RQC function ( Figure 2D ) .", "Current models suggest that collided ribosomes trigger ZNF598 mediated eS10 and uS10 ubiquitylation ( Ikeuchi et al . , 2019; Juszkiewicz et al . , 2018 ) .", "In this case , it remains conceivable that the individual ubiquitylation events on eS10 and uS10 may not be taking place on the same ribosome , but rather occur on neighboring , collided ribosomes .", "For example , upon collision with the trailing ribosome , ZNF598 may mediate ubiquitylation of eS10 on the leading ribosome followed by ubiquitylation of uS10 on the trailing ribosome or vice versa .", "This could require a specific conformation of ZNF598 in order to traverse both ribosomes , or following addition of the first ubiquitin the ligase moves upstream to the next site .", "Support for this model comes from studies in yeast that indicate ribosome collisions are critical events to initiate the no-go RNA decay pathway ( Simms et al . , 2017 ) , and are required for upstream cleavage of the defective mRNA by the endonuclease Cue2 ( D'Orazio et al . , 2019; Guydosh and Green , 2017 ) .", "Having shown that modification of both eS10 and uS10 are required to prevent readthrough of polyA-induced stalls , it is probable that the collision with the leading ribosome triggers combinatorial ubiquitylation events that are required for recruitment of downstream RQC factors .", "Further biochemical analysis is needed to determine the cellular role of RRub events in recruitment of quality control factors and reshaping the translational landscape ." ], [ "The dual-fluorescence translation stall reporter plasmids were described previously ( Juszkiewicz and Hegde , 2017; Sundaramoorthy et al . , 2017 ) .", "All Dub coding regions were cloned into Myc-tagging CMV expression vectors using Gateway cloning system ( Invitrogen ) .", "QuickChange site-directed mutagenesis was done utilizing PCR-based approaches ( primers 5’ to 3’: OTUD3-C76S , GGGGACGGCAATAGCTTGTTCAGAGC; OTUD1-C320S , CATTCCAGACGGCAACAGCCTCTACCGAGCTG; OTUB2-C15S , GGGGATGGGAACAGCTTCTACAGGG; USP10-C424S , GATCAATAAAGGGAACTGGAGCTACATTAATGCTACACTG; USP21-C250S , CCTGGGAAACACGAGCTTCCTGAATGC ) , followed by Dpn1 digestion of template DNA and transformation of the mutated plasmids into TOP10 E . coli cells .", "Plasmids were confirmed by sequencing and screened for expression by immunoblotting .", "All reagents utilized in this study can be found in Supplementary file 1: Key Resources Table .", "HCT116 and HEK293 cells were grown in DMEM ( high glucose , pyruvate and L-Glutamine ) containing 10% fetal bovine serum ( FBS ) and 1% penicillin/streptomycin and maintained in a 5% CO2 humidified incubator .", "Where indicated , cells were exposed to 0 . 02 J/cm2 UV radiation using a Spectorlinker XL-1000 ( Spectronics ) before harvesting or treated with 5 mM DTT or 2 μg/mL Haringtonine for 4 hr before harvesting .", "Ansiomycin was used at a final concentration of 5 μg/ml .", "HCT116 knock-in cells ( uS10 K4R/K8R and eS10 K138R/K139R ) were generated using CRISPR/Cas9 genome engineering approaches ( Biocytogen ) .", "Individual clones were first validated by genomic sequencing .", "Cells containing the desired point mutation were selected for validation by immunoblotting .", "USP21 and OTUD3 knock out cell lines were generated in the 293T cell background using CRISPR/Cas9 genome engineering utilizing three individual guide RNAs ( oligonucleotides 5’ to 3’: USP21: CAAGTATCGGTGGAGCCCGG , GGTAGCTTGGATCCCACTCG , TATGGAGCACGAGGATTCGA; OTUD3: CGGAATCGGCCGGAGTCTGG , CAACGCTTGAGGCGGACGCT , GCTCTTGGTGATCAATTGGA ) .", "293T cells were transfected with the pSpCas9 ( BB ) −2a-GFP plasmid containing individual guide RNAs using lipofectamine 2000 .", "After 2 days GFP positive cells were single cell sorted on a BD FACSAria Fusion ( BD BioSciences ) cell sorter .", "Cells were validated for loss of USP21 and OTUD3 by immunoblotting .", "Stable doxycycline inducible cell lines expressing Flag-HA-tagged proteins were generated using the Flp-In system ( Thermo Fisher ) through single locus integration , and Hygromycin selection .", "Flp-In T-Rex 293 cells were transfected with Flp-In expression vectors for the gene of interest ( listed in resource table ) using TransIT 293 transfection reagent ( Mirus ) according to manufacturer guidelines .", "Cells seeded at 60% confluency were transfected for 24 hr followed by selection of stable expression clones with 100 μg/mL Hygromycin .", "Protein expression was induced with 2 μg/mL doxycycline 16 hr prior to UV exposure , drug treatment , or harvesting .", "All cellular transient transfections were performed using Lipofectamine 2000 ( Thermo Fisher ) and siRNA ( see Supplementary file 1: Key Resources Table ) knockdown transfections were performed using Lipofectamine RNAiMAX ( Thermo Fisher ) according to manufacturer guidelines .", "All dual-fluorescent reporter plasmid cellular transfections were done using Lipofectamine 2000 according to manufacturer guidelines .", "Cellular ChFP and GFP fluorescence intensities for 10 , 000 individual events were measured 48 hr following transfection on a BD LSRFortessa X-20 cell analyzer ( BD Biosciences ) .", "FACS data was analyzed using FlowJo ( v10 . 4 . 1 ) .", "Cell pellets were resuspended in denaturing lysis buffer ( 8 M urea , 50 mM Tris-Cl , pH 8 . 0 , 75 mM NaCl , 1 mM β-glycerophosphate , 1 mM NaF , 1 mM NaV , 40 mM NEM and EDTA-free protease inhibitor cocktail ( Roche Diagnostics ) ) and kept on ice throughout preparation .", "Cell lysates were sonicated for 10 s at output of 3 W with a membrane dismembrator model 100 ( Fisher Scientific ) with a microtip probe followed by centrifugation at 15 , 000 rpm at 4°C for 10 min .", "Supernatant protein concentrations were determined by BCA Protein assay ( Thermo Fisher ) .", "Laemmli sample buffer with β-mercaptoethanol was added to cell lysates and heated at 95°C for 10 min .", "Lysates were resolved on 12 or 15% SDS-PAGE gels then transferred to PVDF membranes ( Bio-Rad ) using Bjerrum semi-dry transfer buffer ( 48 mM Tris Base , 39 mM Glycine-free acid , 0 . 0375% SDS , 20% MeOH , pH 9 . 2 ) and a semi-dry transfer apparatus ( Bio-Rad Turbo Transfer ) at 25V for 30 min .", "Immunoblots were blocked with 5% blotting grade nonfat dry milk ( APEX Bioresearch ) in TBST for 1 hr , followed by diluted primary antibody in 5% BSA over-night .", "Membranes were probed with anti-RPS2 ( Cat# A303-794A , Bethyl ) ; anti-RPS3 ( Cat# A303-840A , Bethyl ) ; anti-RPS10 ( Cat# ab151550 , Abcam ) ( antibody was used in Figures 1E , 3E , 4E , 5A–D and 7C , E , Figure 6—figure supplement 1E , F ) ; anti-RPS10 ( Cat# A6056 , ABclonal , this was used for all other RPS10 ( eS10 ) immunoblots ) ; anti-RPS20 ( Cat# ab133776 , Abcam ) ; anti-ZNF598 ( Cat# HPA041760 , Sigma ) ; anti-OTUD3 ( Cat# ab107646 , Abcam ) ; anti-USP21 ( RRID:AB_10603227 , Cat# HPA028397 , Sigma ) ; anti-USP21 ( Cat# ab171028 , Abcam ) ( antibody was used in Figure 6—figure supplement 1D; anti-phospho-eIF2α ( Ser51 ) ( D9G8 ) ( Cat# 3398S , Cell Signaling Tech ) ; anti-c-Myc ( 9E10 ) ( Cat# sc-40 , Santa Cruz ) ; anti-ubiquitin ( Cat# MAB1510 , EMD Millipore ) ; anti-HA ( Cat# MMS-101P , Biolegend ) ; anti-tubulin ( Cat# 3873S , Cell Signaling Tech ) ; anti-Rabbit IgG , HRP ( Cat# W4011 , Promega ) ; or anti-Mouse IgG , HRP ( Cat# W4021 , Promega ) .", "Immunoblots were developed with Clarity Western ECL Substrate ( Bio-Rad ) and imaged on a Bio-Rad Chemi-Doc XRS+ system .", "Imagelab ( Bio-Rad ) software was used to process all blots with final images prepared in Adobe Illustrator .", "Cells were lysed in 600 ul of ( 50 mM Hepes , pH 7 . 4 , 100 mM KAc , 2 . 5 mM MgAc2 , 0 . 5% Triton X-100 , 1 mM DTT , 1 mM NaF , 1 mM NaV , 40 mM NEM and EDTA-free protease inhibitor cocktail ) buffer and 500 ul of whole cell extracts were pelleted over a 0 . 5 M sucrose cushion ( 500 ul ) by spinning whole cell lysate in a TLA120 . 2 rotor at 100 , 000 rpm at 4°C for 35 min .", "Pelleted material was resuspended in Laemmli sample buffer with β-mercaptoethanol and heated at 95°C for 10 min followed by standard immunoblotting .", "Cells were lysed in 600 ul of ( 500 mM Tris , pH 7 . 8 , 1 . 5M NaCl , 150 mM MgCl2 , 0 . 5% NP-40 , 150 ug/ml cycloheximide , 8 U/ml SUPERase In RNAse inhibitor , 1 mM NaF , 1 mM NaV , 40 mM NEM and EDTA-free protease inhibitor cocktail ) lysis buffer followed by sonication and clarification of lysate at 15 , 000 rpm at 4°C for 10 min . 500 ul of whole cell extract was fractionated over a 10–50% sucrose gradient ( prepared on Gradient Master 108 ( Biocomp ) : 1 min 38 s , 81 . 5 degrees , 18 rpm ) spinning at 41 , 000 rpm at 4°C for 2 hr in an SW41i rotor .", "1 ml fractions were collected using a PGFip piston gradient fractionator ( Biocomp ) .", "Protein fractions were precipitated overnight at 4°C with 10% Trichloroacetic acid ( TCA ) followed by three washes with ice-cold acetone .", "Pellets were dried in Vacufuge plus ( Eppendorf ) at room temperature for 5 min then resuspended in Laemmli sample buffer with β-mercaptoethanol and heated at 95°C for 10 min followed by standard immunoblotting .", "All FACS-based assays , plasmid transfections and siRNA transfections were performed in triplicate ( n = 3 ) as biologically distinct samples .", "The mean ChFP:GFP ratio and SEM were calculated and compared to K20-reporter transfection alone , parental cell type or control siRNA knockdown .", "Immunoblot quantification of the relative ubiquitin modification was calculated by normalization of the individual Ub intensities for each time point to that of the no UV control .", "Significance ( p value ) was calculated using an unpaired two-tailed Student’s t test using GraphPad Prism 7 . 0 ." ] ]

[ "Activation of the integrated stress response ( ISR ) or the ribosome-associated quality control ( RQC ) pathway stimulates regulatory ribosomal ubiquitylation ( RRub ) on distinct 40S ribosomal proteins , yet the cellular role and fate of ubiquitylated proteins remain unclear .", "We demonstrate that uS10 and uS5 ubiquitylation are dependent upon eS10 or uS3 ubiquitylation , respectively , suggesting that a hierarchical relationship exists among RRub events establishing a ubiquitin code on ribosomes .", "We show that stress dependent RRub events diminish after initial stimuli and that demodification by deubiquitylating enzymes contributes to reduced RRub levels during stress recovery .", "Utilizing an optical RQC reporter we identify OTUD3 and USP21 as deubiquitylating enzymes that antagonize ZNF598-mediated 40S ubiquitylation and can limit RQC activation .", "Critically , cells lacking USP21 or OTUD3 have altered RQC activity and delayed eS10 deubiquitylation indicating a functional role for deubiquitylating enzymes within the RQC pathway ." ]

[ "Ribosomes are cellular machines that build proteins by latching on and then reading template molecules known as mRNAs .", "Several ribosomes may be moving along the same piece of mRNA at the same time , each making their own copy of the same protein .", "Damage to an mRNA or other problems may cause a ribosome to stall , leading to subsequent collisions .", "A quality control pathway exists to identify stalled ribosomes and fix the ‘traffic jams’ .", "It relies on enzymes that tag halted ribosomes with molecules known as ubiquitin .", "The cell then removes these ribosomes from the mRNA and destroys the proteins they were making .", "Afterwards , additional enzymes take off the ubiquitin tags so the cell can recycle the ribosomes .", "These enzymes are key to signaling the end of the quality control event , yet their identity was still unclear .", "Garshott et al . used genetic approaches to study traffic jams of ribosomes in mammalian cells .", "The experiments showed that cells added sets of ubiquitin tags to stalled ribosomes in a specific order .", "Two enzymes , known as USP21 and OTUD3 , could stop this process; this allowed ribosomes to carry on reading mRNA .", "Further work revealed that the ribosomes in cells that produce higher levels of USP21 and OTUD3 were less likely to stall on mRNA .", "On the other hand , ribosomes in cells lacking USP1 and OTUD3 retained their ubiquitin tags for longer and were more likely to stall .", "The findings of Garshott et al . reveal that USP21 and OTUD3 are involved in the quality control pathway which fixes ribosome traffic jams .", "In mice , problems in this pathway have been linked with neurons dying or being damaged because toxic protein products start to accumulate in cells; this is similar to what happens in human conditions such as Alzheimer's and Parkinson's diseases .", "Using ubiquitin to target and potentially fix the pathway could therefore open the door to new therapies ." ]

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

[ "cell biology", "genetics and genomics" ]

[ [ "In photosynthetic eukaryotes chloroplasts fulfill many essential functions such as photosynthetic conversion of light into chemical energy , synthesis of essential amino acids , fatty acids and other secondary metabolites .", "Moreover , they act as signaling platforms during plant development and stress adaptation , as they can alter the expression of thousands of nuclear genes and influence many cellular activities that are key to plant performance ( Chan et al . , 2016 ) .", "Selective impairment of protein homeostasis in chloroplasts triggers the chloroplast unfolded protein response ( cpUPR ) , a conserved organelle quality control pathway ( Ramundo et al . , 2014; Llamas et al . , 2017 ) .", "Akin to unfolded protein responses operating from the endoplasmic reticulum ( ER ) and mitochondria ( Walter and Ron , 2011; Shpilka and Haynes , 2018 ) , the cpUPR invokes comprehensive transcriptional changes thought to mitigate an increased burden of proteotoxicity in the organelle .", "As such , the cpUPR comprises the selective up-regulation of nuclear encoded chloroplast-localized small heat shock proteins , chaperones , proteases , and proteins involved in chloroplast membrane biogenesis .", "Furthermore , other pathways , such as autophagy and sulfur uptake are activated to mitigate general cellular stress caused by chloroplast metabolic dysfunctions ( Ramundo et al . , 2014 ) .", "In the single-celled alga Chlamydomonas reinhardtii , the cpUPR is induced after either inactivation of the Clp protease , which degrades misfolded chloroplast proteins in the organelle’s stroma ( Figure 1A ) , or exposure to higher than normal light intensity ( high light ‘HL’ ) , which causes protein damage through the production of reactive oxygen species in the chloroplast ( Ramundo et al . , 2014 ) .", "Similarly , in higher plants , mutants with constitutively reduced levels of the Clp and FtsH proteases selectively upregulate the expression of chloroplast chaperones , such as Cpn60 , Hsp70 , Hsp90 , Hsp100 ( Llamas et al . , 2017; Zybailov et al . , 2009; Sjögren et al . , 2004; Rudella et al . , 2006; Dogra et al . , 2019 ) .", "However , the mechanism by which chloroplast proteotoxic stress is monitored and how the signal is transmitted from the organelle to the nucleus has remained unknown ." ], [ "In summary , our results suggest that , upon onset of chloroplast proteotoxic stress , a signal transduction pathway , originating in the chloroplast , leads to activation of the cytosolic kinase Mars1 , which in turn orchestrates the cpUPR transcriptional program .", "Activation of cpUPR through Mars1 mitigates photooxidative stress and delays photobleaching .", "However , loss of Mars1 does not impair expression of genes involved in non-photochemical quenching .", "Thus , the exact mechanism by which the cpUPR pathway confers photoprotection remains to be deciphered .", "Input conditions that activate the cpUPR , as well as cpUPR target genes identified to date , are phylogenetically conserved from C . reinhardtii to A . thaliana ( Llamas et al . , 2017; Zybailov et al . , 2009; D’Andrea et al . , 2018 ) .", "Thus , despite not yet having identified a functional ortholog of MARS1 in higher plants , it is reasonable to assume that the cpUPR’s previously unknown role in protecting cells against photooxidative stress would likewise be conserved .", "This notion is particularly appealing in light of the observation that basal induction of the cpUPR conferred a protective effect in response to stress and that , conversely , loss of Mars1 activity profoundly sensitized cells towards HL and other chloroplast stressors .", "Hence , engineering plants with constitutive cpUPR activation may be a promising strategy to enhance their tolerance to environmental stresses ." ], [ "All C . reinhardtii strains were maintained on Tris-Acetate-Phosphate ( TAP ) solid media ( 1 . 6% agar , USP grade , Thermo Fisher Scientific ) with revised Hutner’s trace elements ( Kropat et al . , 2011 ) at 22°C in low light ( ~10–20 µmol photons m−2 s−1 ) .", "Lines harboring the ClpP1 repressible gene were maintained in the media supplemented with 100 µg ml−1 spectinomycin ( Sigma ) .", "Lines harboring a mutagenic cassette disrupting the MARS1 gene were maintained in the media supplemented with 20 µg ml−1 paromomycin ( Sigma ) .", "Lines harboring a MARS1 transgene construct were maintained in the same conditions with solid media supplemented with 20 µg ml−1 hygromycin ( Thermo Fisher Scientific ) .", "Sulfur-depleted TAP liquid and agar pates were prepared as previously described ( Davies et al . , 1994 ) .", "Generally , during liquid growth , no antibiotic was supplemented .", "For the experiments shown in Figure 4—figure supplement 2A–C , TAP liquid cultures and agar plates were supplemented with 5 µg/ml or 0 . 2 µg/ml tunicamycin ( EMD Millipore ) , respectively .", "All strains used in this study are listed in Table 1 .", "The cpUPR reporter strain ( CrPW1 ) was generated by nuclear transformation of the C . reinhardtii ClpP1 repressible strain ( DCH16; Ramundo et al . , 2014 ) , using 300 ng of Nde1-linearized pPW3217 plasmid Table 2 for plasmids used in this study ) .", "Nde1 and all the other restriction enzymes described in this publication were purchased from NEB .", "The nuclear transformation was carried out via electroporation as described below ( section on ‘Insertional mutagenesis’ ) .", "Transformants isolated on TAP agar plates containing 20 µg ml−1 hygromycin and tested by Flag immunoblot analysis upon ClpP1 repression and exposure to HL .", "As previously observed ( Ramundo et al . , 2014; Ramundo et al . , 2013 ) , during random insertion of a construct with regulatory regions in its promoter , less than 10% of the hygromycin resistant transformants preserved the correct gene expression pattern of the downstream coding sequence .", "Among them , we selected CrPW1 for further studies .", "The pPW3217 plasmid , containing a minimum region of the VIPP2 gene promoter , its 5’ untranslated region and its first exonexon intrintrone yellow fluorescent protein coding sequence ( YFP CDS ) , C-terminally appended to a triple Flag epitepitope the 3’ untranslated region of the RBCS2 gene , was generated by In-Fusion cloning ( Clontech ) .", "The VIPP2 genomic fragment was amplified from genogenomic with primers SR510 and SR502 ( see Table 3 for primers used in this study ) .", "The YFP CDS fused to a 3x-Flag epitope was amplified from pLM005 ( Chlamydomonas Resource Center ) with primers SR503 and SR504 .", "The 3’ untranslated region of the RBCS2 gene was amplified from pHyg3 ( Berthold et al . , 2002 ) with primers SR505 and SR506 .", "All PCRsPCRse performed using Phusion Hotstart II polymerase ( Thermo Fisher Scientific ) .", "PCR products were gel-extracted using NucleoSpin Gel and PCR Clean-Up Kit ( Takara ) according to manufacturer’s instructions , and they were further purified by ultrapurephenol:chloroform:isoamyl alcohol ( 25:24:1 , v/v/v ) ( Life Technologies ) extraction and ice-cold ethanol ( Sigma ) precipitation .", "These three purified DNA fragments were then mixed with a purified and linearized pHyg3 vector , previously digested by PciI and EcoRV , and incubated with the In-Fusion reagents ( Takara ) as per manufacturer’s instructions .", "The In-Fusion product was transformed in Stellar competent cells ( Takara ) according to manufacturer’s instructions and putative positive clones were selected in LB solid media ( 1 . 7% agar ) supplemented with ampicillin after overnight incubation .", "The resulting plasmid , pPW3217 , was purified using the Kit PureYield Plasmid Miniprep System ( Promega ) and verified by analytical digestion and sequencing .", "All constructs made by In-Fusion cloning for this publication follow this same protocol from transformation through plasmid isolation .", "The mutagenic DNA cassette was isolated by restriction enzyme digestion of pMJ016c ( provided by the Jonikas laboratory ) , which contains the HSP70-RBCS2 chimeric promoter , the paromomycin resistance gene AphVIII , and the PSAD and RPL12 chimeric terminator ( Mackinder et al . , 2016 ) .", "Using the Mly1 enzyme , a blunt fragment of 2204 bp ( containing the mutagenic DNA cassette ) was isolated and extracted from a 1% agarose gel through the NucleoSpin Gel and PCR Clean-Up Kit ( Takara ) per the manufacturer’s instructions .", "To further remove possibly contaminating DNA , the mutagenic DNA cassette was electrophoresed on a 1% agarose gel , extracted and repurified as explained above .", "A 1-liter liquid culture of cpUPR reporter strain ( CrPW1 ) was grown in TAP medium in low light ( ~30 µmol photons m−2 s−1 ) to a density of about 2–4 × 106 cells ml−1 .", "Cells were collected at room temperature ( RT ) by centrifugation at 3000 x g for 5 min and gently resuspended in TAP supplemented with 40 mM sucrose at 2 × 108 cells ml−1 .", "Multiple aliquots of 250 µL of cell suspension were then transferred into Gene Pulser electroporation cuvettes ( 0 . 4 cm gap , Bio-Rad ) and incubated at 16°C for 5–30 min .", "In each cuvette , about 20 ng of mutagenic DNA cassette was added to the cell suspension and quickly mixed by pipetting .", "Electroporation was performed immediately using a Gene Pulser II electroporation system ( Bio-Rad ) with the following parameters: capacitance = 25 μF and voltage = 800 V . Electroporated cells from each cuvette were then diluted into 8 ml TAP supplemented with 40 mM sucrose and allowed to recover overnight by gentle agitation in very dim light ( 5–10 µmol photons m−2 s−1 ) .", "The next day , cells were collected by centrifugation at 1000 x g for 5 min , resuspended in 1 ml of TAP medium , plated on TAP agar plates containing 20 µg ml−1 paromomycin and incubated in darkness for about three weeks before picking colonies .", "Approximately 55 , 000 total mutants were picked and re-arrayed in a 384-colony format on rectangular agar plates ( Singer Instruments ) using a Norgren CP7200 colony-picking robot .", "In each 384-mutant array plate , the last two rows were kept empty to include internal positive and negative controls for the next stage of the screen ( for details , read section ‘Execution of YFP mutant screen on agar plates’ ) .", "This library of mutants ( of approximately 150 agar plates ) was grown in complete darkness at 22°C and propagated every 3–4 weeks by robotically passaging the mutant arrays to fresh 1 . 5% agar solidified TAP medium containing 100 µg ml−1 spectinomycin using a Singer RoToR robot ( Singer Instruments ) .", "Unfortunately , numerous mutants were lost during propagation due to a wide-spread contamination event .", "To screen for YFP silencing or activating mutants , rectangular agar plates , each containing 97 ml of TAP medium -/+ Vit ( 400 μM thiamine-HCl ( Sigma ) and 80 ng ml−1 of vitamin B12 ( Sigma ) ) , were prepared .", "In each agar plate ( -/+ Vit ) , 12 colonies of the cpUPR reporter strain ( CrPW1 ) , 12 colonies of the parental ClpP1 repressible strain ( DCH16 ) and 12 colonies of 2 different positive YFP expressor strains ( CrPW2 and CrPW3 ) were robotically spotted in the last two rows of the 384-colony array to be used as internal positive and negative controls during the YFP screen ( see scheme in section ‘Semi-automated identification of YFP mutants through Image-J macroscripts’ ) .", "Next , insertional mutants ( freshly propagated ) were spotted onto these same plates .", "Plates were incubated in dim light ( 20–30 µmol photons m−2 s−1 ) at 25°C and were imaged after 2 and 6 days using a Typhoon TRIO fluorescence scanner ( GE Healthcare ) .", "For each round , 12 plates of insertional mutants ( 6 without and six with Vit ) were simultaneously scanned with the settings described below .", "Chlorophyll Autofluorescence: Excitation 633 nm , Emission filter 670/30 nm , PMT 300 , Sensitivity Normal , Pixel size 500 μm .", "YFP Fluorescence: Excitation 532 nm , Emission filter 555/20 nm , PMT 800 , Sensitivity Normal , Pixel size 500 μm .", "The focal plane parameter was set at ‘plus 3 mm’ to focus the optics 3 mm higher than the glass plate .", "This last detail and the thickness of the agar plate ( 97 ml of liquid agar ) were critical parameters to successfully detect the YFP signal .", "We used macroscripts in ImageJ64 software ( Schneider et al . , 2012 ) to quantify the intensity values of colonies in the mutant library ( for details , please refer to Source code 1 ) .", "Each plate was imaged in the YFP and in the chlorophyll channel .", "To orient each image , the bottom two rows of each plate were spotted with characterized positive and negative YFP strains ( CrPW1 , DCH16 , CrPW2 , and CrPW3 ) in the specific order outlined in the scheme below , where ( - ) denotes lack of YFP signal and ( + ) denotes presence of YFP signal .", "The ordering of these colonies conferred a reproducible fluorescent pattern in the YFP channel , which was used to identify the bottom two rows of each image .", "Plate ( -Vit ) position123456789101112131415161718192021222324StrainDCH16CrPW1DCH16CrPW2CrPW3CrPW1CrPW3CrPW1CrPW2DCH16CrPW2CrPW3YFP signal------++++--++--++--++++Plate ( +Vit ) position123456789101112131415161718192021222324StrainDCH16CrPW1DCH16CrPW2CrPW3CrPW1CrPW3CrPW1CrPW2DCH16CrPW2CrPW3YFP signal--++--++++++++++++--++++ To quantify the YFP intensity of each mutant , a 16 × 24 array containing 384 Regions-Of -Interest ( ROIs ) was constructed for each image in the chlorophyll channel , where all living colonies exhibited signal .", "The same grid was applied to the corresponding image in the YFP channel .", "From each ROI on autothresholded images , the maximum intensity in the YFP channel was measured .", "To account for variability in the magnitude of the YFP response on different plates ( due to slight variations in agar thickness ) , it was necessary to normalize the YFP intensity of each mutant colony to the YFP intensity of its parental strain ( CrPW1 ) from the same plate ( average of n = 12 ) .", "Colonies exhibiting YFP fluorescence higher than three standard deviations from the average of all colonies were labeled as potential activators , while colonies with YFP intensities below three standard deviations from the average were labeled as potential silencers .", "Of the potential cpUPR silencers , we observed that many of these mutants grew to a larger colony size than the parental CrPW1 strain after 6 days .", "Their robust growth suggested suppression of vitamin-induced ClpP1 inactivation .", "To exclude these suppressor mutants , we analyzed the area of all the colonies at 2 and 6 days by measuring the particle area of autothresholded images in the chlorophyll channel .", "The average colony size increase was 1 . 38-fold for the 10000-plus colonies analyzed .", "Candidate colonies that increased 2-fold in colony area ( more than one standard deviation away from the average ) were regarded as suppressors .", "Of the remaining silencing candidates , mars1-1 exhibited the most attenuated YFP response in the presence of Vit .", "We indicate mars1-2 with an asterisk ( * ) in Figure 2A because this mutant was identified in a secondary screen but its position in the original mutant library was lost due to contamination of the plate .", "To evaluate the YFP response and colony size of mars1-2 in the context of the entire mutant library , we re-spotted mars1-2 on a fresh agar plate containing the characterized cpUPR reporter strain and the other positive and control strains in the bottom two rows as described above .", "The normalized YFP intensity of mars1-2 in the absence and presence of Vit was then mapped onto the quantification of the original mutant colonies .", "With the single exception of the DNA samples submitted for whole genome sequencing , all the other genomic DNA ( gDNA ) extractions were performed as described below .", "A 6 ml aliquot of a liquid TAP culture in mid-log phase were spun down , and the media was decanted .", "The pellet was resuspended in 400 µl of water and then 1 vol of 2x DNA lysis buffer was added ( 200 mM Tris HCl pH 8 . 0 , 6% Sodium Dodecyl Sulfate ( SDS ) , 2 mM EthyleneDiamineTetraAcetic acid ( EDTA ) .", "To digest proteins , 5 µl of 20 mg/ml proteinase K ( Life Technologies ) was added and allowed to incubate at Room Temperature ( RT ) for 15 min . 200 µl of 5M NaCl was then added and mixed gently .", "Next , to selectively precipitate nucleic acids , 160 µl of 10% cetyltrimethyl ammonium bromide ( CTAB ) ( EMD Millipore ) in 0 . 7 M NaCl was added and allowed to sit for 10 min at 65°C with gentle agitation .", "Two or more consecutive rounds of DNA extraction using ultrapure phenol:chloroform:isoamyl alcohol ( 25:24:1 , v/v/v ) were performed to achieve a clean interphase .", "Then , the upper aqueous phase was retained and mixed with 1 vol of 2-propanol ( Sigma ) .", "This was mixed gently for 15 min at RT .", "Then it was spun down for half an hour at 21 , 000 x g at 4°C .", "The supernatant was removed and 1 vol of ice-cold 70% ethanol was added and mixed with the pellet .", "This mixture was spun down for 15 min at 21 , 000 x g .", "The supernatant was removed and the DNA precipitate was dried in a speed-vac for about 10–25 min and resuspended in 40 µl of nuclease-free water ( Ambion ) .", "To ensure complete removal of any potential RNA contamination , in most cases , the gDNA prep was then subjected to in-solution ribonuclease treatment using Rnase A/Rnase T1 mix ( Thermo Fisher Scientific ) according to manufacturer’s instructions .", "Finally , the gDNA was quickly repurified through an additional round of DNA extraction using ultrapure phenol:chloroform:isoamyl alcohol ( 25:24:1 , v/v/v ) and 2-propanol precipitation as described above .", "The purity of the gDNA prep was assessed by Nanodrop ( Thermo Fisher Scientific ) , ensuring absorbance ratios at 260/280 nm and 260/230 nm to be ~1 . 8 and ~2 . 0 , respectively , prior to using the gDNA preparation for most of the follow-up applications .", "For the pooled ( whole genome sequencing ) DNA samples , the genomic DNA extraction was performed with the following protocol adapted from the Qiagen , DNeasy Plant Mini Kit using its proprietary buffers ( Buffer P3 , AW1 , AW2 , AE ) .", "A 25 ml culture of each progeny was grown for ~2 days to about 3 × 106 cells ml−1 .", "Cells were then pelleted and resuspended in 0 . 5 ml of SDS-EB lysis buffer buffer ( 50 mM Tris-HCl , 200 mM NaCl , 20 mM EDTA , nuclease-free H2O , 2% SDS , 1% Polyvinylpyrrolidone -average molecular weight = 40 , 000- , 1 mg/ml of proteinase K ) and allowed to incubate for ~10 min .", "One volume of phenol:chloroform:isoamyl alcohol ( 25:24:1 , v/v ) was added and mixed vigorously .", "The mixture was spun at 13 , 500 x g for 5 min at RT .", "The upper phase was transferred into new Eppendorf tubes and 5 µl of 100 mg/ml of RNase A , was added and incubated at RT for 30 min .", "The lysate was mixed with 130 µl aliquot of Buffer P3 and incubated on ice for 5 min .", "This mixture was spun at 18 , 400 x g for 5 min at RT .", "The lysate was transferred to the QIAshredder Mini column and spun for 18 , 500 x g for 2 min .", "The flow-through fraction was transferred into a new tube and 1 . 5 volumes of Buffer AW1 was added to the cleared lysate and mixed well .", "This mixture was then transferred to a DNeasy Mini column and spun at 7600 x g for 1 min .", "The flow-through fraction was discarded and this step was repeated for any remaining mixture .", "The DNeasy Mini column was transferred to a new collection tube and 500 µl of Buffer AW2 was added , centrifuged at 7600 x g for 1 min and the flow-through discarded .", "Another 500 µl of Buffer AW2 was added and centrifuged at 18 , 400 x g to dry the membrane .", "The column was then transferred to a new Eppendorf tube .", "90 µl of Buffer AE was added onto the DNeasy membrane and incubated for 5 min at RT .", "The DNA was eluted by centrifugation at 7600 x g for 1 min .", "This step was repeated again , using the 90 µl of Buffer AE collected after the first centrifugation .", "The quality of the DNA samples was assessed by Nanodrop as described above .", "The DNA samples were then stored in −20°C until use .", "The protocol was optimized for single-colony DNA sequencing from the original protocol ( Li et al . , 2016 ) .", "A pure genomic DNA preparation was assured by running the DNA on a 1 . 5% agar gel prior to starting .", "A single-stranded DNA fragment was generated by extending a biotinylated primer from the cassette to the flanking DNA using either primer oMJ598 or primer oMJ1234 , which anneals to the 3’ or 5’ end of the mutagenic cassette , respectively .", "The linear extension mix was set in the following way: 500 ng of gDNA , 2 µl of 0 . 25 µM of biotinylated primer , 0 . 5 µl of Phusion Hot Start Polymerase ( Thermo Fisher Scientific ) , 10 µl of Phusion GC buffer , 3 µl of DMSO , 1 µl of 50 mM MgCl2 , 1 µl of 10 mM dNTPs .", "Prior to starting the mix , the GC buffer was thawed , heated to 95°C for five minutes , vortexed and then put back on ice until the solution became completely clear .", "The linear extension reaction was carried out in a thermocycler with the following program: Stage 1 ) 98°C for 3 min; Stage 2 ) 98°C for 10 s , 65°C for 30 s , 72°C for 18 s ( 40 cycles ) .", "This program was run twice and , in between the first run and the second run , 0 . 5 µl of Phusion Hot Start Polymerase was added .", "The Dynabeads kilobase Binder Kit ( Thermo Fisher Scientific ) was used to purify the linear extension product .", "For each reaction , 8 µl of streptavidin-coupled magnetic beads ) were transferred into an Eppendorf tube and washed in 100 µl of phosphate-buffered saline ( PBS ) up to four times using a DynaMag magnet ( Thermo Fisher Scientific ) .", "The Dynabeads were then washed once more in 20 µl of binding solution and gently resuspended in 100 µl of binding solution , pipetting up and down only a few times .", "Next , the beads were transferred in the PCR tube from the linear extension reaction described above .", "To allow efficient binding of the linear extension product to the streptavidin-couple magnetic beads , the samples were incubated overnight at RT on an overhead-rotating platform .", "The following day , the linear extension product was isolated and ligated to a single-strand DNA adaptor , per the following procedure .", "The beads were washed three times with 100 µl of PBS allowing 8 min incubation in between washes .", "At the end of the final wash , we ensured that all PBS was carefully removed to avoid interference with the ssDNA ligation reaction .", "A 20 µl ssDNA ligation reaction was added and gently mixed with the magnetic beads .", "The ligation mix contained 11 . 25 µl of H2O , 1 µl of 25 µM ssDNA adapter primer ( oMJ619 ) , 1 µl of 50 mM MnCl2 , 4 µl of 5 M betaine , 2 µl of CircLigase II reaction buffer , 0 . 75 µl of CircLigase II ( Epicentre ) .", "The beads were transferred to the thermocycler , which was pre-heated to 60°C for 10 min .", "This mixture was incubated for 1 hr at 60°C .", "The beads were then washed three times with 100 µl of PBS as described above .", "Next , the ssDNA was converted to a dsDNA using primers annealing to the ligated adaptors at the ends of the ssDNA sequence .", "1 µl of 25 µM of Primer 1 ( see below ) , 1 µl of 25 µM of Primer 2 ( oMJ621 ) , 0 . 5 µl of Phusion HotStart , 10 µl of Phusion GC buffer , 32 . 5 µl of H2O , 3 µl of DMSO , 1 µl of 50 mM MgCl2 , 1 µl of 10 mM dNTPs and the template DNA ( beads ) were mixed together .", "Primer one depended on whether the original extension from the cassette was in the 5’ or 3’ orientation .", "If the 3’ cassette flanking primer ( oMJ598 ) was used during the linear extension , primer T3_3’oMJ016c 11/23 was chosen .", "Instead , when the 5’ cassette flanking primer ( oMJ1234 ) was used during linear extension , oMJ1239 was chosen .", "Primer 2 ( oMJ621 ) annealed to the ligated adaptor .", "Both primers were designed to contain a binding site for the mutagenic cassette and a binding site for a T3 sequencing primer .", "The following amplification program was used: Stage 1 ) 98°C for 3 min , Stage 2 ) 98°C for 10 s , 63°C for 25 s , 72°C for 20 s ( 10 cycles ) , Stage 3 ) 98°C for 10 s , 72°C for 45 s ( 13 cycles ) .", "The dsDNA products were then run on a 1% gel .", "The DNA smears were cut out of the agarose gel , purified using NucleoSpin Gel and PCR Clean-Up Kit ( Takara ) according to manufacturer’s instructions and subjected to Sanger Sequencing using a standard T3 sequencing primer .", "Finally , to identify the insertion site of the mutagenic cassette , the sequencing results were blasted in Phytozome , v5 . 5 .", "The sequence of the aforementioned primers can be found in Table 3 .", "Note: mars1-1 and mars1-2 proved difficult to mate due to their genetic background .", "Therefore , extra measures were taken to increase the efficiency of mating .", "mars1-3 and mars1-4 , obtained from the Jonikas library , did not have this problem , therefore , these extra measures were not taken .", "The following protocol will indicate the differences .", "Cells were re-streaked onto fresh TAP agar and incubated in low light ( <15 µmol photons m−2 s−1 ) for five days .", "They were then transferred onto TAP agar containing 1/10 of the usual NH4Cl concentration and kept in this medium for four-five days under moderate light ( ~40 µmol photons m−2 s−1 ) to induce starvation .", "The gametes from each strain were then resuspended in a 24-well sterile transparent plate ( Costar ) using 150–200 µl of water or M-N/5 solution till a dark green resuspension is obtained .", "M-N/5 solution was used for the mars1-1 and mars1-2 backcrosses , and water was used for mars1-3 and mars1-4 backcrosses .", "M-N5 solution contained 1 ml of 10% sodium citrate , 0 . 2 ml of 1% FeCl3 , 0 .", "2 ml of 4% CaCl2 , 0 . 34 ml of 10% K2HP04 , 0 . 2 ml of 10% KH2PO4 , 0 . 2 ml of Hutner’s Trace elements ( Chlamydomonas Resource Center ) , H2O to 1 . 25 liter .", "The solution was autoclaved in 100 ml aliquots per bottle .", "The plate was then transferred to a shaker under moderate light ( ~40 µmol photons m−2 s−1 ) and allowed to mix for ~1 hr .", "Gametes of the opposite mating type were mixed ( ~100 µl per gamete ) in a separate well and the plate was placed under light with no shaking .", "For the mars1-1 and mars1-2 strains , after one hour of mixing , dibutyryl cyclic AMP ( Sigma ) was added to each mating mix to a final concentration of 30 mM .", "Mating efficiency was checked periodically ( every 15–30 min ) for fusion events and quadriflagellate formation .", "The gametes were mated for ~3 hr .", "Aliquots ( 100 µl ) of the mating mixture were plated into TAP 4% agar .", "Plates were exposed to light ( ~50 µmol photons m−2 s−1 ) overnight and the next day wrapped in aluminum foil .", "After ~1–2 weeks , the vegetative cells were scraped off using a small rectangular soft razor blade ( Personna , . 009’' , two-facet aluminum blade ) with gentle pressure on the agar .", "Zygotes adhere to the agar surface and can be recognized under a light microscope due to their darker and larger appearance .", "A 100 µl aliquot of liquid TAP medium was then added on top of the zygotes and a more rigid scalpel ( Feather , N . 2 ) was used to scrape the zygotes off the agar .", "A line was drawn onto the center of a fresh TAP 1 . 5% agar plate , and the zygotes were spotted along this line .", "The cells were then allowed to dry .", "For mars1-1 and mars1-2 , but not for mars1-3 and mars1-4 , vegetative cells were killed by treating the plate with chloroform vapor for ~15–30 s .", "The plate was incubated under light ( ~80 µmol photons m−2 s−1 ) overnight to 1 . 5 days .", "At 24°C , germination typically occurred after ~20 hr .", "Under the dissection scope , tetrads and octads were found and dissected .", "Incomplete tetrads , full tetrads and octads were then re-arrayed onto TAP agar in a 96-array format and then replicated onto the appropriate drug resistances .", "When necessary , mating type-specific PCRs ( Werner and Mergenhagen , 1998 ) were carried out to ensure that the progeny were in fact due to a sporulation event and were not mistakenly parental strains .", "Genomic DNA from progeny derived upon crossing mars1-1 to CC-124 was obtained as outlined in ‘Genomic DNA extraction’ section .", "The insertion of the mutagenic cassette in the MARS1 locus was verified by PCR by using primers SR773 and T3_5'_oMJ016c 11/24 , which anneal to exon 17 of MARS1 , and to the 5’ side of the mutagenic cassette , respectively .", "The PCR reaction was run on 1–1 . 5% agarose , cut out of the agarose gel , purified using NucleoSpin Gel and PCR Clean-Up Kit ( Takara ) according to manufacturer’s instructions and subjected to Sanger sequencing to verify the expected sequence identity .", "The sequence of the aforementioned primers can be found in Table 3 .", "Progeny derived upon crossing mars1-2 x CC-124 were re-arrayed onto 96-well plates and replicated onto TAP supplemented with hygromycin , paromomycin or spectinomycin to determine the segregation patterns of the progeny .", "Mating type-specific PCRs were performed on almost all progeny ( Werner and Mergenhagen , 1998 ) .", "The progeny was then tested in high light and by Vipp2 immunoblot analysis two or three times to determine which one had silencing vs . wild-type phenotypes .", "Genomic DNA was extracted as described above in the section ‘Genomic DNA extraction’ and was subsequently pooled per the progeny’s phenotype , i . e . mars1-like or WT-like .", "Additional pools containing the parental strains were also analyzed likewise .", "The size of the pools of gDNA were of different proportions depending on the amount of progeny in that group ( WT vs . mars1-like ) .", "The pooled gDNA was then fragmented using Covaris and Bioruptor Pico .", "The sequencing libraries were prepared with the aid of the PrepX DNA library kit ( Takara ) .", "One cycle of PCR was used to linearize the library molecules .", "Fragment analyzer traces and Qubit values were assessed for each sequencing library as quality control checks .", "Sequencing was performed on the HiSeq2500 Rapid sequencer .", "The C . reinhardtii reference genome was downloaded from Phytozome , v5 . 5 onto the Geneious software ( Kearse et al . , 2012 ) .", "The reads from each library were then aligned to the reference genome .", "Genomic DNA from progeny of meiotic tetrads derived upon crossing mars1-2 to CC-124 was obtained as outlined in ‘Genomic DNA extraction’ section .", "The MARS1 locus was amplified by using primers SR789 and KP235 , which anneal to exon 15 and the intron 19 - exon 20 junction of MARS1 , respectively .", "The MARS1 deletion locus was amplified by using primers KP346 , which anneals to intron 1 of the MARS1 gene , and KP347 , which was derived from the a WGS read found only in the ‘mars1-like’ progeny pool .", "The KP347 primer sequence is a hybrid of telomeric sequence and MARS1 gene sequence - this sequence seems to have arisen after a genomic deletion at the end of chromosome 16 in mars1-2 .", "The PCR reactions were then purified and sequenced as described above .", "The sequence of the aforementioned primers can be found in Table 3 .", "A MARS1 ‘midigene’ was generated by amplifying four different portions of this gene either from gDNA or cDNA using KOD Hot Start DNA Polymerase ( Thermo Fisher Scientific ) or Phusion Hotstart II polymerase ( Thermo Fisher Scientific ) .", "In particular , the region spanning the promoter , the 5’UTR and the first 5 exons of this gene was amplified from gDNA using Phusion polymerase and the following primers: SR828 and SR818; the region spanning exon 5 to exon 15 was amplified from gDNA using KOD polymerase and the following primers: SR819 and HT7; the region spanning exon 15 to exon 28 was amplified from cDNA using KOD polymerase and the following primers: SR789 and SR797 and the 3’UTR was amplified from gDNA using KOD polymerase and the following primers: SR793 and SR829 .", "All PCR products were gel extracted and purified as described above in the section regarding the pPW3217 cloning .", "Next , these 4 PCR fragments were mixed with a purified and linearized and pRAM118/pPW3216 vector , previously digested by EcoRV and Not1 , and incubated in presence of the In-Fusion reagents ( Takara ) as per manufacturer’s instructions .", "The resulting plasmid is notated as pPW3218 .", "The sequence of the aforementioned primers can be found in Table 3 .", "The Phytozome v5 . 5 MARS1 transcript annotation is Cre16 . g692228 . t1 . 1 .", "Given the large size of the MARS1 transgene ( >10 kbp ) , a different electroporation protocol was used to successfully integrate each aforementioned MARS1 transgene into the nuclear genome .", "The electroporation was performed using a NEPA21 electroporator ( Nepagene ) ( Yamano et al . , 2013 ) .", "A 4–8 µl aliquot of purified , non-linearized , plasmid DNA at a concentration of 0 . 5–1 mg ml−1 was used per transformation .", "In each case , the plasmid DNA was mixed together with 5 µl of a ready-to-use , sheared solution of salmon sperm DNA at a concentration of 10 mg ml−1 ( Thermo Fisher Scientific ) prior electroporation .", "The electroporation parameters were set as follows: Poring Pulse ( 300 V; length = 6 ms; Interval = 50 ms; No = 1; D . Rate = 40%; + Polarity ) , Transfer Pulse ( 20 V; length = 50 ms; Interval = 50 ms; No = 5; D . Rate = 40%; +/- Polarity ) .", "Usually , during the electroporation , the impedance was measured to be around 400–700 ohms .", "Transformants were isolated on TAP agar containing 20 µg ml−1 hygromycin and screened by Flag immunoblot analysis to identify MARS1 transgene expressors .", "Due to the low abundance of the Mars1 protein , the Alexa Fluor 488 Tyramide SuperBoost Kit ( Thermo Fisher Scientific ) was utilized to amplify the signal .", "WT ( mock control ) and Flag-tagged strains were grown in TAP medium to logarithmic phase and a 5 ml aliquot of each cell cultures was harvested at 500 x g for 5 min at RT .", "The supernatant medium was decanted and the cell pellet was resuspended in 1 . 5 ml of PBS .", "A 35 µl cell suspension was added to wells on a slide pre-treated with 0 . 1% poly-L-lysine and allowed to adhere for 7 min .", "To solubilize the chlorophyll and maintain cell structure , slides were incubated in 100% methanol 2 times for 4 min at −20°C .", "Excess methanol was removed and the slides were dried for 2 min at RT .", "Slides were incubated in PBS supplemented with 0 . 1% Tween 20 ( PBS-T ) for 10 min to permeabilize the cell before adding the manufacturer-provided 3% H2O2 solution to quench endogenous peroxidase activity for 1 hr .", "Following three washes with PBS , non-specific signal was blocked with 10% goat serum in PBS ( Jackson ImmunoResearch Laboratories ) for 1 hr .", "Samples were incubated with commercial monoclonal mouse anti-Flag antibody ( M2 Sigma , F1804 , diluted 1:500 ) in combination with one of three subcellular markers: anti-Histone H3 ( Agrisera , AS10 710 , diluted 1:500 ) , anti-AtpD ( Agrisera , AS10 1590 , diluted 1:500 ) , or anti-Nab1 ( Agrisera , AS08 333 , diluted 1:500 ) , and left overnight at 4°C in a humid chamber .", "The remaining steps were performed as per manufacturer’s instructions .", "In brief , samples were washed for 10 min in PBS for a total of three washes .", "Alexa Fluor 488 poly- horseradish peroxidase ( HRP ) -conjugated goat anti-mouse and Alexa Fluor 546 goat anti-rabbit secondary antibodies were utilized for detection of the Flag and subcellular marker signals respectively .", "Alexa Fluor 546 was diluted 1:500 in the manufacturer-prepared Alexa Fluor 488 solution and added to each well for 1 hr at RT protected from light .", "Slides were washed 3 times in PBS before the amplification step .", "To amplify the Flag signal , 35 μl of the Tyramide Working Solution was added to the cells and the reaction proceeded for 4 min before an equal volume of Stop Solution was added to end the reaction .", "Slides were rinsed in 1x PBS and covered with a thin layer of 0 . 5% low-melting point agarose dissolved in TAP medium before observation by 3D-Structured Illumination Microscopy ( 3D-SIM ) .", "The microscopy samples were observed using an Elyra PS . 1", "SIM microscope ( Zeiss ) with objective lens alpha Plan-Apochromat 100x/1 . 46 oil ( Immersol 518F/30°C , Zeiss ) , as described previously ( Iwai et al . , 2018 ) .", "The fluorophores Alexa Fluor 488 and Alexa Fluor 546 were excited with a 488 nm laser and 561 nm laser , and the fluorescence was acquired through a 495–550 nm and 570–620 nm bandpass filters , respectively .", "Image acquisition was performed with ZEN software ( Zeiss ) .", "Each focal plane for 3D-SIM image was captured sequentially by the excitation with the patterned light of 3 rotated angled , each of which contains five shifted phases .", "The optimal z-interval distance was set to 101 nm .", "Raw SIM images were processed to reconstruct 3D-SIM images using ZEN software .", "Extraction of the intensity data was done using the SIMcheck plugin for ImageJ software ( Ball et al . , 2015 ) .", "For localization studies , cytosol-enriched and cytosol-depleted fractions were isolated from mars1-3 cells ( a cell-wall deficient strain ) complemented with the MARS1-A transgene , according to the protocol described below , incorporating guidelines previously described in Klein et al . , 1983 and Zerges and Rochaix ( 1998 ) .", "A one liter liquid culture , synchronized by growth in dark-light cycles in minimum media and in early exponential phase ( 1–2 × 106 ml−1 ) , was harvested at 3000 x g for 5 min at RT .", "Upon media removal , the cell pellet was resuspended by gentle hand-shaking of the centrifugation tube without using a pipette in 15 ml of autolysin freshly supplied with 1 mM potassium phosphate buffer ( pH 6 . 0 ) and 0 . 5 mg/ml BSA ( Solution A ) .", "Solution A was pre-warmed at 30°C for 30 min prior to use and , after resuspending the pellet in this solution , cells were transferred to a 200 ml 30°C-prewarmed beaker immersed in a water bath and incubated at 30°C for 50 min .", "Next , cells were transferred to a 50 ml Falcon tube and collected by centrifugation at 2000 x g for 5 min at RT .", "The autolysin was quickly removed using a 25 ml plastic pipette and the cell pellet was very gently resuspended in 20 ml of ice-cold Solution B consisting of 5 mM K-phosphate buffer ( pH 6 . 5 ) , 6% PEG ( w/w ) and 4 mg/ml BSA .", "Cells were then transferred to a 32°C-prewarmed beaker immersed in a water bath at 32°C and 80 µl of freshly-prepared 1% digitonin ( Calbiochem ) ( 0 . 004% final concentration ) was quickly added and well-mixed with the cell suspension .", "Cells were then subjected to two rapid warming-cooling cycles using 32°C pre-warmed or ice pre-chilled beakers to induce plasma membrane rapture and cytosolic protein release without intracellular organelles breakage .", "Cycling was performed for 2 min at 32°C , 5 min on ice , 1 min at 32°C , 5 min on ice .", "Next , this suspension of permeabilized cells and released cytosolic proteins was transferred to a 50 ml pre-chilled Falcon tube and centrifuged at 800 x g for 3 min at 4°C .", "After centrifugation , the cytosol-enriched fraction was further purified from the supernatant fraction , while the cytosol-depleted fraction containing chloroplasts and mitochondria was further purified from the cell pellet .", "To remove potential cell debris and obtain a clean cytosolic fraction , the supernatant was subjected to two further consecutive rounds of centrifugation: first , at 5000 x g for 15 min at 4°C and then at 23 , 000 x g for 1 hr at 4°C .", "Finally , the cytosol-enriched fraction was precipitated in ice cold acetone containing 10% of trichloroacetic acid ( TCA ) .", "To enrich the cytosol-depleted fraction with organelles , the pellet of permeabilized cells was kept in ice and resuspended in 2 ml of ice-cold 2x isotonic solution consisting of 0 . 6 M sorbitol , 10 mM MgCl2 and 20 mM Tricine pH 7 . 8 .", "At this point , a rather dark-green aggregate formed in the falcon tube .", "To resuspend this aggregate , a cut plastic pipette tip was used to gently pipette the pellet up/down for 20 times .", "Then , 2 ml of ice-cold milliQ water were added to bring the isotonic solution to 1x and the aggregates were further dissolved as described above .", "Next , this suspension was loaded on a Percoll step gradient ( 10 ml 75% Percoll in isotonic solution/10 ml 45% Percoll in isotonic solution/4 ml cell lysate ) in a Corex glass tube .", "The gradient was subjected to centrifugation using the HB4 swinging-rotor at 7000 x g for 15 min at 4°C .", "Chloroplasts and contaminating mitochondria were recovered from the interface between 45% and 75% Percoll and diluted in 20 ml of 1x ice-cold isotonic solution .", "The organelles were collected by 5 min centrifugation at 4000 x g at 4°C and , after removing the supernatant , the pellet was resuspended in 2 ml ice-cold isotonic solution and run through a second Percoll gradient as described above .", "Finally , the cytosolic-depleted fraction was precipitated in ice-cold acetone containing 10% TCA .", "Denatured proteins from each fraction were extracted after TCA precipitation , as described below .", "Prior to gel electrophoresis , the protein content of each fraction was normalized using a bicinchoninic acid ( BCA ) assay as described in the section below .", "Proteins were extracted from whole cell lysate using a denaturing SDS extraction protocol for all experiments except for immunoblots that include the Mars1 protein , in which case TCA precipitation was used .", "For the SDS protein extraction , cells from a 5 ml culture in exponential growth phase were harvested at 3000 x g for 5 min and resuspended in 150 µl of SDS-lysis buffer ( 100 mM Tris-HCl pH 8 . 0 , 600 mM NaCl , 4% SDS , 20 mM EDTA , freshly supplied with Roche Protease Inhibitors ) .", "Samples were vortexed for 10 min at RT and centrifuged at maximum speed for 15 min at 4°C to remove cell debris .", "The supernatant , containing a total extract of denatured proteins was transferred to a new Eppendorf tube , a 5 µl aliquot was saved for BCA quantification and 1/4 vol of 5X SDS-loading buffer ( 250 mM Tris-HCl pH 6 . 8 , 5% SDS , 0 . 025% bromophenol blue , 25% glycerol ) , freshly supplied with 500 mM DTT or 5% of 2-mercaptoethanol prior use , was added to the extract and denatured at 37°C for >30 min .", "For the TCA precipitations , cell pellets were resuspended in 1 ml of 10% TCA in acetone , freshly supplemented with 0 . 5% β-mercaptoethanol .", "Samples were vortexed for 10 min at 4°C then left at −20°C for 1–2 hr for efficient protein precipitation .", "Samples were centrifuged at maximum speed for 10 min at 4°C and the TCA solution was carefully aspirated .", "Three washes with 1 ml of cold 100% acetone were performed ( 5 min of vortexing followed by 5 mins of maximum speed centrifugation ) and the remaining pellet was dried for 5–10 min before resuspension in Lysis Buffer ( same as above ) , achieved through vigorously shaking of the Eppendorf tube with the aid of a vortex at RT or with the aid of a thermomixer at 50°C for 10–15 min .", "The resuspended protein extract was isolated by a quick centrifugation and was transferred to a new Eppendorf tube .", "A 5 µl aliquot was saved for BCA quantification and 1/4 vol of 5x SDS loading buffer was added to the rest and denatured at 37°C for at least 30 min .", "Immunoblot analysis was performed on 20–60 µg of denatured protein extract .", "Proteins were separated by SDS-PAGE using Mini-PROTEAN or Criterion Precast Gels ) ( Bio-Rad ) and transferred onto Protran nitrocellulose membrane , 0 . 2 µm pore ( Perkin Elmer ) .", "Non-specific signal was blocked with PBS-T supplemented with 5% instant nonfat dry milk ( Carnation , Nestlè ) for 1 hr at RT or overnight at 4°C .", "All primary and secondary antibodies were diluted in this blocking buffer .", "The following antibodies ( at the indicated dilution ) were used for this publication: monoclonal mouse anti-Flag ( 1:3 , 000 ) ( M2 , Sigma F1804 ) , monoclonal mouse anti-α-tubulin ( 1:5 , 000 ) ( Sigma #T6074 ) , polyclonal rabbit anti-DnaK ( provided by Jean David Rochaix ) ( 1:10 , 000 ) ( H . Naver , K . Wilson and J . D . Rochaix , unpublished results ) ( Dauvillee , 2003 ) , polyclonal rabbit anti-RpoA ( 1:10 , 000 ) ( Ramundo et al . , 2013 ) , polyclonal rabbit anti-ClpP1 ( provided by Francis-André Wollman and Olivier Vallon ) ( 1:5 , 000 ) ( Majeran et al . , 2000 ) , polyclonal rabbit anti-Hsp22E/F ( provided by Michael Schroda ) ( 1:5 , 000 ) ( Rütgers et al . , 2017 ) , polyclonal rabbit anti-Vipp2 ( 1:3 , 000 ) ( raised against a –CDPLERELEELRRRARE- peptide , developed during this study by Yenzym , South San Francisco ) , polyclonal rabbit anti-Aox1 ( 1:2 , 000 ) ( Agrisera AS06 152 ) , polyclonal rabbit anti-Sultr2 ( provided by Arthur Grossman ) ( 1:3 , 000 ) ( Pootakham et al . , 2010 ) , polyclonal rabbit holo-Rubisco ( provided by Jean David Rochaix ) ( 1:10 , 000 ) ( Borkhsenious et al . , 1998 ) , polyclonal rabbit anti-Hsp90C ( 1:10 , 000 ) ( Agrisera AS06 174 ) and anti-Histone H3 ( 1:10 , 000 ) ( Agrisera AS10 710 ) .", "To detect the primary antibodies , HRP-conjugated anti-rabbit and anti-mouse secondary antibodies ( Promega ) were used at dilution 1:10 . 000 in PBS-T supplemented with 5% instant nonfat dry milk for 1 hr at RT .", "In between the incubation with primary and secondary antibody and after the incubation with the secondary antibody , three washes of about 10 min each time , at RT , were performed using PBS-T supplemented with 5% instant nonfat dry milk .", "The membranes were quickly rinsed three times with milliQ-water and a luminol-based enhanced chemiluminescence ( ECL ) method was applied to develop the signal .", "For most immunoblot analysis , the SuperSignal West Dura Extended Duration Substrate kit ( Thermo Fisher Scientific ) was used according to manufacturer’s directions .", "By contrast , for Flag immunoblot analysis to detect Mars1 protein , the SuperSignal West Femto Maximum Sensitivity Substrate kit ( Thermo Fisher Scientific ) was chosen , given the low expression level of this protein .", "The ECL signal was detected with the LI-COR Odyssey imaging system or using clear-blue X-ray films ( CL-Xposure , Thermo Fisher Scientific ) .", "To carry out the BCA assay , 5 µl of protein extract was added to 200 µl of BCA/copper sulfate solution ( 1:50 dilution of 4% CuSO4 into BCA solution , Sigma ) and incubated at 50°C for 5 min .", "Protein concentration was estimated by measuring the absorbance at 562 nm and comparing it to a BSA standard .", "Note: for Figure 6—figure supplement 1B , the denaturing protein extraction was carried out as follows: cell cultures started in 7 ml of TAP were grown to mid-log phase and subsequently spun down at 3000 x g for 8 min .", "The pellets were resuspended in 150 µl of TAP .", "Then , an equal volume of 0 . 2M NaOH was added to the pellets , vortexed at RT for 5 min and pelleted at 15 , 000 x g for 5 min .", "The supernatant was removed , the pellet was resuspended in 280 µl of SDS samples buffer ( 0 . 06 M Tris-HCl pH 6 . 8 , 5% glycerol , 2% SDS , 4% 2-Mercaptoethanol , 0 . 0025% bromophenol blue ) , boiled for 5 min and then pelleted again .", "A 28 µl aliquot was loaded onto a Criterion gel .", "The following protocol was used for the HL assay described in Figure 4A–B .", "Slight modifications were applied during the HL assays described in Figure 6E and will be underscored below .", "Liquid cultures were started from TAP plates that had all been grown in dim light and in the same conditions .", "A small slab of cells was taken from the agar plate and resuspended in 28 ml of TAP media in 50 ml falcon tubes ( Sarstedt ) .", "The Olympus Plastics brand product line from Genesee Scientific was avoided because there was a higher propensity for the cells to adhere to this plastic material .", "An equal slab of cells was used for each culture to approximate the same level of starting cells .", "Typically , the strains had been growing in fresh TAP agar for ~3–5 days .", "The cells were pre-conditioned in low light ( ~20–50 µmol photons m−2 s−1 ) for about 38–44 hr .", "For the HL assays shown in Figure 6E , the cells were preconditioned for slightly longer period , ~3 days .", "The chlorophyll concentration of the cell cultures was measured using the methanol extraction method as described in Porra et al . ( 1989 ) .", "At the above described time points , it was found to be ~13–18 µg/ml ( HL assays described in Figure 4A–B ) , or ~25 µg/ml ( in the HL assays described in Figure 6E ) .", "Cell cultures were then equally diluted to ~10 µg chlorophyll ml−1 ( in the case of the HL assays described in Figure 4A–B ) , or to 7 µg chlorophyll ml−1 ( HL assays described in Figure 6E ) .", "Chlorophyll concentrations were confirmed and , if needed , re-adjusted after dilution before HL was started .", "The final volume of cell culture used for high light treatment was ~26 ml in 50 ml Falcon tubes .", "During the high light treatment , the distance between the light source ( Phlizon 2017 , 2000W Plant LED Growth light ) and the shaker was set to 25 cm .", "The HL intensity was measured at 1100 µmol photons m−2 s−1 at the beginning of the experiment but was reduced to ~900 µmol photons m−2 s−1 by the end of the experiment .", "On the right and left side of the shaker , two fans were turned on to keep the samples at RT and a Smart Sensor ( SensorPush ) was used to monitor temperature in real-time .", "Typically a 4°C increase in temperature ( from 24 . 5°C to about 28 . 5°C ) was measured after the cultures were shifted from the dim light growth setup to this HL setup .", "The light intensity at each position of the culture on the shaker was measured to ensure cells were getting the same number of photons ( ~50 , 000 LUX ) .", "The cultures in the Falcon tubes were taped ( clear tape ) onto the shaker and the shaker was set at 150 rpm .", "Chlorophyll measurements were taken at multiple time points of HL treatment ( during HL assays described in Figure 4A and Figure 4—figure supplement 1A ) and after 27 hr or 50 hr of HL treatment ( during HL assays described in Figure 4B and Figure 6E , respectively ) .", "Serial dilutions performed on cultures before and after the HL treatment were spotted onto TAP plates .", "Photographs of these plates were taken over time to track cell recovery .", "For immunoblot analyses , as shown in Figure 4D , cell cultures were grown in liquid TAP medium in a 50 ml Falcon tube for about two days to a chlorophyll concentration of 11 µg/ml in a volume of 30 ml .", "Cells were spun down and the pellet was resuspended in 1 . 5 ml of TAP .", "0 . 3 ml of the resuspended pellet was then added to 10 ml of TAP with or without 1 . 1 mM metronidazole ( Sigma ) .", "These cultures were then placed under white light ( 20–50 µmol photons m−2 s−1 ) for 12–15 hr and then spun down and saved at −20°C or directly used for denaturing protein extraction as described above .", "For growth tests on TAP agar supplemented with metronidazole , as shown in Figure 4C and Figure 4—figure supplement 1B–E , strains were either manually re-streaked or robotically replicated from TAP agar plate to +/- 1 . 5 mM metronidazole TAP agar plates .", "For dilution spot tests , shown in Figure 6D , cells were freshly inoculed from a 3-4 days old agar plate and grown in in falcon tubes for 2 days in a starting volume of 30 ml of TAP .", "After these 2 days of preconditioning , chlorophyll concentrations were normalized to be at ~18 µg/ml and serial dilutions of 1 . 5-fold were done in liquid TAP using a 96-well plate .", "Finally , 6 µl cells from each dilution series were spotted onto TAP agar plates with or without 2 . 2 mM metronidazole .", "Photographs were taken periodically to track growth over time .", "Metronidazole is rather insoluble in aqueous solutions; therefore , it was always added ( as powder ) directly to the autoclaved liquid TAP medium at final concentrations of 1 . 1 mM , 1 . 5 mM or 2 . 2 mM .", "Mars1-D and Mars1-D KD strains were subjected to metronidazole treatment for 15 hr .", "Culture were then harvested and subjected to Flag affinity purification followed by MS analysis according to the protocol described by {Mackinder , 2017 #30} and publically available through this link: https://docs . google . com/viewer ?", "a=v&pid=sites&srcid=ZGVmYXVsdGRvbWFpbnxjaGxhbXlzcGF0aWFsaW50ZXJhY3RvbWV8Z3g6NzlkNjUzMTM0ZWEyYmI5 .", "Preparation of samples for MS analysis and processing of MS raw data was performed by the Stanford Mass Spectrometry Facility in Palo Alto .", "A 10–ml aliquot of a cell culture in early-mid exponential phase ( 1–5 × 106 ml−1 ) , was harvested at 3000x g for 5 min at RT .", "After decanting the media , 1 ml of Trizol Reagent ( ThermoFisher Scientific ) was added to the cells .", "The cells were lysed in sample by vigorous shaking with the aid of a vortex for 5–8 min .", "Chloroform ( 1/5 vol , ~200 µl ) of was then added to the lysate and the tube was vigorously shaken up and down by hand for 60 s .", "The sample was then centrifuged at 11 , 000 x g for 7 min at RT .", "The upper aqueous phase ( ~350 µl ) was removed with care to not draw any of the organic layer , transferred in a nuclease-free 1 . 5 ml tube ( Ambion ) and mixed well with 1 vol of 100% ethanol ( Fisher Scientific ) at RT .", "From this point onwards , the RNA purification was carried out with the aid of the Direct-zol RNA MiniPrep Plus kit ( ZymoResearch ) following the manufacturer’s protocol , including in-column DNase I treatment prior RNA washing and elution steps .", "A semi-quantitative RT-PCR was carried out to qualitatively determine the presence or absence of MARS1 gene transcripts .", "Total RNAs were extracted as described in the previous section .", "Complementary DNA was synthesized from 1 µg of total RNA using PrimeScript 1 st strand cDNA Synthesis Kit ( Takara ) as per manufacturer’s instructions .", "Subsequently , RNA/DNA hybrids were removed by ribonuclease H treatment ( NEB ) as per manufacturer’s instructions .", "cDNAs were diluted two-fold and 1–2 µl were used as template for a 20 µl PCR reaction by Phusion High-Fidelity DNA Polymerase ( Thermo Fisher Scientific ) with the following parameters: initial melting 98°C for 30 s , amplification cycles 98°C for 10 s , 68°C for 30 s , 72°C for 1 min 15 s ( 35 times ) , final extension 72°C for 5 min .", "Primers SR834 and SR835 were used to amplify a fragment of the MARS1 coding sequence spanning from exon 16 to exon 28 .", "Primers SR836 and SR837 were used to amplify the GBLP coding sequence , used as loading and positive control during the RT-PCR analysis .", "The sequence of the primers can be found in Table 3 .", "For qPCR analyses , the cDNA was prepared as described above but the cDNA was diluted six to eight-fold in nuclease-free water prior to use .", "Primers to amplify the target transcripts are indicated in the Table 3 .", "The qPCR reactions were carried out using the iQ Sybr green supermix as per manufacturer’s instructions ( Bio-Rad ) .", "To determine whether there was DNA contamination in the mix or whether there was primer dimer formation or misannealing , the same volume of the master mix , without cDNA , was added to a well in the 96-well plate .", "The raw Ct values were analyzed per the ‘eleven golden rules’ , as previously described ( Udvardi et al . , 2008 ) .", "GBLP was chosen as reference housekeeping transcript during normalization .", "Standard deviation was obtained for the 2–5 technical replicates and a minimum of 2 biological replicates were done per experiment , except for the experiment in Figure 3—figure supplement 1B , where only one biological replicate was analyzed .", "The Phytozome v5 . 5 gene annotation for the target transcripts is as follows: VIPP2 ( Cre11 . g468050 . t1 . 2 ) , SULTR2 ( Cre17 . g723350 . t1 . 2 ) , LHCBM9 ( Cre06 . g284200 . t1 . 2 ) , HSP22F ( Cre14 . g617400 . t1 . 1 ) , SNOAL ( Cre11 . g478100 . t1 . 2 ) , LHCSR3 . 1 ( Cre08 . g367500 . t1 . 1 ) , PSBS1 ( Cre01 . g016600 . t1 . 2 ) , CPLD29 ( Cre02 . g088500 . t1 . 1 ) , GBLP ( Cre06 . g278222 . t1 . 1 ) .", "Prior to RNA-seq analyses , the mars1-1 strain was backcrossed to the wild-type CC-124 ( Chlamydomonas Resource Center ) .", "A full tetrad was selected and analyzed as shown in Figure 2E .", "A wild-type and MARS1 mutant progeny ( CrPW8 , indicated as E12 , and CrPW9 , indicated as F2 respectively , in Figure 2E and Figure 2—figure supplement 2B ) were chosen for follow-up studies , based on the retention of the ClpP1 repressible system in their genetic background .", "For each strain , two cultures ( biological replicates ) were inoculated in 30 ml liquid TAP medium using 50 ml Falcon tubes , starting from a fresh re-streak of cells propagated on TAP agar .", "These cultures were grown with mild agitation ( 150 rpm ) on a shaker at around 22°C , under an illumination of 30–40 µmol photons m−2 s−1 for about three-four days till they reach mid-late exponential phase ( 4–7 × 106 cells ml−1 ) .", "Next , cells were diluted to about 1 × 106 cells ml−1 in 30 ml of liquid TAP medium using 50 ml Falcon tubes and they were subjected to three different treatments:", "a ) low light , i . e . they were kept at the same light intensity used during conditioning;", "b ) HL , i . e . they were shifted to very high light ( 1200 µmol photons m−2 s−1 ) for 40 min or 70 min; and", "c ) ClpP1 repression , i . e . they were diluted in liquid TAP medium containing 100 µM thiamine and 40 ng ml−1 vitamin B12 and incubated for 68 hr at the same light intensity used during conditioning .", "Cells were then shifted to ice and quickly harvested by centrifugation at 3000 x g for 5 min at 4°C .", "Cell pellets were snap-frozen in liquid nitrogen and saved at −80°C till use .", "RNA extraction was carried as described in the previous section .", "The following extra measures were taken to ensure a complete removal of DNA contaminants: An additional round of in-solution Dnase I treatment was performed using 1 unit of Rnase-free Dnase I ( Roche ) /1 µg of total RNA at RT for 20 min in presence of 1 unit of recombinant ribonuclease inhibitors ( RNaseOUT , Thermo Fisher Scientific ) .", "Next , the RNA was re-purified using the same extraction protocol described above .", "Each total RNA preparation was ran on an Agilent Bioanalyzer RNA 6000 Nano chip for quantification and quality control .", "PolyA mRNAs were purified and RNAseq libraries were prepared using the Kapa mRNA HyperPrep kit ( Roche , KK8540 ) following manufacturer’s protocols .", "Libraries were pooled based on fragment analyzer concentrations .", "Sequencing was performed on Nextseq high-output flowcell , 1 × 75 bp run ( Illumina ) .", "We used a combination of publicly available tools and custom scripts for the processing of the raw demultiplexed Illumina sequencing data .", "Illumina adapter sequences were first trimmed off with TrimGalore !", "( version_0 . 4 . 1 ) ( www . bioinformatics . babraham . ac . uk/projects/trim_galore ) and contaminating ribosomal reads were removed by mapping against the Silva rRNA database using bbduk v37 . 32 ( part of the BBTools suite , https://jgi . doe . gov/data-and-tools/bbtools/bb-tools-user-guide/ ) .", "Quality control of raw and processed fastq files was performed using FastQC version 0 . 11 . 3 ( https://www . bioinformatics . babraham . ac . uk/projects/fastqc/ ) .", "The remaining reads were aligned to the unmasked C . reinhardtii genome ( Phytozome , v5 . 5 ) using STAR v . 2 . 5 . 3a ( Dobin et al . , 2013 ) and bam files were sorted with samtools 1 . 1 ( Li et al . , 2009 ) .", "Count generation and downstream analysis were done in R ( R project v3 . 4 . 0 , www . R-project . org ) using a combination of the packages Rsubreads ( Liao et al . , 2013 ) EdgeR ( Robinson et al . , 2010 ) plyr , ggplot2 , gplots , and heatmap2 .", "For differential expression analysis , genes with less than 0 . 5 counts per million reads in less than two samples were discarded , the data were fit to a negative binomial generalized linear model , and differential expression was determined using the quasi likelihood F-test with Benjamini-Hochberg correction of multiple testing in EdgeR .", "To subdivide genes into groups of MARS1-dependent and -independent genes , weakly-expressed genes ( average RPKM <2 . 5 in at least two conditions ) were discarded .", "Stress-responsive genes ( defined as log2-fold change >= |2| at FDR 0 . 001 upon treatment - high light , ClpP repression , or both as noted – in WT background ) were considered MARS1-dependent when treatment in the MARS1 mutant did not lead to a greater than 2-fold change in expression ( log2-fold change < |1| ) .", "MARS1-independent genes were defined as genes with an at least 4-fold ( log2-fold change >= |2| ) change upon treatment in the mars1 background , in the same direction ( up or down ) as the response in WT cells .", "For analysis of functional categories in MapMan 10 . 0 ( Thimm et al . , 2004 , Usadel et al . , 2005 ) the C . reinhardtii v5 . 5 proteome ( downloaded from Phytozome , v5 . 5 , protein sequences from primary transcript only ) was binned using Mercator4 ( www . plabipd . de/portal/web/guest/mercator4 ) and MARS1-dependent and MARS1-independent gene subsets were mapped onto plant pathways in MapMan ." ] ]

[ "In response to proteotoxic stress , chloroplasts communicate with the nuclear gene expression system through a chloroplast unfolded protein response ( cpUPR ) .", "We isolated Chlamydomonas reinhardtii mutants that disrupt cpUPR signaling and identified a gene encoding a previously uncharacterized cytoplasmic protein kinase , termed Mars1—for mutant affected in chloroplast-to-nucleus retrograde signaling—as the first known component in cpUPR signal transmission .", "Lack of cpUPR induction in MARS1 mutant cells impaired their ability to cope with chloroplast stress , including exposure to excessive light .", "Conversely , transgenic activation of cpUPR signaling conferred an advantage to cells undergoing photooxidative stress .", "Our results indicate that the cpUPR mitigates chloroplast photodamage and that manipulation of this pathway is a potential avenue for engineering photosynthetic organisms with increased tolerance to chloroplast stress ." ]

[ "Life on Earth crucially depends on photosynthesis , the process by which energy stored in sunlight is harnessed to convert carbon dioxide into sugars and oxygen .", "In plants and algae , photosynthesis occurs in specialized cellular compartments called chloroplasts .", "Inside chloroplasts , complex molecular machines absorb light and channel its energy into the appropriate chemical reactions .", "These machines are composed of proteins that need to be assembled and maintained .", "However , proteins can become damaged , and when this occurs , they must be recognized , removed , and replaced .", "When exposed to bright light , the photosynthetic machinery is pushed into overdrive and protein damage is accelerated .", "In response , the chloroplast sends an alarm signal to activate a protective system called the “chloroplast unfolded protein response” , or cpUPR for short .", "The cpUPR leads to the production of specialized proteins that help protect and repair the chloroplast .", "It was not known how plants and algae evaluate the level of damaged proteins in the chloroplast , or which signals trigger the cpUPR .", "To address these questions , Perlaza et al . designed a method to identify the molecular components of the alarm signal .", "These experiments used specially engineered cells from the algae Chlamydomonas reinhardtii that fluoresced when the cpUPR was activated .", "Perlaza et al . mutagenized these cells – that is , damaged the cells’ DNA to cause random changes in the genetic code .", "If a mutagenized cell no longer fluoresced in response to protein damage , it indicated that communication between protein damage and the cpUPR had been broken .", "In other words , the mutation had damaged a piece of DNA that encoded a protein critical for activating the cpUPR .", "These experiments identified one protein – which Perlaza et al . named Mars1 – as a crucial molecular player that is required to trigger the cpUPR .", "Algal cells with defective Mars1 were more vulnerable to chloroplast damage , including that caused by excessive light .", "These discoveries in algae will serve as a foundation for understanding the mechanism and significance of the cpUPR in land plants .", "Perlaza et al . also found that mild artificial activation of the cpUPR could preemptively guard cells against damaged chloroplast proteins .", "This suggests that the cpUPR could be harnessed in agriculture , for example , to help crop plants endure harsher climates ." ]

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

[ "short report", "neuroscience" ]

[ [ "Animals of diverse vertebrate and invertebrate species distinguish between sensations arising from self-generated movements from those arising from other-generated movements ( Crapse and Sommer , 2008; Sommer and Wurtz , 2008 ) .", "To make this distinction between self and other , motor areas produce copies of motor commands ( i . e . , corollary discharges ) that are directly compared with sensory signals arising from self-generated movements ( i . e . , reafference; [Poulet and Hedwig 2006; Sommer and Wurtz 2002] ) .", "At the level of the neural comparator , reafference is blocked when there are no discrepancies between the corollary discharge and reafferent signals ( Poulet and Hedwig 2006; Bell , 2001; Brooks and Cullen , 2014; Bell , 1989 ) .", "As a result , expected reafference arising from voluntary movements is attenuated ( Voss et al . , 2006; Seki et al . , 2003; Haggard and Whitford 2004 ) , thereby increasing the salience of unexpected reafference ( Poulet and Hedwig 2006; Brooks et al . , 2015; Brooks and Cullen , 2013 ) .", "We previously demonstrated in week-old rats that wake-related movements do not trigger substantial reafference in sensorimotor cortex ( SMC ) .", "In contrast , sleep-related twitches—which are produced exclusively and abundantly during active ( or REM ) sleep—trigger robust reafference ( Tiriac et al . , 2014 ) .", "We provided converging evidence that twitches , unlike wake movements , are processed by the nervous system as if they are unexpected .", "Accordingly , we hypothesized that the actions of corollary discharge that normally gate reafference arising from wake movements are either absent or inhibited during twitching .", "If true , there must be a neural structure , located somewhere within the sensorimotor network , that acts as a comparator to specifically gate wake-related reafference .", "One possible comparator is the external cuneate nucleus ( ECN ) , which receives primary muscle spindle afferents from forelimb and nuchal muscles and conveys sensory information to downstream structures including the cerebellum , thalamus , and cerebral cortex ( Campbell et al . , 1974; Cooke et al . , 1971; Mackie et al . , 1999; Boivie and Boman , 1981; Huang et al . , 2013 ) .", "Moreover , the ECN receives projections from such premotor structures as the red nucleus ( Holstege and Tan , 1988; Edwards , 1972; Martin et al . , 1974 ) —which contributes both to the production of wake movements and twitches in infant rats ( Del Rio-Bermudez et al . , 2015 ) —and the premotor C3-C4 propriospinal neurons ( PNs; [Pivetta et al . , 2014] ) , which have been implicated in the conveyance of corollary discharge ( Pivetta et al . , 2014; Alstermark et al . , 2007; Azim et al . , 2014 ) .", "Corollary discharge from the red nucleus or PNs could contribute to sensory gating in the ECN via known GABAergic and glycinergic inputs to that structure ( Galindo and Krnjević , 1967; Heino and Westman 1991; Sato et al . , 1991 ) , perhaps through presynaptic inhibition ( Seki et al . , 2003; Andersen et al . , 1964 ) ." ], [ "We first recorded neural activity in the forelimb region of SMC to confirm that it , like the hindlimb region ( Tiriac et al . , 2014 ) , exhibits state-dependent activity .", "Unanesthetized and head-fixed postnatal day ( P ) 8–10 rats cycled spontaneously between sleep and wake with their limbs dangling freely .", "Figure 1A depicts representative spindle bursts ( recorded from the local field potential; LFP ) and multiunit activity ( MUA ) in relation to both wake movements and twitches .", "Spindle bursts and MUA were particularly prominent during periods of twitching and were virtually absent during periods of wake movements .", "Across all pups , the mean rate of spindle bursts ( t5 = 10 . 2 , p=0 . 0002 , n = 6 pups ) and unit activity ( t7 = 3 . 9 , p=0 . 006 , n = 8 units ) was significantly higher during active sleep than during wake ( Figure 1B ) .", "Moreover , spindle bursts and unit activity were triggered by forelimb twitches with a latency of 100–125 ms ( Figure 1C ) . 10 . 7554/eLife . 18749 . 003Figure 1 . Forelimb twitches , but not wake movements , trigger neural activity in forelimb sensorimotor cortex .", "( A ) Representative data depicting sleep and wake behavior , MUA , LFP , and forelimb and nuchal EMG during spontaneous sleep-wake cycling .", "Red tick marks denote forelimb twitches and red horizontal bars denote forelimb wake movements as scored by the experimenter .", "( B ) Mean ( +SEM ) rate of spindle burst ( n = 6 pups ) and unit activity ( n = 8 units ) during periods of wake and active sleep ( AS ) .", "The mean rate of spindle bursts and unit activity was significantly higher during active sleep than during wake .", "* significant difference from active sleep , p<0 . 01 .", "( C ) Waveform average and event correlation for LFP power and unit activity , respectively , in relation to forelimb twitches ( 2413 and 2943 twitches , respectively ) .", "The blue dashed lines denote upper and lower acceptance bands ( p<0 . 01 ) .", "LFP , Local field potential; MUA , Multiunit activity .", "DOI: http://dx . doi . org/10 . 7554/eLife . 18749 . 003 If the ECN is a comparator of reafference and corollary discharge signals , then it should—like the SMC—exhibit state-dependent activity .", "Therefore , we next recorded from the ECN in P8-10 rats ( Figure 2A ) .", "As expected , ECN firing rate was higher in response to forelimb twitches than to hindlimb ( t8 = 3 . 5 , p=0 . 008 ) or nuchal ( t21 = 3 . 2 , p=0 . 007 ) twitches ( Figure 2B ) ; there was no significant difference in ECN firing rate between hindlimb and nuchal twitches ( t8 = 1 . 3 , p=0 . 23 ) .", "Also , as expected given the relative anatomical locations of the ECN and SMC , the latency to twitch-related ECN activity ( 10–50 ms ) was shorter than that observed in SMC ( 100–125 ms; see Figure 1C ) .", "Finally , ECN firing rate was movement dependent , with greater activity occurring during periods of forelimb twitching than during periods of forelimb wake movement ( t15 = 2 . 8 , p=0 . 012 , Figure 2C ) . 10 . 7554/eLife . 18749 . 004Figure 2 . The ECN exhibits wake-dependent inhibition of sensory reafference .", "( A ) Top: For all ECN recordings , P8-10 rats were instrumented with forelimb ( blue ) and nuchal ( red ) EMGs ( n = 22 ) .", "A subset of these rats also had a hindlimb ( green ) EMG ( n = 9 ) .", "The torso was supported by a platform and the limbs dangled freely .", "Bottom left: Coronal brain section depicting the anatomical location of the ECN in the hindbrain ( inset; red dashed line depicts AP position of the coronal section ) .", "Bottom right: Sample record of a burst of ECN reafference in response to forelimb twitches .", "( B ) Left: Event correlations for unit activity in relation to forelimb ( blue , n = 8146 twitches ) , nuchal ( red , n = 9603 twitches ) , and hindlimb ( green , n = 1243 twitches ) twitches .", "The colored dashed lines denote upper acceptance bands ( p<0 . 01 ) for the event correlations .", "Right: Pairwise comparisons of mean ( +SEM ) peaks in unit activity ( Hz ) in response to forelimb , nuchal , and hindlimb twitches .", "Comparisons are between forelimb and nuchal muscles ( top ) , forelimb and hindlimb muscles ( middle ) , and nuchal and hindlimb muscles ( bottom ) .", "( C ) Left: Representative data depicting sleep and wake behavior , MUA , and forelimb and nuchal EMG during spontaneous sleep-wake cycling .", "Red tick marks denote forelimb twitches , and red horizontal bars denote forelimb wake movements as scored by the experimenter .", "Right: Mean ( +SEM ) unit activity ( n = 16 ) during wake and active sleep ( AS ) periods .", "* significant difference from active sleep , p<0 . 05 .", "( D ) Left: Event correlations for evoked unit activity in response to forelimb stimulations performed during active sleep ( n = 188 stimulations ) and wake ( n = 238 stimulations ) across 6 ECN units in six pups .", "Right: Mean ( +SEM ) peak unit activity ( n = 6 ) derived from event correlations during wake and active sleep .", "ECN , External cuneate nucleus; MUA , Multiunit activity . DOI: http://dx . doi . org/10 . 7554/eLife . 18749 . 004 It is possible that the inhibition of wake-related reafference was due to a global inhibition of all sensory activity during wake .", "If so , then stimulation of the ipsilateral forelimb should produce less exafference in the ECN during wake than during sleep .", "To test for this possibility , we recorded from ECN neurons as the ipsilateral forelimb was stimulated during sleep and wake .", "As shown in Figure 2D , there was no significant effect of behavioral state on ECN activity in response to forelimb stimulation ( t5 = 1 . 2 , p=0 . 286 ) .", "This result is consistent with our earlier finding in the hindlimb region of SMC ( Tiriac et al . , 2014 ) .", "Figure 3A depicts our proposed model to explain movement-dependent modulation of reafference in the ECN .", "To test the model’s predictions , we recorded ECN activity before and during combined iontophoretic infusion of GABAA ( 10 mM bicuculline methiodide ) and glycine ( 10 mM strychnine hydrochloride ) receptor antagonists or saline ( Figure 3B; n = 5 pups per group ) .", "As predicted , inhibitory blockade of the ECN unmasked reafference in response to forelimb wake movements ( t8 = 3 . 7 p=0 . 006 , Figure 3C ) .", "Moreover , and again as predicted , inhibitory blockade had no effect on ECN reafference in response to forelimb twitches ( Figure 3D ) .", "Saline infusions had no effect on either wake or sleep reafference . 10 . 7554/eLife . 18749 . 005Figure 3 . Pharmacological blockade of GABAA and glycine receptors in the ECN specifically unmasks reafference from self-generated wake movements .", "( A ) Proposed circuitry depicting how reafference arising from self-generated movements is modulated at the level of the ECN .", "Left: During wake , ECN inputs arising from motor areas convey a corollary discharge ( i . e . , motor copy ) signal that gates expected reafference , resulting in decreased activity in the ECN and downstream sensory areas .", "Right: During active sleep , the motor copy is absent or inhibited and , therefore , ECN inputs arising from motor areas do not gate reafference , resulting in activity in the ECN and downstream sensory areas .", "VPL: ventral posterolateral nucleus of thalamus; SMC: primary sensorimotor cortex .", "( B ) ECN recordings were performed using multibarrel electrodes filled either with a GABAA antagonist ( 10 mM bicuculline methiodide ) and a glycine antagonist ( 10 mM strychnine hydrochloride ) or saline .", "For all animals , a separate barrel of the electrode was filled with fluorogold ( FG ) to mark the location of the recording site and estimate the spread of the drug ( image at bottom right ) .", "( C ) Left: Event correlations for unit activity in relation to the onset of forelimb wake movements in animals in the GABA/glycine ( blue ) or saline ( red ) groups before ( Pre ) and after ( Post ) infusion .", "Data are pooled across all pups .", "The dashed lines denote upper acceptance bands ( p<0 . 01 ) for the event correlations .", "Right: Event correlations depicting changes in unit activity between the pre-infusion and post-infusion periods for the GABA/glycine ( blue ) and saline ( red ) groups .", "Color-coded shaded regions denote +SEM .", "Histograms depict mean ( +SEM ) peak changes in unit activity .", "n = 5 per group .", "* significant difference from saline , p<0 . 05 .", "( D ) Same as in ( C ) but during active sleep; event correlations are triggered on forelimb twitches .", "ECN , External cuneate nucleus . DOI: http://dx . doi . org/10 . 7554/eLife . 18749 . 005 We next assessed whether inhibitory blockade of the ECN alters motor activity .", "Blocking GABAA and glycine receptors had no effect on the amplitude of wake movements ( Figure 4A ) , the frequency of forelimb wake movements ( Figure 4B ) , or the frequency of forelimb twitches ( Figure 4C ) .", "Moreover , inhibitory blockade had no effect on tonic neural activity in the ECN ( Figure 4D ) , thus providing further evidence that the inhibitory inputs to the ECN function specifically in the context of wake movements . 10 . 7554/eLife . 18749 . 006Figure 4 . Pharmacological blockade of GABAA and glycine receptors in the ECN does not affect forelimb motor activity or tonic ECN unit activity .", "( A ) Waveform averages ( 3-s time windows ) depicting forelimb EMG power in relation to peak forelimb wake activity before and after infusion of GABAA and glycine receptor antagonists ( blue ) or saline ( red ) .", "Shaded regions denote +SEM .", "At right , mean ( +SEM ) forelimb EMG peak power derived from waveform averages .", "( B ) Top row: Mean ( +SEM ) forelimb wake movements/min before and after infusion of GABAA and glycine receptor antagonists ( blue ) or saline ( red ) .", "At right , mean ( +SEM ) percent difference ( pre vs . post ) in forelimb wake movements/min for both experimental groups .", "( C ) and ( D ) : Same as in ( B ) but for twitches/min and unit activity ( in Hz ) , respectively .", "None of the differences in this figure are significant .", "ECN , External cuneate nucleus .", "DOI: http://dx . doi . org/10 . 7554/eLife . 18749 . 006" ], [ "To establish that the ECN is a comparator of corollary discharge and reafferent signals , several criteria must be met ( Poulet and Hedwig , 2007 ) .", "First , to be a comparator , the ECN must be a sensory structure; in fact , the ECN processes proprioceptive inputs from forelimb and nuchal muscles ( Campbell et al . , 1974 ) and recordings from anesthetized cats have demonstrated that the ECN codes , with high fidelity , the stretch on muscle fibers ( Mackie et al . , 1999 ) .", "Second , the ECN must receive motor-related input; in fact , the ECN receives direct and indirect input from premotor areas , including the red nucleus ( Holstege and Tan , 1988; Edwards , 1972; Martin et al . , 1974 ) and C3-C4 PNs ( Pivetta et al . , 2014 ) .", "Third , the ECN should not participate in the production of movement; as shown here , disinhibition of the ECN had no discernible effect on the production of wake movements or twitches ( Figure 4 ) .", "Finally , and most critically , a comparator must gate reafference arising from movements .", "This was demonstrated here by showing that inhibitory blockade of the ECN unmasked reafference exclusively during wake movements ( Figure 3C ) .", "Importantly , inhibitory blockade had no effect on the ECN’s tonic firing rate ( Figure 4D ) , thus demonstrating that inhibitory control of the ECN is not engaged throughout the waking state .", "Furthermore , when we manually stimulated the forelimbs , ECN activity was evoked similarly during sleep and wake ( Figure 2D ) .", "There is no evidence that wake movements and twitches are produced by independent brainstem structures .", "On the contrary , the red nucleus is involved in the production of both types of movements ( Del Rio-Bermudez et al . , 2015; Gassel et al . , 1966 ) .", "Therefore , it may be that", "( a ) premotor structures like the red nucleus are state-dependently modulated such that they convey motor copies to the ECN ( or an intervening structure ) only during wake movements , or", "( b ) the ECN receives motor copies from both types of movements , but state-dependent modulation of the ECN prevents the twitch-related motor copies from engaging the inhibitory mechanism .", "Indeed , in neonatal rats , there are structures , including the noradrenergic locus coeruleus , that could play a state-dependent modulatory role ( Karlsson et al . , 2005 ) .", "Regardless of which mechanism turns out to be correct , the fact remains that wake movements and twitches are processed very differently at the level of the ECN .", "Experimental disruption of corollary discharge pathways has a negative impact on forelimb reaching ( Azim et al . , 2014 ) and control of eye movements ( Sommer and Wurtz 2002 ) .", "In this context , the gating of wake-related reafference within the ECN makes functional sense .", "But if sensory gating is critical for motor function , why disengage this mechanism during sleep-related twitches ?", "The absence of sensory gating during sleep raises the possibility that corollary discharge mechanisms interfere with functional processes in which twitches are involved .", "Indeed , if sensory gating were to occur during active sleep , twitches—which are especially abundant during early development—would trigger little or no reafference .", "In the context of a developing system that relies heavily on activity-dependent processes ( Kirkby et al . , 2013 ) , the gating of twitch-related reafference would suppress the very activity upon which some of these processes depend .", "Because reafference is not gated during twitching , it is permitted to sequentially activate interconnected sensorimotor structures—including the ECN ( as shown here ) , red nucleus ( Del Rio-Bermudez et al . , 2015 ) , cerebellum ( Sokoloff et al . , 2015a; 2015b ) , thalamus ( Tiriac et al . , 2012; Khazipov et al . , 2004 ) , sensorimotor cortex ( Tiriac et al . , 2014; Khazipov et al . , 2004 ) , and hippocampus ( Mohns and Blumberg , 2010 ) .", "Such cascading neural activity provides the opportunity for competitive synaptic interactions that , through such mechanisms as spike-timing-dependent plasticity ( Feldman , 2012; Song and Abbott , 2001 ) , can contribute to the development and refinement of somatotopic organization across the neuraxis .", "The very presence of a neural comparator that is differentially engaged in a movement-dependent manner argues both for the importance of sensory gating during wake and its absence during sleep .", "With regard to the latter , the identification here of a neural mechanism that distinguishes twitches from wake movements reinforces the notion that twitches play a critical role in shaping sensorimotor circuits ( Blumberg et al . , 2013 ) ." ], [ "A total of 42 Sprague-Dawley Norway rats ( RRID:RGD_5508397 ) were used at P8-10 .", "Males and females were used and littermates were always assigned to different experimental groups .", "Litters were culled to eight pups within 3 days of birth .", "Mothers and their litters were housed in standard laboratory cages ( 48 × 20 × 26 cm ) .", "Food and water were available ad libitum .", "All animals were maintained on a 12:12 light-dark schedule with lights on at 0700 hr .", "The electromyographic ( EMG ) electrodes were connected to a differential amplifier ( A-M Systems , Carlsborg , WA; amplification: 10 , 000x; filter setting: 300–5000 Hz ) .", "To record from forelimb SMC , 16-channel silicon depth electrodes were used ( 100 µm vertical separation; NeuroNexus , Ann Arbor , MI ) .", "To record from the ECN , four-channel silicon depth electrodes were used ( 50 µm vertical separation ) .", "Silicon electrodes had impedances ranging from 1 to 4 MΩ .", "To simultaneous perform ECN recordings during iontophoretic application of GABAA and glycine receptor antagonists , multibarrel electrodes were used with an iontophoretic pump ( Kation Scientific , Minneapolis , MN ) .", "Electrodes were connected to a headstage which communicated with a data acquisition system ( Tucker-Davis Technologies , Alachua , FL ) that amplified ( 10 , 000x ) and filtered the signals .", "All cortical recordings were obtained using a 5000 Hz low-pass filter , and all recordings in ECN were obtained using a 500–5000 Hz band-pass filter .", "A 60-Hz notch filter was also used .", "Neurophysiological and EMG signals were sampled at 25 kHz and 1 kHz , respectively , using a digital interface and Spike2 software ( Cambridge Electronic Design , Cambridge , UK ) .", "Prior to insertion of the silicon probe into SMC or ECN , the electrode surface was coated with fluorescent DiI ( Life Technologies , Carlsbad , CA ) for subsequent histological verification of electrode placement .", "A Ag/AgCl ground electrode ( Medwire , Mt . Vernon , NY , 0 . 25 mm diameter ) was placed into the visual cortex ipsilateral to the silicon probe .", "Brain temperature was monitored using a fine-wire thermocouple ( Omega Engineering , Stamford , CT ) placed in the visual cortex contralateral to the ground wire .", "For all experiments , brain temperature was maintained at 36–37 °C .", "Electrode position was established when it was possible to reliably evoke neural activity by gentle stimulation of the contralateral ( for sensorimotor cortex ) or ipsilateral ( for ECN ) forelimb .", "Other parts of the body were also stimulated to confirm selectivity .", "Using procedures similar to those described previously ( Del Rio-Bermudez et al . , 2015; Sokoloff et al . , 2015a; Tiriac et al . , 2012 ) , data acquisition began after local field potentials ( LFP ) and multiunit activity ( MUA ) were identified and had stabilized for at least 10 min .", "At the end of the recording session , the pup was overdosed with sodium pentobarbital ( 1 . 5 mg/g ) and perfused transcardially with phosphate-buffered saline followed by 4% paraformaldehyde .", "Brains were sectioned at 50 µm using a freezing microtome ( Leica Microsystems , Buffalo Grove , IL ) .", "Recording sites were verified by visualizing the DiI tract or fluorogold at 5–20X magnification using a fluorescent Leica microscope .", "Tissue slices were then stained using cresyl violet and the location of the recording site was identified ." ] ]

[ "Nervous systems distinguish between self- and other-generated movements by monitoring discrepancies between planned and performed actions .", "To do so , corollary discharges are conveyed to sensory areas and gate expected reafference .", "Such gating is observed in neonatal rats during wake-related movements .", "In contrast , twitches , which are self-generated movements produced during active ( or REM ) sleep , differ from wake movements in that they reliably trigger robust neural activity .", "Accordingly , we hypothesized that the gating actions of corollary discharge are absent during twitching .", "Here , we identify the external cuneate nucleus ( ECN ) , which processes sensory input from the forelimbs , as a site of movement-dependent sensory gating during wake .", "Whereas pharmacological disinhibition of the ECN unmasked wake-related reafference , twitch-related reafference was unaffected .", "This is the first demonstration of a neural comparator that is differentially engaged depending on the kind of movement produced .", "This mechanism explains how twitches , although self-generated , trigger abundant reafferent activation of sensorimotor circuits in the developing brain ." ]

[ "Many parts of our body twitch while we are asleep and these movements are especially common in babies .", "Unlike the movements we make while awake , twitches during sleep are brief , staccato-like movements that appear to be aimless – but they are not , as traditionally believed , mere remnants of dreams .", "Rather , it is believed that twitches help infants learn about their bodies and how they move .", "When we are awake , the brain routinely compares information from the areas of the brain that produce movements with information coming in from the senses , so that we are better able to anticipate and control the movements we make .", "However , this comparison appears to be suspended during twitching: in 2014 , researchers studying infant rats reported that twitches , although self-produced , are treated by the brain as if they are unexpected or surprising .", "To confirm that the brain treats twitches differently from wake movements , Tiriac and Blumberg – who were involved in the previous study – recorded electrical activity in the brains of infant rats while they were awake and asleep .", "These experiments show that a brain area known as the external cuneate nucleus ( ECN ) was mostly inactive when awake rats vigorously moved their front legs , but became highly active when these same legs twitched during sleep .", "Drugs that disinhibited electrical activity in the ECN unmasked leg movement signals produced by awake rats , but these same drugs had no effect on leg movement signals produced during twitching .", "Thus , these experiments indicate that , when infant rats are awake , the ECN compares signals from the senses with signals from the parts of the brain that produce movements , a key feature of motor control .", "However , when the rats are asleep and twitching , the comparison mechanism is disengaged and sensory signals are allowed to cascade through the ECN to many other structures in the brain .", "Tiriac and Blumberg’s findings open new avenues for understanding how the developing brain learns to distinguish the movements that we produce ourselves from those that occur due to forces in the outside world .", "Also , the challenge remains to identify the specific mechanisms by which twitches help develop and refine the brain circuits that enable mammals to move around as effectively as they do ." ]

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

[ "genetics and genomics" ]

[ [ "Systemic Lupus Erythematosus ( SLE ) is a complex autoimmune disease resulting from a profound loss of immune tolerance to self-antigens ( Olsen and Karp , 2014; Theofilopoulos , 1995a; 1995b; Fairhurst et al . , 2006 ) .", "The disease initiates with the production of autoantibodies against a spectrum of self-antigens ( typically >10 in SLE patients ) , focused on nucleic acids and nucleic-acid-associated proteins .", "Disease pathology begins with the deposition of immune complexes in various target tissues , leading to the activation of inflammatory effector mechanisms that damage critical organ systems .", "Patients with SLE can present with combinations of symptoms , including skin rashes , oral ulcers , glomerulonephritis , neurologic disorders , severe vasculitis , and a distinct form of arthritis ( Tsokos , 2011 ) .", "This extensive heterogeneity in clinical presentation presumably reflects variations in the sites of immune complex deposition and induced inflammation among patients , but also suggests that SLE may be a collection of related diseases , rather than a single pathogenic process .", "A generalized loss in immune tolerance by the humoral immune system and the aberrant activation of inflammatory effector mechanisms at the sites of immune complex deposition , however , are consistent features of SLE .", "Susceptibility to SLE is caused by a combination of genetic and environmental factors ( Fairhurst et al . , 2006; Harley et al . , 2009; Deng and Tsao , 2010; Rai and Wakeland , 2011 ) .", "Current thought postulates that a collection of common risk alleles mediates the development of an autoimmune-prone immune system which , when coupled with poorly-defined environmental triggers , becomes dysregulated , leading to the development of autoantibodies and the initiation of disease pathologies .", "Genome-wide association analyses ( GWAS ) have identified more than 50 SLE risk loci to date , indicating that susceptibility is quite polygenic ( Harley et al . , 2009; Nath et al . , 2008; Harley et al . , 2008; Kim et al . , 2012; Graham et al . , 2006; 2008; 2009; Hom et al . , 2008; Gateva et al . , 2009; Relle et al . , 2015 ) .", "A variety of candidate genes have been identified within these risk loci , including: HLA-DR and HLA-DQ class II alleles , IRF5 , ITGAM ( CD11b ) , STAT4/STAT1 , TNFAIP3 , and BLK .", "The functional effects or 'endophenotypes' that these disease genes contribute to the disease process have not been clearly delineated .", "GWAS utilize a dense array of single nucleotide polymorphisms ( SNP ) to map the positions of risk loci within the human genome to relatively small segments , termed linkage disequilibrium ( LD ) blocks ( typically < 200 Kb in length ) .", "Within these LD blocks , recombination is infrequent and polymorphisms form stable combinations or 'haplotypes' that persist within populations for extended periods ( Balding , 2006; de Bakker et al . , 2005; Frazer et al . , 2007 ) .", "Disease associated 'tagging' SNPs are postulated to be imbedded in specific haplotypes that contain the functional variations that impact disease susceptibility .", "The characteristics of these functional variations and the endophenotypes that they contribute to disease processes are a poorly described aspect of common disease genetics .", "Population sequencing studies have identified extensive variations in both the coding and non-coding regions of the human genome ( Abecasis et al . , 2010; 2012; Barreiro and Quintana-Murci , 2010; Laval et al . , 2010 ) .", "The ENCODE consortium has investigated the functional characteristics of non-coding regions in the human genome in detail and have defined a plethora of regulatory elements impacting transcription levels and cell lineage differentiation , including histone associated regions , transcription factor ( TF ) binding sites , and DNase hypersensitivity clusters ( Gerstein et al . , 2012 ) .", "A parallel series of investigations by several research groups have used expression quantitative trait locus ( eQTL ) analysis to identify common polymorphisms that quantitatively impact gene transcription ( Sheffield et al . , 2013; Vernot et al . , 2012; Dunham et al . , 2012; Bernstein et al . , 2010; Cookson et al . , 2009; Fairfax et al . , 2012; 2014; Gilad et al . , 2008 ) .", "These findings , coupled with data indicating that many disease-tagging SNPs are localized to non-coding regulatory regions ( Maurano et al . , 2012 ) , suggest that the causal variants for common disease risk alleles may impact regulatory processes , rather than protein structure .", "Here we describe the targeted , deep sequencing of 28 risk loci for SLE in a population of SLE patients and controls .", "Our sequencing study identified 124 , 552 high quality sequence variants contained in these risk loci among 1349 Caucasian cases ( 773 ) and controls ( 576 ) .", "Detailed analysis of sixteen of these SLE risk loci demonstrate that haplotypes of functional variations in tight LD with SLE- tagging SNPs often impact the expression of multiple genes , resulting in the association of several transcriptional variations with SLE risk haplotypes .", "Notably , multiple SLE risk haplotypes within the HLA-D region were found to coordinately upregulate HLA-DR , -DQ and a variety of other genes within the antigen processing and presentation pathways for HLA class I and class II molecules .", "These results reveal a new functional diversification mediated by HLA-D polymorphisms and provide important insights into the molecular mechanisms by which HLA-D and other SLE risk loci potentiate disease ." ], [ "Targeted genomic sequencing of twenty-eight GWAS-confirmed SLE risk loci was performed using Illumina ( Illumina Inc . , San Diego , CA ) custom enrichment arrays on genomic DNA from 1775 SLE patients and controls ( Supplementary file 1A , Figure 1A–F ) .", "These procedures resulted in >128 fold coverage of the genomic segments containing these SLE risk loci ( Supplementary file 1B , Figure 1G ) .", "Our bioinformatics pipeline ( Figure 1H ) defined 1349 samples of European American ( EA ) ancestry ( Figure 2A ) that carried 124 , 552 high quality variants of which 114 , 487 are single nucleotide variations ( SNVs ) and 10 , 065 are insertion/deletions ( In/Del ) .", "This sequence-based variant database , which identifies an average of one variant every 39 basepairs in the targeted regions , provides a comprehensive assessment of genomic diversity at SLE risk loci in the EA population .", "The functional properties of these variants were annotated using multiple databases cataloguing the functional properties of human genomic variation ( Figure 2B , C ) , including the phase 3 release from the 1000 genome study ( Auton et al . , 2015 ) , the PolyPhen/SIFT ( Adzhubei et al . , 2010; Ng and Henikoff , 2003 ) coding region database , the ENCODE ( Pazin , 2015 ) and RegulomeDB databases ( Boyle et al . , 2012 ) , and several eQTL databases for immune cell lineages ( Fairfax et al . , 2014; Raj et al . , 2014; Westra et al . , 2013 ) .", "The specific technologies , bioinformatics algorithms , and quality assessments used to generate these data are discussed in Materials and Methods and relevant data are provided in Supplementary file 1B and Figures 1A–H .", "Overall , these sequence analyses identified 70 , 070 previously annotated variations and 54 , 482 novel or unannotated variations within the EA cohort .", "Functional annotation defined about 40% of the variants in the dataset as regulatory , based on their inclusion in eQTL datasets or their localization into ENCODE-defined regulatory segments ( Figures 2B , C ) . 10 . 7554/eLife . 12089 . 003Figure 1 . Sequencing quality metrics and work flow pipeline .", "( A ) Depth of sequence reads across chromosomes 6 , 7 and 8 for three samples , illustrating enrichment efficiency for targeted regions .", "( B ) Zoom in read depth analysis of IRF5-TNPO3 gene region ( ~228 Kb ) for three different samples .", "( C ) Genotype calls for a SNP in IRF5 illustrating read depth across a typical variant position .", "( D ) Examples of data used to genotype a novel SNV in RAVER1 , a novel deletion in ITGAM and a novel insertion in SCUBE1 gene .", "( E ) The distribution of variant calls in forward and reverse sequencing reads .", "( F ) About 35 SNPs from various targeted genes were confirmed by Sanger sequencing .", "Sanger sequencing results were further validated by calculating read depths for reference and alternate alleles in heterozygous samples , as shown for ITGAM and BANK1 .", "( G ) This figure compares fold coverage versus SNP concordance rate for a subset of samples that were both sequenced and genotyped with the Immunochip . v1 SNP array .", "( H ) A diagram of the work flow pipeline for bioinformatics analysis of the sequencing data including quantitative information for the number of variants passing filters at each step . DOI: http://dx . doi . org/10 . 7554/eLife . 12089 . 00310 . 7554/eLife . 12089 . 004Figure 2 . Principal component analysis ( PCA ) and variant summary .", "( A ) Principal component analysis ( PCA ) , showing clustering of study cohort ( orange points ) with the CEU ( blue points ) HAPMAP reference group for Caucasians .", "( B )", "( i ) Pie chart showing percentages of annotated and unannotated variants in common ( MAF≥0 . 05 ) and low frequency ( MAF<0 . 05 ) categories .", "( B )", "( ii ) Pie chart showing percentages of potentially functional single nucleotide variants ( SNVs ) and structural variants ( InDels ) defined by ENCODE and eQTL data .", "( B )", "( iii ) Pie chart showing the distribution of variants in various genomic regions and percentage of potential functional variants in each .", "( B )", "( iv ) Pie chart showing classification of coding variants into various sub-categories .", "( C )", "( i ) Pie chart showing classification of common frequency coding/splice variants .", "( C )", "( ii ) Pie chart showing percentages of ENCODE and/or eQTL defined potentially functional common regulatory variants .", "( C )", "( iii ) Pie chart showing the percentages of un-annotated or novel SNVs and InDels with potentially functional annotations . DOI: http://dx . doi . org/10 . 7554/eLife . 12089 . 004 As shown in Figure 3A , multiple variants in 26 of the 28 risk loci were strongly associated with susceptibility to SLE , with seven loci reaching genome-wide significance ( p≤5 × 10-8 ) , ten reaching suggestive significance ( p≤5 × 10-5 ) , and nine reaching confirmatory significance ( p≤10-3 ) ( tabulated in Supplementary file 1D ) .", "We also replicated associations previously reported in SLE GWAS for 36 SNPs at ten loci ( Supplementary file 1E ) , although the bulk of the strongest associations detected in the sequence dataset were variants that were not previously reported to be associated with SLE .", "As tabulated in Supplementary file 1F , 673 variants in the sequencing data set exhibited similar or stronger associations with disease than published tagging SNPs , and 345 of these were categorized as functional .", "This is presented in Figure 3B , in which functional variants are shown as yellow points , variants with no functional annotations in blue , and previously identified tagging SNPs in red .", "Zoom in Manhattan plots of TNFAIP3 and ITGAM are also shown .", "These results show that multiple , new variants had the strongest disease-associations in 27 of the 28 risk loci and that 14 of the peak variants are annotated as functional . 10 . 7554/eLife . 12089 . 005Figure 3 . Association analysis of sequencing variants from 28 SLE risk loci .", "( A ) Manhattan plot of 15582 common variants ( MAF>0 . 05 ) plotting –log10 p-value of SLE association ( y-axis ) versus chromosomal location ( x-axis ) .", "Horizontal lines mark threshold of significant ( p=10-8 ) and suggestive ( p=10-5 ) genome-wide significance threshold .", "( B ) Same Manhattan plot using color coding to identify functional variants ( yellow ) , variants with no current functional annotation ( blue ) , and previously identified SLE GWAS tagging SNPs ( red ) .", "Zoom in picture of Manhattan plot for TNFAIP3 and ITGAM gene is shown . DOI: http://dx . doi . org/10 . 7554/eLife . 12089 . 005 Sixteen risk loci were selected for more detailed analyses , based predominantly on the presence of multiple variations showing strong associations with disease .", "Table 1 provides association statistics , identifies the strongest associated variants , and tabulates the coding and non-coding functional variants in tight LD with the peak signal ( s ) in each locus .", "As shown , conditional analyses identified four risk loci with multiple , independent signals .", "This indicates that NMNAT2-SMG7 , TNFSF4 , HLA-D , and XKR6 each contained two or more LD blocks with potentially regulatory variants which might be contributing to disease susceptibility independently .", "In this regard , we attribute regulatory characteristics to these variants based on published studies from the ENCODE consortium and other research groups ( see Supplementary file 1F for details ) .", "Additional studies will be required to confirm these regulatory properties and delineate the precise mechanisms impacting disease-relevant mechanisms .", "As shown , 1206 functionally annotated variants were in tight LD ( D’>0 . 8 ) with the 21 peak risk signals and all but 7 of these were non-coding , regulatory variants .", "These results demonstrate that multiple functional variations are in tight LD with the peak disease associated signal in every risk locus . 10 . 7554/eLife . 12089 . 006Table 1 . Characteristics of disease associated variants at sixteen SLE risk loci . DOI: http://dx . doi . org/10 . 7554/eLife . 12089 . 006Risk locusSignalPeak SNPMinor alleleOdds ratio ( Minor allele ) Allele Freq .", "( Cases ) Allele Freq .", "( Controls ) SLE association P-valueSLE associated Annotated variantsVariants in LD with peak SNP ( D' >0 . 8 ) Total variantsTotal potentially functional variantsTotal coding variantsSTAT41rs12612769C1 . 70 . 290 . 195E-10524990HLA-D1rs9271593 ( XL9 ) C1 . 70 . 550 . 427E-1083553039802rs9274678 ( DQB1 ) G2 . 10 . 240 . 136E-097362166903rs36101847 ( DRB1 ) T0 . 50 . 130 . 238E-097602961260ITGAM-ITGAX1rs41476751C1 . 90 . 250 . 158E-09153121623IRF5_TNPO31rs34350562G1 . 80 . 230 . 143E-092451891240UBE2L31rs181366T1 . 50 . 270 . 202E-078279551BANK11rs4699260T0 . 70 . 200 . 289E-06267143292TNIP11rs62382335A1 . 40 . 140 . 106E-054622160TNFAIP31rs57087937T1 . 90 . 100 . 062E-066963401CCL22-CX3CL11rs223889T1 . 50 . 340 . 275E-073225200RAVER1-ZGLP11rs35186095T1 . 30 . 210 . 172E-044324190ICA11rs74787882A0 . 70 . 060 . 092E-03341060TNFSF41rs1819717G0 . 70 . 290 . 362E-0573301402rs4916313C1 . 30 . 390 . 322E-0430210BLK1rs7822109C0 . 80 . 460 . 529E-059761380XKR61rs4840545A2 . 00 . 130 . 071E-07335512302rs7000132C0 . 90 . 420 . 465E-041781180NMNAT2-SMG71rs41272536G2 . 90 . 110 . 052E-08338802rs111487113A0 . 60 . 130 . 185E-041750ETS11rs34516251A0 . 80 . 180 . 217E-03181060 The strategy utilized to assess the association of functional variations with disease is outlined in Figure 1H", "( iv ) and illustrated for the STAT4 risk locus in Figure 4 .", "As shown in Figure 4A and tabulated in Supplementary file 1B , targeted sequencing of the 104 . 2 kb STAT4 risk locus produced an average of 100 . 17-fold coverage and identified 2273 high quality variants .", "The LD structure of this region was assessed using 104 common markers ( MAF>0 . 1 ) .", "As shown , two distinct LD blocks were identified and the ~68 Kb LD block that encompasses the 3’ portion of STAT4 contained the SLE disease-tagging SNPs .", "Figure 4B plots the disease association of all of the common variants within this LD block and Figure 4C demonstrates that conditioning with the strongest SLE tagging SNP ( rs12612769 ) accounts for all of the disease association within STAT4 .", "These results indicate that functional variations in tight LD with rs12612769 are responsible for the disease-associated endophenotypes of the STAT4 locus .", "Figure 4D demonstrates that seven functional variants are in strong LD ( D’>0 . 8 ) with rs12612769 ( strongest tagging SNP in this analysis ) and rs7574865 ( strongest tagging SNP from literature ) .", "Figure 4E presents 4 prevalent ( frequency > 0 . 05 ) haplotypes formed by these functional variants , which in sum account for >90% of the chromosomes found among the 1349 EA samples .", "As shown , HAP2 is strongly associated with susceptibility to SLE ( 6 . 88E-08 ) and HAP1 is associated with protection ( 6 . 00E-04 ) . 10 . 7554/eLife . 12089 . 007Figure 4 . LD structure , haplotypes and MJ networks analysis at STAT4 locus .", "( A ) LD structure of STAT4 sequenced segment is shown above molecular map of the genomic segment showing STAT1 and STAT4 exon structure .", "The locations of GWAS tagging SNPs are shown above LD plot , which was produced with 104 markers ( MAF≥10% ) in 1349 Caucasians .", "( B ) Zoom in Manhattan plot showing SLE association levels of individual sequence variants in STAT4 LD block containing STAT4 tagging SNPs .", "Yellow points indicate functional variants , blue points indicate un-annotated variants and red points identify GWAS and study peak tagging SNPs .", "( C ) Conditional analysis on peak SNP rs12612769 removes all significant associations with SLE within the LD block .", "( D ) LD block based on nine potentially functional SLE associated variants used for haplotype analysis .", "( E ) Derived haplotypes with SLE association results .", "( F ) Median-joining ( MJ ) network analysis of STAT4 haplotypes .", "Spheres ( termed nodes ) represent the locations of each haplotype ( from table in E ) within the network and the size of the node is proportional to the overall frequency of that haplotype in the dataset .", "Each node is overlaid with a pie chart that reflects the frequency of that haplotype in cases ( red ) versus controls ( white ) .", "The lines connecting the nodes are labeled with the variants that distinguish the connected nodes and the length is proportional to the number of variants .", "Haplotypes with significant ( p<0 . 05 ) association with SLE are highlighted with red ( risk ) and blue ( non-risk ) .", "Study peak SNP , SLE GWAS tag SNP and eQTLs are indicated with arrows , boxes and circles within their locations within the network .", "( G ) Presents cis-eQTL effects observed with SNP2 on STAT1 and STAT4 in macrophage RNAseq analysis .", "( H ) Similar eQTL effects observed in published eQTL databases in literature . DOI: http://dx . doi . org/10 . 7554/eLife . 12089 . 007 Figure 4F presents the patterns of sequence divergence that distinguish these haplotypes , utilizing the median neighbor joining ( MJ ) algorithm ( Bandelt et al . , 2000 ) .", "MJ analysis is a phylogenetic algorithm that models sequence-based allelic divergence of haplotypes within species .", "For MJ diagrams , the spheres ( termed nodes ) represent individual haplotypes in the network and their size is proportional to their frequency .", "The pie charts overlaid on each node represent the relative frequency of that haplotype in cases ( red ) and controls ( white ) .", "The individual SNPs that distinguish each node are listed along the line that connects them and the length of the line is roughly proportional to the number of SNPs that distinguish the haplotypes .", "The network is progressive , such that the two nodes at opposite ends of the network are most divergent .", "In essence , MJ analysis provides a visually informative illustration of the relationships of a set of haplotypes segregating within a population .", "Several features of STAT4 polymorphisms within the EA population are apparent from this analysis .", "First , HAP1 , HAP3 , and HAP4 form a clade of protective haplotypes ( nodes highlighted in blue ) , all with decreased frequencies in SLE patients .", "Further , both the peak signal SNP in this analysis ( SNP5 ) and the peak GWAS SNP from the literature ( SNP6 ) together with three functional variants , SNP2 , SNP8 , and SNP9 , distinguish the disease-associated HAP2 ( highlighted in red ) from the haplotypes in the protective clade .", "As listed in Supplementary file 2 , SNP8 ( rs10181656 ) is located within a binding site for the CCCTC-binding factor ( CTCF ) , which is a chromatin insulator that inhibits transcription and plays a role in defining the borders of transcriptional domains .", "SNP9 ( rs7582694 ) is located within an ENCODE-defined segment containing transcription binding sites for ESR1 ( estrogen response elements ) and FOS1 .", "Both of these transcription factors are active in multiple tissues and immune cell lineages and both are annotated by ENCODE with strong effect scores and good regulomeDB scores , suggesting that these variations mediate transcriptional endophenotypes in several cell lineages .", "Finally , SNP2 ( rs11889341 ) is the most potent of several eQTL variants within the STAT4 risk locus is very strongly associated with SLE susceptibility ( p<4 . 8 × 10-9 ) , and impacts the transcription levels of STAT1 and STAT4 ( Supplementary file 2 ) .", "As shown in Figure 4G , our eQTL dataset for monocyte-derived macrophages ( MDM ) identifies a significant increase in baseline STAT1 and STAT4 transcription with the T allele of SNP2 , which associates this phenotype with susceptibility to SLE .", "Several other SNPs distinguishing the protective and risk haplotypes were also associated with STAT1 and/or STAT4 transcription levels in published eQTL databases from ex vivo monocytes or peripheral blood ( Supplementary file 2 and Figure 4H ) .", "These results indicate that the transcription of both STAT1 and STAT4 are impacted by variants in tight LD with the SLE tag variant and that the disease risk allele is associated with increased transcription of both genes in multiple cell types .", "Sequence analysis of the HLA-D region revealed 15129 common variants ( 1 variant/29 . 6 bp ) distributed throughout the 448 Kb segment analyzed .", "These variations occur predominantly in non-coding regions , indicating that the entire HLA-D region is diversified .", "This result is consistent with previous genomic sequencing analyses of HLA-D that defined 4–5 phylogenetic clades with ancient origins for this segment of HLA-D ( Raymond et al . , 2005 ) .", "Figure 5A presents the LD structure of HLA-D within the EA cohort , based on 8062 common variants ( MAF>0 . 15 ) .", "Overall , LD in the region is high and the LD structure is very complex , with multiple partial LD associations exhibited between various blocks throughout the region . 10 . 7554/eLife . 12089 . 008Figure 5 . LD structure , haplotypes and MJ Network analysis of XL9 region .", "( A ) The LD structure of HLA-D region is shown below a molecular map of the region .", "The locations of the genes and five genome-wide SLE association signals are marked .", "Peak association signal is coded blue .", "( A )", "( i ) The HLA-D LD structure in 1349 Caucasians from present study assayed with -8062 common ( MAF>0 . 15 ) variants .", "( A )", "( ii ) The HLA-D LD structure in 2504 samples representing twenty-six cohorts from the world population .", "Data obtained by analysis of the1000 Genome project datasets using the same -8062 variants analyzed in A", "( i ) .", "( A )", "( iii ) SNP content of Immunochip v . 1 across HLA-D region .", "( A )", "( iv ) High quality common variant calls in this region from targeted sequencing in this study .", "Highlighted area boxes the XL9 through DQB1 5’ segment regulatory region .", "Yellow points indicate potentially functional variants and blue points indicate un-annotated sequencing variants .", "( B ) Zoom Manhattan plot of all common ( MAF>0 . 05 ) variants using color coding to identify functional ( yellow ) and non-annotated ( blue ) variants .", "The locations of peak association signals are marked .", "A molecular map of the region and the tiled regions for targeted sequencing are identified at the bottom .", "Gaps reflect the locations of long stretches of highly repetitive regions that cannot be assembled .", "( B )", "( i ) The residual association level after conditioning on peak signal 1 in XL9 .", "( B )", "( ii ) Residual association level after conditioning on both signal 1 ( XL9 ) and signal 2 ( DQB1 5’ segment ) .", "( B )", "( iii ) No significant associations remain after conditioning on signal 1 ( XL9 ) , signal 2 ( DQB1 5’ segment ) , & signal 3 ( DRB1 ) .", "Yellow points identify potentially functional variants and blue points indicate un-annotated variants .", "( C ) Conditional analysis on peak SNP rs9271593 ( XL9 signal ) showing that all significantly associated variants are in tight LD .", "( D ) A 60KB LD block generated with 56 variants from XL9 region with strong regulatory scores and association with SLE .", "( E ) Twelve haplotypes generated with HAPLOVIEW using the 56 regulatory variants .", "Frequencies in cases and controls , association statistics , and odds ratios are provided .", "Protective ( blue ) and risk ( red ) haplotypes are highlighted .", "( F ) Median neighbor-joining ( MJ ) network produced as described in the text .", "Annotation is the same as presented in legend for Figure 2 .", "Variants that disrupt binding sites of CTCF , ZNF143 , and IRF4 are labeled . DOI: http://dx . doi . org/10 . 7554/eLife . 12089 . 008 More common ( 15129 ) than rare ( 12076 ) variants were detected in HLA-D , which differs significantly from the roughly 5-fold excess of rare variants that are typically detected throughout the human genome and at other SLE risk loci in our study ( Abecasis et al . , 2010; 2012; Hu et al . , 2014 ) ( Supplementary file 1B ) .", "Previous studies in many species have demonstrated that much of the extant polymorphisms found in MHC class I and class II genes have ancient origins and persist in populations over evolutionary timespans ( McConnell et al . , 1988; Lawlor et al . , 1988; Gyllensten and Erlich , 1989; Edwards et al . , 1997; She and Wakeland , 1991 ) .", "The preponderance of common variants in this sequence dataset is consistent with an ancient origin of these HLA-D polymorphisms within the human lineage .", "In addition , the LD structure of these HLA-D variations in our EA cohort is very similar to the LD structure obtained for this segment of HLA-D in the 2504 human genomes in the 1000 genome project ( Figure 5Ai and", "ii ) .", "Figure 5B plots the association of variants throughout HLA-D with SLE and uses color-coding to distinguish regulatory variants ( yellow ) from variants with no current functional annotation ( blue ) .", "As shown , 3786 variations in HLA-D are nominally associated with SLE ( p<0 . 05 ) and 1797 of these have annotated regulatory functions .", "Five separate segments within HLA-D contained variants achieving genome-wide significance for association with SLE .", "The peak SLE association was with rs9271593 ( p=6 . 50E-10 ) , which is one of 687 SLE-associated regulatory variants mapping to the XL9 regulatory component within the intergenic region separating DRB1 and DQA1 ( Majumder et al . , 2006; Majumder et al . , 2008 ) .", "This ~50 kb segment is heavily annotated with ENCODE-defined regulatory sequences controlling chromatin structure and/or the binding of specific transcription factors .", "As shown in Figure 5Aiii & iv the XL9 segment of HLA-D is not adequately covered by SNP typing arrays such as the Immunochip v . 1 , possibly accounting for the failure to detect this potent SLE association in previous GWAS analyses .", "The second strongest SLE association signal was with rs9274678 ( p=6 . 21E-09 ) , which is located in a segment extending 5’ from the DQB1 proximal promoter .", "The third genome-wide SLE signal was with rs36101847 ( p=8 . 33E-09 ) , which has no functional annotation and is located in close proximity to DRB1 exon 2 , which encodes the peptide binding segment of DRB1 .", "The fourth signal is rs9269131 ( p=9 . 92E-09 ) , which has no functional annotation and is in close proximity to DRA1 .", "Finally , the fifth signal is rs2076530 ( p=2 . 21E-08 ) , which is a regulatory SNP in proximity to BTNL2 that has previously been associated with sarcoidosis and autoimmunity ( Hofmann et al . , 2013 ) .", "The independence of these five SLE association signals was assessed by conditional analysis , beginning with the peak SNP ( XL9 region , rs9271593 ) and using a forward stepwise regression method ( Figure 5B ) .", "As shown in Figure 5Bi , conditioning with rs9271593 removed the associations of signals 4 and 5 with SLE , consistent with the LD associations revealed in Figure 5A .", "These results indicate that the DRA1 and BTNL2 disease association signals are in strong LD with variants in the XL9 region .", "However , the DQB1 promoter and DRB1 signals , although significantly diminished , were still significantly associated with disease after removal of the peak XL9 signal , indicating that these signals were somewhat independent .", "Removal of the XL9 and DQB1 promoter signals left only the DRB1 signal with marginally significant SLE association and removal of all three signals removed all significant association of HLA-D with disease ( Figure 5Bii &", "iii ) .", "Thus , these conditional analyses identified three independent disease associations , each localized to important regulatory or coding elements within HLA-D .", "Their basic properties and SLE association characteristics are summarized in Table 1 .", "As shown in Figure 5C , conditioning on the XL9 signal ( rs9271593 ) completely removed the SLE association of variants within the DRB1 to DQA1 intergenic segment , indicating that rs9271593 tags the XL9 functional haplotype responsible for this disease signal .", "The content of regulatory variants in strong LD ( D’ >0 . 8 ) with the XL9 signal was very high ( Table 1 ) and consequently detailed haplotype analysis was focused on variants with strong ENCODE functional effect scores ( >500 for at least one transcription factor binding site ) , eQTL effects , and associations with SLE .", "This identified 56 functional variants that formed 12 haplotypes of which HAP3 was strongly associated with SLE susceptibility ( OR 2 . 0 , p<7 . 04 E-08 ) while HAP1 ( OR 0 . 58 , p<1 . 63 E -06 ) and HAP6 ( OR 0 . 34 , p<1 . 69 E-07 ) were protective ( Figure 5D , E , Supplementary file 2 ) .", "As shown in Figure 5F , MJ analysis found that the protective ( shaded in blue ) and risk ( shaded in red ) haplotypes form separate clades at opposite ends of the network , indicating that these two extremes in disease association are also extremes in the divergence of regulatory variations .", "Figure 6A overlays the locations of the three peak disease signals in HLA-D with the multitude of regulatory elements located within the ~130 Kb segment spanning HLA-DRB1 through HLA-DQB1 .", "This genomic segment contains 5 separate regions with dense arrays of sequence elements that regulate chromatin structure ( histone marks , DNAse I clusters ) and transcription factor binding .", "As shown in the top tract within Figure 6A , more than 150 variations within this small genomic segment are eQTLs that have been shown to impact the transcription of 72 genes in various immune cell lineages ( Supplementary file 2 ) .", "Our own RNA-SEQ-based eQTL dataset for MDM associates many of these variations with quantitative variations in the transcription of DRB1 , DQA1 , and DQB1 .", "All of the MDM eQTL variants impacting HLA-DR and DQ expression are associated with variants in XL9 ( Figure 6D ) . 10 . 7554/eLife . 12089 . 009Figure 6 . Chromatin architecture and transcriptional regulation at SLE associated XL9 region .", "( A ) A snap shot of the ~140 Kb DRB1-DQB1 segment that contains three genome-wide association signals for SLE .", "The locations of HLA class II genes and the peak signals are marked .", "The locations of some of the more than 750 eQTLs variants mapped into this region are overlaid onto ENCODE defined regulatory elements ( Histone marks and DNA hyper sensitivity clusters ) .", "( B ) A snap shot of a ~1 Kb segment in the center of the XL9 that contains 13 of the 56 strong regulatory variants that constitutes the XL9 haplotype .", "The positions of the canonical protein binding motifs of CTCF , IRF4 and ZNF143 highlighted in yellow and the peak XL9 SNP highlighted in blue .", "The locations of about 30 binding sites for transcription factor that are located within this same region and are also impacted genetic variation are also listed .", "( C ) The consensus sequence for IRF4 binding in XL9 is shown with the locations of the two nucleotide variants boxed and marked .", "The consensus sequence for IRF4 binding ( GA ) are the alleles present in XL9 risk haplotypes .", "The alternative alleles for these two nucleotides , which are much less frequent in IRF4 binding motifs , are in protective haplotypes .", "The red and blue highlighted paths describe the predicted effects of these variations on IRF4-mediated transcription of HLA-DR and HLA-DQ , with risk haplotypes highlighted in red and protective haplotypes highlighted in blue .", "( D ) shows cis eQTL effects observed with SLE associated XL9 region regulatory variants .", "SNPs were found to impact the expression level of HLA-DRB1 , HLA-DQA1 and HLA-DQB1 gene in monocyte derived macrophages ( MDMs ) .", "In each plot , x-axis shows three genotypes of a given eQTL SNP and y-axis shows RNAseq expression values in RPKM .", "SNP numbers correspond to XL9 variants in Figure 5F .", "( E ) Part i shows LD between peak regulatory SNP and a coding SNP in HLA-DRB1 , DQA1 and DQB1 .", "Part ii highlights the SLE associated coding allele sequence and shows the association statistics on peak regulatory and coding SNP haplotype for above three genes .", "Part iii shows the allelic bias in transcription in DRB1 , DQA1 and DQB1 gene in human macrophages , demonstrated in terms of significantly different number of RNA sequencing reads for SLE risk and non-risk allele .", "Part iv shows the transcriptional bias between risk and protective alleles for HLA class II genes in four heterozygous human donors for these IRF4 variants . DOI: http://dx . doi . org/10 . 7554/eLife . 12089 . 009 XL9 contains binding sites for multiple factors affecting chromatin structure ( CTCF , ZNF143 ) and has been shown to assemble HLA-DR and HLA–DQ proximal promoters into a transcriptional complex that facilitates coordinate , tissue-specific transcription ( Bailey et al . , 2015; Xi et al . , 2007; Liu et al . , 2008; Whitfield et al . , 2012 ) .", "This assembly is mediated by interactions of the transcriptional insulator protein CTCF with cohesion and other chromatin regulatory components such as ZNF143 .", "Current data supports the idea that the XL9 transcriptional complex contains multiple regulatory domains that interact with a variety of transcription factors to control the coordinated expression of HLA-DR and -DQ in lymphoid , myeloid , and thymic epithelial cell lineages .", "For example , recent studies by Singh and co-workers have demonstrated that the level of transcription of HLA class II molecules in dendritic cells is strongly controlled by the transcription factor IRF4 , which is one of several transcription factor binding sites located within XL9 ( Vander Lugt et al . , 2014 ) .", "Notably , IRF4 up-regulation of MHC class II molecules was shown by these investigators to be strongly associated with disease severity in a murine model of experimental autoimmune encephalomyelitis ( EAE ) .", "Figure 6B presents a fine map of variations occurring within the regulatory sequence elements in a 1 Kb segment in the center of XL9 .", "This segment contains more than 30 transcription and chromatin configuration factor binding sites .", "Thirteen of the fifty-six potent regulatory variants in the XL9 haplotypes are located within this small segment .", "Five of these variants are located in the consensus binding sites for CTCF , ZNF143 , or IRF4 and are predicted to impact their binding properties ( Figure 6B ) .", "As shown in the MJ network in Figure 5F , four of the five motif variants occur within the SLE risk clade and differ between the protective and risk clades .", "Interestingly , one of the IRF4 variants occurs in the final link of the risk clade leading to HAP3 ( strongest SLE association ) , while the second IRF4 variant is in the final branch leading to risk HAP2 .", "Figure 6C diagrams the nucleotide changes in the binding motif of IRF4 caused by the two XL9 IRF4 variants , both of which are predicted to strongly impact the binding of IRF4 .", "For both of these variants , alleles carrying the unmodified IRF4 consensus binding sequence are associated with the risk haplotypes , while those with nucleotides that are less common to the consensus motif are present in all of the non-risk haplotypes ( Figure 5F , Figure 6C , and Supplementary file 2 ) .", "As diagrammed in Figure 6C , this suggests that the transcription of HLA-DR and -DQ should be increased in individuals carrying risk HAP2 and HAP3 due to increased IRF4 binding to XL9 regulatory domains .", "As shown in Figure 6D , the HAP2 and HAP3 alleles of variants within this short segment , including an IRF4 motif variant , are strongly associated with increased transcription of HLA-DRB1 , HLA-DQA1 , and HLA-DQB1 in our eQTL panel of MDM cell cultures and in datasets from the literature ( Supplementary file 2 ) .", "The crucial role of the XL9 region in the chromatin configuration of the HLA-DR and DQ transcriptional complex suggests that XL9 variations should impact the transcription of both DR and DQ in a cis-active , chromosome-specific manner .", "To test this , we measured allele-specific transcription in four individuals that are heterozygous for XL9 alleles within our MDM eQTL panel .", "Each of these individuals carries a HAP3 XL9 risk haplotype together with either a HAP1 or HAP6 XL9 protective haplotype .", "We assessed the allelic bias of transcription for DRB1 , DQA1 , and DQB1 in these heterozygotes utilizing coding region SNPs in tight LD with XL9 regulatory variants .", "As shown in Figure 6E , coding region variants linked with XL9 risk associated regulatory variants are present in a significantly higher proportion of RNA-SEQ reads than coding variants linked to protective alleles .", "The allelic bias in RNA-SEQ reads for HLA-DRB1 , HLA-DQA1 , and HLA-DQB1 is presented for a representative coding SNP in Figure 6E", "( iii ) .", "This bias in SNP read depth was a consistent feature for SNP variants throughout exon 2 and exon 3 for all three genes in all four heterozygous individuals [Figure 6E", "( iv ) ] .", "Taken together , these experiments demonstrate that XL9 regulatory variations modulate the level of transcription of HLA-DR and HLA-DQ in a chromosome-specific manner .", "The functional implications of increased transcription of HLA-D by SLE risk associated XL9 alleles are contingent upon their impact on the surface expression levels of HLA-DR and HLA-DQ molecules on immune cell lineages .", "To test this , quantitative flow cytometry was performed on monocyte-derived dendritic cell cultures ( MDDC ) derived from the PBMC of individuals with specific XL9 haplotypes .", "As shown in Figure 7A . 1 and A . 2 , HLA-DR surface expression is roughly 2 . 5-fold higher on unstimulated MDDC from a HAP3 ( risk ) homozygote in comparison to a HAP1 ( protective ) homozygote .", "As shown in Figure 7A . 2 , this statistically significant variation in surface expression was fully reproducible .", "RNA-SEQ analyses of these same MDDC cultures confirmed the increase transcription of HLA-D genes by the XL9 HAP3 donor ( Figure 7B ) .", "Similarly , the surface expression of HLA-DQ on MDDC from a risk/protective heterozygote is greater than the expression levels of those from a homozygote for a protective haplotype ( Figure 7C . 1–C . 2 ) .", "This increased surface expression of HLA-DQ is maintained on dendritic cells following activation with the TLR7/8 ligand R848 in a time course over 18 hours , indicating that HLA-D risk haplotypes drive higher levels of HLA class II molecule surface expression during TLR activation and dendritic cell maturation ( Figure 7C . 3–C . 8 ) .", "Taken together , these findings indicate that variations in the XL9 regulatory region modify chromatin structure and transcription factor binding , leading to a significant increase in the surface expression of HLA class II in the dendritic cell lineage of individuals expressing SLE risk alleles of HLA-D .", "Finally , the transcription of several genes within the HLA complex are strongly upregulated in lymphoblastoid cell lines from risk versus protective XL9 haplotype homozygotes in the 1000 genome RNA-SEQ lymphoblastoid cell line ( Lappalainen et al . , 2013 ) data set ( Figure 7D ) .", "These results , obtained via an identical analysis of a public dataset , are an independent replicate of our findings of increased expression of important HLA genes in individuals carrying HLA-D haplotypes associated with SLE . 10 . 7554/eLife . 12089 . 010Figure 7 . Cell surface expression of HLA-CLASS II genes .", "( A . 1 )", "Monocyte-derived dendritic cell ( MDDC ) surface expression of HLA-DR in a culture produced from a homozygote for protective ( blue ) and homozygote for risk ( red ) HLA-D haplotypes .", "This experiment was repeated in same donors .", "( A . 1 ) shows flow data .", "( A . 2 ) shows the MFIs from repeated experiments .", "p-value shown in ( A . 2 ) was calculated on mean MFIs from two experiments .", "( B ) shows normalized RNAseq expression on HLA-class II genes in dendritic cells on same donors presented in ( A ) .", "( C . 1–C . 8 ) shows HLA-DQ surface expression on MDDC cultures from a homozygote for protective ( blue ) and heterozygote for risk ( red ) HLA-D haplotype .", "Flow data and respective MFIs are shown on MDDCs at steady state ( C . 1 and C . 2 ) , at 4 hr ( C . 3 and C . 4 ) , 8 hr ( C . 5 and C . 6 ) and 18 hr ( C . 7 and C . 8 ) after stimulation with TLR7/8 ligands .", "( D ) heatmap on RNAseq data on lymphoblastoid cell line ( LCL ) from 1000 genome project compare expression level of HLA-class II genes between individuals homozygous for HLA-D protective and risk haplotype . DOI: http://dx . doi . org/10 . 7554/eLife . 12089 . 010 Detailed analyses of the SLE signal within the segment 5’ of HLA-DQ ( signal 2 ) revealed tight LD with twenty eight regulatory variants that are distributed through a ~40 KB segment extending 5’ from the DQB1 transcription start site .", "The properties of these regulatory variants are provided in Supplementary file 2 and conditioning and MJ analyses are provided in Figure 8Ai-vi .", "Interestingly , none of the regulatory variants are correlated with eQTL effects that impact DQB1 , although they are associated with transcriptional effects on other genes within the antigen processing pathway of HLA , such as PSMB8 , PSMB9 , TAP1 , TAP2 , DQA2 , and DQB2 .", "Among the variants in tight LD , seven have strong ( ≥900 ) effect scores from ENCODE and 4 of these are also scored within the 1 or 2 category in the RegulomeDB database , making it likely that they impact transcription factor binding and transcription ( Supplementary file 2 ) .", "ENCODE predicts that these variants will impact the binding of many transcription factors including POLR2A , RELA , BATF , RUNX3 , TBP , TAF1 , PAX5 , RFX5 , EP300 , and NFIC .", "The twenty eight variants formed seven haplotypes of which HAP4 was protective ( OR 0 . 3 , p<3 . 88 E-09 ) and HAP2 condoned risk ( OR 1 . 9 , p<1 . 11 E-06 ) .", "As shown , two of the strongest regulatory variants and all of the variants showing strong associations with SLE are located on the final MJ branch leading to the risk clade .", "These results indicate that the most potent regulatory variants identified by ENCODE and associated with the increased transcription of multiple components of the antigen presentation pathway ( APP ) , are all associated with increased risk for SLE .", "The DRB1 signal ( signal 3 ) is also associated with twenty eight regulatory variations; however , their predicted functional properties are weaker than those found in the XL9 or DQB1 signals ( Supplementary file 2 ) .", "The DRB1 regulatory variants are distributed in a region extending from the peak SNP through DRB1 and about 10 Kb 5’ from the DRB1 start site towards the XL9 regulatory region .", "As shown in Figure 8Biv , they form 8 haplotypes of which HAP6 is protective ( OR 0 . 4 , p=4 . 58E-05 ) and HAP1 is risk ( OR 1 . 6 , p=1 . 50E-06 ) .", "The DRB1 regulatory variants do not have strong ENCODE or RegulomeDB scores and do not contain eQTL associations with DRB1 expression .", "However , associations with DRB5 , DQA1 , and DQB1 transcription have been reported for variants in the DRB1 regulatory haplotypes .", "Also , although the DRB1 peak variant is strongly associated with SLE , only 2 of the regulatory variants in tight LD with this peak variant are strongly associated with SLE ( Figure 8Bv ) and those variants were not included in the MJ network branch proximal to the risk clade .", "Taken together , these results suggest that these DRB1 regulatory variations may not play a dominant role in the endophenotypes causing the association with SLE .", "The SLE associations of regulatory variations spanning the entire HLA-D interval were assessed using composite haplotypes formed with 32 regulatory variants with strong SLE association signals that were derived from the three independent SLE associated signals .", "As shown in Figure 8C , these variants spanned a 116 Kb block containing DRB1 , DQA1 , and DQB1 and are all in tight LD .", "The composite analysis formed eleven haplotypes that accounted for more than 90% of all of the chromosomes identified within the EA panel ( Figure 8D ) .", "As shown , HAP1 ( p=2 . 38E-07 , OR = 0 . 59 ) and HAP6 ( p=1 . 01E-06 , OR = 0 . 42 ) are protective , and HAP3 ( p=4 . 56E-09 , OR = 2 . 1 ) , HAP2 ( p=0 . 032 , OR = 1 . 3 ) , and HAP11 ( p=0 . 0172 , OR = 2 . 3 ) are risk .", "MJ analysis revealed a pattern similar to that obtained for XL9 haplotypes ( Figure 5F versus Figure 8F ) .", "As shown in Figure 8E , an assessment of chromosome-specific transcription in MDM cultures from eleven heterozygotes for the rs9271593 ( peak XL9 signal ) revealed consistent and highly significant increases in transcription of HLA-DRB1 , HLA-DQA1 , and HLA-DQB1 for chromosomes carrying risk-associated variants .", "These results demonstrate that all of the regulatory haplotypes carrying the risk allele of rs9271593 , which is the XL9 peak SNP ( Figure 8F ) , transcribe higher levels of HLA-D class II genes than protective regulatory haplotypes . 10 . 7554/eLife . 12089 . 011Figure 8 . LD structure , haplotypes and MJ network analysis in HLA-DQB1 and HLA-DRB1 region .", "( A )", "( i ) LD structure at HLA-DQB1 5’ region generated with 68 common ( MAF≥10% ) potentially functional variants in 1349 samples .", "( A )", "( ii ) Zoom Manhattan plot showing SLE variant association levels and conditional analysis on peak SNP rs9274678 .", "( A )", "( iii ) LD block structure of 28 potentially functional SLE associated SNPs which are used for downstream haplotype analysis .", "( A )", "( iv ) Haploview generated seven haplotypes from these 28 functional variants .", "Frequencies in cases and controls and association statistics are provided .", "Risk ( red ) and protective ( blue ) haplotypes are color highlighted .", "( A )", "( v ) MJ networks analysis to illustrate divergence of risk and protective regulatory haplotypes .", "( A )", "( vi ) eQTL variations from public databases for variants in strongest risk haplotype .", "( B )", "( i ) LD structure at HLA-DRB1 region generated with 66 common ( MAF≥10% ) potentially functional variants in 1349 samples .", "( B )", "( ii ) Zoom Manhattan plot showing SLE variant association levels and conditional analysis on peak SNP rs36101847 .", "( B )", "( iii ) LD block structure of 28 potentially functional SLE associated SNPs which are used for downstream haplotype analysis .", "( B )", "( iv ) Haploview generated eight haplotypes from these 28 functional variants .", "Frequencies in cases and controls and association statistics are provided .", "Risk ( red ) and protective ( blue ) haplotypes are color highlighted .", "( B )", "( v ) MJ networks analysis to illustrate divergence of risk and protective regulatory haplotypes .", "( B )", "( vi ) eQTL variations from public databases for variants in strongest risk haplotype .", "Panel ( C ) 116 kb LD block generated with 32 SLE associated potentially functional variations from the three independent association signals in HLA-D region .", "( D ) Haplotype association statistics in cases and controls with risk ( red ) and protective ( blue ) haplotypes highlighted .", "( E ) Allelic bias in level of transcription for HLA-class II genes between SLE risk and non-risk alleles in 11 independent heterozygous donors ( measured as shown in Figure 6 ) .", "Number of RNA sequencing reads were compared between chromosome carrying risk ( orange line ) verses non-risk ( blue line ) allele for each class II gene .", "( F ) MJ network analysis illustrating the relationships of risk and non-risk haplotypes based on 32 functional variations .", "SLE associated variants sitting exactly within specific protein binding motifs i . e . IRF4 , CTCF and ZNF143 are highlighted with arrows . DOI: http://dx . doi . org/10 . 7554/eLife . 12089 . 011 The HLA-D sequence data allowed the imputation of standard HLA-DRB1 , HLA-DQA1 , and HLA-DQB1 class II alleles with four digit accuracy for the EA cohort , using algorithms and strategies described previously ( Morris et al . , 2012 ) .", "The imputed HLA class II allele designations were used to assess the associations of HLA class II alleles with SLE in our EA cohort and to assess the relationships of these classical HLA-D class II alleles with the defined HLA-D regulatory haplotypes .", "The table in Figure 9A lists the SLE association statistics for the most strongly associated HLA class II alleles and HLA-D regulatory haplotypes in the EA cohort .", "As shown , HLA-DRB1_0301 and HLA-DQB1_0201 were the most strongly associated HLA-D class II alleles in this analysis , which is consistent with several previous studies of HLA-D associations with SLE ( Morris et al . , 2012; Armstrong et al . , 2014; Fu et al . , 2011; Furukawa et al . , 2014; Kim et al . , 2014; Morris et al . , 2014; Fernando et al . , 2012 ) .", "The disease associations and odds ratios detected for HAP3 of the XL9 signal ( Figure 5F ) and HAP2 of the DQB1 promoter signal are equivalent with these HLA class II alleles .", "Similarly , as shown in the bottom of Figure 9A , both regulatory haplotypes and HLA class II alleles are strongly associated with protection from SLE .", "Thus , HAP4 of the DQB1 promoter signal , HAP6 of the XL9 signal , and HAP7 of the DRB1 signal all have potent association statistics and odds ratios for decreased frequencies in cases , as do the classic HLA-D class II alleles HLA-DQB1_0302 , HLA-DRB1_0402 , and HLA-DQA1_0301 . 10 . 7554/eLife . 12089 . 012Figure 9 . HLA-D regulatory haplotypes and classical HLA alleles .", "( A ) SLE association statistics of regulatory and classical HLA alleles in this study .", "( B ) Conditional analysis on peak regulatory signals in XL9 , DQB1 and DRB1 regions .", "( C ) Median-joining ( MJ ) network analysis of 32 regulatory variants spanning HLA-DRB1 to DQB1 region .", "SLE associated variants sitting directly on canonical binding motif of CTCF , IRF4 and ZNF143 transcription factor are indicated with arrows .", "The HLA DRB1-DQA1-DQB1 haplotypes associated with each of the risk and protective regulatory haplotypes are presented . DOI: http://dx . doi . org/10 . 7554/eLife . 12089 . 012 Figure 9B presents conditional analyses of the SLE associations contributed by the HLA-D regulatory haplotypes and the imputed HLA class II alleles .", "The top plot illustrates that XL9 regulatory polymorphisms can completely remove the associations of HLA-DRB1 and HLA-DQA1 imputation variants with SLE , but does not remove the DQB1 imputation or 5’ region regulatory haplotype associations .", "Similarly , conditioning on the DQB1 regulatory haplotype removes the association of HLA-DQB1 imputation SNPs with disease , but has little effect on the imputation or regulatory variations in DRB1 , DQA1 , or XL9 ( Figure 9B , middle panel ) .", "Finally , conditioning on HLA-DRB1 imputation variants removes the association of XL9 regulatory variants and the HLA-DQA1 and HLA-DQB1 imputation SNPs , but does not significantly impact the association of the DQB1 regulatory haplotype with SLE .", "These analyses indicate that variations in XL9 and HLA-DRB1 class II alleles are in tight LD and represent a combined contribution to SLE , while variations in the segment 5’ of DQB1 independently contribute to SLE susceptibility .", "Finally , Figure 9C presents the MJ network formed by the composite HLA-D regulatory haplotypes ( from Figure 8F ) and overlays the imputed DRB1-DQA1-DQB1 HLA class II alleles present in individuals homozygous for the protective and risk HLA-D regulatory haplotypes .", "As shown , LD is very strong , but incomplete between the classical HLA class II alleles and the regulatory haplotypes .", "Notably , all homozygotes for regulatory HAP3 ( peak risk ) are also homozygous for DRB1_0301 , DQA1_0501 , DQB1_0201 , which is the HLA-D class II haplotype found in the extended DR3 haplotype ( Kachru , 1984; Smolen et al . , 1987; Hohler and Buschenfelde , 1994; Schur et al . , 1990; Niu et al . , 2015 ) .", "Similarly , regulatory risk HAP2 is predominantly associated with the DRB1_1501 , DQA1_0401 , DQB1_0602 haplotype , which has also been previously associated with susceptibility to SLE .", "Overall , the DR and DQ alleles that have been associated with SLE in previous studies of EA cohorts are found among the regulatory risk haplotypes and are absent from the protective clade ( Morris et al . , 2012; 2014; Niu et al . , 2015; Ramos et al . , 2010 ) .", "Taken together , these results are consistent with the strong LD within this small genomic segment of HLA and suggest that the regulatory variations and the peptide binding groove polymorphisms are two aspects of HLA-D diversification that are tightly intertwined within allelic lineages of the HLA-D region .", "Table 2 provides a summary for all sixteen SLE risk loci that have been analyzed in this study .", "Detailed analyses for the fourteen loci not discussed above are presented in Figures 10–13 .", "Several characteristics of the genetic variations that underlie common SLE susceptibility alleles are revealed by these data .", "First , maximal risk for disease is associated with specific haplotypes typically composed of five or more functional variations that are in strong LD with the peak risk variant .", "The overwhelming majority of these variants are in regulatory elements ( 1199 of 1206 , Table 1 ) and carry ENCODE scores indicating that they are potent functional polymorphisms .", "They occur as stable haplotypes within the EA population and are predicted to impact multiple endophenotypes .", "MJ analysis revealed that the risk and protective/non-risk haplotypes are typically at opposite ends of the networks ( 15 of 16 risk loci ) , indicating that significant variations in disease risk are most strongly associated with multiple functional changes .", "Furthermore , for some risk loci , multiple haplotypes are significantly associated with either risk or protection , but with varying odds ratios , indicating that a spectrum of functional haplotypes with varying disease risk contributions underlie the disease association of individual risk loci . 10 . 7554/eLife . 12089 . 013Figure 10 . LD structure , haplotypes and MJ network analysis of ITGAM , IRF5 , UBE2L3 and BANK1 . Panel 10", "( i ) shows ITGAM , Panel 10", "( ii ) shows IRF5 , Panel 10", "( iii ) shows UBE2L3 and Panel 10", "( iv ) shows BANK1 genetic association analysis .", "( A ) LD structure of studied intervals generated with common ( MAF≥10% ) variants in 1349 samples , 221 in case of ITGAM , 400 in case of IRF5 , 84 in case of UBE2L3 and 430 variants in case of BANK1 .", "( B ) Zoom Manhattan plot of all common variants in studied region showing SLE association levels and conditional analysis on peak SNP/s .", "( C ) LD block based on potentially functional SLE associated SNPs which are used for downstream haplotype analysis .", "( D ) Haploview generated haplotypes from functional variants .", "Frequencies in cases and controls and association statistics are provided .", "Risk ( red ) and protective ( blue ) haplotypes are color highlighted .", "( E ) MJ networks analysis to illustrate divergence of risk and protective regulatory haplotypes .", "Haplotype with significant p value ( p<0 . 05 ) are highlighted with red ( risk ) and blue ( non-risk ) color .", "Study peak SNP , previously known SLE GWAS tag SNP and eQTLs are indicated with arrows .", "( F ) eQTL variations from public databases for variants in strongest risk haplotype . DOI: http://dx . doi . org/10 . 7554/eLife . 12089 . 01310 . 7554/eLife . 12089 . 014Table 2 . Summary of SLE association and functional characteristics of peak variants and functional haplotypes for 16 SLE risk loci . DOI: http://dx . doi . org/10 . 7554/eLife . 12089 . 014GeneKnown GWAS ( tag ) SNPGWAS referenceGWAS ( tag ) SNP ORStudy peak SNPStudy peak SNP ORPeak risk- associated functional haplotypeRisk haplotype ORIncrease in OR of haplotype versus GWAS SNPIncrease in OR of haplotype versus study peak SNPRelated Figure in the manuscriptStrongest ENCODE effect cell line/ tissueeQTL data cell type/tissueSTAT4rs7574865Lee et al . , 20121 . 4rs126127691 . 7ATTCCTTGC1 . 70 . 30Figure 4Mammary gland , EpithelialMonocyte , macrophageHLA-Drs1150754Taylor et al . , 20111 . 54rs9271593 ( XL9 ) 1 . 6910364CCCCTCCATC_TAGCGATGGCG AGCATCGTCA2 . 10 . 560 . 4089636Figure 8C-FB-lymphocyte , lymphoblastoidMonocyteITGAM-ITGAXrs9888739Harley et al . , 20081 . 6rs414767511 . 8696398AAGCATC TAGTCTT GTCTACAA TAGTCTCTC1 . 950 . 350 . 0803602Figure 10 . iB-lymphocyte , lymphoblastoidMonocyte , Peripheral bloodIRF5-TNPO3rs12531711Chung et al . , 20111 . 5rs343505621 . 7593583GAGTT TTCAGTCTA AGCAGT GGTCAGAAC1 . 80 . 30 . 0406417Figure 10 . iiEpithelial cell ( Lung ) , B-lymphocyteMonocyte , macrophageUBE2L3rs5754217Chung et al . , 20111 . 3rs1813661 . 5217361TCAGTTCAC TCCTCTG1 . 40 . 10-0 . 1140361Figure 10 . iiiEpithelial cell ( Lung ) , B-lymphocyteMonocyteBANK1rs10516487Kozyrev et al . , 20081 . 3rs46992601 . 25ATCTCGACGCA TGCGGA TTGGAAC1 . 300 . 05Figure 10 . ivHela-S3 , Epithelial , FibroblastMonocyteTNIP1rs10036748Han et al . , 2009; Galimberti et al . , 20081 . 2rs623823351 . 37AATACGGTC1 . 30 . 12-0 . 05Figure 11 . iB-lymphocyte , lymphoblastoidPeripheral bloodTNFAIP3rs5029939Graham et al . , 20082 . 2rs570879371 . 9092441GGGCAATCT TTGGGGCAAAT2 . 20 . 040 . 3307559Figure 11 . iiB-lymphocyte , lymphoblastoid , hepatocyteno dataCCL22-CX3CL1rs223889Galimberti et al . , 20081 . 4rs2238891 . 45TATAAAGC1 . 50 . 050Figure 11 . iiiB-lymphocyte , lymphoblastoidMonocyteZGLIP-RAVER1rs35186095Present study1 . 3rs351860951 . 3173789TATAGTCT GTAGGATG1 . 50 . 20 . 1826211Figure 11 . ivFibroblast , K-562 , HeLa-S3 , B-lymphocyteMonocyteICA1rs10156091Harley et al . , 20081 . 3rs747878821 . 5GGGT1 . 50 . 20Figure 12 . iB-lymphocyte , lymphoblastoidno dataBLKrs13277113Hom et al . , 20081 . 3rs78221091 . 26ATTTGCCCCA1 . 300 . 04Figure 12 . iiB-lymphocyte , lymphoblastoidMonocyte , Peripheral bloodETS1rs7932088Yang et al . , 20101 . 2rs345162511 . 23GGGCGA1 . 40 . 20 . 17Figure 12 . iiiB-lymphocyte , lymphoblastoid , EpithelialMonocyteTNFSF4rs2205960Han et al . , 20091 . 3rs49163131 . 3TCCATCTTCGA1 . 300Figure 13 . iEpithelial cell ( Lung ) , Fibroblast , HeLa-S3no dataNMNAT2-SMG7rs2022013Cunninghame Graham et al . , 20111rs1114871131 . 3TCACTAAC1 . 30 . 30Figure 13 . iiPrimary Th1 T cellsno dataXKR6rs11783247Harley et al . , 20081 . 2rs70001321 . 2TGTCGCGGCTT1 . 20 . 030 . 03Figure 13 . iiiNeuroblastoma , Mammary gland , FibroblastMonocyte As tabulated in Table 2 , peak risk haplotypes have greater odds ratios for SLE susceptibility than individual peak tagging SNPs from the original published GWAS studies and from the targeted association studies performed here .", "Overall , the peak risk haplotype had a higher odds ratio than the peak GWAS tagging SNP for 13 of 16 loci , resulting in an overall 17% increase in the average odds ratio for the sixteen loci tested .", "This result is consistent with theoretical predictions of the increase in odds ratio that would be achieved by specifically identifying causative variants in complex disease risk loci , thus supporting the presence of the causal variants of SLE within the identified functional haplotypes ( Gusev et al . , 2013; Yang et al . , 2010 ) .", "Thirteen of the SLE risk loci characterized in detail here were identified previously by our group and others and the detailed sequence analyses of these risk loci has confirmed and extended these previous findings .", "Several of these loci contain long haplotypes , as discussed for IRF5-TNPO3 , ITGAM-ITGAX , TNFAIP3 , UBE2L3 and BANK1 , while TNFSF4 and XKR6 each contain two independent association signals in separate LD blocks ( Figures 10–13 and Tables 1 and 2 ) .", "Our analyses found that regulatory haplotypes often contain variants impacting several eQTLs , chromatin structure and transcription regulatory elements .", "The ENCODE defined regulatory elements for POLR2A , CTCF , IRF4 , RELA , STAT5A , RFX5 , RUNX3 were the most common regulatory elements affected by SLE associated variants ( Supplementary file 2 ) .", "Finally , three risk loci , CCL22-CX3CL1 ( Figure 11 . iii ) , ZGLP1-RAVER1 ( Figure 11 . iv ) , and ICA1 ( Figure 12 . i ) , which were comparatively less well-studied for SLE association , were detected with strong statistical associations in this EA cohort ( Table 1 , Supplementary file 2 ) .", "Detailed sequence analysis of these loci identified significant associations of these genes with SLE and identified SLE associated haplotypes impacting multiple regulatory components .", "More results on these loci have been incorporated into the relevant Figure legend for each risk locus . 10 . 7554/eLife . 12089 . 015Figure 11 . LD structure , haplotypes and MJ network analysis of TNIP1 , TNFAIP3 , CCL22 and ZGLP1-RAVER1 . Panel 11", "( i ) shows TNIP1 , Panel 11", "( ii ) shows TNFAIP3 , Panel 11", "( iii ) shows CCL22 and Panel 11", "( iv ) shows ZGLP1-RAVER1 genetic association analysis .", "( A ) LD structure of studied intervals generated with common ( MAF≥10% ) variants in 1349 samples , 140 in case of TNIP1 , 356 in case of TNFAIP3 , 30 in case of CCL22 and 126 variants in case of ZGLP1-RAVER1 .", "( B ) Zoom Manhattan plot of all common variants in studied region showing SLE association levels and conditional analysis on peak SNP/s .", "( C ) LD block based on potentially functional SLE associated SNPs which are used for downstream haplotype analysis .", "( D ) Haploview generated haplotypes from functional variants .", "Frequencies in cases and controls and association statistics are provided .", "Risk ( red ) and protective ( blue ) haplotypes are color highlighted .", "( E ) MJ networks analysis to illustrate divergence of risk and protective regulatory haplotypes .", "Haplotype with significant p value ( p<0 . 05 ) are highlighted with red ( risk ) and blue ( non-risk ) color .", "Study peak SNP , previously known SLE GWAS tag SNP and eQTLs are indicated with arrows .", "( F ) eQTL variations from public databases for variants in strongest risk haplotype . DOI: http://dx . doi . org/10 . 7554/eLife . 12089 . 01510 . 7554/eLife . 12089 . 016Figure 12 . LD structure , haplotypes and MJ network analysis of ICA1 , BLK and ETS1 . Panel 12", "( i ) shows ICA1 , Panel 12", "( ii ) shows BLK and Panel 12", "( iii ) shows ETS1 genetic association analysis .", "( A ) LD structure of studied intervals generated with common ( MAF≥10% ) variants in 1349 samples , 370 in case of ICA1 , 258 in case of BLK and 209 variants in case of ETS1 ( B ) Zoom Manhattan plot of all common variants in studied region showing SLE association levels and conditional analysis on peak SNP/s .", "( C ) LD block based on potentially functional SLE associated SNPs which are used for downstream haplotype analysis .", "( D ) Haploview generated haplotypes from functional variants .", "Frequencies in cases and controls and association statistics are provided .", "Risk ( red ) and protective ( blue ) haplotypes are color highlighted .", "( E ) MJ networks analysis to illustrate divergence of risk and protective regulatory haplotypes .", "Haplotype with significant p value ( p<0 . 05 ) are highlighted with red ( risk ) and blue ( non-risk ) color .", "Study peak SNP , previously known SLE GWAS tag SNP and eQTLs are indicated with arrows .", "( F ) eQTL variations from public databases for variants in strongest risk haplotype . DOI: http://dx . doi . org/10 . 7554/eLife . 12089 . 01610 . 7554/eLife . 12089 . 017Figure 13 . LD structure , haplotypes and MJ network analysis of TNFSF4 , NMNAT2 and XKR6 . Panel 13", "( i ) shows TNFSF4 , Panel 13", "( ii ) shows NMNAT2 and Panel 13", "( iii ) shows XKR6 genetic association analysis .", "These three interval showed more than one independent LD block associated with SLE in our analysis .", "( A ) LD structure of studied intervals generated with common ( MAF≥10% ) variants in 1349 samples , 152 in case of TNFSF4 , 411 in case of NMNAT2 and 643 variants in case of XKR6 .", "In case of TNFSF4 , ( B ) shows two SLE associated LD blocks and zoom Manhattan plot of all common variants in studied region .", "( C ) showing SLE association levels and conditional analysis on peak SNP/s .", "( D and E )", "Haploview generated haplotypes from functional variants in block 1 and block2 , respectively .", "Frequencies in cases and controls and association statistics are provided .", "Risk ( red ) and protective ( blue ) haplotypes are color highlighted .", "Similarly , ( F and G ) shows MJ networks analysis to illustrate divergence of risk and protective regulatory haplotypes from block1 and block2 , respectively .", "Haplotype with significant p value ( p<0 . 05 ) are highlighted with red ( risk ) and blue ( non-risk ) color .", "Study peak SNP , previously known SLE GWAS tag SNP and eQTLs are indicated with arrows .", "In case of NMNAT2 ( 13 . ii ) , ( B ) shows zoom Manhattan plot of all common variants in studied region showing SLE association levels and conditional analysis on peak SNP/s .", "Panel C: LD block based on potentially functional SLE associated SNPs which are used for downstream haplotype analysis .", "( D ) Haploview generated haplotypes from functional variants .", "Frequencies in cases and controls and association statistics are provided .", "Risk ( red ) and protective ( blue ) haplotypes are color highlighted .", "( E ) MJ networks analysis to illustrate divergence of risk and protective regulatory haplotypes .", "( F ) LD block based on a low frequency SLE associated variant ( G ) Low frequency haplotype association analysis ( H ) MJ networks analysis with low frequency haplotype and ( I ) eQTL variations from public databases for variants in risk haplotype .", "In case of XKR6 ( 13 . iii ) , ( B ) shows zoom Manhattan plot of all common variants in studied region showing SLE association levels and conditional analysis on peak SNP/s .", "( C ) LD block based on potentially functional SLE associated SNPs which are used for downstream haplotype analysis .", "( D ) Haploview generated haplotypes from functional variants .", "Frequencies in cases and controls and association statistics are provided .", "Risk ( red ) and protective ( blue ) haplotypes are color highlighted .", "( E ) MJ networks analysis to illustrate divergence of risk and protective regulatory haplotypes .", "Haplotype with significant p value ( p<0 . 05 ) are highlighted with red ( risk ) and blue ( non-risk ) color .", "Study peak SNP , previously known SLE GWAS tag SNP and eQTLs are indicated with arrows .", "( F ) eQTL variations from public databases for variants in strongest risk haplotype . DOI: http://dx . doi . org/10 . 7554/eLife . 12089 . 017" ], [ "The most intriguing result of our sequence analyses is the discovery of a strong association between SLE susceptibility and HLA-D polymorphisms that regulate HLA class II gene expression .", "The HLA-D region is consistently a potent susceptibility locus in autoimmunity and significant effort has focused on defining the molecular mechanisms that mediate autoimmunity in the context of specific HLA-D class II alleles ( Morris et al . , 2012; 2014; Armstrong et al . , 2014; Kim et al . , 2014; Niu et al . , 2015; Graham et al . , 2007; Cruz-Tapias et al . , 2012; Todd et al . , 1987 ) .", "Multiple genetic studies have identified coding variations in the peptide binding sites of MHC class II molecules as key genetic components of the disease associations , strongly supporting the hypothesis that allelic variations in the antigen presentation process underlie autoimmune disease ( Kim et al . , 2014; Morris et al . , 2014; Fernando et al . , 2012; Raychaudhuri et al . , 2012 ) .", "The dominant paradigm has been that the peptide binding regions of disease-associated HLA class II alleles have unique peptide binding properties that present a novel spectrum of self-peptides or modified self-peptides in a manner capable of eliciting autoimmunity .", "Solid data supporting this mechanism have been developed by decades of experiments , notably for insulin peptides in autoimmune diabetes ( Unanue , 2014 ) However , many studies have found that multiple self-antigens are recognized by T cells clones isolated from the earliest stages of disease development , suggesting that HLA-D associated autoimmunity is initiated against multiple self-antigens by a heterogeneous T cell response .", "Notably , SLE patients have a profound breach in immune tolerance and typically produce autoantibodies binding more than ten different self-antigens , with the diversity of autoantigens recognized increasing as individuals approach disease diagnosis ( Olsen and Karp , 2014; Arbuckle et al . , 2003; Li et al . , 2005 ) .", "Further , multiple HLA class II DR and DQ alleles are associated with SLE susceptibility , which indicates that HLA class II alleles with highly divergent peptide binding properties are capable of promoting disease development .", "In this regard , classic studies of the association of DR2 and DR3 with susceptibility to SLE have shown that DR2/DR3 heterozygotes are more strongly associated with disease susceptibility than the individual haplotypes ( Graham et al . , 2007 ) .", "Taken together , these data suggest that SLE is associated with an extensive array of divergent HLA class II alleles that would be predicted to present a diverse array of self-peptides .", "Our data indicate that all of the HLA-DR and -DQ alleles that are strongly associated with susceptibility to SLE are in strong LD with XL9 regulatory haplotypes that increase HLA class II gene transcription .", "Boss and co-workers have shown that XL9 contains CTCF elements that interact with cohesion molecules and other chromatin factors to assemble a transcriptional complex that brings multiple HLA class II promoters into close proximity with an array of transcription factor binding sites ( Majumder et al . , 2006; 2008 ) .", "The XL9 model in Figure 15A . i , which is adapted from a model presented by Majumder et al . ( 2008 ) , illustrates the key role of chromatin configuration in the coordinated transcription of HLA class II genes .", "Consistent with the chromatin structure effects of XL9 , our data indicate that transcriptional variations are chromosome specific in HLA-D heterozygotes , with polymorphisms in the XL9 regulatory haplotype modulating transcription of DR and DQ genes in a cis-specific fashion .", "This indicates that the level of transcription dictated by XL9 will be specific for the adjacent HLA-DR or DQ allele , thus making expression levels an additional allele-specific facet of HLA-D class II molecules .", "Classic studies have demonstrated allele-specific variations in the expression levels of MHC class II molecules in murine MHC heterozygotes and shown that these levels strongly impacted the stimulation of antigen-specific T cells in autoimmune disease models ( Ridgway et al . , 1998 ) .", "Our data suggest that these early studies revealed an important facet of MHC diversity that strongly impacts the development of autoimmunity . 10 . 7554/eLife . 12089 . 019Figure 15 . Model of chromatin architecture and transcription regulatory elements in XL9 and DQB1 segments .", "( A ) ( i-ii ) A model showing the XL9 transcription complex and three important proteins ( CTCF , IRF4 and ZNF143 ) which may be impacted by SLE associated genetic variants hitting canonical motifs in XL9 region ( Adapted from Majumdar et al . , 2008 ) .", "The chromatin structure of the regulatory complex produced in the DQB1 5’ segment is hypothetical and currently unknown .", "A chromosomal map of HLA-DRB1 through HLA-DQB1 region showing ENCODE defined regulatory marks , eQTLs and most strongly impacted transcription factors by XL9 and the DQB1 5’segment is shown below these models .", "The transcription factor binding sites impacted by functional variations within these regions are shown below the molecular map .", "( A )", "( iii ) A table listing the numbers of and characteristics of functional variants in these two regulatory regions of HLA-D .", "( B ) : Global distribution of the major risk and protective haplotypes from the composite HLA-D region analysis . DOI: http://dx . doi . org/10 . 7554/eLife . 12089 . 019 XL9 contains the peak association signal with SLE in our sequencing dataset , indicating that these regulatory haplotypes play an important role in the development of SLE autoimmunity .", "We suspect that the failure to detect XL9 associations in previous GWAS reflects the low frequency of many of the associated variants and the paucity of SNPs in this region on the commonly utilized SNP typing arrays ( see Figure 5 . iii &", "iv ) .", "Our data demonstrate that individuals homozygous for XL9 HAP3 ( risk ) variations have more than two-fold higher surface expression for HLA-DR and DQ molecules at baseline which increases to 4-fold after stimulation with TLR-ligand on monocyte-derived dendritic cells than HAP1 ( protective ) homozygotes ( Figure 7 ) .", "This increase is maintained during the maturation of these dendritic cells via TLR7/8 stimulation , thus supporting the functional significance of these transcriptional variations to immune mechanisms known to impact immune response activation .", "Potent polymorphisms in the binding motif of the IRF4 transcription factor are in the final MJ branch to the SLE-associated XL9 HAP2 and HAP3 haplotypes and it is likely that these two polymorphisms are causal and contribute significantly to this expression change .", "Recent studies by Vander Lugt et al ( Vander Lugt et al . , 2014 ) have demonstrated that IRF4 is a key component of the transcriptional regulation of HLA class II molecules in dendritic cells and that upregulation of MHC class II molecules strongly promotes susceptibility to autoimmunity in an animal model .", "Based on these data , we hypothesize that the XL9-mediated increase in surface expression of HLA-DR and DQ in dendritic cells is predominantly responsible for the association of XL9 regulatory haplotypes with susceptibility to SLE .", "The extensive diversification of HLA-D regulatory elements has implications well beyond the association of these polymorphisms with SLE .", "HLA class II molecules are expressed on a variety of immune cell lineages , including monocytes , macrophages , dendritic cells , B cells , activated T cells and thymic epithelial cells .", "Expression levels are tightly controlled by a variety of transcription factors unique to these cell lineages and their expression impacts a variety of functional processes in the immune system ( Cresswell , 1994; Krawczyk et al . , 2004; Steimle et al . , 1994; Reith et al . , 2005 ) .", "For example , increased surface expression of class II molecules is an essential event in the maturation of dendritic cells , in that higher surface expression is crucial to the increased capacity of mature dendritic cells to effectively present antigens to naïve T cells ( Cella et al . , 1997a; 1997b; Banchereau and Steinman , 1998; Pierre , 1997 ) .", "As tabulated in Figure 15A . ii , twenty-three transcription factor binding sites in XL9 and fourteen in the DQB1 5’ segment are strongly modified by the extensive variations present within these genomic segments .", "Overall , we identified a total of 1651 functional variants in XL9 and 1912 functional variants in the DQB1 5’ segment , indicating that the regulatory characteristics of these transcriptional complexes will be highly diversified among HLA-D haplotypes .", "This level of polymorphism is readily comparable to that observed in the codons for the peptide binding regions of the HLA class II molecules .", "Interestingly , as shown in Figure 15B , both the protective and risk haplotypes that we identified for the entire HLA-D region are present with varying frequencies throughout the global population .", "These findings are consistent with the characteristics of other highly polymorphic regions of HLA , including the HLA-D class II coding alleles , and indicate that these regulatory haplotypes have evolved together with the HLA class II coding regions over long periods .", "Given all of these characteristics , we propose that these regulatory variations represent an essential and highly selected characteristic of the diversification of HLA-D that strongly impacts a variety of immune functions .", "These regulatory HLA-D haplotypes probably have divergent effects on the expression levels of HLA class II molecules in different cell lineages .", "That is , XL9 HAP3 clearly upregulates HLA class II expression in the myeloid lineage , however , that may not be true for HAP3 B cells , T cells , or thymic epithelial cells .", "In addition , XL9 variants impact the function of a variety of transcription factors whose activity is modified by innate system activation signals in specific cell lineages , indicating that modulations of HLA class II expression levels by activation signals may also be differentially associated with individual haplotypes .", "Taken together , these features indicate that the regulatory polymorphisms identified in this study will have multifaceted effects on the adaptive immune response and probably result in a significant diversification in the functioning of HLA class II antigen presentation among HLA haplotypes .", "Finally , a variety of eQTL studies have identified the HLA complex as a 'master' regulatory complex that impacts the expression of genes throughout the genome ( Fehrmann et al . , 2011; 2012 ) .", "An analysis of the available eQTL databases identified a total of 72 genes whose transcription levels are reported to be modulated by variations in the HLA-D region .", "Many of these eQTL targets are encoded in other segments of the HLA complex , as well as 35 that are located on other chromosomes .", "A variety of immune system genes are included in this list and Figure 16 illustrates the pattern of up and down expression that distinguishes the HAP3 risk haplotype from the HAP1 and HAP6 protective haplotypes .", "As shown , essentially all of the HLA class II molecules and a variety of gene products involved in antigen processing , peptide loading , and surface expression are up-regulated by the HAP3 risk haplotype .", "This result illustrates the extensive functional variations that are associated with a single HLA-D risk haplotype within the EA population .", "Whether these eQTLs reflect the formation of remarkably intricate transcriptional complexes , or ( more likely ) very strong LD throughout the HLA complex remains to be determined .", "However , these results indicate that regulatory polymorphisms in HLA-D affect a plethora of immune system genes that are involved in various pathways of the adaptive immune system .", "It is likely that these regulatory variations are an integral element of the functional diversification of HLA and that they will ultimately be found to modulate functions throughout the immune system . 10 . 7554/eLife . 12089 . 020Figure 16 . SLE risk haplotype upregulates the antigen presentation pathway ( APP ) .", "All of the composite HLA-D haplotypes within the risk clade ( highlighted in red ) contain eQTL variants reported to impact 72 genes in the publicly available eQTL datasets utilized in this study .", "The patterns of increased or decreased transcription associated with all of these haplotypes is modeled on the left , with red indicating increased expression and green indicating decreased expression relative to the protective haplotypes shaded in blue .", "All of the HLA-DR , HLA-DQ , and HLA-DP class II molecules , along with a variety of gene products involved in the APP pathway are upregulated in all SLE risk haplotypes .", "A variety of other genes in the immune system , including some with known associations with SLE susceptibility ( C2 , C4A ) are also modulated . DOI: http://dx . doi . org/10 . 7554/eLife . 12089 . 020" ], [ "Genomic sequencing libraries were prepared from 1775 SLE patient or control samples contributed by 5 collaborating sites in the U . S . A and Europe ( Supplementary file 1A ) .", "All subjects gave their written informed consent and research protocols and methods employed were approved by the UT Southwestern Institutional Review Board .", "More than 50% of SLE cases and all of the control samples used in the present study were new recruitments and have not been used in any previous association or GWAS on SLE .", "Target enrichment and deep sequencing was carried out in the UT Southwestern Medical Center IIMT Genomics Core .", "1 ug picogreen measured genomic DNA was sonicated using Covaris S220 platform to generate 300–400 bp genomic fragments .", "The sequencing libraries were generated using TruSeq ( Illumina ) or KAPA Biosystem library preparation kits ( KK8232 ) .", "Each sample was ligated with custom designed Illumina-compatible adaptors with unique 6 base barcodes following the kit manufacturer’s protocol .", "The custom target enrichment array ( Illumina Inc . San Diego , CA , www . illumina . com ) was designed to capture the complete genome sequence of 28 confirmed or potential SLE risk loci ( Supplementary file 1B ) .", "The Illumina custom enrichment system theoretically captured sequence information for ~99 . 94% of the ~4 . 4 Mbs of genome targeted in these risk loci .", "The enriched libraries were sequenced using a paired-end 100 bp protocol to produce 1–2 Gb of high quality data per sample .", "Sequence reads were demultiplexed and each sample’s reads were aligned to the human genome ( HG19 ) using BWA-MEM , with base quality recalibration and local realignment performed with the Genome Analysis Toolkit ( GATKv2 ) ( Li et al . , 2009; McKenna et al . , 2010; DePristo et al . , 2011 ) .", "As illustrated in Figure 1A , target enrichment was highly specific and efficient , typically resulting in >70% of reads on target and resulting in >128X average coverage for the 28 risk loci analyzed ( Supplementary file 1B ) .", "Figure 1B illustrates that coverage within the targeted segments was comprehensive , with relatively uniform read depth throughout the non-repetitive regions .", "In general , continuous sequences could be derived for >85% of the targeted intervals in assembled sequences , with only extended regions ( i . e . >1 Kb ) of highly repetitive sequences failing to assemble .", "Variant analysis with the GATK Haplotype caller identified a total of 215880 variations in the targeted regions using analytic technologies and thresholds analogous to those of the 1000 Genomes Project ( Supplementary file 1B ) ( Abecasis et al . , 2010; 2012 ) .", "As listed in Figure 1G , H , we used additional criteria to filter this dataset to create an ethnically-matched , case-control cohort with uniform coverage and high quality variant calls .", "Figure 1C illustrates the read depth and balanced allele representation of variants that passed all filters and Figure 2A presents principal component analysis of sample ethnicity in comparison to standardized populations from the HAPMAP dataset ( International HapMap et al . , 2003 ) .", "We started with 1775 samples , all of which were sequenced with targeted array .", "Of these , 88 samples had missing case/control status information and 249 were PCA outliers as they did not cluster with HapMap CEU reference population in principal component analysis .", "Of the remaining 1438 PCA pass samples , 11 and 5 were excluded due poor call rate ( <85% ) and being duplicate , respectively .", "Furthermore , 73 samples were excluded due to poor sequencing fold coverage ( <25x , n=54 ) and significant p value ( p>0 . 001 ) of HWE in controls ( n=19 ) .", "Thus , 1349 samples which included 773 SLEs and 576 normal controls passed all quality criteria and were used for genetic association analysis .", "Application of these filters defined 1349 samples of EA ancestry and identified 124 , 552 high quality variants , of which 114 , 487 are single nucleotide variations ( SNVs ) and 10 , 065 are insertion/deletion ( In/Del ) polymorphisms .", "Data supporting the accuracy of these variant calls is provided in Figure 1F , which presents Sanger sequence validation of several key variants .", "In addition , the concordance of sequence-based SNV calls versus SNV calls with the Illumina Immunochip was >99 . 8% for samples with >25X average coverage ( Figure 1G ) , which supports the overall accuracy of variant detection throughout the genomic segments assayed .", "This sequence-based variant database , which identifies an average of one variant every 39 basepairs through 4 . 4 Mb of genomic DNA in 28 validated SLE risk loci , provides a comprehensive assessment of genomic diversity at SLE risk loci in the EA population .", "Raw sequencing data ( FASTQ files ) for all targeted intervals in 1349 individuals is available on request ( www . utsouthwestern . edu/labs/wakeland/about/contact . html ) .", "Variants were annotated using multiple databases cataloguing the functional properties of human genomic variation , including the recent phase 3 release from the 1000 genome study , the PolyPhen/SIFT coding region database , the most recent release of the ENCODE database , and several recent eQTL databases for immune cell lineages .", "The outcome of these analyses for all variants in the final dataset is summarized in Figure 2B , C .", "Our sequence analyses identified 70070 previously annotated variations and 54482 unannotated or novel variations .", "All but 76 of the novel variations were low frequency or rare ( MAF<0 . 05 ) within the EA cohort , which is consistent with expectations ( Supplementary file 1C ) .", "Functional annotation determined that about 40% of the variants in the dataset were potentially functional , most of which were categorized as regulatory based on their localization into ENCODE-defined regulatory segments or inclusion in eQTL datasets ( Figure 2B . i-iv ) .", "Figures 2C . i-iii shows summary statistics on just common coding , regulatory and novel variants .", "A subset ( n=536 ) of study samples was also genotyped with the Immunochipv1 , an Illumina infinium genotyping chip which contains 196524 genomewide markers .", "SNP concordance analysis was done between sequencing and immunochip genotypes in order to validate the quality of sequencing calls .", "Raw image files from immunochip array were imported into Genome Studio ( GS version 1 . 9 . 4 ) and SNPs were called .", "The genotype outputs from Genome Studio were then imported into SNP & Variation suite ( SVS version 7 . 6 . 8 win64 ) for further quality control ( QC ) and downstream association analysis .", "In addition , about 35 SLE associated SNPs from various targeted genes were also confirmed by Sanger sequencing method .", "Quality control filters were applied to both samples and markers .", "All the duplicates and those with call rate <85% were excluded from the analysis .", "Principal component analysis ( PCA ) was done to remove any population stratification .", "About 2902 markers ( MAF≥0 . 05 ) present in both Omni1 HapMap data set and our sequencing data were used for PCA ( Figure 2A ) .", "PCA clusters was further confirmed by doing another PCA based on ~26000 markers present on Omni1 HapMap data set and Immunochip v1 . 0 on subset samples ( n=408 ) .", "Samples that did not cluster with HapMap CEU reference population were excluded after visual inspection of plots .", "SNP concordance was done between sequencing and immunochip data on subset of samples ( n=408 ) , which showed a genotype concordance rate ≥98% at ≥25x fold coverage .", "Therefore , samples with fold coverage <25x were excluded from downstream analysis .", "For genetic association tests , autosomal markers showing HWE p<0 . 001 in controls were excluded .", "Of the 23 , 805 common ( MAF≥0 . 05 ) markers detected in 28 targeted loci , 15 , 582 quality pass variants were used for genetic association tests .", "A basic allelic association test was performed with 1349 PCA confirmed and age matched European cases ( n=773 ) and controls ( n=576 ) .", "The association test was controlled for genomic inflation using Golden Helix scripts where we first determined uncorrected genomic inflation factor ʎ value which was 3 . 0 .", "We further corrected data for batch effects and stratification with PCA using numeric association and regression analysis in Golden Helix .", "Finally , we corrected association results using this inflation value .", "This removed observed genomic inflation in association results .", "Chi-square p values were further corrected for gender bias , using the covariate regression module in SVS , Golden Helix software .", "A total of 5561 markers across 28 loci showed significant ( p<0 . 05 ) association with SLE .", "The LD structure and haplotype analysis was performed in SVS , Golden Helix and using Haploview v4 . 2 .", "Regulatory haplotypes were generated on potentially functional variants with strong LD ( ≥0 . 8 ) to the study peak and/or previously known SLE tagging SNPs .", "An allele was defined as potentially functional if it is a coding variation i . e . non-synonymous , synonymous , UTR or splice variant , and or an ENCODE’s histone mark , transcription factor binding site , DNase I hypersensitivity clusters or an expressed quantitative trait loci ( eQTL ) .", "PAR has been used in genome-wide association studies ( Ziegler and studies , 2009; Bonnelykke et al . , 2013; Wang et al . , 2010 ) .", "It combines information on risk allele frequencies and genotypic relative risks to estimate the excess fraction of cases that would not occur if no one in the population carried the risk allele .", "The case/control design of present study provided authors an opportunity to apply odds ratio ( OR ) and PAR to calculate the relative risk of SLE disease .", "As it has already been shown and present study also confirms that SLE is a polygenic disease , assessment of cumulative disease risk by calculating joint PAR from all the loci is a reasonable approach .", "First we performed conditional analysis on peak SNP from all the 16 loci analyzed in the present study .", "We observed residual SLE association after conditioning on each locus ( Supplementary file 3A ) , which suggests significant and cumulative contribution of each locus to SLE risk within this population .", "Then , we used 16 SLE risk haplotypes to assess the percentage of population disease risk that is attributable to the combined effects of all of these risk loci ( Supplementary file 3B ) .", "PAR was calculated using methods applied in other complex diseases ( Zheng et al . , 2008 ) .", "Human peripheral blood mononuclear cells ( PBMCs ) were enriched by density gradient centrifugation of peripheral blood from healthy human donors through a Ficoll-Hypaque gradient .", "Monocytes were isolated from PBMC either by negative selection using an EasySep Human Monocyte Enrichment Kit ( STEMCELL Technologies ) , or by cell culture dish adherence as plastic adherence method .", "For monocyte isolation by adherence , PBMCs were plated in tissue culture treated dishes and incubated for 2 hours at 37ºC in a humidified CO2 incubator .", "Non-adherent cells were discarded by washing three times .", "For the generation of monocyte-derived dendritic cells ( MDDCs ) or human monocyte-derived macrophages ( MDMs ) , monocytes were cultured in RPMI-1640 with 10% FBS , 2 mM L-glutamine , 10 mM HEPES , 1 mM sodium pyruvate , 100 U/ml penicilin , 100 µg/mL streptomycin supplemented with 100 ng/ml GM-CSF and 50 ng/ml IL-4 or 50 ng/ml M-CSF , respectively .", "The culture media which contained fresh GM-CSF and IL-4 or M-CSF were replaced every 2 days .", "MDDCs and MDMs were harvested on day 7 .", "MDDCs or MDMs were seeded in 6 or 12 well plates at a density of 1 × 106 or 5 × 105 cells/ well , respectively , treated and incubated with 10 ug/ml R848 for 18 hr .", "R848 ( InvivoGen , tlrl-r848 ) is an imidazoquinoline compound with potent anti-viral activity .", "This low synthetic molecule activates immune cells via the TLR7/TLR8 MyD88-dependent signaling pathway ( Hemmi et al . , 2002 ) MDDCs were incubated with anti-HLA-DR FITC ( clone G46-6 ) for 20 min on ice and washed three times with PBS .", "MDDCs were acquired on a FACS Calibur ( BD Biosciences ) and data analyzed using FlowJo software .", "For HLA-DQ staining , MDDC’s were stimulated with 1 ug/ml R848 for 4 , 8 and 18 hr .", "Cells were harvested and washed twice with PBS .", "Staining was done with HLA-DQ-FITC ( Clone: Tu169 ) in PBS for 30 min on ice followed by two washes with PBS and ran on BD FACSCALIBUR .", "Data was analyzed using FlowJO software .", "RNA was extracted using TRIZOL ( Life Technologies ) and RNeasy Mini Kit ( QIAGEN ) according to the manufacturer’s protocol .", "RNA quantity and purity was assessed on a NanoDrop 2000 spectrophotometer ( Thermo Fisher Scientific ) , and integrity was measured on an Agilent Bioanalyzer 2100 ( Agilent Technologies ) .", "RNA-seq libraries were prepared with the Illumina TruSeq RNA Sample Preparation kit ( Illumina ) according to the manufacturer’s protocol .", "Libraries were validated on an Agilent Bioanalyzer 2100 .", "Six RNAseq libraries were sequenced on a SE50 ( single end 50 base pair ) Hiseq 2500 lane , which yielded an average of about 30 × 106 reads/sample .", "We used CLC Genomics Workbench 7 for bioinformatics and statistical analysis of the sequencing data .", "This approach used by CLC Genomics Workbench is based on method developed by Mortazavi et al . ( Mortazavi et al . , 2008 ) .", "Human Genome GRCh37 was used as reference sequence .", "The reference has 33615 genes and 30842 transcripts .", "All uniquely mapping reads to the genes were counted .", "Alignment with mismatch cost of '2' , Insertion cost '3' Deletion cost of '3' was used .", "The maximum number of hits for a read was set to 1 meaning that only reads those maps uniquely were considered .", "The steady state expression of various genes was calculated in terms of RPKM values .", "For eQTL analysis RPKM values were normalized as described previously ( Dozmorov and Lefkovits , 2009; Dozmorov et al . , 2011 ) as well as for population stratification or batch effect and cis-eQTL results were corrected for gender and ethnicity .", "We have accessed multiple public databases to validate and functionally annotate sequencing variants identified in the present study .", "We used DNA and RNA sequencing data-based variants from the 1000 Genome Project samples ( http://www . 1000genomes . org/ ) ; and downloaded DNA sequencing data from the phase III dataset ( ftp://ftp . 1000genomes . ebi . ac . uk/vol1/ftp/release/20130502/ ) for haplotype analysis of 2504 genomic samples from the global human population .", "Similarly , FASTQ files of RNA sequencing data ( http://www . geuvadis . org/web/geuvadis/RNAseq-project ) of lymphoblastoid cell lines derived from 369 Europeans were downloaded and used for HLA class II expression analysis .", "Our analysis of this data is shown in Figure 7D as a heatmap .", "The ENCODE database ( www . encodeproject . org ) was used to annotate variants for transcription factors binding motif , DNase hypersensitivity cluster and histone marks .", "Similarly , the RegulomeDB database ( http://regulomedb . org/ ) was used to annotate potentially regulatory variations .", "Finally , UCSC genome browser ( https://genome . ucsc . edu/ ) was used to generate custom tracks on sequencing variants and for the general visualization of study data .", "We used SNP & Variation suite ( SVS ) of Golden Helix ( version 7 . 6 . 8 win64 , Golden Helix , Inc . , Bozeman , MT , www . goldenhelix . com ) for genetic analysis .", "SNP conditioning analysis was done using regression module of SVS .", "Haploview v . 2 software was used for visualization of LD plots and haplotype analysis ( Barrett et al . , 2005 ) .", "GraphPad Prism 6 . 0 software was used for statistical analysis and graphics .", "Correlations between continuous variables were determined using Pearson’s r in GraphPad Prism 6 . 0 .", "Discontinuous variables were compared by Fisher’s exact test .", "P values <0 . 05 were considered significant ." ] ]

[ "Targeted sequencing of sixteen SLE risk loci among 1349 Caucasian cases and controls produced a comprehensive dataset of the variations causing susceptibility to systemic lupus erythematosus ( SLE ) .", "Two independent disease association signals in the HLA-D region identified two regulatory regions containing 3562 polymorphisms that modified thirty-seven transcription factor binding sites .", "These extensive functional variations are a new and potent facet of HLA polymorphism .", "Variations modifying the consensus binding motifs of IRF4 and CTCF in the XL9 regulatory complex modified the transcription of HLA-DRB1 , HLA-DQA1 and HLA-DQB1 in a chromosome-specific manner , resulting in a 2 . 5-fold increase in the surface expression of HLA-DR and DQ molecules on dendritic cells with SLE risk genotypes , which increases to over 4-fold after stimulation .", "Similar analyses of fifteen other SLE risk loci identified 1206 functional variants tightly linked with disease-associated SNPs and demonstrated that common disease alleles contain multiple causal variants modulating multiple immune system genes ." ]

[ "The human immune system defends the body against microbes and other threats .", "However , if this process goes wrong the immune system can attack the body’s own healthy cells , which can lead to serious autoimmune diseases .", "Systemic lupus erythematosus ( SLE ) is an autoimmune disease in which immune cells often attack internal organs – including the kidneys , nervous system and heart .", "Over the past decade , multiple genes have been linked with an increased risk of SLE .", "However , it is largely unknown how the sequences of these genes differ between individuals with SLE and healthy individuals , and the precise changes that lead to an increased risk of SLE are also not clear .", "Now , Raj , Rai et al . have determined the genetic sequences of over 700 people with SLE and over 500 healthy individuals and looked for differences that influence susceptibility to the disease .", "The vast majority of differences were discovered in stretches of DNA that regulate the expression of nearby genes , rather than in DNA that encodes the structures of proteins .", "Notably , extensive differences were found in a region of the human genome that regulates the production of proteins called Human Leukocyte Antigen class II molecules; which are known to play a critical role in activating the immune system .", "Raj , Rai et al . found that slight changes to the regulatory DNA sequences resulted in an overabundance of these proteins , which led to a hyperactive immune system that is strongly associated with SLE .", "Future studies could now ask if the changes to the regulatory DNA sequences highlighted by Raj , Rai et al . increase susceptibility to other autoimmune disorders as well .", "It may also be possible to use the increased understanding of how the immune system is regulated to develop new ways to minimize the rejection of organ transplants ." ]

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

[ "chromosomes and gene expression", "biochemistry and chemical biology" ]

[ [ "Chromatin remodelers are members of the extensive superfamily 2 ( SF2 ) group of ATPase motors found in all kingdoms of life ( Flaus and Owen-Hughes , 2001; Flaus et al . , 2006 ) .", "SF2 ATPases possess a bi-lobed architecture , with a central ATP-binding pocket defined by two RecA-like domains ( reviewed in Singleton et al . , 2007 ) .", "The occupancy of the central pocket – whether empty , ADP-bound or ATP-bound – determines the preferred conformation for the two lobes of the motor .", "Like the architecturally similar SF1 ATPases , both lobes of the motor coordinate in binding to DNA or RNA , with conformational changes driven by cycles of ATP binding and hydrolysis enabling many SF2 enzymes to translocate in an inchworm fashion along nucleic acids ( Singleton et al . , 2007 ) .", "Chromatin remodelers are specialized for altering the organization of DNA on the nucleosome .", "Canonical nucleosomes consist of two copies each of the four core histones ( H2A , H2B , H3 , and H4 ) wrapped by ~146 base pairs ( bp ) of duplex DNA ( reviewed in McGinty and Tan , 2015 ) .", "Several chromatin remodelers have been shown to ratchet DNA past the histone core in single bp steps , known as nucleosome sliding , which is consistent with the basic inchworm mechanism of DNA translocation ( Deindl et al . , 2013; Harada et al . , 2016 ) .", "One of the earliest models for nucleosome motion suggested that DNA could be shifted past the histone core by twist diffusion , where transfer of one or a small number of bps from one DNA segment to the next results in DNA displacement ( van Holde and Yager , 2003; van Holde and Yager , 1985 ) .", "Each segment of nucleosomal DNA that gains or loses a bp relative to the canonical nucleosome experiences a change in both DNA length and twist and is referred to as a twist defect .", "In addition to explaining heat-induced movement of the histone core along DNA , it was proposed that diffusion of twist defects could explain nucleosome movement by chromatin remodelers ( van Holde and Yager , 2003 ) .", "Given the helical nature of the double helix , DNA translocation by a chromatin remodeler would necessarily impart twist into segments of nucleosomal DNA adjacent to the motor , stimulating propagation of single bps onto and off of the nucleosome .", "Indeed , experimental support for twist diffusion comes from the observations that chromatin remodelers shift DNA off the nucleosome in single bp steps ( Deindl et al . , 2013; Harada et al . , 2016 ) and can induce and are sensitive to torsional strain ( Gavin et al . , 2001; Havas et al . , 2000 ) .", "In contrast to INO80 , which appears to translocate on DNA near the edge of the nucleosome around superhelix location 6 ( SHL6 ) ( Ayala et al . , 2018; Brahma et al . , 2017; Eustermann et al . , 2018 ) , Chd1 , ISWI , and SWI/SNF-type remodelers translocate on nucleosomal DNA at the internal SHL2 site , located ~20 bp from the nucleosome dyad ( Deindl et al . , 2013; McKnight et al . , 2011; Saha et al . , 2005; Schwanbeck et al . , 2004; Zofall et al . , 2006 ) .", "Recent work has revealed the organization of Chd1 domains on the nucleosome in the ADP·BeF3– state , which has defined the positioning and orientation of ATPase motor domains on nucleosomal DNA at SHL2 ( Farnung et al . , 2017; Nodelman et al . , 2017; Sundaramoorthy et al . , 2018 ) .", "However , the underlying mechanism of nucleosome sliding – how the remodeler ATPase stimulates DNA translocation from this internal site on the nucleosome – has remained elusive .", "Here we dissect conformational changes coupled to the ATP binding and hydrolysis cycle of the Chd1 remodeler , which reveals an unexpected process of remodeler-catalyzed DNA translocation .", "Using site-specific cross-linking , we show how both Chd1 domains and nucleosomal DNA change position in response to different nucleotide-bound states of the ATPase motor .", "Remarkably , Chd1 can shift the outer turn or gyre of nucleosomal DNA by 1–3 bp in nucleotide-free ( apo ) and ADP-bound states , consistent with stabilization of a small DNA bulge at SHL2 .", "The shift of entry DNA upon Chd1 binding occurs in a sequence-selective fashion , and correlates with the phasing strength for nucleosomal DNA surrounding the SHL2 binding site of the ATPase motor .", "Using a modified Widom 601 nucleosome positioning sequence , we demonstrate that Chd1 binding at SHL2 alters the twist of nucleosomal DNA in a nucleotide-dependent fashion .", "These findings suggest that nucleosome sliding by Chd1 occurs by creating and expelling twist defects at SHL2 , providing insight into the mechanism of chromatin remodeling ." ], [ "In previous work , we identified the arrangement of Chd1 domains on the nucleosome using site-specific cross-linking with 4-azidophenacyl bromide ( APB ) ( Nodelman et al . , 2017 ) .", "Cross-linking requires van der Waals contact between the reactive nitrene moiety of APB and its DNA target , which corresponds to a reach of ~11 Å from the Cα of the APB-modified cysteine ( Pendergrast et al . , 1992 ) .", "Since cross-links do not necessarily represent the closest DNA contacts , multiple independent cross-links are required to determine the position and orientation of a protein domain with respect to DNA .", "Our modeling , which was based on nine different cross-linking sites , yielded a structurally similar placement of Chd1 domains as cryoEM complexes solved in an ADP·BeF3– state ( Farnung et al . , 2017; Sundaramoorthy et al . , 2018 ) .", "While ADP·BeF3– has been cited as a ground-state mimic of ATP , the BeF3– moiety as the gamma phosphate mimic can occupy a range of positions relative to ADP , depending on the system and conditions , and thus can also resemble a hydrolysis intermediate ( Kagawa et al . , 2004; Thomsen and Berger , 2009 ) .", "As an SF2-type translocase , the ATPase motor of Chd1 is expected to oscillate between open and closed states during nucleosome sliding .", "In this work , we use five cross-linking sites on the ATPase motor to monitor how contacts with DNA change in different nucleotide states .", "Single cysteine substitutions were located in lobe 1 ( N459C and E493C ) and lobe 2 ( V721C , N650C , and S770C; Figure 1A ) .", "A summary of where these sites cross-link in the context of the nucleosome is shown in Figure 1—figure supplement", "1 . Chd1 interacts with both strands of the minor groove at SHL2 ( Farnung et al . , 2017; Nodelman et al . , 2017 ) .", "Based on comparisons to SF1 and SF2 ATPases that translocate along single stranded DNA or RNA , these two strands are referred to as the ‘tracking’ and ‘guide’ strands .", "Like the SWI/SNF-type remodeler RSC , Chd1 is positioned to translocate along the tracking strand in a 3’ to 5’ direction ( Farnung et al . , 2017; Nodelman et al . , 2017; Saha et al . , 2005 ) .", "As the SHL2 site is internally located , it is expected that the ATPase motor must remain in place on the nucleosome as it shifts DNA past the histone core .", "Consistent with this notion , cross-links for both ATPase lobes indicate a relatively fixed position of the motor at SHL2 in apo , ADP , and ATP-mimic states ( Figure 1 and Figure 1—figure supplement 1 ) .", "Cross-linking was expected to vary in a nucleotide-dependent fashion , since the two lobes of SF2 ATPases are tightly packed together in ATP-bound states and are more open in apo and ADP states .", "However , we also observed cross-linking differences between the two sides of the nucleosome .", "The Widom 601 nucleosome positioning sequence is non-palindromic , and one sequence feature of the 601 that is asymmetrically distributed on the two sides are periodic TpA dinucleotides ( TA steps ) ( Chua et al . , 2012; Lowary and Widom , 1998 ) .", "Phased relative to other sequence elements of the 601 , the TA steps allow DNA to bend more easily around the histone octamer , with unique geometry that allows for tighter histone binding to both the H3-H4 tetramer and H2A-H2B dimers ( Chua et al . , 2012; Ngo et al . , 2015 ) .", "Given the asymmetric distribution of TA steps , we refer to the two sides of the 601 as TA-poor and TA-rich , with the TA-poor side denoted with positive ( + ) numbering and the TA-rich side with negative ( - ) numbering ( see Figure 1—figure supplement 2 ) .", "The two lobes of the ATPase motor displayed different sensitivities with changes in nucleotide state .", "Variation in nucleotide conditions produced relatively little change in lobe 1 cross-linking .", "Both N459C and E493C cross-linked to the same sites in all nucleosome conditions , though stronger cross-links were often observed in ADP·BeF3– conditions , possibly reflecting higher affinity binding ( Figure 1B , C and Figure 1—figure supplement 3A ) .", "E493C cross-linked to both strands on both sides of the nucleosome , with three of the four sites relatively insensitive to nucleotide .", "The fourth site , a doublet on the tracking strand of the TA-poor side , did show subtle but distinct variation in band intensity in AMP-PNP compared with apo/ADP conditions .", "In contrast to lobe 1 , all cross-links from lobe 2 were sensitive both to the nucleotide state and DNA sequence .", "V721C ( lobe 2 ) failed to cross-link in apo/ADP conditions on either side of the nucleosome , and cross-linking on the TA-poor side was also severely reduced in AMP-PNP ( Figure 1D ) .", "However , the other two lobe 2 cross-linking positions , N650C and S770C , did cross-link in apo and ADP conditions , indicating that lobe 2 was indeed engaged with nucleosomal DNA in all four nucleotide conditions .", "Like V721C , the other two lobe 2 cross-linking sites were also highly sensitive to DNA sequence: N650C failed to cross-link to the tracking strand on the TA-rich side , whereas S770C failed to cross-link to the tracking strand on the TA-poor side ( Figure 1—figure supplement 3B ) .", "The cross-linking patterns for both N650C and S770C also varied depending on the bound nucleotide ( Figure 1E , F ) .", "This sensitivity of lobe 2 cross-linking to DNA sequence suggests that the conformation and/or stability of DNA strongly influence the engagement of lobe 2 at SHL2 .", "In two instances , cross-linking to the guide strand seemed to be somewhat at odds with expected structural interpretations .", "S770C made weak but significant cross-links to the guide strand 23 and 24 nt from the dyad ( Figure 1E and Figure 1—figure supplement 3B ) .", "In contrast with other cross-links mapped onto the cryoEM structure , these guide strand cross-links span a distance of ~17–21 Å and are therefore significantly longer than expected for APB ( Figure 1—figure supplement 1 ) .", "This discrepancy likely reflects conformational variability in the protein and/or DNA not captured by the structure .", "A second instance was guide strand cross-linking to N650C .", "In agreement with lobe 2 moving toward the dyad upon cleft closure , both N650C and S770C showed stronger cross-linking closer to the dyad in ADP·BeF3– compared with apo , ADP and AMP-PNP conditions ( Figure 1E , F ) .", "However , a different nucleotide-dependent cross-linking pattern was obtained for N650C with nucleosomes possessing a different DNA sequence .", "Using a variant of the Widom 601 sequence where segment 24–39 on each side of the dyad had been swapped ( called 601[swap SHL2 . 5/3 . 5] , see Figure 1—figure supplement 2 ) , stronger cross-linking was instead observed closer to the dyad in apo/ADP conditions ( Figure 1—figure supplement 4 ) .", "While these findings indicate that physical interpretations of each cross-linking reaction should be taken with caution , the combined results suggest that lobe 2 and/or the guide strand are dynamic and sensitive to sequence and structural perturbations .", "We and others have used APB cross-linking of single cysteine-substituted histones to monitor changes in nucleosome positioning during ATP-dependent sliding by chromatin remodelers .", "In the course of our studies , we noticed a two nt shift in cross-linking upon addition of Chd1 under conditions where ATP was absent ( Figure 2—figure supplement 1 ) .", "This shift was observed with H2B ( S53C ) , which cross-links to DNA 53 nt on either side of the nucleosome dyad of the Widom 601 ( Kassabov et al . , 2002 ) ( Figure 2A ) .", "To investigate this phenomenon further , we monitored H2B ( S53C ) cross-linking in the presence and absence of Chd1 in four nucleotide conditions ( apo , ADP , AMP-PNP , and ADP·BeF3– ) .", "No changes in H2B ( S53C ) cross-linking were observed with AMP-PNP and ADP·BeF3– , whereas a two nt shift was apparent in a Chd1-dependent manner for both apo and ADP conditions ( Figure 2B and Figure 2—figure supplements 2 and 3 ) .", "This shift was only apparent on the TA-poor side of the Widom 601 sequence .", "We interpreted this new cross-link as a Chd1-induced change in histone-DNA contacts .", "To determine whether other regions of nucleosomal DNA were similarly affected by Chd1 binding , we generated and tested the cross-linking characteristics of six other single-cysteine histone variants ( Figure 2A ) .", "Although H2B ( R30C ) failed to cross-link , the other five variants yielded specific cross-linking patterns ( Figure 2—figure supplements 2 and 4 ) .", "Of these , cross-linking for both H2A ( G28C ) and H2B ( T87C ) was clearly affected by Chd1 , showing patterns similar to H2B ( S53C ) at roughly 10 nt intervals from each other .", "On the TA-poor side of the 601 nucleosome , H2A ( G28C ) cross-linked primarily +43 nt from the dyad , and addition of Chd1 in apo and ADP conditions yielded novel cross-linking at +46 and more weakly at +44 ( Figure 2C ) .", "For H2B ( T87C ) , which cross-linked +32 nt from the dyad , addition of Chd1 produced a new cross-link at +33 ( Figure 2D ) .", "The shifts in cross-linking from these three sites were all nucleotide specific , occurring only in apo and ADP conditions , and also were only apparent on the TA-poor side of the nucleosome .", "Despite their proximity to H2B ( S53C ) , H2A ( G28C ) and H2B ( T87C ) , no clear Chd1-dependent changes in cross-linking were observed for H2A ( A45C ) or H2B ( T85C ) .", "For H2B ( T85C ) , the lack of a Chd1-dependent change may have been due to sensitivity to the 601 sequence , since cross-linking was much more pronounced on the TA-rich compared to the TA-poor side ( Figure 2—figure supplement 4 ) .", "Consistent with this , the neighboring H2B ( T87C ) site was strongly affected by the 601 sequence , showing either single or double cross-links to each strand depending on the side of the nucleosome ( Figure 2—figure supplement 2C ) .", "H2A ( A45C ) , however , produced strong symmetric cross-links on each side of the nucleosome ( Figure 2—figure supplement 4 ) .", "Curiously , all of the Chd1-dependent cross-links occurred on one strand , and H2A ( A45C ) cross-linked to the complementary DNA strand at +40 .", "Like H2A ( A45C ) , H2B ( T87C ) also cross-linked to the complementary strand , at +37/+38 .", "This doublet did subtly change in intensity with Chd1 in apo and ADP conditions , yet no new cross-links were observed ( Figure 2—figure supplement 2C ) .", "This correlation raises the intriguing possibility that the histone-DNA interactions of the two strands may be differently affected by Chd1 binding .", "Alternatively , differences in cross-linking may have arisen from distortion of the histone core .", "The hydrophobic cores of H2A and H4 have been shown to be affected by binding to the Snf2h remodeler ( Sinha et al . , 2017 ) , and recent cryoEM analyses have revealed a remarkable flexibility of the histone core ( Bilokapic et al . , 2018a; Bilokapic et al . , 2018b ) .", "A distortion of the histone dimer might therefore be responsible for the non-uniform cross-linking response to Chd1 binding .", "In addition to the cross-linking histone variants described above , we also tested H3 ( M120C ) , which yields double cross-links at the dyad ( Hota et al . , 2013 ) .", "H3 ( M120C ) cross-linking was also unaffected by the presence of Chd1 ( Figure 2E ) .", "As we describe below , the H3 ( M120C ) cross-linking can show Chd1-dependent changes in certain nucleosome contexts .", "Therefore , here we interpret the absence of cross-link changes as a lack of Chd1-dependent changes in histone-DNA contacts around the nucleosome dyad of Widom 601 nucleosomes .", "The nucleosome can be bound by two Chd1 molecules , one at each SHL2 site ( Nodelman et al . , 2017 ) , and we hypothesized that the DNA shifts we observed were stimulated by Chd1 binding at one of those sites .", "The SHL+2 binding site is adjacent to the segment of DNA where histone-DNA cross-links changed in response to Chd1 binding .", "The SHL–2 site is on the opposite gyre , and therefore almost one superhelical turn of DNA away .", "Recent cryoEM structures , however , show that Chd1 binding at SHL2 unwraps ~20 bp of DNA on the opposite gyre ( Farnung et al . , 2017; Sundaramoorthy et al . , 2017; Sundaramoorthy et al . , 2018 ) .", "Therefore , it is possible that the Chd1-dependent changes in cross-linking could arise from Chd1 binding at either SHL2 site .", "To determine which side might be responsible , we employed a previously devised biotin/streptavidin blocking strategy ( Nodelman et al . , 2017 ) , where a biotin moiety was attached to nucleosomal DNA , 19 nt from the dyad on one side .", "Addition of streptavidin should occlude the biotinylated SHL2 , and thus selectively allow association of Chd1 to the other SHL2 site .", "Chd1 binding was monitored using the Chd1 ( N459C ) cross-linking variant , while shifts in histone-DNA cross-linking were followed with the H2B ( S53C ) variant .", "For the nucleosome construct with biotin on the TA-rich SHL–2 site , the addition of streptavidin significantly diminished Chd1 binding to SHL–2 , as expected , yet a robust Chd1-dependent shift in the H2B ( S53C ) cross-link was still observed ( Figure 3A ) .", "This result indicates that the shift in histone-DNA cross-linking ( +53 to +55 ) was accomplished by Chd1 binding SHL+2 on the same gyre .", "In support of this conclusion , the H2B ( S53C ) cross-linking shift was completely blocked on the nucleosome variant with biotin at SHL+2 , on the TA-poor side ( Figure 3B ) .", "As observed in cryoEM structures , Chd1 unwraps ~2 turns of DNA from the nucleosome edge of the opposite gyre ( Farnung et al . , 2017; Sundaramoorthy et al . , 2017; Sundaramoorthy et al . , 2018 ) .", "Unwrapping by Chd1 appeared to diminish the signal from the H2B ( S53C ) cross-linking site , because the biotin/streptavidin block consistently yielded stronger H2B ( S53C ) cross-linking on the side opposite where Chd1 could no longer bind ( compare Figure 3 to Figure 2—figure supplement 1 ) .", "With a biotin block on the TA-rich side , H2B ( S53C ) cross-linking on the TA-poor side was stronger yet still showed the Chd1-induced shift , indicating that the change in cross-linking resulted from Chd1 binding to DNA on the same gyre .", "Relative to the remodeler bound at the SHL+2 site , the Chd1-dependent cross-links all occur on the tracking strand .", "We propose that these shifts in cross-linking result from Chd1-dependent movement of DNA relative to the H2A/H2B dimer .", "From these experiments , we cannot distinguish between movement of the histone dimer and movement of DNA relative to the rest of the nucleosome .", "However , all of the cross-linking shifts occur in the same direction , and would correspond to movement of DNA toward the Chd1-bound SHL+2 site .", "We propose that Chd1 could accomplish such a shift if binding stabilized a small ~1 bp bulge of nucleosomal DNA at SHL2 .", "Given the helical nature of duplex DNA , a small bulge would reduce local twisting of the double helix and pull DNA toward the remodeler in a corkscrew-like motion ( Figure 3C ) .", "A corkscrew-like motion is also consistent with the ability to still observe histone-DNA cross-linking , which likely requires a canonical path of nucleosomal DNA with respect to the histone core .", "Experiments described in the last section further support this idea that trapping or excluding a bulge at SHL2 allows Chd1 to rotate and translocate DNA on the nucleosome .", "The cryoEM structures of Chd1 and the SWI/SNF ATPase show intimate remodeler-DNA contacts at SHL2 ( Farnung et al . , 2017; Liu et al . , 2017 ) .", "In both structures , the DNA backbone is directly contacted by a tryptophan residue from the ATPase motor , and in the apo SWI/SNF structure , this contact occurs at a location of significant DNA distortion on the guide strand .", "This tryptophan is highly conserved across all remodeler families and therefore appears to be structurally important .", "To investigate the significance of this residue , we generated a Chd1 ( W793A ) variant to see if removing this DNA contact altered the Chd1-dependent shifts in H2A/H2B cross-links .", "Using the double variant Chd1 ( W793A/N459C ) , we found that the W793A substitution did not interfere with ATPase binding to either the TA-rich or TA-poor SHL2 in apo conditions ( Figure 3—figure supplement 3A ) .", "Interestingly , despite the intimate contacts between this tryptophan and DNA in the cryoEM structures , the Chd1 ( W793A/N459C ) also produced a similar shift of the H2B ( S53C ) cross-link ( Figure 3—figure supplement 3B ) .", "Unlike the loss of remodeling activity observed for the corresponding mutation of yeast SWI/SNF ATPase ( W1185A ) ( Liu et al . , 2017 ) , we found only a ~2 fold slower rate of sliding compared to the wild-type background ( Figure 3—figure supplement 3C , D ) .", "We therefore conclude that despite the conservation and central placement of W793 , it is not essential for nucleosome sliding by Chd1 , nor required for the Chd1-dependent DNA shift described here .", "Despite Chd1 binding to both SHL2 sites , appearance of the shifted H2B ( S53C ) , H2A ( G28C ) , and H2B ( T87C ) cross-links were notably asymmetric , observed only on the TA-poor side of the 601 .", "We hypothesized that some sequence element ( s ) of the 601 were responsible for either allowing Chd1-induced shifts on the TA-poor side , or preventing shifts on the TA-rich side .", "To explore this idea , we tested several variants of 601 , where sequence segments were flipped , replaced , or swapped ( Figure 4A and Figure 1—figure supplement 2 ) .", "The binding site for the Chd1 ATPase motor at SHL2 extends between 15–25 bp from the nucleosome dyad .", "To determine if the segment bound by Chd1 is responsible for the observed asymmetry , we tested a 601 variant where the central 61 bp was flipped , thus exchanging the 30 bp on either side of the dyad ( called 601[dyad flip61] ) .", "Despite changing the DNA sequence where Chd1 binds at each SHL2 site , this flip did not alter the Chd1-dependent shift in H2B ( S53C ) cross-linking ( Figure 4B and Figure 4—figure supplement 1 ) .", "To see if the segment adjacent to the Chd1 binding site had an impact , we copied the segment from 24 to 39 bp on the TA-rich side to the same region on the TA-poor side ( called 601[duplicate SHL–2 . 5/–3 . 5] ) .", "Interestingly , this replacement appeared to prevent the shift in H2B ( S53C ) cross-linking on the TA-poor side ( Figure 4B and Figure 4—figure supplement 1 ) .", "We note that this replacement did not alter the DNA sequence surrounding the H2B ( S53C ) cross-link on the TA-poor side , which is 53 nt from the dyad .", "Given the interference observed from the 24–39 bp segment of the TA-rich side of the nucleosome , we analyzed a variant where the 24–39 bp segments on each side of the Widom 601 sequence simultaneously replaced each other ( called 601[swap SHL2 . 5/3 . 5] ) .", "Like the duplicated variant , this swap appeared to block a shift in cross-linking on the TA-poor side , but did yield a Chd1-dependent shift on the TA-rich side by one nt ( Figure 4B and Figure 4—figure supplement 1 ) .", "These results suggest that the 24–39 bp region on the TA-poor side was sufficient for permitting a Chd1-dependent shift in entry DNA .", "As described above , the asymmetry of the Widom 601 sequence also influenced the cross-linking patterns for the Chd1 ATPase motor to nucleosomal DNA ( Figure 1 ) .", "We suspected that some of these sequence-dependent differences also arose from the 24–39 bp region on either side of the dyad .", "To investigate this idea , we performed Chd1 cross-linking with the 601[swap SHL2 . 5/3 . 5] variant .", "Chd1 ( V721C ) in particular was markedly sensitive to DNA sequence in AMP-PNP conditions , showing robust cross-linking on the TA-rich side of the Widom 601 yet virtually no cross-linking on the TA-poor side .", "Interestingly , swapping the 24–39 bp regions partially reversed this effect , showing a significant increase in cross-linking with AMP-PNP on the TA-poor side and a decrease in TA-rich side cross-linking ( Figure 4—figure supplement 2 ) .", "Importantly , Chd1 ( V721C ) cross-links 19 nt from the dyad and is therefore not directly affected by the sequence changes from swapping the 24–39 segment .", "These results suggest that the stability or dynamics of the ATPase motor at SHL2 is affected by the sequence of the 24–39 bp segment of the nucleosome .", "With the 601 variants described so far , it remained unclear whether some unique sequence property of the 601 specifically blocked a Chd1-dependent DNA shift ( on the TA-rich side ) , or was required to observe a Chd1-dependent DNA shift ( on the TA-poor side ) .", "To address this issue , we created two additional 601 variants , where the 24–39 bp segment on the TA-rich side of the 601 was replaced with random DNA sequences ( Figure 1—figure supplement 2 ) .", "Notably , both of these variants enabled a Chd1-dependent DNA shift on the TA-rich side ( Figure 4C ) .", "Thus , the sequence of the 24–39 bp segment on the TA-rich side of the canonical 601 prevented Chd1 binding from inducing DNA movement , and replacing this segment with two unrelated sequences was sufficient to allow a Chd1-dependent shift in DNA .", "The experiments presented above show that the Chd1-dependent shifts depend on sequence context .", "To see if this effect was specific to the Widom 601 positioning sequence , we performed similar histone mapping experiments using nucleosomes made with the sea urchin 5S positioning sequence ( Figure 5—figure supplement 1 ) .", "The 5S sequence yielded a distribution of nucleosome positions , and through native gel purification we were able to enrich for a centered species ( referred to as N0 in Figure 5 ) .", "Notably , the enriched 5S species exhibited Chd1-dependent changes in both H2B ( S53C ) and H3 ( M120C ) cross-linking patterns .", "For H2B ( S53C ) experiments , Chd1 promoted new faster-migrating cross-links for 5S nucleosomes in both apo and ADP conditions , similar to shifts seen for the Widom 601 ( Figure 5A ) .", "Unlike the Widom 601 nucleosomes , however , Chd1 also altered the enriched 5S cross-linking patterns for H3 ( M120C ) ( Figure 5B ) .", "The H3 ( M120C ) cross-linking pattern was nucleotide-dependent , most clearly showing differences in cross-linking intensity in apo and ADP conditions .", "The shifts in band intensity for both H3 ( M120C ) cross-links on both DNA strands correlated with each other , suggesting a concerted change in DNA positioning at the nucleosome dyad .", "These results show that Chd1 binding alone is sufficient to affect histone-DNA contacts on either side of SHL2 , suggesting that structural changes due to Chd1 binding in nucleotide-specific states can impact the global positioning of DNA on the histone core .", "In contrast to the enriched species , several of the less populated 5S nucleosome positions failed to show shifts in cross-linking .", "These results reinforce our findings that this Chd1-dependent shift is sensitive to DNA sequence , and yet also suggest that such DNA shifts are a general phenomenon that can occur in various sequence contexts .", "Our model proposes that the Chd1-stimulated corkscrew motion of DNA is coupled to a local bulge at SHL2 ( Figure 3C ) .", "We suspected that a requirement to twist DNA may explain why shifts were not observed on the TA-rich side of the Widom 601 sequence .", "The phased TA-steps on the TA-rich side give the 601 sequence high flexibility , and the precise positioning of these periodic TA steps where the DNA is most geometrically constrained makes it less costly to wrap around the histone octamer ( Chua et al . , 2012; Ngo et al . , 2015 ) .", "Twisting the DNA to form a bulge at SHL–2 would disrupt the phasing of TA steps on the TA-rich side , and would therefore be more energetically expensive compared to the TA-poor side , which has only one such positioned TA step .", "To test the hypothesis that a DNA bulge was coupled to DNA twisting , we designed a variant 601 with a single bp insertion to disrupt phasing of TA-steps on the TA-rich side ( Figure 6A ) .", "This variant , with an A:T base pair insertion 22 bp from the dyad , is referred to as 601[insert ( +1 ) TA-rich] ( Figure 1—figure supplement 2 ) .", "A prediction of our model was that Chd1 binding to this altered SHL–2 site in apo and ADP conditions would bring the TA-steps back into phase by effectively absorbing the extra twist from the single bp insertion .", "Our initial experiments with 601[insert ( +1 ) TA-rich] showed that Chd1 binding changed both the cross-linking pattern of H2B ( S53C ) on the TA-rich side , as well as H3 ( M120C ) cross-linking around the dyad ( Figure 6—figure supplement 1 ) .", "The shifts in dyad cross-links were particularly intriguing , since the dyad is surrounded by the strongest histone-DNA contacts that appear to ultimately dictate nucleosome positioning ( Hall et al . , 2009 ) .", "A complication of interpreting the dyad shifts , however , was that Chd1 can bind to both SHL2 sites , and therefore it was unclear if binding to one or a combination of both sites may have been responsible for the observed shifts .", "We therefore introduced a biotin/streptavidin block on the TA-poor side of the 601[insert ( +1 ) TA-rich] variant ( Figure 6B ) , allowing us to determine how the positioning of nucleosomal DNA might be affected by Chd1 binding to the altered SHL–2 site .", "We generated two different biotinylated 601[insert ( +1 ) TA-rich] nucleosomes to separately monitor H2B ( S53C ) and H3 ( M120C ) cross-linking in the presence of streptavidin .", "Cross-linking reactions in the presence and absence of Chd1 in different nucleotide conditions are shown in Figure 6C , D and Figure 6—figure supplement", "2 . For H2B ( S53C ) , the predominant cross-link for the nucleosome alone occurred at −52 instead of −53 ( numbering refers to DNA positioning on the canonical 601 ) , which is consistent with the +1 insertion at SHL–2 shifting the DNA sequence past the H2B ( S53C ) site by 1 bp .", "As shown by H3 ( M120C ) cross-linking , the dyad for 601[insert ( +1 ) TA-rich] nucleosomes alone was in the same location as canonical 601 .", "Consistent with the idea that Chd1 binding affects DNA twist on the nucleosome , addition of Chd1 influenced the length of DNA between H2B ( S53C ) and H3 ( M120C ) cross-linking sites in a nucleotide-dependent manner .", "Interestingly , in all nucleotide conditions , Chd1 binding shifted the H2B ( S53C ) cross-link by one nt , corresponding to DNA movement toward the remodeler .", "This shift resulted in a cross-link at the original −53 site of the 601 in spite of the 1 bp insertion ( Figure 6C , E ) .", "In contrast , distinct nucleotide-dependent patterns were observed for H3 ( M120C ) , which cross-linked on the other side of SHL–2 ( Figure 6D ) .", "In apo , ADP , and AMP-PNP conditions , the dyad cross-links of 601[insert ( +1 ) TA-rich] were unchanged by Chd1 , whereas with ADP·BeF3– , Chd1 stimulated a shift of the dyad cross-links by 1 bp .", "This shift corresponded to movement of DNA away from the the +1 insertion site on the TA-rich side .", "To further examine how ATP-mimics influence Chd1 , we also tested ATPγS and the transition state analogs ADP·AlFX and ADP·MgFX .", "Both transition state analogs behaved similarly to ADP·BeF3– , with cross-link shifts corresponding to DNA being pushed away from the SHL–2 site where the remodeler bound ( Figure 6D; Figure 6E ) .", "We interpret these cross-linking patterns as Chd1 promoting two types of DNA structures that either stabilize or disfavor a small DNA bulge at SHL–2 ( Figure 6F ) .", "In apo and ADP conditions , Chd1 shifted the H2B ( S53C ) cross-link by one nt but had no effect on dyad cross-links , consistent with creating a small bulge that effectively absorbed the +1 bp insertion at SHL–2 .", "With ADP·BeF3– and the transition state analogs , Chd1 shifted cross-links at both H2B ( S53C ) and H3 ( M120C ) , thereby maintaining separation of these sites expected for the +1 bp insertion .", "We interpret the effect of Chd1 with ADP·BeF3– and transition state analogs as stabilizing the canonical structure of nucleosomal DNA , in agreement with cryoEM structures of Chd1-nucleosome complexes ( Farnung et al . , 2017; Sundaramoorthy et al . , 2018 ) .", "In addition to the predominant 1 bp shift at the dyad , significant levels of cross-linking in ADP·BeF3– , ADP·AlFX and ADP·MgFX were also observed at the original 601 position .", "The shifted and non-shifted cross-links may reflect an equilibrium of the bulged and non-bulged states of DNA at SHL2 .", "Interestingly , ATPγS gave a small but reproducible cross-link representing the +1 shift at the dyad .", "While one interpretation is that the ATP-bound Chd1 may support either bulged or non-bulged DNA at SHL2 ( Figure 6F ) , the dyad shift with ATPγS was more clearly apparent on one strand than the other , perhaps reflecting an intermediate structure ." ], [ "SF1 and SF2 translocases travel along nucleic acids using an inchworm-type mechanism: opening of the bi-lobed ATPase motor in the apo state extends contacts by one nt in the direction of translocation , and domain closure upon ATP binding ratchets the motor along the nucleic acid substrate by one nt ( reviewed in Singleton et al . , 2007 ) .", "Here we describe a related but distinct process for DNA translocation on the nucleosome , where DNA movement occurs during both open and closed states of the motor .", "In the inchworm model , the front and back halves of the motor take turns shifting past the nucleic acid substrate .", "For translocases like chromatin remodelers that move in a 3’ to 5’ direction , lobe 2 is the leading half , reaching forward in the open state to engage with DNA ahead of the motor .", "As monitored by site-specific cross-linking , lobe 2 of the Chd1 remodeler changed contacts with DNA in a nucleotide-dependent fashion and was highly sensitive to DNA sequence ( Figure 1 ) .", "Remarkably , the open state of the Chd1 ATPase showed the capability of pulling DNA toward itself .", "On the TA-poor side of the Widom 601 nucleosome , Chd1 shifted a large segment of DNA across the histone surface by 1–3 nt .", "This DNA shift toward the remodeler occurred in apo and ADP-bound states and was independent of nucleotide hydrolysis ( Figures 2 and 3 ) .", "We propose that such a shift could be accomplished if the open conformation of the ATPase motor trapped a small ~1 bp bulge of DNA .", "A bulge or DNA distortion at SHL2 could induce a concerted , corkscrew-like shift of DNA toward the remodeler .", "Recently , the cryoEM structure of the SWI/SNF ATPase bound to the nucleosome provided a first view of how an open ATPase motor engages with the nucleosome ( Liu et al . , 2017 ) .", "This structure , which preferentially bound the TA-poor SHL+2 site of the Widom 601 nucleosome , revealed significant distortions in DNA at the binding site , with 3–6 Å displacements of the sugar-phosphate backbone from its canonical path and some disruptions in base stacking interactions .", "Such disruptive interactions are consistent with an ability to favor a ~ 1 bp bulge at SHL2 when both lobes of the ATPase motor bind in an open , active state .", "The nucleotide-free states of SF1 ( RecBCD ) and SF2 ( NS3 ) ATPases were previously found to unwind DNA duplexes , independently of nucleotide-driven translocation ( Farah and Smith , 1997; Levin et al . , 2005; Singleton et al . , 2004 ) .", "For NS3 , it was proposed that a nucleotide-free unwinding step is achieved by favorable protein-DNA interactions , with the motor acting as a Brownian ratchet to capture DNA in a transiently melted state ( Levin et al . , 2005 ) .", "For Chd1 , instead of duplex melting , the open state of the ATPase motor appears to promote a bulged DNA duplex .", "Compared to the open apo and ADP-bound states of the motor , nucleotide-bound states of Chd1 containing ATP mimics and transition state analogs favored distinct organizations of nucleosomal DNA at SHL2 .", "With ADP·BeF3– and transition state analogs , Chd1 appeared to reinforce the canonical structure of nucleosomal DNA at SHL2 , without a bulge .", "As shown with the 601[insert ( +1 ) TA-rich] nucleosome variant , Chd1 binding at SHL–2 shifted DNA over the dyad by ~1 bp with transition state analogs , indicating an incompatibility with the SHL–2 bulge favored by apo and ADP-bound states ( Figure 6 ) .", "These observations agree with Chd1-nucleosome complexes bound to ADP·BeF3– and visualized by cryoEM , which show only modest perturbations of nucleosomal DNA at the bound SHL2 sites ( Farnung et al . , 2017; Sundaramoorthy et al . , 2018 ) .", "Like ISWI- and SWI/SNF-type remodelers , Chd1 binds at SHL2 and translocates DNA toward the nucleosome dyad ( Farnung et al . , 2017; McKnight et al . , 2011; Nodelman et al . , 2017; Sundaramoorthy et al . , 2018 ) .", "We propose that the core mechanism for translocating nucleosomal DNA past the histone core arises from the ATPase motor enforcing unique geometries of DNA at its binding site ( Figure 7 ) .", "According to this model , DNA translocation is initiated by the open form of the remodeler ATPase motor binding at SHL2 , which draws ~1 bp into a bulge and results in DNA on the entry gyre of the nucleosome being shifted toward the remodeler by ~1 bp .", "Subsequent closure of the ATPase cleft upon ATP binding and hydrolysis forces a redistribution of the bulged DNA , with collapse of the bulge pushing ~1 bp onto the DNA segment toward the dyad , on the other side of the motor .", "In this manner , the segments of DNA on either side of SHL2 alternately shift toward and away from the motor , with transitions between open and closed forms of the ATPase effectively transferring ~1 bp from one DNA segment to the other in the process .", "While our data reveal that the ATPase motor can stabilize two distinct conformations of nucleosomal DNA that differ by ±1 bp , the ATPase motor may also favor intermediate DNA conformations , particularly in an ATP-bound state .", "The ATP analog states of Chd1 showed characteristics of apo/ADP and also ADP·BeF3– , depending on the context .", "For lobe 2 cross-linking , AMP-PNP conditions were more similar to apo and ADP and different from ADP·BeF3– ( Figure 1 ) , yet for experiments showing movement of DNA on the TA-poor side of the 601 , no effect was seen for Chd1 with AMP-PNP nor ADP·BeF3– , unlike apo/ADP states ( Figure 2 ) .", "In the context of the 601[insert ( +1 ) TA-rich] nucleosome , however , the Chd1-stimulated shifts of nucleosomal DNA with AMP-PNP and ATPγS predominantly resembled apo and ADP , though a minor cross-link with ATPγS hints that nucleosomal DNA may occupy an intermediate state between bulged and non-bulged structures ( Figure 6 ) .", "The ATP-bound state may accommodate a range of local DNA perturbations and thus bridge the transition from bulged to non-bulged conformations of nucleosomal DNA .", "Future high resolution studies will be necessary for defining how intermediate states of the ATPase motor alter and respond to the structure and energetics of nucleosomal DNA during translocation .", "Nucleosomes are expected to respond to DNA translocation by twist diffusion .", "Our data reveal that twist diffusion also defines the core mechanism by which remodelers shift nucleosomal DNA .", "For DNA to be ratcheted forward , nucleotide-dependent conformations of the ATPase motor must be coupled to pre- and post-shifted positions of nucleosomal DNA .", "Our data indicate that the pre- and post-shifted states correspond to distinct twist states of nucleosomal DNA .", "By accommodating ~1 additional bp , the DNA bulge caused by the apo/ADP states of the ATPase motor results in DNA undertwisting at the binding site ( using the definition that more bp/turn means less twist per bp ) .", "Returning DNA conformation to a more canonical form , favored by restructuring of the motor as it hydrolyzes ATP , forces the twist defect onto the neighboring DNA segment .", "In this manner , distinct twist states on the nucleosome oscillate in response to ATP-driven changes of the motor , resulting in DNA being effectively pumped toward the dyad .", "This model differs from the conventional expectations of inchworm-type translocation , where DNA on either side of the motor coordinately moves as a unit during translocation .", "Instead , with twist defects the DNA moves as two alternating segments .", "As a Brownian ratchet , the motor achieves directionality through the ATP hydrolysis cycle yet relies on thermal fluctuations to transport DNA .", "By generating a twist defect , the motor uncouples the two segments of DNA , allowing the segment in front of the motor to shift without requiring simultaneous movement of the DNA segment on the exiting side of the motor .", "Subsequent elimination of the twist defect upon ATP hydrolysis presumably locks in the shifted position of entry DNA , necessary for directional diffusion of DNA away from the motor .", "One consequence of action via twist defects is that the motor need not have a strong external contact for shifting DNA around the histone core .", "In the traditional inchworm model , a translocase would need to be held in place so that its attempt to walk along nucleosomal DNA would result in pulling DNA in a corkscrew motion relative to the histone core .", "By creating twist defects , the DNA in front of the remodeler would shift relative to the DNA behind , with the remodeler maintaining contact with both portions of DNA during alternating movement .", "Although remodelers contact the H4 tail and interact with histone H3 via Snf2-specific insertions when bound at SHL2 ( Farnung et al . , 2017; Liu et al . , 2017; Sundaramoorthy et al . , 2018 ) , we predict that these contact are likely not necessary for force generation and instead are important for properly orienting and positioning the motor at SHL2 .", "Nucleosomes have long been recognized to satisfy the core requirement of twist diffusion .", "The first and many subsequent crystal structures of the nucleosome showed that ±1 bp differences in DNA length readily occur at SHL2 and SHL5 ( Luger et al . , 1997; reviewed in McGinty and Tan , 2015; and Muthurajan et al . , 2003 ) .", "Biochemically , it was shown that these differences in DNA length , corresponding to twist defects , are dynamic and easily diffuse to other SHLs on the nucleosome ( Edayathumangalam et al . , 2005 ) .", "Twist diffusion has recently been visualized in molecular simulations of nucleosomes , where DNA segments bounded by minor groove contacts spontaneously gain or lose single bps in coordination with adjacent DNA ( Brandani et al . , 2018 ) .", "In addition to changes in DNA , cryoEM structures have recently shown that deformations of the histone core can also be coupled to distortions at SHL2 ( Bilokapic et al . , 2018b ) .", "In these structures , histone dimers appeared to pivot to maintain contact with displaced DNA , and in some cases formed distinct histone-DNA contacts at SHL0 . 5 and SHL1 . 5 .", "The ability of SHL2 to easily accommodate ±1 bp may be why Chd1 and other chromatin remodelers utilize this site for shifting DNA around the histone core .", "While defects can form spontaneously at SHL2 , the nucleotide-dependent conformations of the ATPase motor both accelerate and add directionality to this process , first creating/capturing individual DNA bulges and then expelling excess DNA toward the dyad .", "As Brownian ratchets , we anticipate that the actions or efficiency of remodelers may be impacted by the ease that twist defects can form at SHL2 .", "We found a correlation between Chd1-dependent formation of a bulge at SHL2 and the sequence of the DNA segment that shifts toward the motor .", "Previously , Widom proposed that DNA sequence would affect DNA diffusion around the histone core , with faster DNA movement predicted for sequences that twist more easily ( Widom , 2001 ) .", "Our results are in agreement with this idea , and indicate that the ease of forming a bulge at SHL2 is coupled to the strength of phasing for the SHL2 . 5/SHL3 . 5 segment .", "With strong phasing , as seen with the periodic TA-steps , a corkscrew-like displacement of DNA is more expensive and disfavors the SHL2 bulge ( Figure 4 ) .", "We speculate that the influence of DNA sequence on bulge formation could impact remodeling activity .", "We previously showed that Chd1 preferentially shifts 601 nucleosomes toward the TA-poor side , and that swapping the SHL2 . 5/SHL3 . 5 segment ( 24 to 39 bp ) on each side of the 601 resulted in a more even distribution toward TA-rich and TA-poor sides ( Winger and Bowman , 2017 ) .", "Interestingly , nucleosome sliding was also perturbed when the 24–39 bp region was replaced by poly[dA:dT] .", "Although the full impact of this poly[dA:dT] segment on nucleosome structure and dynamics is not presently clear , this substitution produced several phased positions of neighboring DNA segments differing by ~1 bp , which could potentially influence formation or directional elimination of a twist defect at SHL2 ( Winger and Bowman , 2017 ) .", "Future studies with other 601 variants and distinct positioning sequences will be necessary to clarify how particular DNA motifs on the nucleosome impact DNA translocation by chromatin remodelers .", "By proposing that DNA translocation by Chd1 is intimately tied to twist diffusion , the work described here should stimulate further investigations of both sequence-dependent repositioning and the basic mechanism of nucleosome sliding ." ], [ "All Chd1 constructs spanned residues 118–1274 of Saccharomyces cerevisiae Chd1 .", "The 118–1274 portion includes the chromodomains , ATPase , and DNA-binding domains , as previously described ( Patel et al . , 2011 ) and is referred to throughout this manuscript as Chd1 .", "A cysteine-free version of Chd1 was used as a template to introduce single cysteine mutations at specific positions: N459C , E493C , N650C , V721C , and S770C ( Nodelman et al . , 2017 ) .", "The W793A substitution was introduced through site directed mutagenesis into the cysteine variant Chd1 ( N459C ) , in which all native cysteines were mutated to alanine , and separately into an otherwise wildtype Chd1 background .", "All constructs were His-tagged , and were recombinantly expressed in E . coli and purified as described previously ( Patel et al . , 2011 ) .", "Briefly , cells were lysed via sonication , clarified , and the cell extract loaded onto a HisTrap HP ( GE Healthcare ) column .", "After washing , His-tagged Chd1 was eluted using 175 mM imidazole and then further separated from contaminants via a HiTrap SP-FF cation exchange column ( GE Healthcare ) .", "Following removal of the His-tag by overnight digestion with Precission protease , Chd1 was cleaned up by a second passage over a HisTrap HP column ( collected in the flow through ) and further purified using a HiLoad 16/600 Superdex 200 column ( GE Healthcare ) .", "The expression constructs for H3 ( C110A ) and wild type H2A , H2B , and H4 Xenopus laevis histones were kindly provided by Karolin Luger .", "Single cysteine histone variants were generated by site-directed mutagenesis: H2A ( G28C ) , H2A ( A45C ) , H2B ( R30C ) , H2B ( S53C ) , H2B ( T85C ) , H2B ( T87C ) , and H3 ( M120C ) .", "H3 ( M120C ) was made in the H3 ( C110A ) background , which removed the native cysteine .", "Previous work by the Bartholomew group reported histone cross-linking for three sites used here: H2B ( S53C ) and H2A ( A45C ) ( Kassabov et al . , 2002 ) , and H3 ( M120C ) ( Hota et al . , 2013 ) .", "All histones were expressed and purified as previously described ( Luger et al . , 1999 ) , and stored as 2 mg lyophilized aliquots at −20 ˚C .", "Histones were combined into tetramers , dimers or octamers by refolding equimolar amounts of the appropriate histones and then purified using a HiLoad 16/600 Superdex 200 size exclusion column as previously described ( Luger et al . , 1999; Nodelman et al . , 2017 ) .", "DNA fragments were generated via PCR using two primers that contained different fluorophores , thus resulting in doubly-labeled DNA .", "The template used for PCR was either the canonical Widom 601 sequence ( Lowary and Widom , 1998 ) , a 601 variant sequence ( Figure 1—figure supplement 2 ) , or the 5S nucleosome positioning sequence ( Figure 5—figure supplement 1 ) .", "Nucleosome constructs used for sliding reactions were end-positioned , with the 145 bp Widom 601 sequence flanked by 80 bp on the TA-rich side and 0 bp on the TA-poor side ( called 80N0 ) .", "Biotinylated DNA was produced using long primers containing an internal dT-biotin located 19 nt and 5’ relative to the 601 dyad .", "On the TA-rich side , the biotinylated construct was FAM-19- ( +biotin ) 601-29-Cy5 , with biotin on the FAM strand .", "On the TA-poor side , two constructs were made: Cy5-40-601 ( +biotin ) -19-FAM and Cy5-40-[insert ( +1 ) TA-rich]601 ( +biotin ) -19-FAM , each with biotin on the FAM strand .", "All other nucleosome constructs were Cy5-40N40-FAM .", "For each construct , the desired DNA product was purified away from primers by separation on 6% ( 60:1 ) acrylamide:bisacrylamide native gels .", "To produce nucleosomes , each DNA fragment was first combined in a 1:1 ratio with octamer , or a 1:1:2 . 2 ratio with tetramer and dimer in 2M KCl .", "To deposit the histone octamer on the nucleosome positioning sequence , the DNA and histone mixture was gradually dialyzed from 2 M to 200 mM KCl at 4°C over a 24 hr period , with a final transfer to 2 . 5 mM KCl buffer ( Luger et al . , 1999 ) .", "Centered nucleosomes were purified away from free DNA , hexasomes , and off-positioned nucleosome species by separation on 7% ( 60:1 ) acrylamide:bisacrylamide native gels .", "Chd1 constructs containing a single cysteine were diluted to a concentration of 7 . 5 µM and labeled in the dark at room temperature with 4-azidophenacyl bromide ( APB , dissolved in DMF; Sigma , cat# A6057 ) for 2 . 5 hr , Cf = 400 µM .", "Labeling and cross-linking to the nucleosome were previously described ( Nodelman et al . , 2017 ) .", "Briefly , samples to be crosslinked were assembled in 1x Slide buffer ( 20 mM Tris-HCl , pH 7 . 5 , 50 mM KCl , 5 mM MgCl2 , 0 . 1 mg/ml bovine serum albumin ( BSA ) , 5 mM dithiothreitol ( DTT ) , 5% sucrose ) for either apo , 2 mM ADP , or 2 mM AMPPNP; or slide buffer minus magnesium for ADP·BeF3– ( 2 mM ADP , 15 mM NaF , 3 mM BeCl2 , and 6 mM MgCl2 ) .", "Nucleotide was added to tubes , followed by either nucleosome or naked DNA ( naked DNA was equivalent to that used in nucleosome reconstitutions ) to a final concentration of 150 nM , and finally APB-labeled Chd1 ( Cf = 450 nM ) .", "Chd1 was incubated with nucleosome in the dark for 30–45 min , UV irradiated for 15 s ( λ = 302 nm; VWR UV transilluminator 89131–440 ) , followed by the addition of 100 µl quench ( 20 mM Tris-HCl , pH 7 . 5 , 50 mM KCl , 0 . 1 mg/mL BSA , 5 mM EDTA , and 5 mM DTT ) and 150 µl post-irradiation buffer ( 20 mM Tris-HCl , pH 8 . 0 , 0 . 15% SDS , 50 mM NaCl ) .", "Samples were processed as described ( Kassabov and Bartholomew , 2004; Nodelman et al . , 2017 ) .", "Histone mapping followed the published methods of Kassabov and Bartholomew ( Kassabov and Bartholomew , 2004 ) .", "Nucleosomes were first buffer exchanged into cold 20 mM Tris-HCl , pH 7 . 5 , 5% glycerol to remove DTT , and then labeled with ~220 μM APB in the dark for ~2 . 5 to 3 hr at room temperature .", "During nucleosome labeling , sample tubes were prepared at room temperature in the absence ( apo ) or presence of 2 mM nucleotide in 1X Slide buffer .", "ADP·BeF3– , ADP·AlFX , and ADP·MgFX conditions all contained 2 mM ADP , 15 mM NaF , and 6 mM MgCl2 , with ADP·BeF3– and ADP·AlFX , also containing either 3 mM BeCl2 or 3 mM AlCl3 , respectively .", "For experiments with 5S nucleosomes ( Figure 5 ) , ADP stocks ( 100 mM ) were pre-treated to remove residual ATP by incubation with 100 Units/ml of S . cerevisiae hexokinase ( Sigma-Aldrich , cat# H4502 ) , 1 M glucose , and 5 mM MgCl2 for 20 min at 25°C .", "Where indicated , Chd1 was added to a final concentration of 600 nM immediately before nucleosomes .", "After labeling , nucleosomes were added to the sample tubes to a final concentration of 150 nM in the dark .", "The samples were incubated for 10–30 min and then given a 15 s exposure to UV light to induce cross-linking .", "After UV exposure , samples were quenched 2:1 with quench buffer , diluted 1:1 with post-irradiation buffer ( 20 mM Tris-HCl , pH 8 . 0 , 0 . 15% SDS , 50 mM NaCl ) , and then heated at 70°C for 20 min .", "Subsequent phenol clean up of cross-linked DNA and NaOH cleavage steps were followed as described ( Kassabov and Bartholomew , 2004; Nodelman et al . , 2017 ) .", "DNA samples , resuspended in deionized formamide loading buffer , were separated on an 8 M urea denaturing gel ( 8% ( 19:1 ) acrylamide:bisacrylamide ) and run at 65 Watts , typically 1 . 5 to 3 hr depending on fragment size .", "The DNA fragments were visualized via their fluorophores on a 9410 Typhoon variable mode imager ( GE ) .", "Experiments using biotinylated nucleosomes were APB-labeled on either H2B-S53C or H3-M120C .", "Nucleosomes for Figure 3 were incubated at room temperature for 15 min with a final concentration of either 9 . 6 µM streptavidin ( Pierce , cat# 21125 ) , a 64-fold excess over nucleosome ( 150 nM ) , or 1x Slide buffer for the no streptavidin samples .", "For Figure 6 experiments , all samples were pre-incubated with 9 . 6 µM streptavidin under the same conditions as Figure 3 in the presence of nucleotide analog or apo conditions .", "Chd1 was then added where appropriate to a final concentration of 600 nM ( 4-fold over nucleosome ) , and 50 µl samples were further incubated for 20 min .", "Samples were UV irradiated and processed as described above for histone mapping .", "Nucleosome sliding assays were conducted similar to Patel et al . ( 2011 ) .", "Remodeling reactions ( 20 µl ) were performed at room temperature with 50 nM of either Chd1 or Chd1 ( W793A ) and 150 nM FAM-labeled 80-601-0 nucleosomes in 1x Slide buffer ( 20 mM Hepes-KOH , pH 7 . 6 , 50 mM KCl , 5 mM MgCl2 , 0 . 1 mg/ml BSA , 1 mM DTT , 5% sucrose ) .", "After removal of a t = 0 time point , time-keeping was started upon addition of 2 . 5 mM ATP , with 1 µl of remodeling reaction transferred to 20 µl quench buffer ( 20 mM Hepes-KOH , pH 7 . 6 , 50 mM KCl , 0 . 1 mg/ml BSA , 1 mM DTT , 5% sucrose , 5 mM EDTA , pH 8 . 0 , and 150 ng/µl ultrapure salmon sperm DNA ( Thermo Fisher Scientific , cat#15632011 ) ) and tubes placed on ice .", "Time point samples were loaded onto 6% ( 60:1 ) acrylamide:bis-acrylamide native PAGE gels and run at 100V in 0 . 25xTBE for 100 min .", "Gels were scanned on a 9410 Typhoon variable mode imager ( GE ) and quantitated using ImageJ software ( RRID:SCR_003070 ) .", "Using Mathematica ( RRID:SCR_014448 ) , data were fit to the equation y = a1* ( 1-exp ( -k1*x ) ) + a2* ( 1-exp ( -k2*x ) ) +c , where k1 and k2 are apparent rate constants , a1 and a2 are corresponding amplitudes , and c is a constant ." ] ]

[ "As superfamily 2 ( SF2 ) -type translocases , chromatin remodelers are expected to use an inchworm-type mechanism to walk along DNA .", "Yet how they move DNA around the histone core has not been clear .", "Here we show that a remodeler ATPase motor can shift large segments of DNA by changing the twist and length of nucleosomal DNA at superhelix location 2 ( SHL2 ) .", "Using canonical and variant 601 nucleosomes , we find that the Saccharomyces cerevisiae Chd1 remodeler decreased DNA twist at SHL2 in nucleotide-free and ADP-bound states , and increased twist with transition state analogs .", "These differences in DNA twist allow the open state of the ATPase to pull in ~1 base pair ( bp ) by stabilizing a small DNA bulge , and closure of the ATPase to shift the DNA bulge toward the dyad .", "We propose that such formation and elimination of twist defects underlie the mechanism of nucleosome sliding by CHD- , ISWI- , and SWI/SNF-type remodelers ." ]

[ "DNA is shaped like a spiral staircase , twisting around itself to create a double helix .", "This results in a long string-like molecule that needs to be carefully packaged to fit inside the cells of organisms as diverse as fungi or humans .", "This packaging process starts when a portion of DNA tightly wraps around a spool-like core of proteins called histones .", "The resulting structure is known as a nucleosome .", "Like the beads on a necklace , nucleosomes exist at regular intervals along DNA .", "The DNA sequence around the histones cannot be accessed by a cell , and so the nucleosomes need to be ‘shifted’ along DNA to free up the genetic information .", "Enzymes known as chromatin remodelers perform this role by binding to a nucleosome , and then using energy to fuel a change in their structure that makes them ‘crawl’ on DNA like an inchworm .", "During this process , chromatin remodelers slide nucleosomes along the DNA , but it was unclear how exactly the inchworm motions pushed DNA around the histones .", "Here , Winger et al . look into the details of this mechanism by focusing on the chromatin remodeler Chd1 , which is conserved from yeast to humans .", "Experiments show that , first , the enzyme slightly untwists the DNA double helix; this untwisting causes the DNA to pucker a little on the nucleosome .", "The puckering creates tension and ‘pulls’ DNA towards the remodeler .", "Then , Chd1 changes its structure and twists DNA in the opposite direction , which forces the puckered DNA onto the other side of the remodeler .", "This extra bit of DNA then propagates around the rest of the nucleosome , like the wave created by flicking the end of a long rope .", "This sheds light on how these enzymes can ratchet DNA past the histones .", "As the gatekeepers of our genetic information , chromatin remodelers are key to the health of the cell – in fact , they are often affected in cancers .", "The work by Winger et al . creates a framework that will help to understand how exactly chromatin remodelers help cells access the genetic information that the body needs to function properly ." ]

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

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

[ [ "Cell biology seeks to understand how nanometer-scale molecules organize micron-scale cells , a question well-suited to theory and modeling ( Marshall , 2017 ) .", "As quantitative cell biology has grown , modeling has expanded in scope ( Mogilner et al . , 2006 ) .", "Theory and simulation can now predict cellular phenomena across length and time scales , giving new insight into cellular self-organization .", "In the cytoskeleton , an important challenge is understanding how a relatively small number of building blocks can produce diverse structures and machines .", "Quantitative modeling has contributed to our understanding of cytoskeletal functions including mitosis ( Mogilner and Craig , 2010; Civelekoglu-Scholey and Cimini , 2014 ) , cytokinesis ( Akamatsu et al . , 2014; Stachowiak et al . , 2014 ) , and cell motility ( Allard and Mogilner , 2013; Barnhart et al . , 2017 ) .", "Chromosome segregation in eukaryotes is performed by the mitotic spindle , a self-organized microtubule ( MT ) -based machine ( Bray , 2000; McIntosh et al . , 2012 ) .", "Dynamic spindle MTs are typically organized with their plus-ends toward the center of the spindle , forming a bipolar array as the spindle poles move apart ( Figure 1; Taylor , 1959; McIntosh et al . , 2012 ) .", "Motor proteins and crosslinkers that bundle and slide MTs create , extend , and stabilize MT bundles ( Figure 1A , B; Mann and Wadsworth , 2019; Pidoux et al . , 1996; Chen et al . , 2012; Hepperla et al . , 2014; Hueschen et al . , 2019; Yamashita et al . , 2005; Janson et al . , 2007; Braun et al . , 2011; Lansky et al . , 2015 ) .", "As the spindle assembles , MTs attach to duplicated chromosomes at kinetochores and align them at the spindle midzone ( Figure 1A–C; Musacchio and Desai , 2017; Hinshaw and Harrison , 2018; Hamilton et al . , 2019 ) .", "Biorientation occurs when sister kinetochores are attached to sister poles , but is often preceded by erroneous attachment ( Figure 1D; Cimini et al . , 2001; Salmon et al . , 2005; Rumpf et al . , 2010; Gregan et al . , 2011; Lampson and Grishchuk , 2017 ) .", "Kinetochores therefore perform multiple functions: they link chromosomes to MTs , maintain attachment to MT ends under force and as MTs grow and shrink , sense MT attachment and tension between sisters , and regulate correction of attachment errors and the spindle-assembly checkpoint ( Sacristan and Kops , 2015; Musacchio and Desai , 2017 ) .", "It is not fully understood how kinetochores , microtubules , and associated proteins robustly assemble a bipolar spindle and align chromosomes .", "In particular , it is unclear which kinetochore functions are most important for error correction and proper chromosome segregation ( Lampson and Grishchuk , 2017; Sacristan and Kops , 2015 ) .", "Error correction is affected by kinetochore geometry ( Gregan et al . , 2007; Paul et al . , 2009; Rumpf et al . , 2010; Magidson et al . , 2015; Zaytsev and Grishchuk , 2015 ) and attachment/tension sensing ( Sacristan and Kops , 2015; Musacchio , 2015; Musacchio and Desai , 2017; Salmon and Bloom , 2017 ) , although the relative contribution of different effects is not established ( Nannas and Murray , 2014; Tauchman et al . , 2015; Kuhn and Dumont , 2017; Yoo et al . , 2018 ) .", "Destabilization of incorrect attachments by Aurora B kinase appears to be particularly important for high-fidelity chromosome segregation ( Cheeseman et al . , 2002; Cimini et al . , 2006; Liu et al . , 2009; Liu et al . , 2010a ) .", "Therefore , further insight into the minimal mechanisms required for spindle assembly and chromosome biorientation could be gained from a computational model .", "Once the spindle assembles and attaches to chromosomes , it achieves a consistent length ( Dumont and Mitchison , 2009; Goshima and Scholey , 2010; Nannas et al . , 2014; Rizk et al . , 2014; Lacroix et al . , 2018 ) .", "The force-balance model proposes that outward-directed forces from plus-end directed sliding motors separate spindle poles , while inward-directed forces from minus-end directed sliding motors and chromosomes pull the poles together ( Saunders and Hoyt , 1992 ) .", "This model helps explain perturbations that alter spindle length ( Syrovatkina et al . , 2013; Hepperla et al . , 2014; Chacón et al . , 2014; Nannas et al . , 2014 ) .", "However , a change in spindle length may occur from a direct change in force production or from indirect effects such as alteration in MT dynamics or alignment ( Hepperla et al . , 2014; Gergely et al . , 2016 ) .", "In addition , the steady-state force-balance model requires extension to address spindle length fluctuations , in which the bipolar spindle assembles , but then undergoes large , dynamic length changes ( Bratman and Chang , 2007; Griffiths et al . , 2008; Choi et al . , 2009; Hsu and Toda , 2011; Masuda et al . , 2013; Wälde and King , 2014; Syrovatkina et al . , 2013; Gergely et al . , 2016 ) .", "Computational modeling can be a valuable tool to dissect force generation and spindle length changes .", "To better understand the key mechanistic requirements for chromosome biorientation and how kinetochore number and attachment affect spindle length stability , we developed a computational model of fission-yeast mitosis .", "Schizosaccharomyces pombe cells are amenable to genetic manipulation and quantitative experiments ( Ward et al . , 2015; Mary et al . , 2015; Klemm et al . , 2018; Blackwell et al . , 2017b; Blackwell et al . , 2017a ) and the spindles are small enough that full 3D simulations are computationally tractable ( Glunčić et al . , 2015; Ward et al . , 2015; Blackwell et al . , 2017a; Lamson et al . , 2019 ) .", "We were motivated by previous work modeling spindle function and chromosome segregation ( Mogilner and Craig , 2010; Civelekoglu-Scholey and Cimini , 2014 ) .", "Because we study de novo spindle assembly and chromosome alignment , we could not use previous models that started with an already-bipolar structure and/or chromosomes attached to the spindle .", "Therefore , we extended a previous model of spindle assembly in the absence of chromosomes and kinetochore-microtubule attachments ( Blackwell et al . , 2017a; Rincon et al . , 2017; Lamson et al . , 2019 ) to include chromosomes and kinetochores .", "Our model successfully accomplishes spindle assembly and chromosome biorientation .", "The results give insight into key requirements for error correction and long-lived biorientation , emphasizing the importance of progressive restriction of attachment , destabilization of misaligned attachments , and force-dependent attachment lifetime .", "The turnover of kinetochore-MT attachments affects spindle mechanics , because models with larger attachment lifetime exhibit larger fluctuations in spindle length .", "The spindle components which contribute most to force generation change over time: initial spindle -pole separation is due to the outward force from kinesin-5 motors overcoming the passive crosslinker braking force , while interkinetochore stretch is the main inward force after biorientation .", "Finally , properly constructed metaphase spindles are able to robustly segregate chromosomes in the model ." ], [ "The simulation takes place within a sphere that represents the fission-yeast nucleus .", "Two SPBs are embedded in the nuclear envelope but free to move on the surface of the sphere ( although we also consider effects of allowing SPBs to move radially due to a soft nuclear envelope in one variant of the model , as discussed below ) .", "Each SPB nucleates 14 MTs , with their minus-ends tethered to the SPBs by a spring and which undergo dynamic instability at their plus-ends .", "Steric interactions are mediated by short-range hard repulsion between MTs , SPBs , and the nuclear envelope ( Figure 1A , B , Appendix 1 ) .", "Three classes of motors and crosslinkers assemble the spindle ( Figure 1A , B ) .", "Kinesin-5 motors ( representing Cut7 ) move bidirectionally on MTs ( Edamatsu , 2014; Edamatsu , 2016; Britto et al . , 2016; Singh et al . , 2018 ) , with plus-end directed movement on antiparallel MTs exerting force to slide apart the SPBs .", "Kinesin-14 motors ( representing Pkl1 and Klp2 ) crosslink MTs and one head walks toward the MT minus-ends , aligning MTs and exerting force that shortens the spindle ( Pidoux et al . , 1996; Troxell et al . , 2001; Chen et al . , 2012; Olmsted et al . , 2014; Hepperla et al . , 2014; Yukawa et al . , 2015; Yukawa et al . , 2018 ) .", "Crosslinkers ( representing Ase1 ) preferentially bind antiparallel MTs ( Yamashita et al . , 2005; Loïodice et al . , 2005; Janson et al . , 2007; Kapitein et al . , 2008; Courtheoux et al . , 2009; Fu et al . , 2009 ) and stabilize MT overlaps when crosslinking near the end of an MT , an effect which mimics the recruitment of stabilizing proteins such as CLASP ( Bratman and Chang , 2007 ) to MT ends .", "We represent the multiple outer kinetochore proteins involved in MT binding ( Sacristan and Kops , 2015; Musacchio and Desai , 2017 ) by a single attachment factor that can be bound or unbound to an MT . Because fission-yeast kinetochores can bind up to 3 MTs ( Ding et al . , 1993 ) , each kinetochore has three attachment factors in the model separated by 40 nm along the kinetochore plate ( Figure 1C , Appendix 1—figure 1 ) .", "Attachments are constrained so that no more than one attachment factor can bind to the same MT plus-end .", "The attachment factor is a 54-nm-long spring that exerts force on the MT and kinetochore when stretched or compressed ( Tables 4 and 5 ) .", "Attachment factors can make both lateral and end-on attachments to MTs , with different binding kinetics that favor end-on attachment .", "Importantly , the model includes tip tracking: a tip-bound attachment factor tracks MT ends by maintaining end-on attachment during MT growth and shrinking .", "The attachment factor also includes a plus-end-directed kinetochore motor , representing the measured contribution of kinetochore-localized dimeric Cut7 to chromosome alignment ( Akera et al . , 2015 ) .", "End-on attachment alters MT dynamic instability and is force-dependent , as measured previously ( Akiyoshi et al . , 2010; Miller et al . , 2016 ) .", "Physically each kinetochore is a rectangular plate of length 150 nm , width 50 nm , and zero thickness ( Figure 1C; Ding et al . , 1993 ) with a steric repulsion with MTs .", "Sister kinetochores are linked via springs that resist stretching and rotation , to maintain the distance and alignment of the kinetochores ( Figure 1C , Appendix 1—figure 1; Mary et al . , 2015; Smith et al . , 2016 ) .", "The pericentric DNA is modeled as a spherocylinder of length 200 nm and diameter 75 nm , which has a soft repulsion with MTs that allows MT-chromatin overlap with an energy penalty ( Appendix 1 ) .", "With these ingredients , the model can achieve both correct and erroneous kinetochore-MT attachment states ( Figure 1D ) .", "To achieve error correction and persistent biorientation , we found three key model ingredients were required: progressive restriction of attachment ( Figure 1E ) , destabilization of misaligned attachment ( Figure 1F ) , and stabilization of attachment by force ( Figure 1G , Appendix 1 ) .", "With these mechanisms , the model exhibits both spindle assembly and chromosome biorientation ( Figure 1H , Video 1 ) .", "To constrain model parameters , we developed multiple tests of simulation performance based on live-cell imaging , electron microscopy , and biorientation .", "First , we quantified the dynamics of spindle length and kinetochore position by confocal fluorescence light microscopy ( Figure 2; Gergely et al . , 2016; Blackwell et al . , 2017a ) .", "Cells with low-level labeling of MTs with mCherry-atb2 ( Yamagishi et al . , 2012; Blackwell et al . , 2017a ) and the cen2-GFP marker on the centromeric DNA of chromosome 2 ( Yamamoto and Hiraoka , 2003 ) allowed imaging of spindle length and centromere position ( Appendix 1 ) .", "The Cen2 marker is displaced only 125 nm on average from the kinetochore ( Gay et al . , 2012 ) , allowing quantification of the position of a single pair of sister kinetochores .", "We measured spindle length and kinetochore position by fitting Gaussian spots and lines to detect features , and then tracked spindle length and kinetochore position over time using previous methods ( Appendix 1; Jaqaman et al . , 2008 ) .", "Second , we used previously published electron tomographic reconstructions of fission yeast spindles ( Grishchuk and McIntosh , 2006; McIntosh et al . , 2013 ) to measure spindle structure ( Blackwell et al . , 2017a ) .", "Third , we quantified how successfully the models biorient chromosomes , measured by the fraction of simulation time during which all the chromosomes are bioriented and the average number of end-on attachments .", "We combined these measures of simulation performance in a fitness function which quantifies the overall success of each simulation run with a set of model parameters .", "We then varied poorly constrained model parameters to maximize the fitness function .", "The optimized parameters defined the reference model ( Appendix 1 ) ." ], [ "To understand the mechanisms most important for proper chromosome alignment on the spindle , we developed a computational model of fission-yeast mitosis ( Figure 1 ) that includes spindle MTs nucleated from SPBs , crosslinking motors , passive crosslinkers , pericentric chromatin , and kinetochores , all contained within a spherical nucleus ( Materials and methods , Figure 1A , B ) .", "Kinetochore-MT binding occurs via attachment factors that represent MT-binding kinetochore proteins ( Figure 1C ) , which can form both correct and erroneous MT-kinetochore attachments ( Figure 1D ) .", "Kinetochore-MT attachments progressively restrict in angle as MTs bind ( Figure 1E ) , a mechanism motivated by previous work on kinetochore geometry and chromosome rotation in error correction ( Gregan et al . , 2007; Rumpf et al . , 2010; Paul et al . , 2009; Magidson et al . , 2015; Zaytsev and Grishchuk , 2015 ) .", "In particular , work on the S . pombe monopolin complex has proposed that monopolin acts as a site-clamp that co-orients MTs bound to the same kinetochore ( Gregan et al . , 2007 ) .", "To correct attachment errors , we included destabilization of improper attachments and tip-enhanced catastrophe ( Figure 1F ) , mimicking the effectsof Aurora B kinase ( DeLuca et al . , 2006; Cimini et al . , 2006; Gay et al . , 2012 ) and recapture of lost kinetochores by MT depolymerization ( Grishchuk and McIntosh , 2006; Franco et al . , 2007; Gachet et al . , 2008; Gao et al . , 2010; Gergely et al . , 2016 ) .", "To maintain biorientation , we implemented force-dependent kinetochore-MT attachment kinetics ( Figure 1G ) , based on previous work that demonstrated an increase in attachment lifetime with tension when kinetochores are attached to depolymerizing MTs ( Akiyoshi et al . , 2010; Miller et al . , 2016 ) .", "For further details of the construction of the model , see Materials and methods and Appendix 1 .", "With these ingredients , the model is able to spontaneously assemble a bipolar spindle starting with side-by-side SPBs , form MT-kinetochore attachments , correct attachment errors , and biorient the chromosomes ( Figure 1H , Video 1 ) .", "To refine and test the model , we measured spindle assembly and chromosome alignment in fission yeast ( Figure 2 , Materials and methods , Appendix 1 ) .", "We quantified spindle length , SPB-kinetochore separation , and interkinetochore stretch from the onset of mitosis until chromosome segregation ( Figure 2A–D ) and used these data to adjust model parameters ( Materials and methods , Appendix 1 ) .", "After refinement , simulations of the reference model showed dynamics of SPB separation , kinetochore movement along the spindle , and interkinetochore stretch similar to the experimental data ( Figure 2E–H , Video 2 ) .", "As occurs in cells , the dynamics varied from simulation to simulation , but were similar on average ( Figure 2I , Appendix 1—figure 2 ) .", "After developing the reference model , we verified that single model perturbations recapitulate results from fission-yeast genetics .", "Kinesin-5 motors are essential for spindle assembly in S . pombe , and temperature-sensitive mutants of the kinesin-5/Cut7 fail to separate spindle-pole bodies ( Hagan and Yanagida , 1990; Hagan and Yanagida , 1992; Yukawa et al . , 2018; Toda et al . , 2018 ) .", "Consistent with this , when we remove kinesin-5 from the model , SPBs do not separate ( Figure 2J ) .", "Similarly , the microtubule-associated protein CLASP is essential for spindle assembly in fission yeast , where it is recruited to MT antiparallel overlaps by Ase1 and stabilizes MT dynamics ( Bratman and Chang , 2007 ) .", "When the stabilization of dynamics of crosslinked MTs is turned off in the model , SPBs do not separate ( Figure 2K ) .", "Chromosome biorientation is abolished in models where the SPBs do not separate ( Figure 2L , Video 2 ) .", "We further studied combined perturbations ( Figure 2—figure supplement 1 ) by varying kinesin-5 and crosslinker number in the absence of kinesin-14 ( Figure 2—figure supplement 1A ) and by varying kinesin-5 and −14 number in the absence of crosslinkers ( Figure 2—figure supplement 1B ) .", "Kinesin-14 in our models combines the functions of fission-yeast Pkl1 and Klp2 , neglecting the anchoring of MT minus-ends to SPBs by Pkl1 previously measured ( Olmsted et al . , 2014; Syrovatkina and Tran , 2015; Yukawa et al . , 2015; Yukawa et al . , 2018 ) .", "Experimentally , cells lacking Klp2 or both Pkl1 and Klp2 do not show altered average spindle length ( Syrovatkina et al . , 2013; Troxell et al . , 2001 ) .", "Consistent with this , model spindles formed and bioriented chromosomes in the absence of kinesin-14 , and spindle length depended on the ratio of kinesin-5 to crosslinkers .", "In fission yeast , Ase1 deletion cells assemble spindles ( Yamashita et al . , 2005; Syrovatkina et al . , 2013; Yukawa et al . , 2019 ) .", "To test if our model correctly reproduced these results , we removed the crosslinker from the model and varied the number of kinesin-5 and kinesin-14 molecules present ( Figure 2—figure supplement 1B ) .", "Removing crosslinkers in the reference model abolished spindle assembly because spindles cannot maintain robust antiparallel MT overlaps .", "However , in the reference model the kinesin-14 motors are highly sensitive to force-dependent unbinding: the characteristic distance that quantifies this is 3 . 2 times larger for kinesin-14 motors than kinesin-5 motors .", "This leads to kinesin-14 motors that unbind relatively easily under force , and they fail to maintain microtubule antiparallel overlaps necessary for bipolar spindle assembly .", "When we model the kinesin-14 motors with the same force sensitivity to unbinding as for the kinesin-5 motors , spindle formation and chromosome biorientation are rescued ( Figure 2—figure supplement 1C ) .", "Most of our simulations represent the nuclear envelope as a rigid sphere with the SPBs constrained to move on the surface of this sphere .", "However , constraining SPBs to a fixed radius alters force balance on the spindle and may alter spindle length .", "Therefore , we tested a model of a soft nuclear envelope by allowing the SPBs to move radially in a potential that mimics the energy required to deform the nuclear envelope ( Rincon et al . , 2017; Lamson et al . , 2019 ) ( Materials and methods , Appendix 1 ) .", "The results show that a soft nuclear envelope leads to slightly longer spindles ( Figure 2—figure supplement 1D , Video 3 ) , but for a physically realistic nuclear envelope force of around 17 pN , spindle length remains near 3 μm , as measured experimentally .", "Our simulations start in a state mimicking early mitosis with monotelic chromosomes , then spontaneously assemble a bipolar spindle and biorient chromosomes .", "Biorientation requires the model to correct attachment errors and maintain correct attachments .", "This occurs in the simulations primarily through progressive restriction of attachment angle , misaligned destabilization , and force-dependent kinetochore-MT attachment .", "To facilitate correct initial attachment of MTs to kinetochores , the model progressively restricts the angle at which binding can occur as more MTs bind ( Figure 1E ) .", "This is motivated by previous work demonstrating that kinetochore geometry and chromosome rotation play an important role in promoting correct kinetochore-MT attachment and correcting errors ( Gregan et al . , 2007; Rumpf et al . , 2010; Paul et al . , 2009; Magidson et al . , 2015; Zaytsev and Grishchuk , 2015 ) .", "We have extended previous work to include both multiple MT binding sites per kinetochore and changes in kinetochore geometry upon binding .", "In our model , unattached kinetochores have a wide angular range over which attachments can form ( modeled as an angular spring constant for binding , represented by the three wide cones in Figure 1E left ) .", "Each attachment formed narrows the angle allowed for the subsequent attachment , favoring attachment to MTs that are more perpendicular to the kinetochore plate ( represented by the narrower cones in Figure 1E right ) .", "Attachments exert an alignment force/torque on kinetochores and MTs based on the stiffness of this angular spring .", "To illustrate the importance of progressive restriction , we removed it , making the angular range identical for all three kinetochore-MT attachment events ( Figure 3A , Video 4 ) .", "Doing this nearly abolishes biorientation in the model: the fraction of simulation time for which all three chromosomes are bioriented is below 10% , independent the value of the angular spring constant from 1 kB⁢T ( almost any angle of attachment is allowed ) to 100 kB⁢T ( attachment is highly restricted in angle ) .", "These failures occur for different reasons as the angular spring constant varies .", "When attachment angle is most permissive , merotelic attachments form and are not corrected sufficiently rapidly to biorient the chromosomes .", "When the attachment angle is highly restricted , attachments are unlikely to form at all .", "Overall , this result shows that in our model progressive restriction of attachment is essential for biorientation .", "The progressive restriction model requires that the first binding event be relatively permissive in angle , the second more restricted , and the third highly restricted .", "To study this , we varied the angular spring constant of each attachment independently ( Figure 3B , C , Figure 3—figure supplement 1 , Video 4 ) .", "The model achieves a high fraction of simultaneous biorientation around 70% when the first attachment is maximally permissive ( spring constant is 1 kB⁢T ) ; an increase in this spring constant restricts the angle and decreases simultaneous biorientation to below 20% ( Figure 3B ) .", "This means that for the first attachment , promoting kinetochore binding to any MT is important: initial attachments should be established easily , even if erroneous .", "By contrast , biorientation is increased when the third ( final ) binding event is highly restricted ( Figure 3C ) : chromosomes are bioriented in the model <10% of the time when the third attachment is most permissive , but the fraction of simultaneous biorientation increases with the angular stiffness of the third binding site .", "The second value of the angular potential for progressive restriction was less important ( Figure 3—figure supplement 1A ) : varying it did not significantly change the fraction of simultaneous biorientation .", "Because of the importance of progressive restriction in our model , we additionally examined whether varying the number of allowed kinetochore-MT attachments might affect how easily biorientation is achieved , but found no significant effect ( Figure 3—figure supplement 1B ) .", "In these simulations , we chose how to vary the angular spring stiffness as the number of attachment sites varies .", "For fewer attachment sites , we chose the lower values of angular spring stiffnesses for progressive restriction that matched the reference stiffness .", "For increased number of attachments , the later attachments were fixed at an upper limit of 100 kB⁢T .", "In all cases , chromosome biorientation was not compromised .", "Progressive restriction of attachment reduces but does not eliminate erroneous kinetochore-MT attachments .", "Previous experimental work has shown that merotelic attachments are common in early mitosis and are corrected over time ( Cimini et al . , 2003 ) due to increased turnover of kinetochore MTs from the activity of Aurora B kinase ( DeLuca et al . , 2006; Cimini et al . , 2006; Gay et al . , 2012 ) .", "To study this , we considered two different error correction models: biorientation-dependent stabilization and force-dependent stabilization .", "First , we implemented the rule-based model of misaligned destabilization by accelerating the detachment of kinetochore-MT attachments that are not amphitelic ( Figure 1F ) .", "Because experimental work has demonstrated a decrease in kinetochore MT turnover by up to a factor of 65 in the presence of Aurora B inhibitors ( Cimini et al . , 2006 ) , we varied the misaligned destabilization factor in the model , which quantifies the increased turnover of incorrect attachments , over a similar range from 1 to 100 ( Figure 3D , Video 4 ) .", "Consistent with experimental results , biorientation is nearly eliminated in the absence of misaligned destabilization .", "Biorientation time in the model is maximum when the misaligned destabilization factor is 70 , comparable to the experimental value .", "This demonstrates the importance of error correction in the model .", "The biorientation-dependent model has the disadvantage that it cannot test any mechanisms by which incorrect attachments are destabilized .", "We therefore additionally tested a force-dependent error correction model , based on previous results that kinetochore-MT attachments are stabilized by force ( Nicklas and Koch , 1969; Cane et al . , 2013 ) .", "We modeled the kinetics of kinetochore-MT attachments as a function of interkinetochore tension , with the rates decreasing with force ( Figure 3E , Video 5 ) , controlled by a a characteristic force for significant stabilization .", "The force-stabilization model of error correction that we implemented experiences the initial problem of biorientation ( IPBO ) : a bioriented attachment that has just formed is not under tension , and therefore is not stable ( Zhang et al . , 2013; Kalantzaki et al . , 2015; Tubman et al . , 2017 ) .", "Consistent with this , we found implementing force-dependent stabilization alone did not lead to biorientation .", "Recent work has suggested that the IPBO may be solved by initial syntelic-like attachments that are end-on between the kinetochore face near a pole , and lateral to the kinetochore farther from that same pole ( Kuhn and Dumont , 2017 ) .", "Therefore , we varied parameters in the model that might facilitate tension generation before biorientation , including the angular spring constants of the interkinetochore spring , the characteristic angular factor for binding high angles to the kinetochore plate , the effective concentration for binding laterally , and the number of kinesin-5 motors , which affect overall spindle force generation .", "We were able to achieve long-lived biorientation in the force-dependent error correction model with model parameters that favored end-on over lateral attachments , inhibited attachments at high angle , and allowed sister kinetochores to more easily reorient ( Table 6 ) .", "In this version of the model , we then varied the characteristic force that controls how much attachments are stabilized by force ( Figure 3E , Video 5 ) .", "The characteristic force is the value of the interkinetochore force at which attachments are stabilized by a factor of two , so a small value reflects rapid variation of attachment stability with force , while an infinite value means that attachments are force independent .", "We found that the model is sensitive to the value of this characteristic force , with best performance of the model at a characteristic force of 1 . 67 pN .", "Higher or lower values decrease cumulative biorientation by up to a factor of two .", "Once amphitelic kinetochore-MT attachments are formed , they must be maintained for biorientation to persist .", "Attachments between single MTs and purified budding-yeast kinetochores were altered by force applied to the kinetochore , even in the absence of Aurora kinase ( Akiyoshi et al . , 2010; Miller et al . , 2016 ) .", "In particular , the kinetochore-MT attachment lifetime increased with tension when kinetochores were attached to depolymerizing MTs , an effect dependent on a TOG protein ( Akiyoshi et al . , 2010; Miller et al . , 2016 ) .", "Consistent with this , we implemented force dependence of attachments in the model ( Figure 1G ) .", "This effect is required to maintain biorientation: if we eliminate the force dependence of attachment kinetics , biorientation is nearly abolished in the model ( Figure 3F , Video 4 ) .", "To understand which force-dependent rate is most important for this effect , we added them back to the model one at a time .", "The increase in attachment lifetime of a kinetochore bound to a shrinking MT is the key force-dependent rate , because making this the only force-dependent lifetime in the model restores nearly all biorientation compared to the model with all rates force-dependent ( Figure 3F ) .", "This demonstrates that maintenance of biorientation requires kinetochore-MT attachments to persist during MT depolymerization .", "Spindle length regulation ( Dumont and Mitchison , 2009; Goshima and Scholey , 2010; Syrovatkina et al . , 2013; Hepperla et al . , 2014; Nannas et al . , 2014; Rizk et al . , 2014 ) can be understood using the force-balance model of Saunders and Hoyt in which plus-end directed sliding motors produce outward force , and minus-end directed sliding motors and chromosomes produce inward force ( Saunders and Hoyt , 1992; Nabeshima et al . , 1998; Goshima et al . , 1999; Severin et al . , 2001; Tolić-Nørrelykke et al . , 2004; Bouck and Bloom , 2007; Stephens et al . , 2013; Syrovatkina et al . , 2013; Costa et al . , 2014; Zheng et al . , 2014; van Heesbeen et al . , 2014; Syrovatkina and Tran , 2015 ) .", "The force-balance model has been used in mathematical models of spindles in yeast ( Gardner et al . , 2005; Gardner et al . , 2008; Chacón et al . , 2014; Hepperla et al . , 2014; Ward et al . , 2015; Blackwell et al . , 2017a; Rincon et al . , 2017; Lamson et al . , 2019 ) , and Drosophila ( Cytrynbaum et al . , 2003; Cytrynbaum et al . , 2005; Wollman et al . , 2008; Civelekoglu-Scholey and Scholey , 2010 ) cells .", "This work has focused on spindle length at steady state , not dynamic changes .", "However , some fission-yeast mutants exhibit large fluctuations in spindle length in which the bipolar spindle assembles , but then shortens or falls apart , known as spindle collapse ( Bratman and Chang , 2007; Griffiths et al . , 2008; Choi et al . , 2009; Hsu and Toda , 2011; Masuda et al . , 2013; Wälde and King , 2014; Syrovatkina et al . , 2013; Gergely et al . , 2016 ) .", "Remarkably , fission-yeast double mutants can have wild-type average metaphase spindle length , but much larger fluctuations than wild-type ( Syrovatkina et al . , 2013 ) .", "The underlying mechanisms of large spindle length fluctuations have remained unclear , in part because apparently contradictory changes can cause it .", "For example , deletion of proteins known either to stabilize ( Bratman and Chang , 2007 ) or destabilize MTs ( Gergely et al . , 2016 ) can both lead to large spindle length fluctuations .", "In recent work we examined how deletion of the kinesin-8 motor proteins could contribute to large spindle length fluctuations in fission yeast ( Gergely et al . , 2016 ) , but a general understanding of this phenomenon is lacking .", "Therefore , we sought to understand what mechanisms might lead to large length fluctuations .", "One key determinant of the magnitude of spindle length fluctuations is the lifetime of kinetochore-MT attachments ( Figure 4 , Video 6 ) .", "We quantified the magnitude of length fluctuations by determining the standard deviation in spindle length over time after spindle elongation for each individual simulation of the model , then averaging that standard deviation over multiple model runs with the same parameters .", "This measure of length fluctuations increases with kinetochore-MT attachment lifetime: the longer the lifetime , the larger the fluctuations ( Figure 4A–D ) .", "To understand this result , note that for long-lived attachment , the force exerted by a stretched kinetochore can grow over time to a larger value: long-lived attachment allows multiple MTs to bind per kinetochore , exert greater force , and stretch apart the sisters .", "This allows larger inward force to be exerted on the spindle by attached kinetochores .", "Indeed , the average interkinetochore distance increases with kinetochore-MT attachment lifetime ( Figure 4D ) .", "Thus , slow cycles of attachment and detachment lead to slowly varying force on the spindle that causes its length to fluctuate .", "In the opposite limit , short-lived kinetochore-MT attachment causes relatively quick turnover , limiting interkinetochore stretch , inward force , and variation in inward force .", "Alteration in kinetochore-MT attachment lifetime could occur through multiple molecular mechanisms .", "To illustrate how this could occur , we considered two perturbations to the model that have downstream effects on both lifetime and length fluctuations ( Figure 4E ) .", "The first perturbation is a restricted attachment model , in which the angular spring constant of attachment discussed above ( Figure 3A ) is set to 100 kB⁢T for all attachments .", "In this case , attachments rarely form and when formed , their lifetime is short ( <0 . 05 min on average ) .", "As a result , the force produced by interkinetochore stretch is small and does not vary much , leading to small length fluctuations in the model ( <0 . 01μ μm on average ) .", "The opposite limit can occur in a model in which the force-dependent rescue of kinetochore MTs is greatly reduced , by increasing the force constant from 6 . 4 pN to 12 . 8 pN ( this reduces the force sensitivity of rescue , see Appendix 1 ) .", "This causes kinetochore MTs to depolymerize for longer time , and because kinetochore-MT attachments are stabilized during depolymerization , this change dramatically increases the attachment lifetime to 0 . 2 min .", "As a result , interkinetochore stretch can increase , and length fluctuations correspondingly increase ( 0 . 3 μm ) .", "This analysis suggests that altered kinetochore-MT attachment lifetime could be a downstream effect that may result from the diverse mutations observed to cause spindle length fluctuations in S . pombe .", "We note that the effect of lifetime may not be the only source of spindle length fluctuations: other mutations that lead to slow changes in force exerted on the spindle could have similar effects .", "The force-balance model can explain why multiple perturbations alter steady-state spindle length , including mutation of motors and microtubule-associated proteins ( Syrovatkina et al . , 2013; Hepperla et al . , 2014 ) , and chromosome/kinetochore number and chromatin stiffness ( Chacón et al . , 2014; Nannas et al . , 2014 ) .", "However , it can be challenging to distinguish direct from indirect effects of altering force balance .", "For example , the force-balance model posits that minus-end-directed kinesin-14 motors contribute inward force that shortens the spindle , so their deletion would be expected to lead to longer spindles .", "However , in budding yeast , kinesin-14 deletion instead leads to shorter spindles , because kinesin-14 helps bundle spindle MTs , allowing kinesin-5 motors to generate greater outward force when kinesin-14 is present ( Hepperla et al . , 2014 ) .", "Similarly , kinesin-8 deletion in fission yeast leads to longer spindles , but this is likely due to effects of this motor on MT dynamics rather than direct inward force generation by kinesin-8 ( Syrovatkina et al . , 2013; Gergely et al . , 2016 ) .", "To better understand direct and indirect changes in spindle length , we examined the force produced by spindle molecules as the spindle elongates , averaged over many simulation runs ( Figure 5 , Video 7 ) .", "In this analysis , we considered each half-spindle separately , and calculated the total force exerted along the spindle axis produced by separate force-generating elements: outward force by kinesin-5 motors , and inward force by kinesin-14 motors , passive crosslinkers , and kinetochore-MT attachments ( Figure 5A ) .", "We computed spindle length as a function of time ( Figure 5B , E , H ) , force as a function of time ( Figure 5C , F , I ) and spindle length ( Figure 5D , G , J ) in the reference , restricted attachment , and weak rescue models .", "Force generation by kinesin-5 motors , kinesin-14 motors , crosslinkers , and chromosomes changes significantly as the bipolar spindle assembles .", "For early time ( up to 5 min ) when spindles are short ( up to 1 μm ) , motors and crosslinkers exert force that slowly increases in magnitude up to a few tens of pN , but chromosomes exert almost no force ( Figure 5C , F , I , Video 7 ) .", "Because chromosomes are not bioriented on the spindle during initial SPB separation , they do not exert significant inward force .", "This result is consistent with our previous work , which demonstrated that initial bipolar spindle assembly can occur in a model lacking chromosomes ( Blackwell et al . , 2017a; Rincon et al . , 2017; Lamson et al . , 2019 ) .", "The outward sliding force produced by kinesin-5 motors increases approximately linearly with spindle length , as the length of antiparallel MT overlaps increases during spindle assembly ( Figure 5D , G , J ) .", "This agrees with the experimental result that the sliding force generated by kinesin-5 motors is proportional to overlap length ( Shimamoto et al . , 2015 ) .", "The inward force generated by kinesin-14 motors is small , as in previous work that has shown that kinesin-14 is less effective at force generation that kinesin-5 ( Hentrich and Surrey , 2010 ) and that in the spindle kinesin-14 may be more important to align spindle MTs than to generate force directly ( Hepperla et al . , 2014 ) .", "During initial spindle assembly , crosslinkers play the primary role of maintaining antiparallel MT overlaps in opposition to the sliding activity of kinesin-5 .", "Remarkably , we find that the inward force generated by passive crosslinkers initially increases with spindle length to approximately 25 pN when the spindle is 0 . 75 μm long .", "Beyond this point , the crosslinker force steadily decreases , dropping to near zero within a few minutes ( Figure 5C , F , I ) .", "This is consistent with previous results on force generation by the crosslinker Ase1 , which found large force for small overlaps that drops significantly as overlaps become larger ( Lansky et al . , 2015 ) .", "Therefore , our results support a picture of early spindle assembly in which high braking force by crosslinkers on short antiparallel MT overlaps oppose the outward force generated by kinesin-5 .", "This highlights the key role of crosslinkers in early spindle assembly suggested previously ( Blackwell et al . , 2017a; Rincon et al . , 2017; Lamson et al . , 2019 ) .", "Once the spindle elongates sufficiently to separate SPBs by 1 μm , there is a transition in the primary contributer to spindle force .", "In this regime , chromosomes biorient and the inward force from interkinetochore stretch becomes significant , balancing outward force from kinesin-5 motors ( Figure 5C , F , I ) .", "This balance is crucial to setting metaphase spindle length .", "To perturb this force balance , we considered two additional models discussed above ( Figure 4E ) with restricted attachment and weak rescue .", "When attachment is restricted , chromosomes rarely biorient and the inward force from chromosomes is small for spindles of all length .", "This leads to unbalanced force from kinesin-5 motors and long spindles ( Figure 5E–G , Video 7 ) .", "When MT rescue is reduced , interkinetochore stretch is larger and the inward force from stretched sister kinetochores increases ( Figure 5H–J , Video 7 ) .", "This leads to shorter metaphase spindle length and a corresponding increase in force from stretched kinetochores .", "After developing the model of spindle assembly and chromosome biorientation , we examined what additional mechanisms were required for the model to segregate chromosomes to the poles .", "Relatively few changes are required for robust chromosome segregation , suggesting that significant new mechanisms are not required in anaphase for chromosome segregation .", "The rules added to the model for anaphase A include severing the chromatin spring between kinetochores ( based on cumulative time the chromosomes are bioriented ) , stabilization of kinetochore-MT attachment , and depolymerization of MTs ( Table 7 ) .", "With these additions to the model , simulations consistently segregate chromosomes to the poles ( Figure 6A–D , Video 8 ) .", "We compared our simulations to experimental measurements of chromosome segregation , and found similar speed of chromosome movement to the poles and separation of sisters ( Figure 6E–G ) , as expected from the choice of MT depolymerization speed in the anaphase model ." ], [ "The computational model of mitosis presented here can biorient chromosomes as the spindle assembles .", "This framework allows us to examine which functions are most important to assemble a bipolar spindle , attach kinetochores to spindle MTs , biorient chromosomes , and segregate them to the poles ( Figure 1; Video 1 ) .", "Our model was refined with experimental data on spindle structure , spindle elongation , and chromosome movements in fission yeast , leading to quantitative agreement with the data ( Figure 2; Video 2 ) .", "The reference model results match previous genetics that found that kinesin-5 motors and CLASP are essential for bipolar spindle assembly ( Hagan and Yanagida , 1990; Hagan and Yanagida , 1992; Bratman and Chang , 2007; Blackwell et al . , 2017a ) , which suggests that the model captures key features needed to provide insight into mitotic mechanism .", "Three ingredients are required for long-lived biorientation in the model ( Figure 3; Video 4 ) .", "Kinetochores shield themselves from merotely by progressive restriction of attachment .", "Inclusion of this effect in the model was motivated by recent work on the monopolin complex in fission yeast ( Gregan et al . , 2007 ) and attachment-driven compaction of mammalian kinetochores ( Magidson et al . , 2015 ) .", "Progressive restriction has two key effects: it promotes proper attachment by favoring binding of microtubules from the same pole that is already attached to the kinetochore , and simultaneously creates a torque that helps to reorient the kinetochore on the spindle .", "In previous work , the monopolin complex components Pcs1/Mde4 were found not to be essential in fission yeast ( Gregan et al . , 2007 ) , but in our model completely removing progressive restriction abolishes biorientation ( Figure 3 ) .", "This suggests the possibility that in fission yeast , other molecules may contribute to progressive restriction in addition to monopolin .", "Mimicking the effects of Aurora B kinase by including destabilization of misaligned attachments allows the model to achieve robust error correction .", "Destabilization by approximately a factor of 70 gives the highest degree of biorientation the model .", "This is similar to the degree of destabilization previously estimated to occur due to Aurora B ( Cimini et al . , 2006 ) , further suggesting that the model produces biologically relevant results .", "To maintain long-lived biorientation in the model , kinetochore-MT attachment lifetime must increase with tension during microtubule depolymerization .", "This catch-bond behavior has been previously measured for purified budding-yeast kinetochores attached to single microtubules ( Akiyoshi et al . , 2010; Miller et al . , 2016 ) .", "Without this force dependence , kinetochores frequently detach from depolymerizing MTs and lose biorientation .", "Our model achieves biorientation for the longest time with an increased force-sensitivity of attachment compared to experimental measurements , a difference that would be of interest to explore in future work .", "The timing of spindle assembly and biorientation in the model were consistent with those quantified experimentally .", "A current difference between the model and experiment is that we find ongoing turnover of kinetochore-MT attachments , so that biorientation can be lost once established .", "This is in contrast to previous experimental work , which suggests that for metaphase spindles , once biorientation is established it is rarely lost ( Waters et al . , 1996; Nicklas , 1997; Yoo et al . , 2018 ) .", "The mechanisms underlying this difference are an open question .", "Using our model , we studied the origins of large spindle length fluctuations ( Figure 4; Video 6 ) .", "While previous work has examined regulation of spindle length ( Syrovatkina et al . , 2013; Hepperla et al . , 2014; Nannas et al . , 2014; Rizk et al . , 2014 ) , what mechanisms might drive large fluctuations in spindle length over time have been less well-studied .", "We identified the lifetime of kinetochore-MT attachment as a determinant of the degree of spindle length fluctuations .", "Long attachment lifetime allows bioriented chromosomes to become more stretched , leading to large , slowly varying inward force on the spindle .", "Our results suggest why large spindle length fluctuations have not been seen in larger spindles in vertebrate cells: in S . pombe , a relatively small number of kinetochores and MTs contribute to spindle length , and therefore the changing force on the three chromosomes can have a significant effect on the spindle .", "In vertebrate spindles with tens of thousands of MTs , changes in force on a small number of kinetochores contribute only a small fractional change to overall force on the spindle , leading to smaller fluctuations .", "To understand how force generation changes as the spindle assembles , we quantified the force generated by different classes of spindle molecule ( Figure 5; Video 7 ) .", "The early spindle has almost no force generation from interkinetochore stretch because chromosomes are rarely bioriented at this stage .", "Instead , the early spindle is characterized by outward force from kinesin-5 motors that is resisted by crosslinkers .", "Consistent with earlier work ( Lansky et al . , 2015 ) , the force from crosslinkers is highest when MT antiparallel overlaps are short and drops as the spindle elongates .", "Once the bipolar spindle is formed and chromosomes are bioriented , attached chromosomes provide significant inward force that opposes the outward force of kinesin-5 motors .", "These results suggest that the many mutations that alter spindle length in fission yeast ( Syrovatkina et al . , 2013 ) might act indirectly by altering kinesin-5 force generation or interkinetochore stretch .", "Remarkably , the model is able to transition to anaphase A and robustly segregate chromosomes to the poles with a small number of additional rules ( Figure 6; Video 8 ) .", "Overall , our work provides a powerful framework for testing spindle assembly mechanisms that can inform future experimental studies ." ] ]

[ "The essential functions required for mitotic spindle assembly and chromosome biorientation and segregation are not fully understood , despite extensive study .", "To illuminate the combinations of ingredients most important to align and segregate chromosomes and simultaneously assemble a bipolar spindle , we developed a computational model of fission-yeast mitosis .", "Robust chromosome biorientation requires progressive restriction of attachment geometry , destabilization of misaligned attachments , and attachment force dependence .", "Large spindle length fluctuations can occur when the kinetochore-microtubule attachment lifetime is long .", "The primary spindle force generators are kinesin-5 motors and crosslinkers in early mitosis , while interkinetochore stretch becomes important after biorientation .", "The same mechanisms that contribute to persistent biorientation lead to segregation of chromosomes to the poles after anaphase onset .", "This model therefore provides a framework to interrogate key requirements for robust chromosome biorientation , spindle length regulation , and force generation in the spindle ." ]

[ "Before a cell divides , it must make a copy of its genetic material and then promptly split in two .", "This process , called mitosis , is coordinated by many different molecular machines .", "The DNA is copied , then the duplicated chromosomes line up at the middle of the cell .", "Next , an apparatus called the mitotic spindle latches onto the chromosomes before pulling them apart .", "The mitotic spindle is a bundle of long , thin filaments called microtubules .", "It attaches to chromosomes at the kinetochore , the point where two copied chromosomes are cinched together in their middle .", "Proper cell division is vital for the healthy growth of all organisms , big and small , and yet some parts of the process remain poorly understood despite extensive study .", "Specifically , there is more to learn about how the mitotic spindle self-assembles , and how microtubules and kinetochores work together to correctly orient and segregate chromosomes into two sister cells .", "These nanoscale processes are happening a hundred times a minute , so computer simulations are a good way to test what we know .", "Edelmaier et al . developed a computer model to simulate cell division in fission yeast , a species of yeast often used to study fundamental processes in the cell .", "The model simulates how the mitotic spindle assembles , how its microtubules attach to the kinetochore and the force required to pull two sister chromosomes apart .", "Building the simulation involved modelling interactions between the mitotic spindle and kinetochore , their movement and forces applied .", "To test its accuracy , model simulations were compared to recordings of the mitotic spindle – including its length , structure and position – imaged from dividing yeast cells .", "Running the simulation , Edelmaier et al . found that several key effects are essential for the proper movement of chromosomes in mitosis .", "This includes holding chromosomes in the correct orientation as the mitotic spindle assembles and controlling the relative position of microtubules as they attach to the kinetochore .", "Misaligned attachments must also be readily deconstructed and corrected to prevent any errors .", "The simulations also showed that kinetochores must begin to exert more force ( to separate the chromosomes ) once the mitotic spindle is attached correctly .", "Altogether , these findings improve the current understanding of how the mitotic spindle and its counterparts control cell division .", "Errors in chromosome segregation are associated with birth defects and cancer in humans , and this new simulation could potentially now be used to help make predictions about how to correct mistakes in the process ." ]

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

[ "evolutionary biology", "medicine" ]

[ [ "APOE has three functionally polymorphic allelic variants: E4 , E3 , and E2 ( Demarchi et al . , 2005; Safieh et al . , 2019 ) .", "The most prevalent , APOE3 , arose ~200K years ago from a single nucleotide polymorphism ( SNP ) ( C → T ) at locus 19q13 from the ancestral APOE4 ( Fullerton et al . , 2000 ) .", "The evolutionary success of APOE3 has been attributed to its greater plasticity in response to environmental changes compared to the ancestral APOE4 allele ( Huebbe and Rimbach , 2017 ) .", "However , APOE4 is maintained at frequencies up to 40% , such as those in some central African populations .", "Maintenance and distribution of APOE variants may in part be due to the distinct functional capabilities of allelic variants ( Trotter et al . , 2010 ) .", "Eisenberg and colleagues have suggested that the benefits of APOE4 may be most appreciable in environments where cholesterol requirements are increased to meet higher metabolic demands , such as at high and low latitudes where there are climatic extremes ( e . g . northern Europe and South America ) ( Eisenberg et al . , 2010 ) .", "Another leading explanation for APOE4 persistence is based on the theory of antagonistic pleiotropy ( Williams , 1957 ) , which posits that the APOE4 allele may persist due to the fitness benefits of lipid buffering in early life relative to APOE3 , outweighing any harmful health effects that manifest in a post-reproductive life stage ( i . e . ‘selection’s shadow’ ) ( Smith et al . , 2019 ) .", "Consistent with the notion of early life fitness advantage , the APOE4 variant is associated with lower infant mortality and higher fertility among rural Ghanaians experiencing high-pathogen burden ( van Exel et al . , 2017 ) .", "Innate immune responses with fever and systemic inflammation are also energetically expensive ( Muehlenbein et al . , 2010 ) , and cholesterol and other lipids are necessary for fueling these responses ( Tall and Yvan-Charvet , 2014 ) .", "Helminths and some protozoal parasites also require lipids to fuel their own metabolic activities , either pulling them from the host bloodstream , or modulating host metabolic activities like LDL endocytosis or lipid remodeling to gain access to lipids ( Bansal et al . , 2005 ) .", "Thus , in high-pathogen and energy-limited environments , where there may be persistent pathogen-driven immune activation , the ability to maintain peripheral cholesterol levels would presumably be a benefit throughout life ( Finch and Martin , 2016; Gurven et al . , 2016 ) .", "Despite APOE4 being associated with neuroinflammation among those with AD ( Kloske and Wilcock , 2020 ) , in ‘healthy’ individuals the APOE4 allele is associated with downregulated innate immunity .", "Specifically , blood levels of C-reactive protein ( CRP ) in APOE4+ carriers are lower in several post-industrial populations ( Lumsden et al . , 2020; Martiskainen et al . , 2018 ) , as well as the Tsimane , an Amerindian population in rural Bolivia ( and focus of the present study ) ( Vasunilashorn et al . , 2011 ) .", "Moreover , among the Tsimane , APOE4+ carriers had lower levels of blood eosinophils ( Trumble et al . , 2017; Vasunilashorn et al . , 2011 ) .", "Other studies have documented lower levels of certain proinflammatory cytokines ( e . g . IL-6 , TNF-alpha ) in APOE4+ carriers ( Olgiati et al . , 2010 ) , and downregulated expression of biomarkers mediating innate immune sensing ( TLR-signaling molecules ) ( Dose et al . , 2018 ) .", "Experimental studies also showed the associations of APOE4 with heightened innate and complement inflammatory responses to lipopolysaccharide stimulation in Gale et al . , 2014; Tzioras et al . , 2019 .", "Maintaining lower levels of baseline innate immunity may minimize the accumulative damage caused by low-grade innate inflammation over the long term , while still enabling strong targeted immune responses to pathogens following exposure ( Franceschi et al . , 2000; Trumble and Finch , 2019 ) , particularly in environments with a diversity of species of pathogens .", "Further , pathogens like helminthic parasites , may moderate inflammation by triggering Th2-mediated ( anti-inflammatory ) immunological pathways ( Maizels and McSorley , 2016; Motran et al . , 2018 ) , which may be important for counterbalancing a strong proinflammatory response .", "In energy-limited , pathogenically diverse environments , APOE4+ carriers may thus be better able to tolerate energetic costs imposed by infection by having higher concentrations of circulating lipids to fuel immune responses , while also minimizing damage from exposure to generalized systemic inflammation through downregulation of innate immune function .", "By contrast , in post-industrialized contexts , without the moderating influences of parasites on both cholesterol and immune functions , non-pathogenic stimuli ( e . g . obesity ) may be more likely to trigger systemic low-grade inflammatory pathways and , in the absence of a brake , lead to arterial and vascular damage and disease .", "Thus , the link between APOE4 and inflammatory diseases in post-industrialized contexts may in part be due to an environmental mismatch .", "In pathogenically diverse environments , innate immune defenses are likely to be activated owing to more frequent encounters with novel pathogens .", "This more diverse pathogenic setting may increase selective pressure to favor stronger immune regulation .", "We hypothesize that in such a context , an APOE4 variant is less harmful because it", "( a ) minimizes damage caused by chronic innate inflammation and", "( b ) maintains higher circulating cholesterol levels , which buffer energetic costs of pathogen-driven innate immune activation .", "In post-industrial contexts , where there is a relative absence of diverse pathogens and thus reduced pathogen-mediated lipid regulation , coupled with an overabundance of calories , the effect of APOE4 on circulating lipids may instead incur a cost .", "Lifestyle factors that promote obesity and excessive circulating lipids may lead to sterile endogenous inflammation ( Trumble and Finch , 2019 ) that overshadows any potentially positive effects of APOE4 on immune function .", "Thus , the APOE4 variant has greater potential to lead to hyperlipidemia and coincide with related inflammatory diseases in high-calorie , low-pathogen , environments ( Figure 1 ) .", "This study describes the immunophenotypes and lipid profiles of individuals with APOE3/3 and APOE4+ genotypes living in a pathogenically diverse , energy-limited environment .", "For the purpose of hypothesis testing , we focus on testing genotype-related differences in components of innate immunity ( CRP , neutrophils , eosinophils , and erythrocyte sedimentation rate [ESR] ) and blood lipids linked to inflammation ( total cholesterol , LDL , and oxidized LDL [ox-LDL] ) .", "We evaluate the extent to which body mass index ( BMI ) moderates the association between lipids and innate inflammation ( CRP , neutrophils , ESR ) .", "Finally , we test whether the APOE4 allele has a moderating effect on the relationship between BMI and lipids to evaluate the role of APOE4 in the maintenance of stable lipid levels under energetic restriction .", "This research focused on the Tsimane , an Amerindian population in the Bolivian tropics that faces high exposure to a diverse suite of pathogens , and endemic helminthic infections .", "Tsimane have high rates of infection across all ages , with 70% helminth prevalence and >50% of adults with co-infections from multiple species of parasite or protozoan ( Blackwell et al . , 2015; Garcia et al . , 2020 ) .", "Compared to U . S . and European reference populations , the Tsimane have also been found to have upregulated immune function across the life course ( Blackwell et al . , 2016 ) .", "In most villages , there is little or no access to running water or infrastructure for sanitation ( Dinkel et al . , 2020 ) .", "The Tsimane are primarily reliant on foods acquired through slash-and-burn horticulture , fishing , hunting , gathering , and small animal domestication , supplemented with market goods ( e . g . salt , sugar , cooking oil ) ( Kraft et al . , 2018 ) .", "Tsimane are rarely sedentary , instead engaging in sustained low and moderate physical activity over much of their life course ( Gurven et al . , 2013 ) , and have minimal atherosclerosis ( Kaplan et al . , 2017 ) .", "However , with greater globalization and improvements in technology , the Tsimane are experiencing ongoing lifestyle changes; there is variation in participation in the market economy , and related variation in diet ( e . g . access and uptake of processed foods ) and activity level .", "Despite increasing access to markets and towns , infections remain the largest source of morbidity ( Gurven et al . , 2020 ) ." ], [ "Relative to APOE3/3 homozygotes , APOE4 carriers have higher BMI ( β = 0 . 15 [CI: 0 . 02–0 . 28] , p = 0 . 02 ) , total cholesterol ( β = 0 . 15 [CI: 0 . 04 , 0 . 27] , p = 0 . 009 ) , and oxidized LDL ( β = 0 . 16 [CI: –0 . 00 , 0 . 32] , p = 0 . 05 ) , yet lower levels of innate immune blood biomarkers: CRP ( β = –0 . 29 [CI: −0 . 44 , –0 . 14] , p < 0 . 001 ) , eosinophils ( β = –0 . 16 [CI: −0 . 24 , –0 . 08] , p < 0 . 001 ) ( Table 1 , Figure 3 ) .", "Similarly , in constrained models that excluded all individuals with CRP >10 mg/L and >5 mg/L , APOE4 carriers maintained lower levels of CRP relative to APOE3/3 homozygotes ( β = –0 . 23 [CI: −0 . 38 , –0 . 09] , p < 0 . 001; β = –0 . 22 [CI: −0 . 36 , –0 . 08] , p = 0 . 002 , respectively ) .", "Adjusting for multiple testing , CRP ( FDR adj . p < 0 . 001 ) and eosinophils ( FDR adj . p < 0 . 001 ) remain significantly different between APOE genotypes .", "APOE4 is also associated with a lower eosinophil to lymphocyte ratio ( β = –0 . 14 [CI: −0 . 23 , –0 . 05] , p = 0 . 001 ) and lower total leukocytes ( β = –0 . 08 [CI: −0 . 15 , –0 . 01] , p = 0 . 02 ) , but not with LDL , high-density lipoprotein ( HDL ) , or triglycerides ( Figure 3—figure supplement 1 ) .", "Full models shown in Supplementary file 1b-e .", "For Tsimane with higher ( >28 ) BMI , total cholesterol and LDL did not associate with CRP; however , for individuals with median ( 21 ≤ BMI ≤ 28 ) or low ( <21 ) BMI , higher total cholesterol and LDL associate with lower CRP ( Figure 4 ) .", "When considered as a continuous variable , BMI significantly moderates these associations ( total cholesterol: β = 0 . 15 [CI: 0 . 08 , 0 . 21] , p < 0 . 001; LDL: β = 0 . 16 [CI: 0 . 10 , 0 . 22] , p < 0 . 001 ) .", "BMI also interacts with ox-LDL ( β = 0 . 14 [CI: 0 . 08 , 0 . 19] , p < 0 . 001 ) in predicting CRP; however , this relationship is distinct from the other lipids tested .", "For Tsimane with high BMI , ox-LDL positively associates with CRP , while for those with low BMI the inverse is true ( Figure 3C ) .", "For ESR , total cholesterol ( β = 0 . 05 [CI: 0 . 01 , 0 . 08] , p = 0 . 008 ) and LDL ( β = 0 . 03 [CI: –0 . 00 , 0 . 07] , p = 0 . 07 ) positively associate with ESR only among Tsimane with higher BMIs .", "After adjusting for multiple testing , relationships between cholesterols and CRP all remain significant ( all FDR adj . p < 0 . 001 ) , as does the relationship between total cholesterol and ESR ( FDR adj . p = 0 . 02 ) .", "Concerning independent relationships , there are no direct relationship between ox-LDL and CRP , whereas higher total cholesterol and LDL are associated with lower CRP ( total cholesterol: β = –0 . 13 [CI: −0 . 20 , –0 . 05] , p < 0 . 001; LDL: β = –0 . 11 [CI: −0 . 18 , –0 . 03] , p = 0 . 006 ) ( Figure 4—figure supplement 1 ) .", "Total cholesterol is also negatively associated with ESR ( β = –0 . 06 [CI: −0 . 10 , –0 . 02] , p = 0 . 002 ) .", "There are no independent or interactive relationships between BMI , cholesterols , and neutrophils .", "For full models , see Supplementary file 1f-h .", "Finally , we assess whether APOE genotypes differentially moderate associations between cholesterol and BMI for lean and high BMI individuals .", "To evaluate the effects of the APOE4 allele on lipid levels across the range of BMI , we added an interaction term between BMI and APOE genotype to the mixed effects linear regression models assessing relationships between BMI and lipids ( Table 2 ) .", "These analyses show that APOE4 carriers maintain similar levels of total cholesterol and LDL across BMIs ( cholesterol: β = –0 . 08 [CI: −0 . 18 , –0 . 02] , p = 0 . 11; LDL: β = –0 . 09 [CI: –0 . 19 , 0 . 00] , p = 0 . 06 ) , whereas APOE3/3 homozygotes show higher cholesterol with BMI .", "Specifically , APOE4 carriers maintain higher levels of total and LDL cholesterol at lower BMIs , but have lower levels of both at higher BMIs , relative to individuals that are homozygous APOE3/3 ( Figure 5 ) .", "However , neither of these associations are significant .", "Ox-LDL , HDL , and triglycerides do not vary by APOE alleles across BMIs .", "For full models , see Supplementary file 1 ." ], [ "Though our findings draw from a large sample size and are robust to various controls and model specifications , there are several limitations .", "First , our findings are correlative and limit causal inference .", "Because these findings may be important for furthering evolutionary ( i . e . why the APOE4 allele may be maintained ) and clinical ( i . e . the role of APOE in disease pathogenesis ) understanding , they require replication and warrant experimental testing .", "The central thesis presented here – that persistent exposure to pathogens and obesogenic diets moderate the relationship between blood lipids and inflammation – is amenable to experimental manipulation under lab conditions .", "Specifically , a mammalian model system could be split into two treatments: those raised under sterile conditions versus regimented exposure to non-lethal pathogens .", "These treatments may then be crossed with dietary or physical activity conditions that produce differential levels of adiposity .", "Our hypothesis predicts that both decreased adiposity and increased life course pathogen exposure will reduce or even eliminate positive associations between blood lipids and chronic inflammation .", "Importantly , inflammatory biomarkers can be measured at more frequent intervals in lab conditions to assess long-term differences in the function of both pro- and anti-inflammatory pathways between experimental treatments .", "Second , there is some evidence that the APOE4 allele is positively associated with HDL cholesterol levels ( Hopkins et al . , 2002 ) , and that higher HDL levels reduce risk of severe infection ( measured by infectious hospitalizations ) ( Trinder et al . , 2020 ) .", "Inversely , acute infections and systemic inflammation ( e . g . acute phase reaction ) are associated with a decrease in HDL and HDL remodeling that results in lower cholesterol efflux capacity and higher peripheral levels of LDL cholesterol ( Ronsein and Vaisar , 2017; Zimetti et al . , 2017 ) .", "While we did not find differences in HDL by APOE status among the Tsimane , we cannot completely discount the possibility that HDL remodeling plays a role in the higher lipid levels , in addition to APOE allelic variation .", "Further , given the relative lack of , and difficulty in accessing , medical care facilities , it is difficult to assess degrees of infection severity , and thus it is also possible that APOE4 may mitigate infectious disease burden .", "However , the current data cannot provide evidence for either of these potentials , and further research is needed to disentangle the roles of APOE and lipids in infection .", "Third , proxies for energy availability and pathogen exposures are imperfect .", "BMI and fat free mass may have a variable relationship across the degree of market integration .", "However , given that our main goal in the paper – with regard to energy availability – is to investigate APOE and lipids at the extreme tails of BMI ( lean versus obese ) , BMI should adequately capture broad differences in energetic availability between these two groups .", "Because patterns of immune response vary depending on pathogen type and species , the use of a high white blood cell count cutoff as a proxy for current infection overly simplifies immune variation due to different types of infection .", "To this end , we also adjust for seasonality and cluster by community residence in our models , which should capture additional variation in exposures .", "Finally , we were not able to fully adjust for the time of day that samples were collected for the biomarkers used in this paper .", "Given that peripheral levels of most immune biomarkers vary diurnally to some extent , it is possible that not adjusting for exact time of day may have introduced some noise into analyses .", "However , CRP ( which the main findings centered around ) does not appear to follow a circadian rhythm in healthy individuals ( Meier-Ewert et al . , 2001 ) .", "Further , the largest differences in levels ( peak to trough amplitudes ) tend to coincide with sleep and wake cycles ( Labrecque and Cermakian , 2015 ) .", "Because blood draws routinely occur in the morning , samples are constrained to a narrow window , and thus we are not comparing values across the full range of diurnal variation .", "In post-industrial settings , APOE4 is generally considered a purely deleterious allele , increasing inflammation and lipids , and escalating CVD and neurological disease risk .", "Yet , in a high-pathogen environment with minimal obesity , we find that APOE4 is associated with lower levels of innate inflammation .", "While APOE4 carriers do have higher lipid levels , these may be beneficial for immune response and child survival , and unlikely to increase CVD risk in a population without other cardiometabolic risk factors ." ], [ "The Tsimane are an Amerindian population that live in the tropical lowlands of Bolivia .", "As of 2015 , the Tsimane Health and Life History Project ( THLHP ) census estimated a total population size of about 16 , 000 individuals living across 90+ villages ( Gurven et al . , 2017 ) .", "Data come from the THLHP , a longitudinal study of health and behavior that has run continuously since 2002 ( Gurven et al . , 2017 ) .", "Biomarker data used in this paper were collected by the THLHP between 2004 and 2015 ( see Gurven et al . , 2017; Kraft et al . , 2020 for details ) .", "A Bolivian and Tsimane mobile medical team travel annually or biannually among study communities conducting clinical health assessments and collecting biochemical and anthropometric information from community members that want to participate .", "This sample includes all data from individuals for whom we have APOE genotyping and measurements of age , sex , and BMI – which is the base criteria for this study .", "Sample size varies by biomarker and over time for several reasons: sampling strategy varies by data type , absent or sick team personnel needed to collect data , the number of study villages and thus enrolled participants has increased over time , and the data types collected have changed over time ( see Kraft et al . , 2020 ) .", "There are also fewer repeat measurements for a subset of biomarkers ( i . e . CRP and ox-LDL ) that were assayed in the United States , due to them being analyzed as part of a prior project .", "Specific sample sizes are reported in Table 1 , and full tables report sample size for each model .", "Whole blood samples were stored in cryovials ( Nalgene , Rochester , NY ) and frozen in liquid nitrogen before transfer on dry ice to the University of California-Santa Barbara , where they were stored at –80°C until genotyping .", "SNP genotyping was used to identify APOE allelic variants in blood samples .", "Samples were shipped on dry ice to University of Southern California ( 2010 and 2013 ) and University of Texas-Houston ( 2016 ) , where DNA was extracted , quantified , and haplotype-coded for APO- E2 , E3 , and E4 alleles using the TaqMan Allelic Discrimination system ( Thermo Fisher Scientific , Carlsbad , CA ) .", "Determination of the APOE2/E3/E4 alleles in the Tsimane derived from two SNPs of 20–30 bp oligonucleotides surrounding the polymorphic site ( Cys112Arg/rs429358 and Cys158Arg/rs7412 ) ( Trumble et al . , 2017; Vasunilashorn et al . , 2011 ) .", "Biomarkers were assayed either in the field at the time of collection or in the Human Biodemography laboratory at UC Santa Barbara in 2016 .", "Blood was collected by venipuncture in a heparin-coated vacutainer .", "Immediately following the blood draw , total leukocyte counts and hemoglobin were determined with a QBC Autoread Plus dry hematology system ( QBC Diagnostics ) , with a QBC calibration check performed daily to verify QBC performance .", "Relative fractions of neutrophils , eosinophils , and lymphocytes were determined manually by microscopy with a hemocytometer by a certified Bolivian biochemist .", "ESR was calculated following the Westergren , 1957 , method .", "Serum was separated and frozen in liquid nitrogen before transfer to the University of California-Santa Barbara where a commercial immunoassay was used to measure ox-LDL ( Mercodia Cat# 10-1143-01 , Winston Salem , NC ) .", "Serum high sensitivity C-reactive protein ( hs-CRP ) was assessed via immunoassay ( Brindle et al . , 2010 ) and was cross-validated by the University of Washington laboratory , using the protocols utilized for the National Health and Nutrition Evaluation Survey ( NHANES ) ( Meridian Life Sciences Cat# M86005M , RRID:AB_150654 ) .", "Ox-LDL and hs-CRP assays use materials from the same lot across all measures .", "Total and LDL cholesterol levels from serum samples were measured ( Stat Fax 1908 , Awareness Technology , Palm City , FL ) in the THLHP laboratory in San Borja , Beni , Bolivia .", "Birth years were assigned based on a combination of methods including using known ages from written records , relative age lists , dated events , photo comparisons of people with known ages , and cross-validation of information from independent interviews of kin ( Gurven et al . , 2007 ) .", "Each method provides an independent estimate of age , and when estimates yielded a date of birth within a 3-year range , the average was used .", "Individuals for whom reliable ages could not be ascertained are not included in analyses .", "Weight and height were measured in the field by a member of the THLHP medical team , using a basic digital scale ( Tanita , Arlington Heights , IL ) and stadiometer to the nearest 0 . 1 cm .", "BMI was calculated as weight ( kg ) /height2 ( m2 ) .", "Mixed effects linear regressions with restricted maximum likelihood estimation are used for all analyses .", "Models adjust for age , sex , seasonality , and current infection ( leukocyte count >12 , 000 cells per microliter of blood ) ( McKenzie and Williams , 2010 ) , with random intercept effects for individual ID and community .", "Because Tsimane villages vary in sanitation infrastructure , including access to soap and other hygienic products , and potentially prevalence by pathogen type ( e . g . some living very close to the river versus farther out in the forest ) , individuals were clustered by community to account for variation in such community-level factors .", "Household-level variation ( e . g . cohabitating individuals may have similar exposures ) was also tested by including a random household ID as a random variable .", "However , because inclusion of household ID as a random effect did not alter parameter estimates or p-values , and fit ( measured by AIC ) was not substantially improved across any model , it was omitted from final analyses .", "To model moderation effects ( sections Does BMI moderate the association between lipids and inflammation ? and Does APOE genotype moderate the association between BMI and lipids ? ) interaction terms are included between the main predictor and moderator .", "Immune and lipid measures required transformation to normalize their skewed distributions .", "Variables were transformed as follows: CRP , BMI , and triglycerides were natural log-transformed; total leukocytes and subsets ( lymphocytes neutrophils , eosinophils ) , ESR , and remaining cholesterols ( total cholesterol , LDL , HDL , ox-LDL ) were square root-transformed .", "To compare across models , all dependent variables were then z-scored for analyses , and thus all betas are standardized estimates ." ] ]

[ "In post-industrial settings , apolipoprotein E4 ( APOE4 ) is associated with increased cardiovascular and neurological disease risk .", "However , the majority of human evolutionary history occurred in environments with higher pathogenic diversity and low cardiovascular risk .", "We hypothesize that in high-pathogen and energy-limited contexts , the APOE4 allele confers benefits by reducing innate inflammation when uninfected , while maintaining higher lipid levels that buffer costs of immune activation during infection .", "Among Tsimane forager-farmers of Bolivia ( N = 1266 , 50% female ) , APOE4 is associated with 30% lower C-reactive protein , and higher total cholesterol and oxidized LDL .", "Blood lipids were either not associated , or negatively associated with inflammatory biomarkers , except for associations of oxidized LDL and inflammation which were limited to obese adults .", "Further , APOE4 carriers maintain higher levels of total and LDL cholesterol at low body mass indices ( BMIs ) .", "These results suggest that the relationship between APOE4 and lipids may be beneficial for pathogen-driven immune responses and unlikely to increase cardiovascular risk in an active subsistence population ." ]

[ "Genes contain the instructions needed for a cell to make molecules called proteins , which perform various roles in the body .", "Different variants of a gene can affect how the protein works , and in some cases , can increase a person’s risk to develop certain diseases .", "For example , people who carry a version of the apolipoprotein E gene called APOE4 have a greater risk of developing Alzheimer’s disease or heart disease .", "Individuals with two copies of this genetic variant have a 45% higher risk of heart disease and 12 times higher risk of Alzheimer’s disease .", "Studies in industrialized countries suggest this increased risk may be the result of higher cholesterol and inflammation in people with APOE4 .", "But if APOE4 is harmful , why does it continue to be so common worldwide ?", "One potential explanation is that APOE4 , which has been around since before modern humans , may be beneficial in some contexts .", "Cholesterol is essential for many vital tasks in the body .", "In physically demanding environments where parasitic infections are common – conditions similar to those experienced by early humans – APOE4 might be beneficial .", "Under those circumstances , having more cholesterol might help fuel metabolic activities , fight infections , or reduce inflammation caused by infections .", "Garcia et al . investigated the link between the APOE4 genetic variant , cholesterol and inflammation in 1 , 266 Indigenous Tsimane people from 80 villages in Bolivia .", "Tsimane people live an active lifestyle foraging and farming for food .", "Parasite infections are a common problem in their communities , but obesity rates are very low .", "Garcia et al . found that Tsimane people with at least one copy of the APOE4 have lower levels of inflammation and higher levels of cholesterol than those who have two copies of the APOE3 version of the gene .", "Very lean people with APOE4 had especially high levels of the so called “bad” low density lipoprotein ( LDL ) cholesterol compared to people with APOE3 only .", "However , in this situation , storing a little extra cholesterol may not be so bad .", "The findings contradict other studies that have linked obesity to higher LDL levels and APOE4 to higher levels of inflammation .", "For the majority of human history , humans lived in more physically strenuous and calorically restrictive environments , with less access to clean water .", "Garcia et al . suggest that the harmful effects of APOE4 seen in studies in more industrialized societies – where people tend to be more sedentary and have less exposure to pathogens – may reflect a mismatch between a person’s environment and their genes .", "More studies that capture the diversity of environmental conditions under which people live will help clarify the role of APOE4 health and disease ." ]

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

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

[ [ "African Animal Trypanosomiasis is one of the major constraints on the productivity of pastoralists in sub-Saharan Africa and can be caused by infection by a range of trypanosome species ( Shaw , 2004 ) , while infections of humans are caused by only two subspecies of Trypanosoma brucei ( Laveran , 1902; Pays and Vanhollebeke , 2009 ) .", "The disease is persistent as the host immune system is usually unable to clear the infection .", "This is due to the trypanosome having evolved a population survival strategy based on autoregulation of parasitaemia and antigenic variation ( MacGregor et al . , 2011; Horn , 2014 ) .", "The trypanosomes also internalize and degrade surface bound immunoglobulin ( Pal et al . , 2003; Engstler et al . , 2007 ) , increasing the survival of an individual cell and thereby increasing the likelihood of transmission .", "Both of these strategies require a densely packed cell surface coat of variant surface glycoprotein ( VSG ) that acts as a barrier , preventing access of host immunoglobulins to the plasma membrane ( Schwede and Carrington , 2010 ) .", "This coat also undergoes antigenic variation through expression of a single VSG gene from a genomic repertoire of hundreds ( Horn , 2014 ) .", "Although the VSG coat restricts immunoglobulin access , it must be permissive for receptor-mediated binding and uptake of macromolecular ligands .", "T . brucei , and the closely related T . congolense , have receptors for both transferrin ( TfR ) for iron ( Steverding et al . , 1994; Schell et al . , 1991; Jackson et al . , 2013 ) and haptoglobin-haemoglobin ( HpHbR ) for haem ( Vanhollebeke et al . , 2008; Higgins et al . , 2013 ) .", "These are held on the external face of the plasma membrane by covalent attachment of the C-terminal carboxyl group to a glycosylphosphatidyl inositol to form a GPI-anchor .", "All have free movement in the lateral plane of the membrane , although the receptors are concentrated in the flagellar pocket , an invagination of the plasma membrane at the base of the flagellum and the site of all endocytosis ( Mussmann et al . , 2004; Vanhollebeke et al . , 2008 ) .", "Humans , together with a few other primates , display innate immunity to most trypanosome species ( Laveran , 1902 ) through the action of trypanolytic factors-1 and -2 ( TLF1 and TLF2 ) ( Hager et al . , 1994; Raper et al . , 1996 , 1999 ) .", "Although containing different scaffold components , these factors both include apolipoprotein L1 ( ApoL1 ) together with complexes of haemoglobin bound to haptoglobin-related protein ( HprHb ) ( Vanhamme et al . , 2003; Pérez-Morga et al . , 2005 ) .", "TLF1 enters trypanosomes via receptor-mediated endocytosis , through binding of the HprHb component to HpHbR ( Drain et al . , 2001; Widener et al . , 2007; Vanhollebeke et al . , 2008 ) .", "This delivers ApoL1 to the endosome where it causes lysosomal swelling and cell death ( Pérez-Morga et al . , 2005 ) .", "In contrast , the uptake route for TLF2 is unclear as , unlike TLF1 , it is able to kill HpHbR null mutants ( Capewell et al . , 2013; Uzureau et al . , 2013 ) .", "Just two subspecies of T . brucei ( T . b . rhodesiense and T . b . gambiense ) have evolved counter measures to the trypanolytic factors , allowing them to cause Human African Trypanosomiasis ( Pays et al . , 2014 ) .", "In the case of human-infective group 1 T . b .", "gambiense , a unique point polymorphism is found in HpHbR ( Symula et al . , 2012 ) that reduces the monovalent affinity for ligand by 20-fold ( Higgins et al . , 2013 ) .", "This contributes to resistance to TLF1 , illustrating the importance of HpHbR .", "Haptoglobin-haemoglobin is an elongated ‘dumbell-shaped’ complex consisting of a dimer of haptoglobin molecules , each joined to an αβ haemoglobin dimer ( Andersen et al . , 2012 ) .", "Trypanosomes take up this HpHb complex but not the individual components ( Vanhollebeke et al . , 2008 ) .", "The structure of the T . congolense HpHbR is an elongated three-helical bundle with a small membrane distal head ( Higgins et al . , 2013 ) .", "Residues involved in HpHb binding are part of a small conserved patch ∼25 Å below the tip of the receptor , but details of ligand binding and uptake were not characterized .", "Here , we present the structure of T . brucei HpHbR .", "We show that the receptor adopts a similar architecture to its T . congolense homologue , but with a ∼50° kink a third of the way along from the membrane proximal end .", "We also present the structure of TbHpHbR in complex with HpHb , revealing the molecular basis for ligand binding and selectivity .", "Finally , we show that the kink allows two independent membrane attached receptors to interact with a single dimeric HpHb molecule and confirm using cell uptake experiments that this causes dimeric ligand to be taken up with greater efficiency than monomeric ligand .", "This reveals the molecular basis for the uptake of HpHb and trypanolytic factor-1 and identifies adaptations in the trypanosome receptor that allow efficient ligand uptake in the context of the tightly packed VSG coat ." ], [ "To provide detailed molecular knowledge of the mechanism of uptake of haptoglobin-haemoglobin and trypanolytic factor-1 ( TLF1 ) , we aimed to determine the structure of T . brucei HpHbR ( TbHpHbR ) alone and bound to a human haptoglobin-haemoglobin complex .", "TbHpHbR is longer than its homologue from T . congolense due to the presence of an additional C-terminal membrane-proximal domain .", "We therefore used the previously determined structure of T . congolense HpHbR ( Higgins et al . , 2013 ) to design a construct containing the corresponding region of TbHpHbR ( residues 36–299 ) .", "This region of the protein is identical in the human infective T . b .", "rhodesiense .", "Haptoglobin-haemoglobin consists of a dimer of haptoglobin chains , each interacting with an αβ dimer of haemoglobin , and adopts a dimeric ‘dumbell-shaped’ architecture ( Andersen et al . , 2012 ) .", "At each end , a serine protease ( HpSP ) domain of haptoglobin forms a stable complex with a haemoglobin dimer .", "Dimerisation occurs through an interface formed by the CCP domains of haptoglobin , linking together these HpSPHb ‘heads’ .", "Previous studies have shown that TbHpHbR interacts with the HpHb complex but not with either haptoglobin or haemoglobin alone ( Vanhollebeke et al . , 2008 ) , suggesting that that the receptor most likely binds to the heads of HpHb , where its two constituent components come together .", "We therefore designed a human haptoglobin construct containing just the SP domain ( residues 148–406 ) .", "This was expressed in baculovirus-infected insect cells and was combined with haemoglobin extracted from human blood to assemble HpSPHb complexes .", "We used surface plasmon resonance to determine the affinity of these HpSPHb complexes for TbHpHbR , and showed binding with an affinity of 0 . 7 μM ( Figure 1—figure supplement 1 ) , similar to the 1 μM affinity observed for intact human HpHb ( Higgins et al . , 2013 ) .", "Proteolytic cleavage of haptoglobin normally occurs in the endoplasmic reticulum after residue R102 but this cleavage event did not occur in the insect cell expressed HpSP domain .", "However , this did not affect the affinity for TbHpHbR .", "The shortened TbHpHbR construct and the HpSPHb complex therefore interact together with the same affinity as the full-length components , providing reagents for structural determination .", "These findings also confirm that TbHpHbR binds to the ‘head’ structure of dimeric HpHb , raising the possibility of two receptors simultaneously interacting with one HpHb complex .", "To investigate the molecular basis for HpHb binding by TbHpHbR , crystallisation plates were set up for HpSPHb , TbHpHbR and a complex containing TbHpHbR bound to HpSPHb .", "Crystals of HpSPHb diffracted to 2 . 05 Å and were of space group P3121 with one complex in the asymmetric unit .", "Crystals of TbHpHbR diffracted to 1 . 85 Å resolution and were of space group P21 with two molecules in the asymmetric unit .", "Crystals of the TbHpHbR:HpSPHb complex were of space group C2 and diffracted to 3 . 1 Å resolution with a single complex in the asymmetric unit ( Table 1 ) . 10 . 7554/eLife . 05553 . 003Table 1 . Crystallographic data collection statisticsDOI: http://dx . doi . org/10 . 7554/eLife . 05553 . 003HpSPHbTbb HpHbRTbbHpHbR:HpSPHbBeamlineDiamond I04-1Diamond I03Diamond I03Space Groupp3121p21c2Cell dimensions ( Å ) a = b = 96 . 6 , c = 132 . 77a = 27 . 90 , b = 47 . 79 , c = 203 . 38 , β = 92 . 79a = 223 . 4 , b = 56 . 59 , c = 65 . 29 , β = 92 . 99Resolution ( Å ) 2 . 051 . 853 . 1Wavelength ( Å ) 0 . 9160 . 97630 . 9750RPIM ( % ) 8 . 1 ( 37 . 4 ) 4 . 5 ( 42 . 9 ) 6 . 3 ( 72 . 6 ) I/ σ ( I ) 8 . 7 ( 2 . 3 ) 10 . 2 ( 2 . 0 ) 9 . 8 ( 1 . 6 ) Completeness ( % ) 99 . 8 ( 100 ) 97 . 4 ( 96 . 5 ) 96 . 9 ( 97 . 1 ) Multiplicity9 . 6 ( 10 . 2 ) 3 . 1 ( 3 . 1 ) 3 . 2 ( 3 . 3 ) The structure of human HpSPHb was determined using molecular replacement with the equivalent region of porcine HpHb ( pdb: 4F4O ) as a search model .", "The structure of the TbHpHbR:HpSPHb complex was then determined through molecular replacement using HpSPHb as a search model , allowing a poly-alanine model of TbHpHbR to be built .", "This model was then used as a molecular replacement search model to determine the structure of TbHpHbR using higher-resolution data obtained from crystals of the receptor alone .", "Both structures were then completed using iterative cycles of model building and refinement ( Table 2 ) . 10 . 7554/eLife . 05553 . 004Table 2 . X-ray refinement statisticsDOI: http://dx . doi . org/10 . 7554/eLife . 05553 . 004ComplexHpSPHbTbb HpHbRTbbHpHbR:HpSPHbResolution ( Å ) 2 . 051 . 853 . 1No .", "reflections43 , 17044 , 68517 , 302Rwork / Rfree ( % ) 18 . 0 / 22 . 419 . 84 / 23 . 9519 . 5 / 21 . 7No .", "of protein residues in model544523782rmsd bond lengths ( Å ) 0 . 0200 . 0170 . 012rmsd bond angles ( ° ) 2 . 01 . 61 . 5Ramachandran plotAllowed region89 . 0%98 . 8%92 . 5%Additional allowed region11%1 . 2%7 . 5%Generously allowed region0%0%0%Disallowed region0%0%0% Like T . congolense HpHbR , the T . brucei receptor is elongated , consisting primarily of a three-helical bundle ( Figure 1 ) : helix I ( red; residues 42–110 ) , helix II ( orange; residues 116–182 ) , and helix V ( dark blue; residues 224–296 ) with a total length of 118 Å .", "At the membrane distal end , the receptor widens to form a compact head structure that includes the N-terminus and a 42-residue loop containing two further helices , helix III ( yellow: residues 186–196 ) and helix IV ( green: residues 207-–218 ) .", "The upper part of the structure is extremely similar to that from T . congolense , with the membrane distal halves of the two receptors aligning with a root mean square deviation of 1 . 1 Å ( Figure 1—figure supplement 2 ) . 10 . 7554/eLife . 05553 . 005Figure 1 . The structure of the T . brucei haptoglobin-haemoglobin receptor .", "( A ) The structure of the T . brucei haptoglobin-haemoglobin receptor , with helix I ( red ) , helix II ( orange ) and helix V ( blue ) .", "These three helices form an elongated bundle with a ∼50° kink towards the membrane proximal C-terminal end .", "The inset shows a molecular envelope derived from small angle x-ray scattering .", "( B ) The structure of the T . congolense haptoglobin-haemoglobin receptor ( Higgins et al . , 2013 ) for comparison .", "( C ) A change in the pattern of hydrophobic residues results in a rigid kink in the three helical bundle of the TbHpHbR .", "Corresponding regions of the structures of TbHpHbR and TcHpHbR are shown with side chains of the hydrophobic residues that pack in the core of the bundle coloured red and residues at the kink sites in TbHpHbR coloured green .", "Also shown are sequence alignments of TbHpHbR and TcHpHbR for these regions of each helix , coloured in the same way . DOI: http://dx . doi . org/10 . 7554/eLife . 05553 . 00510 . 7554/eLife . 05553 . 006Figure 1—figure supplement 1 . Surface plasmon resonance analysis of the binding of HpSPHb to TbHpHbR . Surface plasmon resonance signals for twofold dilutions of HpSPHb complex from a maximum concentration of 16 μM , binding to a surface coated with the truncated version of T . brucei HpHbR . DOI: http://dx . doi . org/10 . 7554/eLife . 05553 . 00610 . 7554/eLife . 05553 . 007Figure 1—figure supplement 2 . Alignment of the TbHpHbR and TcHpHbR structures . Structural alignment of T . brucei HpHbR ( blue ) with T . congolense HpHbR ( red ) .", "The membrane distal ( upper ) halves of the receptors align with a root mean square deviation of 1 . 1 Å while the membrane proximal ( lower ) halves differ due to the presence of a ∼50° kink in TbHpHbR . DOI: http://dx . doi . org/10 . 7554/eLife . 05553 . 007 The most dramatic difference between the T . brucei and T . congolense receptors is a ∼50° kink in TbHpHbR , located approximately one-third of the way along the receptor from the membrane proximal end .", "Each of the three helices is affected , with the backbone carbonyl groups of Asp88 , Ala89 , Glu123 , Asn124 , Asp270 and Ala271 no longer forming hydrogen bonds .", "This kink is not caused by flexibility , but is a rigid feature of the receptor , as it adopts the same confirmation in crystals of receptor alone , and in crystals of its complex with HpSPHb ( Figure 2A ) , and is also observed in molecular envelopes derived from small angle x-ray scattering ( Figure 1 , Table 3 ) .", "Instead it is caused by changes in the pattern of hydrophobic and hydrophilic residues around the kink site in each of the three helices .", "The three long helices of the T . congolense receptor are characterised by an alternating pattern of hydrophobic and hydrophilic residues , leading to continuous hydrophobic strips along the length of each helix that pack in the core of the helical bundle , stabilising its fold .", "In the T . brucei receptor , this pattern is disturbed at each kink site , breaking the organisation of the helix and leading to an alteration in the surface that displays the hydrophobic patch ( Figure 1C ) .", "This stabilises the kink and makes it a rigid feature of the receptor structure . 10 . 7554/eLife . 05553 . 008Figure 2 . The structural basis for haptoglobin-haemoglobin binding by TbHpHbR .", "( A ) The structure of the complex between T . brucei HpHbR ( blue ) bound to its ligand , HpSPHb ( haptoglobin is yellow , the β-subunit of haemoglobin is red and the α-subunit of haemoglobin is orange ) .", "( B ) The complex viewed from the membrane proximal end , showing the contacts made by haptoglobin and the β-subunit of haemoglobin .", "( C ) A view of the haemoglobin-binding site showing direct contacts between the haem and the receptor .", "Residues from the receptor that directly contact the haemoglobin subunit are shown as sticks and are numbered . DOI: http://dx . doi . org/10 . 7554/eLife . 05553 . 00810 . 7554/eLife . 05553 . 009Figure 2—figure supplement 1 . Stereoview of the TbHpHbR in complex with HpHb . DOI: http://dx . doi . org/10 . 7554/eLife . 05553 . 00910 . 7554/eLife . 05553 . 010Figure 2—figure supplement 2 . Small angle x-ray scattering of complexes of TcHpHbR and TbHpHbR with HpSPHb .", "( A ) The structure of the TbHpHbR:HpSPHb complex docked into an ab initio molecular envelopes calculated from scattering data .", "( B ) The theoretical scattering calculated from ab initio reconstructions ( blue for HpSPHb , red for TbHpHbR and purple for TbHpHbR:HpSPHb ) , superimposed into experimental scattering data .", "Guinier plots are shown as an insert .", "( C ) Distance distribution functions of HpSPHb ( blue ) , TbHpHbR ( red ) and TbHpHbR:HpSPHb ( purple ) derived from small angle x-ray scattering .", "( D ) A model of the TcHpHbR:HpSPHb complex docked into an ab initio molecular envelope calculated from scattering data .", "( E ) The theoretical scattering calculated from an ab initio reconstruction of the TcHpHbR:HpSPHb complex .", "( F ) Distance distribution function of TcHpHbR:HpSPHb derived from small angle x-ray scattering . DOI: http://dx . doi . org/10 . 7554/eLife . 05553 . 01010 . 7554/eLife . 05553 . 011Figure 2—figure supplement 3 . Clashes between TbHpHbR and a haemoglobin tetramer explain why the receptor does not bind to haemoglobin . A model for a complex of TbHpHbR bound to haemoglobin .", "This was derived by docking a haemoglobin tetramer onto the receptor with the β-subunit binding to the receptor as in the TbHpHbR:HpSPHb complex .", "TbHpHbR is shown in blue , the α-subunits of haemoglobin are orange and the β-subunits are red .", "A close up of the model is shown in the right hand panel with side chains involved in clashes shown as sticks . DOI: http://dx . doi . org/10 . 7554/eLife . 05553 . 01110 . 7554/eLife . 05553 . 012Figure 2—figure supplement 4 . The region affected by haptoglobin cleavage is not involved in interaction with TbHpHbR .", "( A ) The structures of the HpSPHb region of porcine HpHb ( red ) aligned to the equivalent region of human HpSPHb from the structure of the TbHpHbR:HpSPHb complex ( yellow ) .", "The structures align with a root mean square deviation of ∼0 . 5 Å .", "The major difference is circled and lies around the site at which haptoglobin is cleaved during a processing event in the endoplasmic reticulum , which is disordered in the TbHpHbR:HpSPHb complex .", "( B ) A structural alignment of the porcine HpSPHb structure onto the TbHpHbR:HpSPHb structure .", "The region that is structurally altered by cleavage is circled and is not involved in contacts with the receptor .", "This is confirmed by surface plasmon resonance data ( Figure 1—figure supplement", "1 ) which shows that TbHpHbR binds with similar affinity to HpSPHb as to previously measured native , cleaved HpHb . DOI: http://dx . doi . org/10 . 7554/eLife . 05553 . 01210 . 7554/eLife . 05553 . 013Table 3 . Small angle x-ray scattering statisticsDOI: http://dx . doi . org/10 . 7554/eLife . 05553 . 013MW ( kDa ) RG ( nm ) Dmax ( nm ) Volume ( nm3 ) Mwapp ( kDa ) HpSPHb59 . 72 . 67 . 57536TbHpHbR32 . 23 . 511 . 54422TbHbHbR:HpSPHb91 . 83 . 210 . 811055TbHpHbR:HpSPHb89 . 63 . 812 . 014070HpHb1525 . 618 . 2214107TbHpHbR:HpHb2176 . 316 . 5370185 The structure of the TbHpHbR:HpSPHb complex reveals an unexpected binding mode in which the ligand-binding surface extends along more than half of the length of the receptor ( Figure 2 , Figure 2—figure supplement 1 ) .", "Residues previously identified as playing a role in HpHb binding in TcHpHbR , such as S59 ( Higgins et al . , 2013 ) , lie ∼35 Å from the membrane distal tip of the receptor and directly contact haemoglobin .", "However , this is the upper part of the binding site , with residues from haptoglobin interacting as far as 70 Å from the membrane distal tip .", "This arrangement is confirmed by small angle x-ray scattering , with complexes of HpSPHb bound to either T . brucei or T . congolense receptors showing a similar architecture to that observed in the crystal ( Figure 2—figure supplement 2 , Table 3 ) .", "The haptoglobin-haemoglobin complex covers a total area of ∼1250 Å2 of the receptor and can be divided into two distinct regions ( Figure 2B ) .", "The membrane distal part , ( ∼745 Å2 ) contacts the β-subunit of the haemoglobin dimer with no contacts between the receptor and the haemoglobin α-subunit .", "The membrane proximal region ( ∼505 Å2 ) forms a binding surface for haptoglobin .", "The involvement of both haemoglobin and haptoglobin in binding explains why the receptor binds HpHb but not haptoglobin alone .", "Modelling suggests that the lack of haemoglobin binding is due to steric clashes of the receptor with the second αβ dimer of haemoglobin when the β-subunit of a haemoglobin tetramer is docked onto the receptor with the binding mode observed in the TbHpHbR:HpSPHb complex ( Figure 2—figure supplement 3 ) .", "Therefore , the conformation of the receptor and the presence of two distinct binding sites allow the receptor to specifically select HpHb over its two constitutive components .", "The haemoglobin β-subunit makes a number of direct interactions , mostly hydrogen bonds , with the receptor ( Figure 2C , Table 4 ) .", "Side chains from helix I of the receptor make the majority of these contacts , with additional interactions from helix II and the loop that links helices III and IV .", "These features lie along a groove on haemoglobin that is formed by helices C and F of the β-subunit .", "The haem group also makes direct contacts with the receptor , with the propionate chains contacting residues K56 , S59 , K164 , R199 and Y200 of the receptor .", "These interactions , mediated by haem , form ∼140 Å2 of the ∼745 Å2 total contact area of Hb . 10 . 7554/eLife . 05553 . 014Table 4 . Interactions between TbHpHbR and HpSPHbDOI: http://dx . doi . org/10 . 7554/eLife . 05553 . 014ReceptorHpSPHbResidueGroupChainResidueGroupInteractionHbβK56side chainBHaem144O1DHydrogen bondE57side chainBK96Side chainSalt bridgeS59side chainBHaem144O1D/O2DHydrogen bondI60side chainBPatchHydrophobicR67side chain NH1BR41Backbone COHydrogen bondE70side chain OE1/OE2BR41Side chain NE/NH2Salt bridgeS161side chainBK60Side chainHydrogen bondS161side chainBS45Backbone COHydrogen bondK164side chainBHaem144O2DHydrogen bondR199side chain NEBHaem144O2AHydrogen bondY200side chain OHBHaem144O2AHydrogen bondS203backbone COBK96Side chainHydrogen bondHpSPS73side chainCK345Side chainHydrogen bondV74hydrophobicCPatchHydrophobicQ75OE1CG276Backbone COHydrogen bondA78side chainCPatchHydrophobicA82side chainCPatchHydrophobicK85side chainCD305Side chain O2DSalt bridge The haptoglobin subunit also interacts with helix I of the receptor , through a predominantly hydrophobic contact , mediated by three loops that emerge from the C-terminal β-sheet of haptoglobin ( Figure 2B , Table 4 ) .", "The structure of human haptoglobin from this complex aligns with that from porcine Hp with a root mean square deviation of just 0 . 5 Å and reveals no significant structural change on receptor binding ( Figure 2—figure supplement 4 ) .", "The alignment also confirms that the natural cleavage of Hp does not affect TbHpHbR binding , as residues in the loop that contains the cleavage site are not close to the receptor .", "Rather than haptoglobin , trypanolytic factor-1 ( TLF1 ) contains haptoglobin-related protein ( Hpr ) and binding of HprHb complex to TbHpHbR results in TLF1 uptake .", "The HprSP domain contains a total of sixteen amino acid substitutions when compared with the HpSP domain .", "Mapping these onto the structure shows that none of these differences lie in residues that contact the receptor ( Figure 3A ) .", "Indeed HprSPHb complexes , prepared using the same protocols as HpSPHb complexes , bound to the receptor with an affinity of 1 . 7 μM , as determined by surface plasmon resonance ( Figure 3B ) , comparable to the 0 . 7 μM affinity of the receptor for HpSPHb .", "This suggests that HprHb , and as a result , TLF1 , will have a shared binding mode with HpHb . 10 . 7554/eLife . 05553 . 015Figure 3 . Differences between haptoglobin and haptoglobin-related protein do not alter affinity for TbHpHbR .", "( A ) The structure of the TbHpHbR:HpSPHb complex is shown with the receptor in blue and haptoglobin in yellow .", "Side chains in haptoglobin that are different in haptoglobin-related protein are highlighted in pink and are not involved in making interactions with the receptor .", "( B ) Surface plasmon resonance signals for two-fold dilutions of HprSPHb complex from a maximum concentration of 8 μM , binding to a surface coated with T . brucei HpHbR .", "The measured affinity of 1 . 7 μM can be compared with the affinity of 0 . 7 μM for HpSPHb . DOI: http://dx . doi . org/10 . 7554/eLife . 05553 . 015 The haptoglobin-haemoglobin receptor operates in the context of the VSG layer , a dense coat of surface protein that covers the trypansosome surface .", "It is therefore initially surprising that the location of the binding site for bulky HpHb complexes extends some 70 Å from the membrane distal tip of the receptor and below the surface of the VSG layer .", "However , one consequence of the kink in the T . brucei receptor is to increase its effective diameter , pushing apart VSG molecules .", "In addition , the orientation of the kink is precisely arranged to increase exposure of the HpHb binding site to the surface , making it more accessible for ligand binding .", "Docking of TbHpHbR:HpSPHb structures onto the structure of dimeric porcine HpHb reveals another consequence of the kink .", "This modelling suggests that two receptors can bind simultaneously to a single HpHb dimer , resulting in a C-shaped complex with a parallel arrangement of the membrane proximal parts of the two receptors ( Figure 4A ) .", "Indeed , this arrangement was confirmed in solution by small angle x-ray scattering .", "Native ( dimeric ) human HpHb was mixed with TbHpHbR , and gel filtration was performed , with SAXS data collected from samples as they emerged from the column .", "The resultant scattering curves confirmed the assembly of a complex containing two receptors and one HpHb in vitro .", "These data were used to generate a molecular envelope for the complex , which confirmed the C-shaped architecture ( Figure 4B , Figure 4—figure supplement 1 , Table 3 ) .", "Additional support for the formation of this complex in solution came from multi-angle laser light scattering ( SEC-MALLS ) , which revealed masses of 30 kDa for the receptor , 150 kDa for HpHb and 210 kDa for the complex , showing that two receptors bind to each HpHb in solution ( Figure 4—figure supplement 2 ) .", "This arrangement , in which two independent GPI-anchored receptors can bind simultaneously to an HpHb dimer , would increase avidity for the ligand and decrease the ligand concentration required for efficient uptake . 10 . 7554/eLife . 05553 . 016Figure 4 . Simultaneous binding of two receptors to each HpHb dimer leads to more efficient uptake into trypanosomes .", "( A ) A model for a complex of one HpHb dimer bound to two receptors , generated by docking the structure of the TbHpHbR:HpSPHb complex onto that of porcine HpHb ( Andersen et al . , 2012 ) .", "The receptors are organized such that two receptors , both associated with the membrane through attachment at their C-termini , can simultaneously bind to one HpHb dimer .", "( B ) An ab initio molecular envelope derived from small angle x-ray scattering analysis of the TbHpHbR:HpHb complex supports the formation of a complex containing one HpHb dimer bound to two receptors .", "( C ) Uptake of fluorescently labelled dimeric HpHb into live cells was monitored via flow cytometry across a range of 1–62 . 5 nM .", "Uptake saturated by 4 nM in wild-type cells whereas no uptake was observed in the HpHbR null cell line .", "No fluid phase uptake of labelled BSA was observed at these concentrations .", "( D ) Uptake of fluorescently labelled monomeric HpSPHb was not readily detected until 62 . 5 nM , at which point uptake had not saturated .", "HpSPHb uptake at 62 . 5 nM was lost in the HpHbR null cell line .", "Each uptake assay was carried out in triplicate .", "Error bars represent standard error of the mean , n = 3 . DOI: http://dx . doi . org/10 . 7554/eLife . 05553 . 01610 . 7554/eLife . 05553 . 017Figure 4—figure supplement 1 . Small angle x-ray scattering of HpHb , alone and in complex with TbHpHbR .", "( A ) An ab initio molecular envelopes calculated from scattering data from the HpHb complex .", "( B ) The theoretical scattering calculated from ab initio reconstructions for HpHb superimposed into experimental scattering data .", "Guinier plots are shown as an insert .", "( C ) Distance distribution functions of HpHb derived from small angle x-ray scattering .", "( D ) An ab initio molecular envelopes calculated from scattering data from the TbHpHbR:HpHb complex .", "( E ) The theoretical scattering calculated from ab initio reconstructions for TbHpHbR:HpHb superimposed into experimental scattering data .", "Guinier plots are shown as an insert .", "( F ) Distance distribution functions of TbHpHbR:HpHb derived from small angle x-ray scattering . DOI: http://dx . doi . org/10 . 7554/eLife . 05553 . 01710 . 7554/eLife . 05553 . 018Figure 4—figure supplement 2 . SEC MALLS data to assess the stoichiometry of the TbHpHbR:HpHb complex . Multi-angle light scattering ( MALLS ) measurements of TbHpHbR ( red ) , HpHb ( blue ) and the TbHpHbR:HpHb complex ( green ) .", "The molecular weights determined from scattering data ( ∼30 kDa for TbHpHbR , ∼150 kDa for HpHb and ∼210 kDa for the TbHpHbR:HpHb complex ) show the formation of a complex containing two receptors bound to a single HpHb . DOI: http://dx . doi . org/10 . 7554/eLife . 05553 . 01810 . 7554/eLife . 05553 . 019Figure 4—figure supplement 3 . Establishment and characterization of an HpHb−/− cell line of T . brucei . TbHpHbR null cell lines were generated in T . b .", "brucei Lister 427 bloodstream form ( BSF ) cells .", "( A ) The TbHbHbR gene was knocked out in Lister 427 BSF cells by replacement of one allele with a blasticidin resistance gene and the other allele with a neomycin resistance gene in four independent clones , as confirmed by Southern blot ( P = Parental cell line , 1–4 = TbHpHbR null clones 1–4 ) .", "The schematic depicts the original ( top ) and replacement ( middle and lower ) TbHpHbR loci and the positions of Southern blot probes and restriction enzyme sites used .", "Expected fragment sizes are annotated .", "( B ) Growth of the TbHpHbR null clones was monitored in vitro over 192 hr .", "Parental L427 cells grew with a mean doubling time of 8 . 4 hr whereas the TbHpHbR null clones had an increased mean doubling time of 11 . 5–12 . 0 hr .", "Error bars represent standard deviation , n = 8 . DOI: http://dx . doi . org/10 . 7554/eLife . 05553 . 019 To test this hypothesis , we performed uptake experiments using T . brucei .", "HpHb ( dimeric ) , HpSPHb ( monomeric ) and bovine serum albumin ( BSA , as a control to assess fluid phase uptake ) , were each fluorescently labelled .", "In addition , we prepared a null cell line , TbHpHbR−/− , in which both copies of the receptor were disrupted ( Figure 4—figure supplement 3 ) .", "These reagents allowed us to investigate the concentration dependence of ligand uptake .", "In wild-type T . brucei cells , uptake of HpHb reached saturation below a concentration of 4 nM ( Figure 4C ) .", "In contrast , the uptake of HpSPHb was negligible at a concentration of 4 nM and continued to increase at 62 . 5 nM ( Figure 4D ) .", "Uptake of HpSPHb and HpHb observed in wild-type cells was due to the TbHpHbR , as expected ( Vanhollebeke et al . , 2008 ) , as uptake of both ligands into TbHpHbR−/− cells was comparable to that of BSA in the range of ligand concentrations assayed ( 0–62 . 5 nM ) .", "Therefore , uptake of dimeric HpHb into trypanosomes occurs efficiently at a significantly lower concentration than that of monomeric HpSPHb .", "As the monovalent affinities of the receptor for HpHb and HpSPHb are indistinguishable , as measured by surface plasmon resonance , this suggests that more efficient uptake of HpHb is caused by the dimeric ligand simultaneously binding to two receptors .", "Indeed , measurements of the binding of HpHb to immobilised receptor , using a very high surface density of HpHbR to measure bivalent binding , gave an affinity of 4 . 5 nM ( Higgins et al . , 2013 ) , which is in the same range as the concentration at which HpHb uptake becomes saturated in live parasites .", "Therefore , it appears as though TbHpHbR has evolved a kink to increase accessibility of its ligand-binding site and to allow simultaneous binding of two receptors to one HpHb ligand , increasing ligand avidity and uptake efficiency ." ], [ "The external surface of an African trypanosome is covered with a tightly packed layer of variant surface glycoprotein that shields epitopes that lie close to the plasma membrane from antibody binding ( Schwede et al . , 2011 ) .", "Receptors such as those required for the uptake of transferrin and haptoglobin-haemoglobin complexes must operate within the context of this coat , with their structures organised such that ligand binding sites are not masked by the VSG layer .", "Here , the first structure of a trypanosome receptor in complex with its ligand is presented: that of the T . brucei haptoglobin-haemoglobin receptor bound to haptoglobin-haemoglobin .", "Remarkably , the ligand-binding site extends more that half the way along the receptor , forming distinct binding surfaces for the β-subunit of haemoglobin and for haptoglobin .", "The simultaneous binding of both components explains the specificity of the receptor for haptoglobin-haemoglobin complexes over each individual component .", "However , the extent of the binding surface places it below the top of the VSG layer , apparently increasing the likelihood that it will be masked by VSG .", "However , a ∼50° rigid kink occurs as an adaptation in the three helical bundle of the T . brucei receptor , and we propose that it has two main functional consequences .", "Firstly , the direction of the kink is precisely arranged to bend the receptor such that the ligand-binding site becomes more exposed at the membrane surface .", "The kink will also increase the effective diameter of the receptor in the plane of the membrane .", "This combination is likely to increase the separation of VSG molecules in the region of the receptor and to increase the accessibility of the binding site for bulky macromolecular ligands such as HpHb and trypanolytic factors .", "Increased separation of VSG molecules by a trypanosome receptor is not a novel phenomena , with bulky glycan chains attached to the transferrin receptor proposed to have a similar effect ( Mehlert et al . , 2012 ) , suggesting that different receptors increase the accessibility of binding sites for bulky ligands by different means .", "The precise nature of the integration of TbHpHbR into the VSG layer remains unresolved as the effect of the C-terminal domains of both the receptor and VSG on the vertical disposition of each molecule remains unknown .", "An attractive model is that the ligand , whether HpHb or TLF1 , is bound above the top of the VSG .", "However , the dimensions of the structures of VSG and the TbHpHbR:HpHb complex suggest that this may not be the case and that the HpHb ligand is held at least partially within the VSG layer ( Figure 5 ) .", "The TLF1 ligand is ∼4 times the size of HpHb and this must , at least partially , protrude above the top of the VSG layer .", "10 . 7554/eLife . 05553 . 020Figure 5 . A comparison of the dimensions of the TbHpHbR:HpHb complex with those of the N-terminal domains of the variant surface glycoproteins ( shown in grey ) .", "This suggests that HpHb will lie at least partially within the VSG layer when bound to two receptors . DOI: http://dx . doi . org/10 . 7554/eLife . 05553 . 020 A second consequence of the kink is to allow two receptors to simultaneously bind to one dimeric HpHb complex when their membrane-proximal C-termini are membrane-attached .", "The affinity of a single receptor for HpHb is modest , at ∼1 μM , with a rapid off-rate .", "By enabling two receptors to bind simultaneously , the kink will increase the ligand avidity , changing the effective affinity to something in the low nanomolar range , as previously measured for bivalent binding by TbHpHbR ( Higgins et al . , 2013 ) and to live cells ( Drain et al . , 2001 ) .", "What is the likelihood of two receptors interacting with a single ligand on the cell surface ?", "The receptor copy number is 200–400 and it is concentrated in the flagellar pocket ( Vanhollebeke et al . , 2008 ) .", "The surface area of the flagellar pocket in live cells is 4 . 3 µm2 ( Grünfelder et al . , 2002 ) .", "If there are 200 receptor molecules in the membrane of the flagellar pocket then the density is one receptor per 0 . 022 µm2 .", "The diffusion constant for HpHbR is unknown , but the diffusion constant for another GPI-anchored protein , VSG , has been measured by fluorescence recovery after photobleaching to be 0 . 01 µm2/s ( Bulow et al . , 1988 ) ; this means that the receptor will contact a receptor molecule approximately every 2 s .", "The t1/2 for release of monovalently bound HpHb is 70–100 s ( Higgins et al . , 2013 ) ( Figure 1—figure supplement 1 ) , so if it is assumed that the receptor has a similar diffusion coefficient to the VSG , then it is very likely that a monovalently bound ligand will become bivalently bound .", "Indeed our analysis of uptake of dimeric HpHb and monomeric HpSPHb into T . brucei confirmed that this increased avidity does occur in vivo , with HpHb uptake saturating at a concentration below 4 nM , while HpSPHb uptake is far from saturation at 62 . 5 nM .", "Whether other trypanosome receptors use a similar avidity-increase mechanism to improve the efficiency of ligand uptake remains to be seen , and whether it is required will depend upon the receptor affinity for monomer and the sera concentration of the nutrient .", "However , it is clear from the example of TbHpHbR that this mode of ligand binding is potentially applicable to other GPI-anchored cell surface proteins .", "While the evolution of the kink allows increased accessibility of the binding site for HpHb , it would also increase accessibility for the large TLF1 complex .", "One mechanism used by human infective T . b .", "gambiense to avoid TLF1-mediated innate immunity is a point polymorphism in HpHbR that reduces the monovalent affinity for HpHb by 20-fold ( Higgins et al . , 2013 ) and reduces TLF1 uptake ( Kieft et al . , 2010 ) .", "It remains to be seen whether TLF1 contains one or multiple HprHb complexes , and whether these are in a suitable arrangement to allow bivalent binding .", "However , even if bivalent binding does occur in TLF1 , the difference in affinity due to the T . b .", "gambiense polymorphism will be amplified under conditions where two receptors bind to a single ligand .", "As the kink in the receptor appears to have a number of functional consequences that facilitate ligand uptake , it is surprising that the T . congolense receptor lacks such a kink .", "One reason for this difference might be the lack of a C-terminal domain in T . congolense surface proteins ( Higgins et al . , 2013 ) .", "Perhaps the direct attachment of the ligand-binding domain to a GPI-anchor provides enough flexibility to allow the receptors to adopt an angle that allows simultaneous uptake , or perhaps the VSG coat of T . congolense parasites is less densely packed .", "These questions will need further study .", "In conclusion , we present the first structure of a trypanosome receptor in complex with its ligand and reveal a number of adaptations that are tailored to facilitate efficient ligand binding in the context of the VSG coat .", "These will decrease the packing of VSG molecules in the immediate vicinity of the receptor and increase accessibility of the ligand-binding site .", "They also allow two receptors to bind to a single ligand , thereby increasing avidity and dramatically decreasing the ligand concentration at which efficient uptake occurs .", "While different adaptations might facilitate each of these goals in different receptors , we would expect them to be general principles , frequently used by the parasite to aid nutrient uptake and survival ." ], [ "Full-length T . b .", "brucei HpHbR , without the N-terminal signal sequence and C-terminal GPI-anchor addition sequence , had been previously cloned for expression in a modified pET-15b to generate a polypeptide with an N-terminal hexahistidine tag and a cleavage site for TEV protease ( Higgins et al . , 2013 ) .", "To produce a truncated construct for expression of the N-terminal ligand-binding domain , a stop codon was inserted after residue R299 using a polymerase chain reaction based mutagenesis protocol , using oligonucleotide GAGATGAAGCGCTAGGGGAACCCGATC and its reverse-complement .", "Mutagenesis was carried out as described for the Quikchange mutagenesis method ( Stratagene , La Jolla , CA ) and the plasmid was sequence verified .", "The protein was expressed in E . coli Origami B , induced with 1 mM IPTG and incubated overnight at 18°C .", "The protein was purified by Ni2+-NTA affinity chromatography and cleaved overnight with his-tagged TEV protease at 4°C in PBS with 3 mM oxidized glutathione , 0 . 3 mM reduced glutathione , followed by reverse Ni2+-NTA affinity chromatography .", "The protein was concentrated by Amicon Ultra centrifugal filter device ( 10 , 000 MWCO ) ( EMD Millipore , Billerica , MA ) and gel filtered using a Superdex 75 16/60 column ( Life Technologies , Carlsbad , CA ) into 20 mM HEPES pH 7 . 5 , 150 mM NaCl .", "Synthetic genes encoding the SP domains of human Hp ( 148–406 ) and Hpr ( 90–348 ) were cloned into a modified pAcGP67A vector to generate a polypeptide with an N-terminal hexahistidine tag and a cleavage site for TEV protease .", "These were transfected into Sf9 insect cells using the BaculoGold Baculovirus DNA transfection protocol ( BD Biosciences , Franklin Lakes , NJ ) .", "Following selection of virus using plaque assays , the third amplification of recombinant virus was used to infect Sf9 insect cells .", "After 3 days , the cells were centrifuged for 15 min at 6000×g .", "After filtering , the supernatant was buffer exchanged into 20 mM Tris pH 8 , 300 mM NaCl using a tangential flow apparatus ( Pall Corporation , Port Washington , NY ) , followed by Ni2+-NTA affinity chromatography .", "The protein was concentrated using an Amicon Ultra centrifugal filter device ( 10 , 000 MWCO ) ( EMD Millipore ) .", "When used for crystallisation , HpSP was deglycosylated by incubation with endoglycosidase Hf ( Sigma-Aldrich , St Louis , MO ) and endoglycosidase F3 at enzyme:protein ratios of 1:25 in 1 mM CaCl2 , 1 mM MgCl2 , 100 mM HEPES pH 7 . 5 at 37°C for 3 hr .", "Hb was isolated from human blood by sonication , followed by anion exchange chromatography using a Mono Q column ( Life Technologies ) .", "HpHb was made by mixing full-length Hp 1-1 ( Sigma-Aldrich ) with purified Hb and isolating the complex by gel filtration using a Superdex 200 16/60 column ( Life Technologies ) in 20 mM HEPES pH 7 . 5 , 150 mM NaCl .", "HpSP or HprSP at a threefold molar excess was mixed with Hb and diluted fivefold into 20 mM Tris pH 8 , 500 mM NaCl , 15 mM imidazole .", "The complex was purified by Ni2+-NTA affinity chromatography , washed using the dilution buffer , and eluted into PBS containing 200 mM imidazole .", "The complexes were then concentrated using an Amicon Ultra centrifugal filter device ( 10 , 000 MWCO ) ( EMD Millipore ) , and purified by gel filtration using a Superdex 200 16/60 column ( Life Technologies ) in 20 mM HEPES pH 7 . 5 , 150 mM NaCl .", "HpSPHb was concentrated to 15 mg ml−1 for crystallization .", "Crystals were obtained after 8 hr in sitting drops with a well solution containing 0 . 2 M NaCl , 0 . 1 M sodium cacodylate pH 6 . 5 and 2 M ammonium sulphate .", "These were cryoprotected by transfer into well solution with the addition of 30% vol/vol glycerol before cryo-cooling using liquid nitrogen .", "Data were collected on beamline I04-1 at the Diamond light source and were integrated and scaled using iMosflm ( Battye et al . , 2011 ) and scala ( Evans , 1993 ) from the CCP4 suite ( Winn et al . , 2011 ) , giving a final resolution of 2 . 05 Å .", "The structure was determined by molecular replacement using Phaser ( McCoy et al . , 2007 ) with the equivalent region of the porcine HpHb ( pdb: 4F4O ) as a search model .", "The structure was rebuilt and refined using Coot ( Emsley et al . , 2010 ) and REFMAC ( Murshudov et al . , 2011 ) .", "TbHpHbR was mixed with HpSPHb and purified by gel filtration using a Superdex 200 16/600 column ( Life Technologies ) into a buffer containing 150 mM NaCl and 20 mM HEPES pH 7 . 5 .", "It was concentrated to a final concentration of 15 mg ml−1 and crystallised at 18 °C using sitting drops with a well solution containing 12 . 5% vol/vol MPD , 0 . 03 M NaBr , 0 . 03 M NaI , 0 . 03 M NaF , 0 . 1 M MES/imidazole pH 6 . 5 , 12 . 5% wt/vol PEG 1000 , 12 . 5% wt/vol PEG 3350 from the Morpheus screen ( Molecular Dimensions , UK ) .", "Crystals formed after 10 days .", "Seed beads ( Hampton Research , Aliso Viejo , CA ) were used to create seeds from these crystals .", "These were used to seed a plate containing 100 nl of protein , 50 nl of the Morpheus well solution , and 50 nl of the Silver Bullet additive screen ( Hampton Research ) .", "Crystals grew after 8 days in the well containing additives 0 . 2% wt/vol 2 , 2′-thiodiglcolic acid , 0 . 2% wt/vol apidic acid , 0 . 2% wt/vol benzoic acid , 0 . 2% wt/vol oxalic acid anhydrous , 0 . 2% wt/vol terephthalic acid .", "These were cryo-cooled in liquid nitrogen in the Morpheus well solution .", "Data were collected on beamline I03 at the Diamond light source .", "Data reduction was performed using XDS ( Kabsch , 2010 ) and the structure was solved by molecular replacement with the HpSPHb structure as a search model using Phaser ( McCoy et al . , 2007 ) .", "Automatic model building in Buccaneer ( Cowtan , 2006 ) was used to identify the positions of the receptor helices , leading to a cycle of model building and refinement in Coot ( Emsley et al . , 2010 ) and Buster ( Bricogne et al . , 2011 ) .", "The coordinates from the higher resolution structures of both and TbHpHbR , also determined during this study , were used to provide restraints during refinement , leading to improved stereochemistry of the resultant model .", "The receptor was concentrated to 12 . 5 mg ml−1 for crystallization .", "Crystals were obtained at 18 °C after 7 days using sitting drops with a well solution of 0 . 15 M KBr , 30% wt/vol PEG 2000 MME from the JCSG+ screen ( Molecular Dimensions ) .", "These were partially dehydrated and cryoprotected by transfer into the well condition with addition of 30% vol/vol glycerol before cryo-cooling in liquid nitrogen .", "Data were collected on beamline I03 at the Diamond light source .", "Data reduction was performed using iMosflm ( Battye et al . , 2011 ) and scala ( Evans , 1993 ) from the CCP4 data processing suite ( Winn et al . , 2011 ) .", "Molecular replacement was performed using Phaser ( McCoy et al . , 2007 ) , with the structure of TbHpHbR taken from the TbHpHbR:HpSPHb complex as a search model .", "A cycle of refinement and model building was carried out using REFMAC ( Murshudov et al . , 2011 ) and Coot ( Emsley et al . , 2010 ) .", "SAXS data for TbHpHbR alone and in complex with HpSPHb , and for the complex of TcHpHbR with HpSPHb , were collected at the PetraIII P12 beamline at Deutsches Elektronen-Synchrotron using a wavelength of 1 . 24 Å .", "SAXS data for the complex of the receptor with dimeric HpHb were collected at beamline BM29 at the European Synchrotron Radiation Facility using a wavelength of 0 . 9 Å .", "SAXS data for HpHb alone was collected at beamline B21 at the Diamond Light Source with a wavelength of 1 . 0 Å .", "In all cases , scattering was detected using a Pilatus image reader at 20°C .", "The receptors alone and in complex with HpSPHb , as well as HpHb alone , were prepared at concentrations of 2 . 0 , 1 . 0 , 0 . 5 , 0 . 25 and 0 . 125 mg ml−1 in 20 mM HEPES pH 7 . 5 , 150 mM NaCl .", "Twenty consecutive frames of 10 s each were recorded for each protein sample with a buffer sample measured between each , except HpHb , for which 180 consecutive 1 s frames were taken .", "Any images where the data had been affected by protein radiation damage were excluded from further processing .", "The complex with dimeric HpHb was prepared and analysed by size-exclusion column ( SEC ) -SAXS , using a Superdex 200 10/300 column ( Life Technologies ) in 20 mM HEPES pH 7 . 5 , 150 mM NaCl with a running speed of 0 . 4 ml min−1 .", "One frame was collected every 2 s .", "Frames corresponding to the peak seen on the UV trace were selected and a curve representing the scattering due to buffer was produced by averaging ten frames from the beginning of the run .", "For each data set , PRIMUS ( Konarev et al . , 2003; Petoukhov et al . , 2007 ) was used to normalize data to the intensity of the incident beam , for averaging of equivalent images and to subtract scattering due to buffer .", "Where Guinier plots revealed aggregation due to high concentration , data were removed ( Guinier and Fournet , 1955 ) .", "Composite curves were generated by scaling and merging the data sets .", "AutoRg calculated the distance distribution function ( P ( r ) ) using an indirect Fourier transform , allowing estimation of the radius of gyration ( Rg ) , the maximum particle dimension ( Dmax ) and the Porod volume ( Porod , 1982 ) by GNOM ( Petoukhov et al . , 2007 ) .", "Initial models of the shape were generated using DAMMIF ( Franke and Svergun , 2009 ) and averaged using the DAMAVER programme suite ( Konarev et al . , 2006 ) .", "DAMMIN then produced a final model by minimising differences between experimental data and scattering of the model .", "The envelope model was produced using Situs ( Birmanns et al . , 2011 ) , and feature-based docking of the crystal structures was completed using Sculptor ( Birmanns et al . , 2011 ) .", "Measurements were performed on a Biacore T200 ( Life Technologies ) instrument with a constant flow rate of 30 μg ml−1 .", "A CM5 chip was prepared by flowing over a 1:1 mixture of ethyl-dimethylaminopropyl-carbodiimide and N-hydroxysuccinimide .", "TbHpHbR was diluted into 10 mM sodium acetate pH 5 to a final concentration of 0 . 1 μM .", "Ligands were diluted into HBS ( 20 mM HEPES pH 7 . 5 , 150 mM NaCl , 0 . 005% vol/vol Tween 20 ) .", "Both channels were equilibrated with HBS before injection of binding partner , and the level of specific binding obtained from a subtraction of the response from channel 2 from that of channel 1 .", "Values for KD were obtained by equilibrium binding analysis using the BIAevaluation software .", "Purified samples were loaded onto a Superose 6 10/300 column ( GE Healthcare ) , then analysed using laser light scattering detected at 662 nm wavelength at 8 scattering angles between 20 . 6° and 149 . 1° using a Heleos 8 instrument ( Wyatt Technology , Germany ) .", "ASTRA 6 . 1 ( Wyatt Technology ) was used to calculate molecular weights using the Zimm equation .", "The samples were loaded at concentrations of 10 μM for HpHb and 40 μM for TbHpHbR .", "T . brucei blood stream form cells were grown in HMI-9 at 37 °C with 5% CO2 ( Hirumi and Hirumi , 1989 ) .", "The linear DNAs used to replace HpHbR genes by homologous recombination were produced by PCR .", "First , one allele was replaced in procyclic form Lister 427 cells using a PCR product that contained 80 bp from upstream of the HpHbR gene , followed by the blasticin resistance cassette , followed by 80 bp from downstream of the HpHbR gene .", "The same approach was used with a G418 resistance cassette .", "Genomic DNA was prepared from blasticin or G418 resistant cell lines and used as a template for a PCR using oligonucleotides 500 bp upstream and downstream of the HpHbR gene .", "These second PCR products were used to serially transfect Lister 427 bloodstream form cells expressing VSG118 .", "When used for uptake experiments , Hp , HpSP and BSA were labelled with Alexa Fluor 488 using the protein labelling kit ( Life Technologies ) .", "The manufacturer's protocol was adapted , extending the reaction time to overnight at 4 °C to increase labelling efficiency .", "Hp and HpSP were then subsequently mixed with Hb to form complex as above .", "For each assay , 5 × 106 wild type Lister 427 or HpHbR −/− cells were resuspended in 100 µl of serum free HMI-9 with 1% BSA and incubated with 2 µM protease inhibitor FMK-024 for 10 min at 37 °C .", "Cells were then incubated with 1–62 . 5 nM fluorescently labelled protein for 2 hr at 37 °C before being washed twice in serum free HMI-9 with 1% BSA .", "Cells were fixed in 4% paraformaldehyde for 10 min at room temperature and resupended in PBS .", "Uptake was assayed by flow cytometry using a FACScan ( BD Biosciences ) and quantified on FlowJo software .", "Mode increase in fluorescence was measured relative to a no ligand negative control , and all assays were carried out in triplicate ." ] ]

[ "The haptoglobin-haemoglobin receptor ( HpHbR ) of African trypanosomes allows acquisition of haem and provides an uptake route for trypanolytic factor-1 , a mediator of innate immunity against trypanosome infection .", "In this study , we report the structure of Trypanosoma brucei HpHbR in complex with human haptoglobin-haemoglobin ( HpHb ) , revealing an elongated ligand-binding site that extends along its membrane distal half .", "This contacts haptoglobin and the β-subunit of haemoglobin , showing how the receptor selectively binds HpHb over individual components .", "Lateral mobility of the glycosylphosphatidylinositol-anchored HpHbR , and a ∼50o kink in the receptor , allows two receptors to simultaneously bind one HpHb dimer .", "Indeed , trypanosomes take up dimeric HpHb at significantly lower concentrations than monomeric HpHb , due to increased ligand avidity that comes from bivalent binding .", "The structure therefore reveals the molecular basis for ligand and innate immunity factor uptake by trypanosomes and identifies adaptations that allow efficient ligand uptake in the context of the complex trypanosome cell surface ." ]

[ "African Trypanosomes are a group of single-celled parasites that are a major concern for livestock farmers in sub-Saharan Africa .", "They are carried by the tsetse fly and can cause disease in domestic livestock that diminishes productivity through reduced growth , and may ultimately lead to death .", "The parasites are coated in a dense layer of protein that help them evade the host’s immune system by preventing immune cells from identifying them .", "Humans have evolved immunity to many trypanosome species by exploiting a weakness in their lifestyle .", "Trypanosomes need to get haem—a molecule found in the protein haemoglobin—from their host to survive .", "In blood plasma , haemoglobin is found associated with a carrier protein called haptoglobin .", "To acquire haem , the parasites have a protein called HpHbR that binds to these haptoglobin-haemoglobin ‘complexes’ .", "However , in humans there are two complexes of proteins called TLFs that contain haemoglobin and a protein related to haptoglobin .", "The TLFs are also able to bind to HpHbR and are taken into the parasite .", "Once inside , TLFs cause internal compartments called lysosomes to swell , which leads to the death of the trypanosome .", "Two subspecies of Trypanosoma brucei are the only trypanosomes that infect humans as they can overcome the TLF1 defense .", "However , the details of how TLFs cause cell death at the molecular level are not clear .", "Lane-Serff et al . used a technique called x-ray crystallography to generate 3-D images of the HpHbR protein from T . brucei bound to the haptoglobin-haemoglobin complexes .", "These images show that HpHbR is elongated so that it only binds to haemoglobin and haptoglobin when they are together as a complex .", "The images also reveal that the shape of HpHbR enables it to hold apart the proteins in the protective layer that coats the trypanosome .", "This allows the haptoglobin-haemoglobin complex to bind to HpHbR , but in humans also makes HpHbR more likely to bind to TLF1 .", "These findings may help to guide future efforts to protect humans and livestock from the diseases caused by trypanosomes ." ]

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

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

[ [ "Cilia ( or eukaryotic flagella , terms essentially referring to the same organelle ) are hair-like organelles that extend from the plasma membrane of nearly all mammalian cells .", "The core structural component of a cilium is the axoneme , a ring of nine unipolar doublet microtubules surrounding a central pair of singlet microtubules .", "Cilia play essential roles in cell motility , generate the movement of fluids over multiciliated surfaces , and sense extracellular signals ( Satir et al . , 2010 ) .", "To assemble and maintain functional cilia , the IFT machinery ( Kozminski et al . , 1993 ) transports axonemal precursors and sensory proteins bidirectionally between the cell body and the ciliary tip .", "Defects in IFT are linked to a wide range of human diseases including Bardet-Biedl syndrome , retinal degeneration , and polycystic kidney disease ( Brown and Witman , 2014 ) .", "Intraflagellar cargoes interact with multiprotein complexes known as IFT particles that are organized into larger IFT trains as they enter the flagellum ( Cole et al . , 1998; Lechtreck et al . , 2017; Pigino et al . , 2009 ) .", "In most species , the IFT trains are transported anterogradely from the base to the tip of a flagellum by heterotrimeric kinesin-II ( Kozminski et al . , 1995 ) , but some species also use a second homodimeric kinesin to build more specialized sensory cilia ( Snow et al . , 2004 ) .", "Once the trains reach the tip , they are reorganized and transported retrogradely to the ciliary base by dynein-1b ( Pazour et al . , 1999; Porter et al . , 1999; Signor et al . , 1999 ) .", "Along the length of a cilium , the activities of kinesin and dynein motors are reciprocally coordinated , such that only one type of a motor is active at a time ( Shih et al . , 2013 ) .", "As a result , trains move between the tip and base of the cilium without significant pauses or back-and-forth motion ( Dentler , 2005; Engel et al . , 2009 ) and switch directions at the turnaround zones ( Ishikawa and Marshall , 2011; Laib et al . , 2009; Shih et al . , 2013 ) .", "Dynein-1b requires kinesin-II activity to reach the ciliary tip , suggesting that it travels on anterograde trains ( Iomini et al . , 2001; Pedersen et al . , 2006; Rompolas et al . , 2007 ) .", "Retrograde traces of kinesin-II have not been frequently observed in Chlamydomonas , and it remains unclear how kinesin-II returns to the basal body ( Engel et al . , 2009; Wingfield et al . , 2017 ) .", "Because anterograde and retrograde IFT trains have different sizes and depart at different frequencies ( Dentler , 2005; Iomini et al . , 2001 ) , IFT trains must be remodeled at the distal tip and the flagellar base .", "The mechanism underlying the remodeling of IFT complexes at the ciliary tip and base cannot be directly observed by conventional microscopy methods because multiple trains coexist in these turnaround zones ( Buisson et al . , 2013; Iomini et al . , 2001; Wren et al . , 2013 ) .", "In this work , we adapted PhotoGate ( Belyy et al . , 2017 ) to control the number of fluorescent IFT trains entering the flagellum of the unicellular algae Chlamydomonas reinhardtii .", "Using this approach , we monitored the turnaround behavior and remodeling of single IFT trains at the flagellar tip .", "We also elucidated the mechanisms by which the kinesin and dynein motors are recycled in this process and IFT trains reverse their direction of motion .", "The dynamics of IFT motor turnover at the tip suggest a new mechanism for how Chlamydomonas controls the length of its flagella ." ], [ "To monitor IFT movement , we tracked the dynamic behavior of IFT27 , a core component of the IFT complex B , in a pf18 IFT27-GFP strain ( Engel et al . , 2009; Qin et al . , 2007 ) .", "This strain has paralyzed flagella ( pf ) that readily adhere to the glass surface , enabling us to monitor IFT under total internal reflection ( TIR ) illumination ( Engel et al . , 2009 ) .", "Consistent with previous studies ( Dentler , 2005; Engel et al . , 2009; Iomini et al . , 2001; Kozminski et al . , 1993; Qin et al . , 2007; Shih et al . , 2013 ) , IFT trains moved processively along the length of flagella , reversing direction at the flagellar tip and base ( Figure 1a , Video 1 ) .", "Pausing and reversals of anterograde trains before reaching the tip were very rare .", "The velocity of IFT27-GFP was 2 . 1 ± 0 . 4 µm s−1 in the anterograde direction and 3 . 0 ± 0 . 7 µm s−1 in the retrograde direction ( Figure 1—figure supplement 1 , mean ± s . d . , N = 80 trains in each direction ) .", "Because a large number of GFP-labeled trains accumulated at the tip , the dwell and departure of individual trains at the tip could not be resolved by conventional TIR imaging ( Figure 1a ) .", "To monitor the turnaround behavior of individual IFT trains at the flagellar tip , we developed the one-dimensional PhotoGate assay ( Belyy et al . , 2017 ) to track single fluorescent complexes at the flagellar tip .", "In this assay , fluorescent trains located at distal parts of a flagellum were initially photobleached by moving a focused laser beam from the tip of the flagellum to near its base .", "We next opened the ‘gate’ by turning off the focused beam until a single fluorescent train entered the flagellum .", "The gate beam was repeatedly turned on for 0 . 2 s at 1 Hz to photobleach any additional anterograde trains entering the flagellum ( Figure 1b , Video 2 ) .", "Under these conditions , less than 1% of anterograde IFT trains were able to pass the gate unbleached .", "This approach revealed the full range of movement of single fluorescent IFT trains within the flagellum .", "IFT movement can be divided into three stages: anterograde movement toward the tip , pausing at the tip , and returning to the base by retrograde transport .", "We directly observed that a single anterograde train splits into multiple retrograde trains at the tip ( Figure 1c–e , Figure 1—figure supplement 2a ) .", "On average , 2 . 4 retrograde trains were detected departing from the tip after the arrival of a single fluorescent anterograde train ( Figure 1f , N = 97 ) , consistent with higher frequencies of retrograde IFT trains than anterograde IFT trains ( Dentler , 2005; Iomini et al . , 2001; Qin et al . , 2007 ) .", "However , the number of retrograde trains per fluorescent anterograde train in PhotoGate assays ( 2 . 4 , Figure 1c ) was significantly higher ( Welch’s t-test , p=10−20 ) than the ratio of retrograde to anterograde train frequencies ( 1 . 15 , Figure 1a ) ( Dentler , 2005; Iomini et al . , 2001; Reck et al . , 2016 ) .", "These observations suggested that IFT complexes from different anterograde trains recombine with each other to form retrograde trains at the tip .", "To test this possibility , we closed the gate after two or three fluorescent anterograde trains entered the flagellum ( Figure 1d , e , Videos 3 and 4 ) and measured the number and return frequency of retrograde trains departing from the tip .", "If individual trains split and return without mixing with each other , the number and frequency of fluorescent retrograde trains would be proportional to the number of fluorescent anterograde trains .", "In contrast , we observed 2 . 4 , 3 . 6 , and 4 . 2 returning trains on average for one , two , and three incoming trains , respectively ( N = 97 , 60 , 42 , Figure 1f ) .", "The return frequencies for one , two , and three incoming fluorescent trains were 0 . 57 , 0 . 71 , and 0 . 76 s−1 , respectively .", "Because the increase was sub-proportional with the number of anterograde trains , we concluded that the fluorescent complexes in the anterograde trains disassemble and mix with a pool of ‘dark’ complexes from the other photobleached trains at the tip before they reorganize into retrograde trains ( Figure 1f ) .", "Monte Carlo simulations revealed that our conclusions are not markedly affected by the limited number of IFT27-GFPs per anterograde train ( ~6 ) or GFP photobleaching under TIR illumination ( 0 . 07 s−1 , Figure 1g , Figure 1—figure supplement 3 , see Materials and methods ) .", "To understand how IFT trains are processed at the tip , we analyzed the time between the arrival of an anterograde train and the departure of fluorescent retrograde trains ( referred to as tip resting time ) at the tip ( Figure 2a ) .", "When only a single fluorescent anterograde IFT27-GFP train was left unbleached near the base of the flagellum , the tip resting time of the first retrograde train was 3 . 1 ± 0 . 3 s ( mean ± s . e . m . , N = 97 , Figure 2b ) , comparable to that of IFT cargos ( Craft et al . , 2015; Reck et al . , 2016; Wren et al . , 2013 ) .", "Tip resting time was independent of flagellar length ( Figure 2—figure supplement 1 ) .", "If IFT tip turnaround was rate-limited by a single process , we would expect a single exponential distribution of tip resting times .", "However , the tip resting time histogram of first retrograde trains fit well to a Gamma distribution with a shape parameter of 3 and a rate constant of ~1 s−1 , indicating that tip turnaround of IFT trains occurs rapidly through a multistep process ( Figure 2b ) .", "We also observed that the tip resting time of first , second , third , and fourth fluorescent retrograde trains increases linearly ( Figure 2c ) .", "Because the average time between successive retrograde trains ( Δt = 1 . 7 s ) is the same , we concluded that the tip departure is a purely stochastic process .", "The linear fit to the tip resting times has a y-intercept of 1 . 3 s ( Figure 2c ) , revealing that the departure of the first train takes longer than Δt .", "Therefore , the complexes dwell at the tip through another rate limiting process before they can depart from the tip .", "When two or three fluorescent anterograde trains were allowed to pass through the gate , Δt became shorter ( 1 . 4 and 1 . 3 s , Welch’s t-test , p=0 . 03 and 0 . 02 for two and three trains , respectively; Figure 2c ) .", "This is because providing a higher number of fluorescent GFPs available at the tip increases the likelihood of retrograde trains to have at least one fluorescent GFP .", "Remarkably , the y-intercept remained constant when we allowed one to three more fluorescent anterograde trains to enter the flagellum ( Figure 2c ) , suggesting that this duration corresponds to processing and breakdown of anterograde trains at the tip ( referred to as tip remodeling time ) .", "The time between tip remodeling and departure of IFT trains is defined as the tip departure time ( Figure 2a ) .", "We also tested whether extracellular calcium and the dynein inhibitor ciliobrevin D ( Firestone et al . , 2012 ) affect the duration of IFT tip turnaround .", "Calcium regulates pausing of IFT trains along the flagellum ( Collingridge et al . , 2013; Shih et al . , 2013 ) , and disrupting calcium-dependent kinesin-II phosphorylation causes abnormal accumulations of IFT proteins at the ciliary tip ( Liang et al . , 2014 ) .", "When extracellular calcium in media ( 0 . 34 mM ) was chelated using 0 . 5 mM EGTA , the tip departure time increased to 2 . 8 s ( Welch’s t-test , p=0 . 01 ) , whereas tip remodeling time ( 1 . 4 s ) remained unaltered ( Figure 2d , e ) .", "Therefore , calcium has minimal effect on the breakdown of anterograde trains , but may have a regulatory role in the assembly or departure of retrograde trains .", "Addition of 0 . 1 mM ciliobrevin D to media results in 50% reduction in the frequency of retrograde and anterograde trains , and 50% and 28% reduction in retrograde and anterograde train velocities , respectively ( Shih et al . , 2013 ) .", "In this case , the tip resting time of IFT27 increased over two-fold ( Figure 2e , p<10−4 ) , suggesting that rapid turnover of IFT trains depends on dynein activity .", "We next turned our attention to the movement of the IFT motors and their exchange at the flagellar tip .", "Dynein-1b was tagged with GFP at its light intermediate chain ( D1bLIC ) , which assembles into the dynein-1b complex and rescues d1blic mutant phenotypes ( Reck et al . , 2016 ) .", "In the d1blic::D1bLIC-GFP strain , D1bLIC moved continuously in the anterograde and retrograde directions at velocities similar to that of the IFT trains ( Reck et al . , 2016 ) ( Figure 3a , Figure 1—figure supplement 1 ) .", "Kinesin-II was tagged with GFP at its non-motor subunit KAP that localizes kinesin-II to the flagellar base ( Mueller et al . , 2005 ) .", "In the fla3::KAP-GFP strain , KAP moved primarily in the anterograde direction to the flagellar tip at a similar speed to anterograde IFT27 ( Figure 3b , Figure 1—figure supplement 1 ) .", "Unlike D1bLIC , retrograde traces of KAP were not frequently observed ( Engel et al . , 2009; Wingfield et al . , 2017 ) , suggesting that kinesin-II dissociates from IFT trains at the tip ( Engel et al . , 2009; Engel et al . , 2012 ) .", "PhotoGate assays revealed that D1bLIC-GFP has similar tip turnaround dynamics to IFT27-GFP ( Figure 3c , Figure 1—figure supplement 2b , Video 5 ) .", "After arrival of a single anterograde D1bLIC-GFP train at the tip , we detected on average 2 . 5 retrograde D1bLIC trains .", "The average tip resting time until the departure of the first retrograde train was 1 . 8 ± 0 . 2 s ( mean ± s . e . m . , N = 60 , Figure 3d ) , with additional ~1 . 1 ± 0 . 1 s between subsequent departure events ( Figure 3e ) .", "PhotoGate imaging of KAP-GFP cells showed that single KAP-GFP trains moved anterogradely to the tip and rested at the tip for 2 . 2 ± 0 . 2 s ( N = 95 ) .", "Unlike D1bLIC , individual KAP-GFP particles moved away from the tip by rapid saltatory motion ( Figure 3f , g , Figure 1—figure supplement 2c , Video 6 ) .", "Mean square displacement ( MSD ) analysis showed that KAP undergoes one-dimensional diffusion at 1 . 68 ± 0 . 04 µm2 s−1 ( mean ± s . e . m . , N = 27 traces ) within the flagellum after it departs from the tip ( Figure 3h ) , consistent with the values measured for tubulin and EB1 that undergo diffusion within the ciliary space ( Craft et al . , 2015; Harris et al . , 2016 ) .", "The tip resting time of KAP remained nearly constant at different steady-state flagellar lengths ( Figure 2—figure supplement 1 ) and was shorter than that of IFT27 , indicating that departure of kinesin from the tip is independent of flagellar length and departure of retrograde trains ( Figure 2—figure supplement 1 ) .", "Unlike IFT trains , the majority ( 89% , N = 95 ) of KAP-GFPs simultaneously departed from the tip in a single step ( Figure 3—figure supplement 1 ) .", "Because each anterograde train contains multiple ( 6 ) KAP-GFPs on average ( Engel et al . , 2009 ) , this observation indicates that kinesin-II departs from the tip in the same oligomeric state as it arrives .", "34% of kymographs clearly showed a single diffusing fluorescent spot after departure ( Figure 3f ) , suggesting that KAPs are held together in a single complex .", "In 30% of kymographs , the KAP signal spread quickly along the length of a flagellum after departure , suggesting that kinesin-IIs can also diffuse alone ( Figure 3—figure supplement 2 , Video 7 ) .", "The rest of the kymographs were ambiguous .", "Similar to IFT27 , the tip resting time of KAP increased ~50% when the cells were treated with ciliobrevin D ( p=0 . 0016 ) , but EGTA had no significant effect on tip resting time of KAP ( Figure 3—figure supplement 1 ) .", "We next investigated whether KAP slides linearly along the microtubule track , similar to the non-processive , microtubule-depolymerizing kinesin MCAK ( Helenius et al . , 2006 ) .", "In this case , KAP clusters are expected to move along the microtubule long axis , so the fluctuation in KAP position in the perpendicular axis would be similar to the error of single particle tracking .", "The KAP-GFP particles had lateral fluctuations of 19 ± 2 nm ( mean ± s . d . ) when moving in the anterograde direction .", "After departing from the tip , lateral fluctuations of diffusing spots increased to 65 ± 7 nm ( Figure 3i , j ) , comparable to the radius of the axoneme .", "The intensity of fluorescent spots stayed relatively constant during anterograde transport and diffusion , suggesting that the measured lateral fluctuations are due to diffusive motion rather than decreased tracking precision .", "We concluded that after KAP detaches from the flagellar tip , it freely explores the space between the flagellar membrane and the axonemal surface rather than sliding along microtubules .", "To investigate how kinesin-II and dynein-1b motors interact with anterograde and retrograde trains and how they are recycled back to the basal body , we transformed a d1blic mutant with both D1bLIC-crCherry and KAP-GFP constructs and simultaneously tracked the movement of KAP and D1bLIC subunits in the rescued cells ( Figure 4—figure supplement 1 , Video 8 ) .", "The D1bLIC-crCherry transgene rescued the flagellar assembly defects in the d1blic mutant , increasing the flagellar length to 12 . 2 ± 1 . 6 µm ( mean ± s . d . , N = 100 flagella ) .", "Both tagged motors were expressed at near wild-type levels ( Figure 4—figure supplement 1 ) .", "The velocities of anterograde and retrograde D1bLIC-crCherry trains were similar to those observed with IFT27-GFP ( Figure 4a , Figure 1—figure supplement 1 ) .", "We observed strong co-localization of D1bLIC-crCherry and KAP-GFP on anterograde trajectories ( Figure 4a ) , demonstrating that dynein-1b is carried to the flagellar tip by kinesin-II .", "Only D1bLIC-crCherry trains showed robust retrograde transport , while retrograde traces of KAP-GFP were rarely observed , consistent with dissociation of kinesin-II from IFT trains at the tip .", "To determine which motor first departs from the tip after the arrival of an anterograde train , we performed two-color Photogate experiments to simultaneously track KAP-GFP and D1bLIC-crCherry from individual anterograde trains ( Figure 4b ) .", "Out of 21 cells , KAP began diffusive motion before the retrograde movement of D1bLIC in 10 cells ( Figure 4b ) , D1bLIC left the tip before KAP in 8 cells ( Figure 4—figure supplement 2 ) , and both appeared to exit the tip simultaneously ( within 0 . 24 s ) in 3 cells .", "These results suggest that kinesin-II and dynein-1b exit the flagellar tip independently from each other .", "Dissociation of KAP from IFT trains at the tip is consistent with the recycling of kinesin-II to the cell body in the absence of active retrograde IFT ( Engel et al . , 2012; Pedersen et al . , 2006; Reck et al . , 2016 ) .", "However , it remained unclear how kinesin-II achieves this long-range movement without active transport .", "To test whether diffusion from the tip effectively recycles KAP to the cell body , we performed fluorescence recovery after photobleaching ( FRAP ) assays in the middle sections of full-length flagella of fla3::KAP-GFP cells ( ~12 µm , Figure 5a , Video 9 ) .", "Directional movements of KAP-GFP labeled trains into the photobleached area were seen from the anterograde direction , whereas recovery of GFP fluorescence from the retrograde direction was primarily due to diffusion of KAP-GFP from the tip .", "These results suggest that the high levels of background observed in KAP-GFP flagella were caused by kinesin-II motors dissociated from IFT trains at the tip .", "The diffusion constant calculated from the fluorescence recovery ( 1 . 8 ± 0 . 1 μm2 s−1 , Figure 5b ) was similar to the result of the MSD analysis ( Figure 3h ) .", "The fluorescent background in KAP-GFP flagella increased towards the tip , suggesting a net efflux of diffusing KAP-GFP towards the cell body ( Figure 5c , d ) .", "During flagellar regrowth , the KAP-GFP gradient was maintained for all flagellar lengths ( Figure 5—figure supplement 1a , see Materials and methods ) .", "The influx of KAP-GFP fluorescence to the flagellum through anterograde IFT was statistically indistinguishable from the efflux of KAP-GFP to the base through one-dimensional diffusion in flagella ( Welch’s t-test , p=0 . 80 , N = 57 , Figure 5—figure supplement 2 , see Materials and methods ) .", "These results strongly indicate that KAP-GFP returns to the cell body by diffusing from the flagellar tip .", "We ran Monte Carlo simulations to estimate the accumulation of KAP in a flagellum at a steady-state using the measured values of IFT train loading ( Engel et al . , 2009 ) , diffusion coefficient , flagellar length , and IFT train frequency .", "The model assumes that KAP is released from anterograde IFT trains at the tip , diffuses within a flagellum , and is taken up by the basal body .", "Under these conditions , simulations confirmed the build-up of a linear concentration gradient of KAP in the flagellum ( Figure 5—figure supplement 1b ) .", "In fully-grown flagella , the return of KAP to the flagellar base takes 42 s on average , an order of magnitude longer than the travel of retrograde trains ( 4 s ) to the base .", "This delay leads to a ~4 fold higher amount of KAP inside the flagellum compared to a case in which KAP returns to the base with retrograde trains ( Figure 5—figure supplement 1c ) .", "Unlike KAP , IFT27-GFP cells have a low fluorescence background without an obvious concentration gradient along the length of the flagellum ( Figure 5d ) due to active transport of the IFT trains in both directions .", "KAP-GFP loading on IFT particles has been shown to decrease with increasing flagellar length ( Engel et al . , 2009 ) , but the underlying mechanism remained unclear .", "Because a larger amount of KAP builds up in the flagellum as the flagella elongate ( Figure 5—figure supplement 1 ) , loading of KAP onto the subsequent IFT trains may be reduced by depletion of KAP at the flagellar base .", "To test this model , we deflagellated fla3::KAP-GFP cells and measured the GFP fluorescence at the basal body and in the flagellum during flagellar regrowth using confocal microscopy ( Figure 6a ) .", "The total amount of KAP localized to the base and flagellum increased by two-fold with flagellar length , indicating the upregulation of IFT components during flagellar growth .", "The fluorescence intensity at the flagellar base was highest for short flagella ( 1–4 µm ) and decreased ~4 fold as cells grew full-length flagella ( ~10 µm , Figure 6b ) , significantly larger than ~1 . 6 fold reduction reported previously ( Ludington et al . , 2015 ) .", "We also observed that the KAP fluorescence in the flagellum was low in short flagella and increased ~10 fold as the flagellar length reached the steady-state ( Figure 6b ) .", "Changes in the amount of IFT complexes were markedly different from that of KAP during flagellar regrowth ( Figure 6a ) .", "Unlike KAP-GFP , basal body fluorescence of IFT20-GFP remained nearly constant across all flagellar lengths in IFT20::IFT20-GFP cells ( Figure 6c ) , presumably because they are rapidly returned to the base through active transport .", "We also observed an increase of the GFP signal in the flagellum with elongation ( Figure 6c ) , in contrast to the previous observation that the total amount of IFT components remains constant during flagellar regeneration ( Marshall and Rosenbaum , 2001 ) .", "This discrepancy may be related to differences in methods for quantifying IFT components in flagella .", "We next determined the localization of KAP-GFP to the basal body and flagellum in cells that grow abnormally long and short flagella .", "Chlamydomonas grows ~1 . 5X longer flagella in the presence of Li+ ( Nakamura et al . , 1987 ) by recruiting flagellar proteins from the cell body pool into the flagella ( Nakamura et al . , 1987 ) rather than requiring new protein synthesis ( Wilson and Lefebvre , 2004 ) .", "Consistent with previous observations , the KAP-GFP strain grew longer flagella in 50 mM Li+ .", "After reaching the steady state length , we calculated the total KAP fluorescence at the basal body and the flagellum ( Figure 6d , Figure 6—figure supplement 1 ) .", "In agreement with our model , we observed that KAP gets depleted at the basal body at equilibrium .", "The KAP fluorescence localized to a flagellum correlated strongly with flagellar length ( Pearson’s R = 0 . 86 ) , similar to untreated cells .", "The total KAP fluorescence in the flagellum was 50% higher than untreated cells .", "In the absence of new protein synthesis , Chlamydomonas can grow half-length flagella after deflagellation , suggesting that the cytoplasmic pool of flagellar proteins is at least one half of that localized to the flagellar compartment ( Rosenbaum et al . , 1969 ) .", "We deflagellated KAP-GFP cells with pH shock and regrew their flagella in the presence of the protein synthesis inhibitor cycloheximide .", "In agreement with untreated and Li+ treated cells , we observed that the KAP fluorescence at the flagellum correlates strongly with the flagellar length ( Pearson’s R = 0 . 86 ) , whereas the KAP intensity was depleted at the basal body ( Figure 6d , Figure 6—figure supplement 1 ) .", "Total KAP fluorescence was one half of untreated cells , consistent with the fact that a large amount of KAP is lost during deflagellation .", "Therefore , over a wide range of flagellar lengths ( 2–22 µm ) , KAP gets depleted at the basal body when the flagella reach their equilibrium length ." ], [ "Using PhotoGate , we have visualized the turnaround behavior of individual components of the IFT machinery at the flagellar tip .", "We present evidence that when IFT trains arrive at the tip , the complexes split apart and mix with complexes from other trains at the flagellar tip before initiating retrograde transport ( Figure 4c ) .", "This dynamic disassembly and reassembly process may lead to differences in size , shape , and structure of anterograde and retrograde trains , as previously suggested by transmission electron microscopy ( Dentler , 2005; Stepanek and Pigino , 2016 ) .", "Remarkably , remodeling of IFT trains is completed within 1 . 3 s , with a 1 . 7 s average waiting time between the departures of successive trains , consistent with previously measured values for IFT complex subunits , dynein-1b , and other axonemal cargoes ( Craft et al . , 2015; Qin et al . , 2007; Reck et al . , 2016; Wren et al . , 2013 ) .", "Kinetic analysis of the tip resting time revealed that disassembly of anterograde trains and reassembly of the retrograde trains is a multistep process regulated by extracellular calcium and the concentration of active dynein motors .", "Simultaneous tracking of the anterograde motor kinesin-II ( with KAP-GFP ) and the retrograde motor dynein-1b ( with D1bLIC-Cherry ) has further revealed significant differences in how these motors are recycled back and forth within a flagellum .", "Kinesin-II drives anterograde trains in a highly processive fashion and then dissociates from the IFT trains when they reach at the flagellar tip .", "KAP-GFP then returns to the flagellar base by diffusing within the flagellum , similar to the diffusion of kinesin-1 in mammalian neurons ( Blasius et al . , 2013 ) and in contrast to the retrograde transport of kinesin-II observed in other cilia ( Signor et al . , 1999; Broekhuis et al . , 2014; Williams et al . , 2014; Prevo et al . , 2015 ) , see below ) .", "We propose that the diffusion of KAP-GFP represents the movement of the entire heterotrimeric kinesin-II complex because KAP and the kinesin-II motor subunits co-sediment in sucrose density gradients of purified flagella extracts ( Cole et al . , 1998; Mueller et al . , 2005 ) and neither KAP nor FLA10 accumulate in flagella during inactivation of retrograde transport ( Engel et al . , 2012; Pedersen et al . , 2006; Reck et al . , 2016 ) .", "In certain cases , KAP-GFP appears to diffuse in an oligomeric form .", "It remains to be studied what holds KAPs together and whether other components of the IFT trains diffuse with KAP clusters after splitting and mixing at the tip .", "The retrograde motor dynein-1b is transported to the flagellar tip on anterograde trains ( Reck et al . , 2016 ) .", "Because kinesin and dynein motors do not compete against each other in a tug-of-war on anterograde trains ( Shih et al . , 2013 ) , we concluded that dynein-1b is carried as an inactive motor complex ( Toropova et al . , 2017; Zhang et al . , 2017 ) , and it actively engages with microtubules only when it reaches the flagellar tip ( Figure 4c ) .", "The average tip resting time of dynein-1b is similar to kinesin-II ( Welch’s t test , p=0 . 05 ) , but the initiation of retrograde transport by dynein-1b does not require departure of kinesin-II motors from the tip , suggesting that these processes are independent from each other .", "Several studies have revealed differences in IFT in the cilia and flagella of different organisms ( Prevo et al . , 2017 ) .", "First , the microtubule tracks can vary considerably .", "In Chlamydomonas , the axoneme contains nine doublet microtubules , each composed of a complete A-tubule ( with 13 protofilaments ) and an incomplete B-tubule ( with 10 protofilaments ) .", "The B-tubule terminates before the A-tubule less than 1 µm from the flagellar tip ( Ringo , 1967; Satish Tammana et al . , 2013 ) .", "Recent electron microscopy studies have revealed that anterograde IFT trains are transported primarily on the B-tubule , whereas retrograde IFT trains are transported on the A-tubule ( Stepanek and Pigino , 2016 ) .", "The pausing of IFT particles observed at the flagellar tip in Chlamydomonas may therefore reflect not only the time involved in the re-organization of the IFT particles , but also the time required for switching between microtubule tracks .", "In other cilia , such as C . elegans sensory and mammalian olfactory cilia , the proximal doublet microtubule segment is shorter , and the distal singlet MTs can vary significantly in length ( ~2 . 5 µm in C . elegans to >100 µm in mouse olfactory cilia ) .", "These cilia also employ two different kinesin-II motors for anterograde IFT ( Prevo et al . , 2015; Snow et al . , 2004; Williams et al . , 2014 ) .", "In C . elegans , heterotrimeric kinesin-II is concentrated near the basal body region and transports anterograde IFT particles into the proximal doublet segment , where they are gradually handed over to a homodimeric kinesin-II , OSM-3 , for transport into the distal singlet segment ( Prevo et al . , 2015; Snow et al . , 2004 ) .", "Unlike Chlamydomonas , both the heterotrimeric and homodimeric kinesin-II motors are recycled to the ciliary base by retrograde IFT , not by diffusion , in these and other metazoan cilia studied to date ( Broekhuis et al . , 2014; Mijalkovic et al . , 2017; Prevo et al . , 2015; Signor et al . , 1999; Williams et al . , 2014 ) .", "Another important difference between IFT in Chlamydomonas and metazoan cilia is the dynamic behavior of the motors themselves .", "IFT particles and motors move processively in the Chlamydomonas flagellum ( Dentler , 2005 ) , with little or no evidence for the frequent pausing and reversal along the axoneme previously described in C . elegans or mouse olfactory sensory cilia ( Mijalkovic et al . , 2017; Prevo et al . , 2015 ) .", "We also did not observe acceleration and deceleration of IFT motors near turnaround zones , nor the instantaneous ( <600 ms ) reversal of dynein-1b at the ciliary tip described in C . elegans ( Mijalkovic et al . , 2017; Prevo et al . , 2015 ) .", "The reasons for these differences in IFT dynamics and turnover remain unknown , but they may be related to the variations in ciliary structure and organization described above , differential phosphorylation of kinesin-II motors ( Liang et al . , 2014 ) , posttranslational modification of the microtubule tracks ( Stepanek and Pigino , 2016 ) , and other unidentified factors .", "In addition , both the frequency and speed of IFT is higher in Chlamydomonas ( Dentler , 2005; Engel et al . , 2009; Engel et al . , 2012; Iomini et al . , 2001; Reck et al . , 2016; Snow et al . , 2004; Williams et al . , 2014; Wingfield et al . , 2017 ) than that measured thus far in metazoan cilia ( Li et al . , 2015; Yi et al . , 2017 ) .", "This may allow Chlamydomonas to rapidly adjust the length of its flagella in response to internal or external stimuli , whereas most cilia in C . elegans sensory neurons or mammalian cells do not undergo extensive structural rearrangements once formed .", "Cilia and flagella serve as a model system to study how cells precisely control organelle size because they elongate only in one direction .", "According to the balance point model , flagellar length is set when flagellar assembly and disassembly rates reach equilibrium ( Marshall et al . , 2005 ) .", "While the disassembly rate is independent of flagellar length ( Kozminski et al . , 1995 ) , the assembly rate is determined by the injection of IFT trains ( Marshall et al . , 2005 ) .", "The amount of material being transported by these trains to the tip is correlated strongly with the amount of material localized to the flagellar base ( Ludington et al . , 2013; Ludington et al . , 2015; Wren et al . , 2013 ) , which serves as a loading dock .", "Previous studies showed that IFT train size and the number of ciliary cargos per train scales inversely with flagellar length ( Craft et al . , 2015; Engel et al . , 2009 ) , consistent with the reduction of the assembly rate as flagella elongate ( Marshall et al . , 2005 ) .", "However , it remained unclear which essential component of the IFT machinery limits the assembly of IFT trains at the basal body during elongation .", "We propose that dissociation of kinesin-II from IFT trains serves as a negative feedback mechanism to control the assembly rate in Chlamydomonas .", "Our results show that the majority of kinesin-II dissociates from IFT trains at the flagellar tip and diffuses within the flagellum .", "Diffusion leads to a large accumulation of kinesin-II in the flagellum as the flagellum grows longer , while the amount of kinesin-II at the base decreases several-fold .", "As a result , lower amounts of kinesin-II are available to bring new anterograde IFT trains to the flagellar tip .", "This may lead to a reduction in the IFT train size and the rate of flagellar assembly as the flagella elongate ( Figure 6e , f ) .", "Indeed , a recent theoretical study demonstrated that increased diffusion time of kinesin-II in longer flagella can explain the inverse relationship between length and IFT motor recruitment rate ( Hendel et al . , 2017 ) .", "Consistent with this model , previous studies showed that KAP intensity at the basal body correlates with KAP loading on IFT trains and the assembly rate during flagellar regeneration ( Ludington et al . , 2013 ) .", "In the temperature-sensitive mutant strain fla10ts , inactivation of kinesin-II motility ceases IFT and leads to resorption of the flagellum at a constant rate ( Kozminski et al . , 1995; Marshall et al . , 2005 ) .", "At intermediate temperatures , flagellar length correlates strongly with the estimated fraction of active kinesin-II motors in fla10ts cells ( Marshall and Rosenbaum , 2001 ) , indicating that the amount of active kinesin-II limits flagellar growth .", "The experiments performed under Li+ and cycloheximide treatments ( Figure 6d ) also support the idea that altering the amount of KAP available for the flagellar compartment positively correlates with the flagellar length and that the equilibrium length is set when KAP gets depleted below a certain threshold at the basal body .", "We note that the KAP intensity at the flagellar base was lower ( 34–40% ) in Li+ and cycloheximide treated cells than in control cells at equilibrium ( p<0 . 0001 , Figure 6d , left ) .", "Although the reason for this difference remains unclear , it could be due to a reduction of KAP concentration in the cytoplasm or changes in the flagellar disassembly rate under treatment ( Wilson and Lefebvre , 2004 ) .", "Unlike kinesin-II , IFT components are rapidly recycled to the cell body by dynein-1b and the amount of these components at the flagellar base remains nearly constant as the flagella elongate .", "Therefore , the abundance of IFT components and dynein-1b at the flagellar base is not limiting to maintain the length , consistent with previous observations that a small amount of IFT complexes and dynein-1b is sufficient to maintain fully grown flagella ( Reck et al . , 2016; Wang et al . , 2009 ) .", "Diffusion is also proposed to play a role in setting the length of bacterial flagella ( Renault et al . , 2017 ) , long polymers made from a single protein flagellin .", "Similar to the flagellar length control model originally proposed for Chlamydomonas ( Levy , 1974 ) , flagellins are injected into the channel of the filament and they diffuse to reach the assembly site at the filament tip , generating a concentration gradient decreasing towards the tip .", "As the filament elongates , it grows more slowly because it takes longer for the components to reach the tip .", "In contrast to bacterial flagellin , structural components are carried to the tip by IFT in eukaryotic flagella .", "In Chlamydomonas , we showed that diffusion of kinesin-II from the tip sets a concentration gradient decreasing towards the basal body , and its return to the flagellar base is delayed as the flagella elongate .", "This delay limits the amount of kinesin-II available for building longer flagella .", "Our model is challenged by studies showing that the kinesin-II mutant strains fla10ts and fla3 maintain nearly full-length flagella at permissive temperatures although they accumulate significantly lower amounts of kinesin-II in the flagellar compartment ( Kozminski et al . , 1995; Mueller et al . , 2005; Pedersen et al . , 2006 ) .", "Remarkably , fla3 cells exhibit slower flagellar regeneration ( Mueller et al . , 2005 ) , consistent with our prediction that the lower amount of kinesin-II negatively affects the assembly rate .", "However , a more recent study showed that fla10ts flagella contain wild-type levels of kinesin-II at permissive temperatures ( Wang et al . , 2009 ) .", "Given these apparent discrepancies , more quantitative approaches will be required to address whether the amount of kinesin-II correlates with flagellar length .", "According to the balance-point model , flagella that contain lower amounts of kinesin-II can still maintain nearly full-length if they also have a lower disassembly rate .", "The studies that reported a reduction in kinesin-II expression in fla10ts and fla3 cells also noted significantly reduced anterograde IFT frequency and IFT particle subunits in flagella ( Mueller et al . , 2005; Pedersen et al . , 2006 ) .", "This could negatively affect the disassembly rate because IFT is required for efficiently removing certain axonemal precursors ( Qin et al . , 2004 ) and resorbing the flagellum prior to mitosis .", "Indeed , flagellar resorption before mitosis occurs at a faster rate than flagellar disassembly after inactivation of IFT ( Marshall et al . , 2005; Pan and Snell , 2005 ) .", "The mechanisms that control the expression of IFT components after deflagellation , regulate the exchange of material between the basal body and cytoplasm , and load material onto IFT trains must also play a major role in determining the length of flagella .", "Several studies have shown that IFT components are upregulated and accumulate in large numbers at the flagellar base after deflagellation ( Albee et al . , 2013; Lefebvre and Rosenbaum , 1986; Stolc et al . , 2005 ) .", "Additionally , a large pool of IFT components in the cytoplasm partially exchanges with the flagellar pool ( Buisson et al . , 2013; Engel et al . , 2009; Wingfield et al . , 2017 ) because cells can grow half-length flagella after deflagellation under complete inhibition of protein synthesis ( Rosenbaum et al . , 1969 ) .", "However , molecular cues that govern these processes remain poorly understood and further studies in mutant cell lines that have abnormally long ( Nguyen et al . , 2005; Tam et al . , 2007 ) or short flagella may provide new insight for the mechanism of flagellar length control ." ], [ "The pf18 IFT27-GFP strain was obtained from the Marshall laboratory ( University of California San Francisco ) after crossing the IFT27-GFP transgene into the pf18 background as previously described ( Engel et al . , 2009; Qin et al . , 2007 ) .", "The ift20::IFT20-GFP strain ( Lechtreck et al . , 2009 ) was obtained from the Lechtreck laboratory ( University of Georgia ) .", "The fla3::KAP-GFP ( Mueller et al . , 2005 ) and d1blic::D1bLIC-GFP ( Reck et al . , 2016 ) strains are available from Chlamydomonas Resource Center at the University of Minnesota ( RRID: SCR_014960 , Minneapolis , MN ) .", "The d1blic::D1bLIC-crCherry KAP-GFP strain was generated as described below .", "These strains were not authenticated or tested for mycoplasma contamination .", "Strains were maintained on plates of TAP media containing 1% agar .", "For light microscopy , vegetative cells were resuspended in liquid TAP media at 22°C for 24–48 hr and passaged to fresh liquid TAP before introduction into a flow chamber .", "The D1bLIC-crCherry construct was generated by subcloning a Chlamydomonas codon-optimized version of the Cherry tag into a genomic copy of the D1bLIC gene ( Reck et al . , 2016 ) .", "The Cherry tag was amplified by PCR from the plasmid pBR9 mCherryCr ( Rasala et al . , 2013 ) and inserted into a unique AscI site located in the last exon of D1bLIC .", "The D1bLIC-crCherry construct was linearized with BamHI and co-transformed into d1blic ( CC-4487 ) with the selectable marker pSI103 and plated on TAP medium plus 10 μg/ml paromomycin .", "960 transformants were picked into TAP media and screened for changes in colony morphology .", "84 colonies were further examined by both phase contrast and fluorescence microscopy for rescue of flagellar assembly and expression of Cherry .", "Isolated flagella from four colonies were analyzed by Western blot for the presence of full-length D1bLIC-Cherry .", "A single colony was selected for a second round of transformation using the KAP-GFP construct ( Mueller et al . , 2005 ) and the plasmid pHyg3 ( Berthold et al . , 2002 ) and selection on 10 μg/ml of hygromycin B . Two out of 96 transformants were identified as positive for both GFP and Cherry by fluorescence microscopy , and western blots of isolated flagella confirmed the presence of both D1bLIC-Cherry and KAP-GFP in the rescued strains .", "Antibodies used included a rat antibody against Chlamydomonas KAP ( Mueller et al . , 2005 ) , a mouse antibody against GFP ( Covance , Inc . , Princeton , NJ ) , a rabbit antibody against Chlamydomonas D1bLIC ( Perrone et al . , 2003 ) , and a rabbit antibody against mCherry ( Rockland Immunochemicals , Limerick , PA ) .", "0 . 34 mM Ca2+ in TAP media was depleted by adding 0 . 5 mM EGTA , which resulted in a free Ca2+ concentration of 1 . 5 µM .", "The concentration of free Ca2+ in the assay buffer as a function of added EGTA was calculated from the Chelator program ( http://maxchelator . stanford . edu ) .", "For dynein-1b inhibition assays , a final concentration of 100 µM ciliobrevin D was added to the TAP media , and the data was collected within 5–10 minof the treatment .", "For cycloheximide treatment , cells were deflagellated by pH shock and cycloheximide was added to a final concentration of 1 . 5 µg/ml immediately afterwards .", "Cells were allowed to regrow flagella for 2 hr before fixation and imaging .", "For Li+ treatment , 50 mM LiCl was added to liquid cell suspensions , and cells were incubated for 2 hr before fixation and imaging .", "For imaging the diffusion gradient in live fla3::KAP-GFP cells , we deflagellated cells in TAP media using shear force by rapidly pushing them through a 20G1 ½ syringe .", "Cells regenerating flagella were imaged in the following hour .", "For imaging the accumulation of GFP signal at the basal body region and in regenerating flagella , fla3::KAP-GFP and IFT20::IFT20-GFP cells were deflagellated with pH shock by adding 60 µl 0 . 5 N acetic acid to 1 ml of cells in TAP media , waiting 45 s , and adding 60 µl 0 . 5 N KOH .", "Cells were fixed 15 , 30 , 45 , 60 , and 75 min after pH shock .", "Fixation was done by pipetting 200 µl of liquid TAP cell culture onto a poly-lysine treated coverslip for 1 min , then gently treating the coverslip with 4% paraformaldehyde in water for 10 min .", "Afterwards , the coverslip was treated twice with 100% methanol chilled to −20 °C for 5 min .", "Coverslips were dipped in water to remove methanol , mounted in a flow chamber with TAP media , and then imaged immediately .", "A custom-built objective-type TIR fluorescence microscope was set up using a Nikon TiE inverted microscope equipped with a perfect focusing unit , bright-field illumination , and a 100-X 1 . 49 NA PlanApo oil immersion objective ( Nikon , Melville , NY ) .", "488 nm and 561 nm solid state lasers ( Coherent , Santa Clara , CA ) were used for GFP and crCherry excitation , respectively .", "The angle of incident light was adjusted lower than the critical angle to illuminate a deeper field ( ~300 nm ) near the coverslip surface .", "The fluorescent signal was recorded by an iXon 512 × 512 electron-multiplied charge-coupled device ( EM-CCD ) camera ( Andor , Belfast , United Kingdom ) .", "1 . 5x extra magnification was used to obtain an effective pixel size of 106 nm .", "Data were collected at 10 Hz .", "Excitation laser beams were controlled by shutters ( Uniblitz , Rochester , NY ) .", "Because the CCD image saturates under intense laser illumination of the focused gate beam , shutter timing was synchronized with the camera acquisition by a data acquisition card ( NI , USB-6221 ) to minimize the number of saturated frames in recorded movies .", "For two-color imaging , GFP and crCherry fluorescence were separated into two channels on a CCD camera using Optosplit II ( Cairn , Kent , United Kingdom ) .", "To avoid bleed-through between channels , movies were acquired using time-sharing between the 488 nm and 561 nm laser beams , synchronized with camera acquisition at 60 ms frame time .", "The effective pixel size was 160 nm .", "The PhotoGate system was assembled as previously described ( Belyy et al . , 2017 ) .", "Briefly , a 488 nm laser beam was split into two paths using a half-wave plate and a polarizer beamsplitter cube .", "The first path was used for objective-type TIR imaging .", "The second path was focused ( 2 MW cm−2 ) to the image plane and steered with a fast piezo-driven mirror ( S-330 . 8SL , Physik Instrumente , Karlsruhe , Germany ) .", "The piezo-driven mirror was mounted at a position conjugate to the back-focal plane of the objective to ensure that the tilting of the mirror resulted in pure translation of the focused beam in the image plane .", "The mirror provided a usable travel range of 30 µm x 30 µm area at the image plane .", "The mirror’s angle was updated via analog output channels of a data acquisition card ( National Instruments , Austin , TX , USB-6221 ) and controlled by software custom-written in LabVIEW .", "Flagellar orientation of surface adhered cells was visualized by TIR imaging .", "Initially , the gate beam was placed at the tip of flagellum and moved along the flagellar orientation to prebleach the distal half of the flagellum .", "The gate beam was turned off when it was positioned near the base of the flagellum to allow a single fluorescent anterograde train to enter the flagellum .", "Occasionally ( <5% ) , two anterograde trains overlapped and entered the flagellum simultaneously .", "The gate beam was then turned on for 0 . 2 s of every 1 s to bleach other anterograde trains .", "Based on the size of the gate beam in the image plane , gating frequency was adjusted such that less than 1% of anterograde IFT trains moved faster than the cutoff speed ( 3 . 0 µm s−1 ) .", "The trajectories of these trains can be distinguished from each other as they move at different speeds along the flagellum .", "The locations of flagellar tips were determined by brightfield imaging ( data not shown ) .", "In two-color photogate experiments , the focused 488 nm laser beam was used to bleach both GFP and crCherry , and 488 and 561 beams were used in a time-sharing mode for TIR excitation .", "FRAP assays on the fla3::KAP-GFP strain were performed by photobleaching the center part of the flagellum ( 5 µm in length ) for 200 ms at 25 kW cm−2 in the epifluorescence mode .", "The recovery of fluorescence signal in the bleached area was simultaneously monitored by imaging with a 100 W cm−2 TIR excitation .", "The analysis was performed by measuring the total fluorescence intensity within the bleached area .", "Fluorescent signal of anterograde transport was manually excluded from the analysis .", "Thirteen different recovery traces were used in the MSD analysis .", "The intensity of each trace was normalized according to the initial and final intensity .", "fla3::KAP-GFP and IFT20::IFT20-GFP cells were fixed with paraformaldehyde and imaged on a confocal microscope using 488 nm laser excitation ( Zeiss , Oberkochen , Germany ) .", "Images were recorded with 560 nm z step , 63 nm pixel size , and 1 . 58 µs photon collection per pixel .", "Fluorescence in basal body and flagellum was quantified using ImageJ .", "The ratio of flagellar to basal-body KAP-GFP fluorescence in confocal images was similar to that of live cell imaging under TIR excitation , indicating that the fixation protocol did not result in the loss of diffusing KAP-GFP signal from the flagella .", "Anterograde and retrograde trajectories were manually assigned from kymographs .", "After the arrival of a single anterograde particle at the tip , the departure of fluorescent retrograde trains was determined at single pixel and frame resolution .", "The tip resting time for each retrograde train was defined as the duration between the arrival of the fluorescent anterograde train and the departure of the retrograde train from the tip .", "Tip resting time histograms were constructed and fitted to a Gamma function using MATLAB .", "The Gamma function was defined as Γ ( t ) = tα-1e-λt , where α and λ are shape and rate parameters , respectively .", "For single particle tracking analysis , the positions of fluorescent spots were determined by a 2D Gaussian fitting algorithm .", "The positions were fitted throughout the movie except at the frames when the gate beam was on or the frames in which the tracked particle overlapped with other fluorophores .", "The intensity of the fluorescent spots was estimated by the volume of the 2D Gaussian peak .", "In a typical assay , we adjusted excitation power to achieve 20 nm localization accuracy at 10 Hz image acquisition rate .", "Individual GFP particles were tracked for 5 s on average before photobleaching and the diffusion constant was obtained by MSD analysis of individual spots .", "In certain kymographs , diffusion of individual KAPs within a flagellum could not be resolved due to the diffraction limit .", "To determine the distribution of the KAP-GFP background in flagella , anterograde trajectories in kymographs of fla3::KAP-GFP cells were manually removed using custom ImageJ plugins .", "The remaining pixels were averaged over the kymograph’s time axis , giving a time-averaged plot of the KAP-GFP background over the flagellum length .", "The cells were grouped by flagellar length .", "The background intensity and flagellum length of each cell were normalized .", "The average background intensity along the length of the flagellum was calculated for each group of cells .", "KAP-GFP efflux from the flagellum was calculated using Fick’s law .", "The slope of the KAP-GFP background over the length of a flagellum was multiplied by the diffusion constant ( 1 . 7 µm2 s−1 ) .", "To calculate KAP-GFP influx , the KAP-GFP background was subtracted from the kymographs .", "Then , the average intensity of anterograde trains was multiplied by the train frequency ( 1 . 3 trains s−1 ) to calculate the influx .", "GFP photobleaching rate under TIR illumination was estimated by heavily decorating the coverslip surface with eGFP and calculating the rate of decrease in GFP fluorescence .", "To estimate the live-cell GFP photobleaching rate in pf18 IFT27-GFP cells , the fluorescent intensities of 94 anterograde trains from 9 cells were quantified at each time point en route to the flagellar tip .", "Each train’s intensity profile was normalized by the mean intensity .", "The normalized intensity values were plotted against time and fit to a single exponential decay .", "The decay constant was used as the photobleaching rate .", "Monte Carlo simulations were performed to test the effect of limited number of GFPs per train and GFP photobleaching in PhotoGate experiments using the pf18 IFT27-GFP strain .", "Experimentally measured values were used for the velocity and frequency of anterograde and retrograde trains .", "Simulations assumed that anterograde trains arrive and retrograde trains depart from the tip through a purely stochastic process , adding and subtracting particles to a mixed flagellar tip pool .", "We estimated that each anterograde train contains six fluorescent GFPs by comparing the fluorescent intensities of anterograde trains in the pf18 IFT27-GFP strain to those of KAP-GFP spots in the fla3::KAP-GFP strain under the same imaging conditions and calibrating the number of molecules based on previous photobleaching analysis of the fla3::KAP-GFP strain ( Engel et al . , 2009 ) .", "Each retrograde train was constructed by a random selection of IFT particles available at the tip .", "Tip intensity measurements revealed that the signal of the IFT complexes located at the tip is approximately three times brighter than an average anterograde train .", "The photobleaching of GFPs ( 0 . 07 s−1 ) under TIR illumination was accounted for in simulations and trains with at least one fluorescent GFP upon leaving the tip were marked detectable .", "Simulations were also run to estimate the distribution of diffusing KAP molecules in the flagellum at a steady-state .", "In these simulations , previously reported values for the anterograde train injection rate ( 1 . 3 trains s−1 ) ( Mueller et al . , 2005 ) and the average number of KAP bound to a single anterograde train for each flagellar length ( Engel et al . , 2009 ) were used to estimate the number of KAP that arrives at the flagellar tip per second .", "KAP dissociated from the trains at the tip and immediately started one dimensional diffusion in the flagellum .", "The resting time of KAP at the tip was insignificant , and was not accounted for .", "The flagellum was modeled as a 5–12 µm long linear grid with spacing defined as the MSD of KAP diffusing at 1 . 7 µm2 s−1 ( Figure 3h ) during the time-step of the simulation ( 5 ms ) .", "At every time point , each active molecule had its grid position changed by +1 or −1 .", "The molecules at the extreme terminus of the tip only moved towards the base .", "The diffusing KAP molecules were perfectly absorbed to the cell body as they arrived at the flagellar base ( i . e . perfect sink ) and exited the simulation .", "The simulations were run for 100 , 000 time points to allow molecules to reach a steady-state .", "The number of molecules at each grid position was calculated to plot the distribution of KAP molecules diffusing along the length of the flagellum .", "The total number of KAP was calculated by integrating the number of KAP diffusing along the entire flagellum and KAP on the anterograde trains .", "This number was compared to a hypothetical scenario that KAP returns to the cell body with active transport .", "The simulations were run 10 times to calculate the error .", "Simulation codes are available on https://github . com/SingleMoleculeAC/IFT-Dynamics ( Chien , 2017 ) .", "A copy is archived at https://github . com/elifesciences-publications/IFT-Dynamics ." ] ]

[ "Intraflagellar transport ( IFT ) is essential for the elongation and maintenance of eukaryotic cilia and flagella .", "Due to the traffic jam of multiple trains at the ciliary tip , how IFT trains are remodeled in these turnaround zones cannot be determined by conventional imaging .", "Using PhotoGate , we visualized the full range of movement of single IFT trains and motors in Chlamydomonas flagella .", "Anterograde trains split apart and IFT complexes mix with each other at the tip to assemble retrograde trains .", "Dynein-1b is carried to the tip by kinesin-II as inactive cargo on anterograde trains .", "Unlike dynein-1b , kinesin-II detaches from IFT trains at the tip and diffuses in flagella .", "As the flagellum grows longer , diffusion delays return of kinesin-II to the basal body , depleting kinesin-II available for anterograde transport .", "Our results suggest that dissociation of kinesin-II from IFT trains serves as a negative feedback mechanism that facilitates flagellar length control in Chlamydomonas ." ]

[ "Cilia and flagella are hair-like structures that protrude from nearly every human cell and play a number of roles including transmitting signals and enabling cells to move .", "These structures lengthen when new material is deposited at their tip by a process called intraflagellar transport ( IFT ) .", "In this process , protein complexes known as IFT trains carry cargo along tracks that run along the length of each flagellum .", "Different motor proteins power the IFT trains in different directions: kinesin moves IFT trains from the base to the tip , while dynein moves them back in the opposite direction .", "When IFT trains arrive at the tip of the flagellum , they release their cargo and undergo a major reorganization process in which the trains switch motors in order to move back to the base .", "Because the many IFT trains at the tip form a kind of ‘traffic jam’ , standard imaging techniques have been unable to distinguish exactly what happens during this reorganization .", "A new imaging method called PhotoGate microscopy can track individual molecules inside crowded cells .", "Chien , Shih et al . have now used this method to visualize the full range of movements made by IFT trains and motors in the flagella of a species of single-celled algae .", "This revealed that at the tip of the flagellum , IFT trains split apart and mix with each other to assemble into new trains , which move back to the base .", "In addition , kinesin carries dynein to the tip as inactive cargo , detaches from IFT trains at the tip and diffuses back to the base of the flagellum .", "This delays kinesin’s return and causes it to accumulate in the flagellum , which helps to explain why flagella assemble more slowly as they lengthen: as the flagellum grows longer , less kinesin is available at the base to transport material to the tip .", "Thus kinesin diffusion helps the algae to regulate the length of their flagella .", "Further research is now needed to study whether similar mechanisms control the length of flagella in other organisms .", "Defects in the intraflagellar transport process have been linked to a range of human diseases known as ciliopathies , and so the results presented by Chien , Shih et al . could also help us to uncover the causes and potential treatments for these conditions ." ]

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

[ "neuroscience" ]

[ [ "The nervous system shows complex organization at many spatial scales: from genes and molecules , to cells and synapses , to neural circuits .", "Ultimately , the electrical and chemical signaling at all these levels must give rise to the behavioral and cognitive processes seen at the whole-organism level .", "When trying to understand prevalent brain disorders such as autism and schizophrenia , a natural question to ask is: where is the most productive level of neuroscientific investigation ?", "Traditionally , most major disorders are diagnosed entirely at the behavioral level , whereas pharmaceutical interventions are targeted at correcting alterations at the molecular level .", "However , even for the most successful drugs , we have little understanding of how pharmaceutical actions at the molecular level percolate up the organizational ladder to affect behavior and cognition .", "This classic bottom-up approach may even be further confounded if phenotypic heterogeneity in disorders such as autism turn out not to reflect a unique cellular pathology , but rather ‘a perturbation of the network properties that emerge when neurons interact’ ( Belmonte et al . , 2004 ) .", "These considerations imply that a more promising level of analysis might be at the level of neural circuits , since the explanatory gap between circuits and behavior is smaller than the gap between molecules and behavior .", "This circuit-level viewpoint argues for a reverse-engineering approach to tackling brain disorders: rather than start at the molecular level and working up , we should instead start by asking how cognitive and behavioral symptoms manifest as alterations at the circuit level , then interpret these changes at the levels of cells , synapses , and molecules as appropriate .", "One prominent circuit-level hypothesis for brain disorders has been the idea of an imbalance in excitatory and inhibitory signaling .", "First proposed as a model for autism ( Rubenstein and Merzenich , 2003 ) , the concept has since been applied to many other brain disorders , including Schizophrenia , Rett syndrome , fragile-X syndrome , tuberous sclerosis , and Angelman Syndrome .", "However , a major drawback of this model is that it only considers overall activity , which is one-dimensional .", "It implies that either too much excitation or too much inhibition is unhealthy ( Figure 1A ) .", "Although several studies have found evidence that the E/I balance is indeed upset in multiple brain disorders ( Bateup et al . , 2011; Dani et al . , 2005; Gibson et al . , 2008; Kehrer et al . , 2008; Wallace et al . , 2012 ) , a model’s usefulness should not be judged on whether it is nominally true or false , but on its explanatory and predictive powers as compared with competing alternative models .", "In this study , we argue that that even if the E/I imbalance model proves correct , its unidimensionality might ultimately limit its applicability , for three reasons .", "First , by placing all disorders on the same single axis , the E/I imbalance model implicitly lumps together some vastly different disorders , such as epilepsy , schizophrenia and autism ( Figure 1A ) because they share an excess of excitation .", "By extension it implies that the symptoms of diverse disorders could be normalized solely by either enhancing or reducing the level of , say , GABAergic signaling as appropriate .", "Although clinical trials for such GABAergic-based interventions are ongoing ( Braat and Kooy , 2015 ) , no treatment for a neurodevelopmental disorder based on this principle has yet been approved .", "A second issue with the unidimensionality of the E/I imbalance model is that it lumps together all excitatory and inhibitory neural circuit components .", "In Figure 1B , we show a schematic diagram of a generic neural circuit with excitatory components colored red and inhibitory components colored blue .", "The E/I imbalance model implies that varying any of the excitatory components , such as the strength of recurrent excitatory synapses or the input resistances of excitatory neurons , would have the same overall effect on circuit function .", "In contrast , theorists have found that these equivalences often do not hold even in very simple circuit models ( Wilson and Cowan , 1972 ) .", "Third , because the standard E/I imbalance model is given in terms of circuit components , not circuit function , it does not specify which aspect of a neural circuit’s activity should be maintained for healthy performance .", "For example , it leaves unclear which of neuronal firing rates , synchrony , or reliability of responses might be altered if E/I balance is upset .", "To motivate our study , we began by investigating which circuit activity properties are altered in a model brain disorder .", "We re-analyzed published in vivo two-photon Ca2+ imaging data we previously recorded from somatosensory cortex in Fmr1 knockout mice ( Gonçalves et al . , 2013 ) , a well-studied animal model for fragile-X syndrome ( The Dutch-Belgian Fragile X Consortium , 1994 ) .", "We compared the data from wild-type ( WT ) mice with Fmr1 KO mice , across three different developmental time points: just before ( P9–11 ) and after ( P14–16 ) the critical period for heightened activity-dependent synaptic plasticity in L2/3 barrel cortex , and a more mature timepoint ( P30–40 ) .", "Example ΔF/F raster plots from each group are shown in Figure 1C , top left .", "We binned the data into 1 s timebins ( originally imaged at 4 Hz ) , then transformed each neuron’s timeseries of ΔF/F values into a probabilistic sequence of binary ON/OFF values by assuming a Poisson firing model ( Materials and methods ) .", "We then summarized the neural population activity from each animal with three statistics: the mean ON probability across all recorded neurons , the standard deviation ( s . d . ) in ON probability across neurons , and the mean correlation between all pairs of neurons ( Figure 1C , bar charts right and scatter plots lower left ) .", "Together these measures capture both the statistics of the bulk population activity and some indication of the heterogeneity across neurons .", "For mean firing rates , the only change we detected was a decrease in firing probability in WT between P14–16 and P30–40 ( p=0 . 027 ) , which was coupled with an increased s . d . of firing rates ( p=0 . 015 ) .", "We also detected a higher firing rate s . d . in P9–11 KO animals than WT ( p=0 . 031 ) .", "Finally , as previously reported ( Golshani et al . , 2009; Gonçalves et al . , 2013; Rochefort et al . , 2009 ) , we found a substantial decrease in pairwise correlations in both genotypes across development , with slightly higher correlations in KO animals than WT at P9–11 ( p=0 . 029 ) and P14–16 ( p=0 . 047 ) .", "These results show that multiple statistics of cortical circuit activity are altered in Fmr1 KO mice .", "However , two questions remain: ( 1 ) Which circuit components are responsible for these activity alterations ?", "( 2 ) What aspects of these activity alterations impact circuit computation ?", "In the remainder of this study , we used computational simulations and further data analysis to ask whether the E/I imbalance model could help address these questions .", "We first built a detailed spiking neural circuit model of mouse L2/3 somatosensory cortex to explore how its various excitatory and inhibitory components affect the circuit’s spiking output , and found that the E/I imbalance model was not flexible enough to capture key aspects of this relationship .", "We then derived an abstract two-dimensional circuit model that captures more features of the circuit function than the 1-dimensional E/I imbalance model did .", "Using this new model , we found that certain sets of circuit components have redundant effects on circuit function , and that circuit function is vastly more sensitive to changes in some components over others .", "To ask how this 2D model could help interpret brain circuit abnormalities in a particular test case , we fit a version of the model to the Ca2+ imaging data from Fragile-X mouse models presented above .", "We found that the model predicts opposite changes in Fmr1 KO circuit properties at different developmental ages .", "Finally , we applied a new large-scale neural population analysis method ( O'Donnell , 2017b ) to the same Ca2+ imaging data , and found systematic shifts in the distribution of neural circuit activity patterns in Fragile-X that were not predictable from neural firing rates or correlations alone ." ], [ "In the above analysis , we investigated how low-level circuit components affect a high-level circuit input-output function , as parameterized by the slope and threshold of fitted logistic functions .", "But how is this logistic input-output function related to more common measures of neural population activity , such as firing rates and pairwise correlations between neurons ?", "To investigate this , we considered the following reduced statistical model of cortical activity .", "We assumed for simplicity that the magnitude of the total input to the L2/3 circuit can be described by a Gaussian distributed random variable , with zero mean and unit standard deviation ( Figure 4A lower left ) .", "Then we described each L2/3 neuron’s input-output as a logistic function as before ( Figure 4A upper left ) , with threshold and slope defined relative to the Gaussian input’s mean and standard deviation , respectively .", "Given this model , we can numerically calculate the probability distribution over a neuron’s firing probability , which in general is skewed and non-Gaussian ( Figure 4A upper right ) .", "From this function , we compute ( Materials and methods ) both the neuron’s mean firing probability and the pairwise correlation of two identical neurons following this profile ( Figure 4A lower right ) .", "Example samples from the model are illustrated in Figure 4B .", "Neural firing rates and correlations had qualitatively different dependencies on the underlying logistic model’s slope and threshold .", "Neural firing rate was greatest when threshold was low and slope was high ( top left of phase plot , Figure 4C left ) , whereas correlations were greatest when both threshold and slope were high ( top right of phase plot , Figure 4C right ) .", "This implies that any change in the circuit’s input-output function slope or threshold will in general have distinct effects on firing rate versus correlations , and so could not be captured by a 1-dimensional E/I balance model that sought to , for example , normalize firing rates alone .", "To illustrate this fact , we plot the calculated correlation values along a contour where firing probability is fixed at 0 . 1 ( Figure 4D ) .", "In the region of parameter space where both the slope and threshold are low ( Figure 4C bottom left ) , correlations are low , ~0 . 01 .", "However , as we move along the contour for firing rate = 0 . 1 toward the region of parameter space where slope and threshold are high ( Figure 4C top right ) , the pairwise correlations increase to ~0 . 4 .", "This shows that a one-dimensional E/I balance rule that exclusively sought to normalize neural firing rates would leave neural correlations free to achieve arbitrary values .", "Previous studies have found evidence for an E/I imbalance in ASD ( Lee et al . , 2017; Nelson and Valakh , 2015 ) .", "Fragile-X syndrome is the leading inherited cause of ASD , and also carries alterations in excitability ( Contractor et al . , 2015 ) .", "We aimed to interpret our Fmr1 KO Ca2+ imaging data ( Figure 1C ) via the 2D logistic model .", "Since our earlier analysis found substantial within-animal heterogeneity in neural activity levels in both genotypes ( Figure 1C ) , we extended the 2D logistic model for single neurons to a 5D neural population version that captured cell-to-cell heterogeneity .", "The three extra parameters represented the standard deviations and correlation in slope and threshold parameters across the neural population ( see Materials and methods for details ) .", "We fit the parameters of the logistic model to reproduce the same neural population Ca2+ imaging data presented in Figure 1C .", "Given the three summary statistics from each animal ( firing rate mean and s . d . , and mean pairwise correlation ) , we used a gradient descent algorithm to find the five parameters of the population-level version of the logistic model that best matched the activity statistics ( see Materials and methods ) .", "The output statistics of the fitted models matched well those of the target data ( Figure 5–figure supplement 1 ) .", "Example neural population activity patterns drawn from the mean model fits for each group are shown in Figure 5A , along with the fitted slope-threshold functions ( Figure 5A insets ) , to be compared with the Ca2+ imaging rasters in Figure 1C .", "We also plot the full 5D parameter fits for all animals in Figure 5–figure supplement 2 .", "For the rest of the analysis , we focus on the mean slope and mean threshold parameters , which showed the most prominent changes .", "In Figure 5B , we plot the mean slope and mean threshold fits on top of the previously calculated ( Figure 4C ) 2D slope-threshold maps of firing rate and correlation .", "We found that in young animals , P9–11 , most points were scattered at high values of both slope and threshold ( Figure 5B left ) .", "With age , the parameter fits for both genotypes moved south-west toward the low slope and low threshold region of parameter space ( Figure 5B center and right ) .", "The mean location of the cloud of points at each developmental age differed between WT and KO .", "We plot the direction of shift in group mean from WT to KO in Figure 5C .", "In young animals , P9–11 and P14–16 , the KO group had both higher slope and higher threshold than WT , whereas in adult animals , P30–40 , the KO group had a lower slope and lower threshold than WT .", "These results demonstrate an opposite direction of circuit parameter change in young Fragile-X mice compared to adults , which was not be uncovered by measures of neural firing rates and correlations ( Figure 1C ) .", "Earlier we asked how sensitive the logistic model slope and threshold parameters were to alterations in the many underlying neural circuit components ( Figure 3 ) .", "In a similar way , we can also ask how sensitive the neural firing rates and correlations are to alterations in the logistic slope and threshold parameters .", "This is important since inspection of the two-dimensional maps in Figure 3C shows that these sensitivities will differ depending on starting location within the slope-threshold space .", "To quantify this effect , we calculated the sensitivity of both the firing rate and correlations to small changes in the slope and threshold ( Figure 6A–B , see Materials and methods ) , quantified as the partial derivatives local to the fitted logistic parameter values for each animal ( black and red circles in Figure 5B ) .", "In general , increasing the slope or decreasing the threshold always increased both firing rates and correlations , as can be predicted from Figure 5B .", "However , the magnitude of sensitivities varied across animals .", "We found only minor differences in sensitivities between genotypes ( Figure 6C–D ) , and as a result we pooled the sensitivity measurements across genotypes to test for statistical differences in sensitivity with developmental age .", "In young animals , P9–11 , changes in the logistic threshold ( solid bars in Figure 6 ) had substantial effects on both firing rates and correlation .", "This sensitivity decreased with age ( p≤0 . 013 for firing rates , p<0 . 01 for correlations from P9–11 to P14–16 ) , so that in adult animals , P30-40 , changes in threshold had relatively little effect on neural activity statistics .", "A different picture emerged for the logistic slope parameter ( striped bars in Figure 6 ) .", "There , the firing rate sensitivity increased from P9–11 to P14–16 ( p<1e-6 ) ( Figure 6C ) , while correlation sensitivity stayed approximately constant ( p≥0 . 18 ) ( Figure 6D ) .", "These results show that the quantitative relationships between neural activity statistics and the parameters of the logistic model , and perhaps also the underlying circuit components , are not fixed across development .", "What are the functional implications of these alterations in firing rates and correlations in Fragile-X mice across development ?", "To address this , we calculated the entropy of the neural population activity for the data from each animal .", "Entropy is a quantity from information theory , measured in bits , that puts a hard upper bound on the amount of information that can be represented by any coding system ( Cover and Thomas , 2006 ) .", "Intuitively , the entropy measures how uniform the neural population activity pattern distribution is: it is large if the circuit exhibits many different activity patterns over time , and small if only a few activity patterns dominate .", "Entropy is an appealing measure for the present problem because it is sensitive both to neural firing rates and to correlations at all orders .", "It is typically highest when firing rates are high and correlations are low .", "Although entropy is notoriously difficult to calculate for large neural populations because most estimation methods require impractically long data recordings ( Quian Quiroga and Panzeri , 2009 ) , we recently developed a new statistical method for this purpose , called the population tracking model , that scales well to large numbers of neurons , even for limited data ( O'Donnell , 2017b ) .", "This model matches both the synchrony distribution for the number of neurons simultaneously active , and the variations in individual cell-to-cell firing rates .", "We fit this population tracking model to the same Ca2+ imaging data as analyzed above ( Figure 7 ) .", "An intermediate step in estimating the neural entropy involves calculating a low-parameter approximation of the entire probability distribution over all 2N neural population activity patterns , where N is the number of neurons .", "The cumulatives of these probability distributions calculated for 50-neuron subsets of the recordings are shown in Figure 7A .", "In young animals P9–11 , a small number of activity patterns accounts for a large fraction of the probability mass ( Figure 7A left ) .", "For example , based on these curves , 50% of the time we would expect to see the same 1000–10 , 000 patterns out of a possible total 250 ≈ 1015 patterns .", "In contrast , in older animals P14–16 and P30–40 the cumulative distributions shift rightwards so that more patterns are typically observed ( Figure 7A center , right ) .", "In these cases , around 1 , 000 , 000 patterns are needed to account for 50% probability mass .", "Instead of attempting to quantify these shifts by asking how many patterns are needed to cross an arbitrary threshold of probability mass , we instead calculated the entropy , which takes into account the shape of the entire probability distribution .", "The entropy depends on the number of neurons analyzed , so we normalized all estimates to calculate the entropy per neuron ( Figure 7B–C ) .", "Since we are treating neurons as binary , the entropy/neuron was bounded between 0 and 1 bits .", "For all age groups , and for both WT and Fmr1 KO animals , entropy/neuron progressively decreased with the number of neurons analyzed ( Figure 7B ) .", "Because each imaging session captured a different number of neurons ( range 40–198 , median 97 ) , we fit the entropy/neuron versus number of neurons data with a double exponential function ( see Materials and methods ) and use the fit to provide a standardized estimate of the entropy/neuron for 100-neuron populations ( Figure 7C ) .", "In WT animals , entropy/neuron showed a non-monotonic trajectory across development ( O'Donnell , 2017b ) .", "At P9–11 it was low , 0 . 38 bits ( 95% c . i . [0 . 35:0 . 41] ) , before increasing at P14–16 ( p<0 . 001 ) to 0 . 50 bits ( 95% c . i . [0 . 48:0 . 52] ) , before decreasing again at P30–40 ( p=0 . 028 ) to 0 . 45 bits ( 95% c . i . [0 . 42:0 . 48] ) .", "We found a different entropy trajectory in Fmr1 KO animals .", "There , although entropy/neuron also began low at 0 . 34 bits ( 95% c . i . [0 . 30:0 . 39] ) , not different from WT ( p=0 . 19 ) , when it increased at P14–16 ( p<0 . 001 ) to 0 . 465 bits ( 95% c . i . [0 . 45:0 . 48] ) it remained lower than for WT ( p=0 . 048 ) .", "Finally , instead of decreasing as in the WT case , entropy continued to increase in P30–40 Fmr1 KO animals ( p=0 . 033 ) to 0 . 51 bits ( 95% c . i . [0 . 47:0 . 55] ) , higher than WT ( p=0 . 034 ) .", "These entropy values estimated directly from Ca2+ imaging data agreed well with entropy estimates for synthetic data sampled from the previously fit logistic models ( Figure 7–figure supplement 1 ) .", "In summary , unlike WT animals , Fmr1 KO mice showed a monotonically increasing entropy/neuron from P9–11 to P30–40 .", "Furthermore , the direction of change in entropy between P14–16 and P30–40 was opposite for WT and Fmr1 KO animals , decreasing in the former and increasing in the latter ." ], [ "The one-dimensional E/I imbalance model has been widely used for interpreting neural circuit changes observed in animal models of diverse brain disorders ( Bateup et al . , 2011; Dani et al . , 2005; Gibson et al . , 2008; Kehrer et al . , 2008; Wallace et al . , 2012 ) .", "In the case of Fragile-X syndrome , the hyperexcitability prediction of the E/I imbalance model is consistent with many of the symptoms of the disease ( e . g . seizures , hyperarousal , hyperactivity , hypersensitivity to sensory stimuli ) and the known pathogenic defects implicated in Fmr1 KO mice ( diminished GABA signaling , exaggerated intrinsic excitability , increased neuronal firing rates; reviewed by Contractor et al . , 2015 ) .", "Here , we tested the hypothesis that the E/I imbalance model can account for alterations in other neural activity statistics beyond the mean firing rates; however , our results demonstrated that it was inadequate .", "The model was too inflexible to account for the joint alterations in both neural firing rates and correlations observed in Fragile-X model mice .", "This suggests that future studies of brain disorders may need to consider higher-dimensional models of neural circuit dysfunction .", "To test how cellular components affect their circuit function , we built computational models of mouse L2/3 somatosensory cortex at two levels of abstraction: a detailed , 100-parameter biophysical model , and a two-parameter logistic response model .", "The purpose of the detailed model was to build a representation of the circuit where each parameter has a one-to-one mapping with something that could be experimentally measured in a real animal - indeed many of these parameters have been shown to be altered in Fmr1 KO mice .", "The purpose of the logistic model was different: it simple enough to be both derivable from the complex model , and provide a direct link with measurable activity variables in our in vivo Ca2 +imaging data , firing rates and correlations .", "The disadvantages of detailed models are that they contain many parameters , and so are hard to constrain to data – in this case it was only possible because of the large dataset from Petersen et al . , for P17-22 WT mice ( Avermann et al . , 2012; Lefort et al . , 2009; Tomm et al . , 2014 ) .", "The disadvantages of the simple 2D model is that its logistic input-output structure implies a very strong and specific assumption about the functional purpose of the circuit – to generate single spikes across a subset of neurons .", "Although this may be a physiologically relevant computation for this particular brain circuit ( Clancy et al . , 2015; Kerr et al . , 2007; Sato et al . , 2007 ) , it is not immediately obvious how to extend this approach to include temporal correlations , for example , or to apply it to other brain circuits where we may have less insight into their natural computations .", "Nevertheless , our approach demonstrates a new way to tackle such problems .", "After building the detailed computational model of L2/3 of mouse somatosensory cortex ( Figure 2 ) , we asked how sensitive the spiking responses of the overall circuit were to changes in its underlying neural components , many of which are known to be altered in Fmr1 KO mice ( Bureau et al . , 2008; Gibson et al . , 2008; Gonçalves et al . , 2013; Harlow et al . , 2010; Hays et al . , 2011; Paluszkiewicz et al . , 2011; Patel et al . , 2013; Testa-Silva et al . , 2012 ) .", "We found that while changing some neural parameters did have a large effect , changing other parameters had little or no effect on circuit function ( Figure 3B ) .", "This redundancy property has been reported as widely prevalent in computational models of biological systems ( Gutenkunst et al . , 2007; O'Leary et al . , 2015 ) .", "Its existence has two important implications for studies of brain disorders: first , many of the physiological component changes discovered in animal models may be entirely benign at the circuit level .", "Second , any treatment designed to correct circuit function is free to push the system by arbitrary amounts along insensitive directions in parameter space without consequence , as long as it makes the correct perturbations along the sensitive directions .", "The insensitive directions form a null space , which is a subspace of the parameter space .", "An important caveat to our parameter sensitivity analysis is that it was linear and local to a particular point in the high-dimensional model parameter space , corresponding to WT P17-22 mice .", "Since the circuit dynamics are nonlinear , it is likely that the particular parameter sensitivities would be different in other parts of parameter space , especially near bifurcations where qualitatively different dynamics emerge ( Hirsch et al . , 2013 ) .", "However , as long as the redundancy property is widely preserved , as suggested by studies on computational models of other biological systems ( Fisher et al . , 2013; Gutenkunst et al . , 2007; Machta et al . , 2013; Panas et al . , 2015 ) , then our conclusions for brain disorders remain valid .", "In addition to the varying magnitudes of circuit components’ effect on circuit function , we also found that different components shifted the circuit input-output function in different directions , as defined by our 2D logistic response model ( Figures 1 and 2 ) .", "Even circuit parameters that are nominally of the same type , such as the strength of glutamatergic synapses between excitatory ( E ) neurons in L4 to E neurons in L2/3 or synaptic strength between E neurons within L2/3 , had qualitatively different effects on the circuit response to stimulation ( Figure 3 ) .", "According to the standard E/I imbalance model ( Rubenstein and Merzenich , 2003 ) , both of these parameters should have similar effects on circuit function; but according to the logistic response model we studied , their differing effects on slope and threshold parameters must necessarily lead to different magnitudes of change in neural firing rates and correlations ( Figure 4C ) .", "Indeed , no 1-dimensional model of circuit function could ever capture the heterogeneity in parameter sensitivities that we observed ( Figure 3B ) .", "Next , we fit the parameters of the logistic response model to match the in vivo firing statistics of neural populations from WT and Fmr1 KO mice of varying age ( Figure 5 ) .", "Previous studies had found that neural correlations decrease during development ( Golshani et al . , 2009; Rochefort et al . , 2009 ) , and that early postnatal Fmr1 KO mice had higher correlations and firing rates than WT mice ( Gonçalves et al . , 2013; La Fata et al . , 2014 ) .", "Circuit hypersynchrony may be a general defect in autism disorders , as it is also found in mouse models of Rett syndrome ( Lu et al . , 2016 ) .", "However , the relationship between these changes in firing statistics and the underlying neural circuit components were unclear .", "Our logistic model helps bridge this gap , leading to two findings: first , the direction of circuit parameter change from WT to KO was opposite in young ( P9–11 and P14–16 ) versus mature ( P30–40 ) animals ( Figure 5C ) .", "Similar opposing switches in sensory cortex properties with age were also recently reported in Fmr1 KO and WT rats ( Berzhanskaya et al . , 2016 ) .", "Second , we found that the sensitivity of neural firing rates and correlations to changes in underlying circuit components depends on developmental age ( Figure 6 ) .", "Taken together , these findings imply that qualitatively different interventions may be needed at different stages of development in Fragile-X , and perhaps other neurodevelopmental disorders , to shift cortical circuit function towards typical wild-type operation .", "Spontaneous , intrinsic activity is ubiquitously present in mammalian cerebral cortex .", "It is highly structured at multiple spatiotemporal scales ( Mitra et al . , 2015; Ringach , 2009 ) and interacts strongly with the signals evoked by sensory stimulation ( Ringach , 2009 ) .", "Cellular-resolution recordings in animals have shown that the patterns of spontaneous activity in neural populations are representative of the ensemble of activity patterns used by the brain to represent sensory stimuli ( Berkes et al . , 2011; Luczak et al . , 2009; Miller et al . , 2014 ) .", "Here , we found that the entropy of spontaneous activity in WT mouse somatosensory cortex follows an inverted-U shaped trajectory across development , and that this trajectory is dramatically altered in the Fmr1 KO mouse model of Fragile-X ( Figure 7 ) .", "Although we saw no reliable differences across genotypes in early postnatal animals ( P9–11 ) , Fmr1 KO animals showed lower entropy than WT after the second postnatal week ( P14–16 ) , while surprisingly switching to show higher entropy than WT in adult ( P30–40 ) .", "Notably , this switch in the direction of entropy change from WT to KO during development mirrors the reversing we saw in logistic model parameter changes in Figure 5C .", "Together , these findings suggest a perturbed trajectory of cortical development during the critical period in Fmr1 KO mice ( Meredith et al . , 2012 ) .", "However , our results cannot distinguish whether the observed perturbation in L2/3 activity statistics reflects a developmental delay , or a permanently altered developmental trajectory .", "Further studies at later developmental time points are needed .", "What is the functional significance of these shifts in population entropy ?", "Previous work suggested that the entropy of neural circuit activity may be optimally tuned at intermediate levels as a trade-off between maximizing representational capacity at high entropy , versus maintaining error correction and regularization at low entropy ( Schneidman et al . , 2006 ) .", "These properties can also be thought of as trading off between discrimination and generalization , respectively ( Qian and Lipkin , 2011 ) .", "If we assume that WT mice are optimally tuned , our findings predict that young Fmr1 KO mice should show poorer somatosensory discrimination in behavioral tasks than wild-type animals , while in contrast adult Fmr1 KO mice should perform more poorly on tasks involving generalization across somatosensory stimuli .", "If the unidimensional E/I imbalance model is not sufficiently rich to capture the circuit changes observed in neurodevelopmental disorders , what should replace it ?", "How many dimensions or degrees of freedom should a working model for a brain disorder have ?", "Theoretical neuroscientists have long studied E/I balance in generic models of recurrent neural circuits ( Brunel and Brunel , 2000; Tsodyks and Sejnowski , 1995 ) .", "These models have uncovered important distinctions between ‘loose’ balanced regimes , where E and I inputs to a neuron are equal only on average , and fine-tuned ‘tight’ balanced regimes where E and I inputs to a neuron track each other closely on fast timescales ( Denève and Machens , 2016; Hennequin et al . , 2017 ) .", "In principle , these generic models could be used to investigate multidimensional E/I imbalances in brain disorders ( Vogels and Abbott , 2007 ) .", "However , it is currently difficult to directly fit these many-parameter network models to data ( although see Arakaki et al . , 2017; Fisher et al . , 2013; Stringer et al . , 2016 ) , and they are agnostic to circuit function .", "Instead we suggest an alternative , complementary approach: start by assuming a computational function for the particular neural circuit under study , then work backwards to design a model that is both sophisticated enough to capture the key information processing features of the circuit , but simple enough to interpret and link to physiological data .", "In this study , we considered a two-parameter model of L2/3 somatosensory cortex’s input-output function , which could account for both neural firing rates and correlations .", "Other brain circuits may demand models with more degrees of freedom .", "Crucially , the most informative models need not be those that include the highest level of physiological detail .", "All models are ultimately wrong in the sense that they make abstractions about their underlying parts , and detailed models carry the additional burden of fitting many parameters , which may be difficult to adequately constrain ( O'Leary et al . , 2015 ) .", "Nonetheless , some models are useful ( Box , 1979 ) .", "One potential use of simple parametric circuit models such as the ones we employed here may be as a tool for rationally designing candidate intervention compounds and then screening their effects on neural population activity .", "For example , the current study could have been extended to fit the logistic model to neural activity data from another cohort of Fmr1 KO mice that had received a candidate treatment , then ask if the fitted model parameters were closer in value to those from WT animals or Fmr1 KO controls .", "Approaches like this could complement the traditional strategy of designing drugs based on reversing molecular deficits and then assessing the drug’s impact on animal model behavior .", "Indeed , our results suggest that given the multi-dimensionality of circuit properties , it may prove difficult or impossible to find a single compound that can correctly reverse deficits at any age .", "This scenario might require a combination of drugs chosen to push circuit-level properties towards the ‘correct’ region of parameter space .", "The framework we have introduced in this study can facilitate this type of high-dimensional intervention analysis for diverse neurodevelopmental disorders ." ], [ "All Ca2+ imaging data were published previously ( Gonçalves et al . , 2013 ) .", "Briefly , data were collected from male and female C57Bl/6 wild-type and Fmr1 KO mice at P9–40 .", "For each group the animal numbers were: P9-11 , n = 13 WT and n = 9 Fmr1 KO; P14-16 , n = 8 WT and n = 10 Fmr1 KO; P30-40 , n = 7 WT and n = 6 Fmr1 KO .", "There were variations in the number of cells recorded from each animal .", "The range of cell numbers for each group were: P9-11 , 49–198 cells in WT and 84–144 cells in Fmr1 KO; P14-16 , 65–119 cells in WT and 40–149 cells in Fmr1 KO; P30-40 , 60–114 cells in WT and 69–105 cells in Fmr1 KO .", "Mice were anesthetized with isoflurane , and a cranial window was fitted over primary somatosensory cortex by stereotaxic coordinates .", "Mice were then transferred to a two-photon microscope and headfixed to the stage while still under isoflurane anesthesia .", "2–4 injections of the Ca2+ sensitive Oregon-Green BAPTA-1 ( OGB ) dye and sulforhodamine-101 ( to visualize astrocytes ) were injected 200 um below the dura .", "Calcium imaging was performed using a Ti-Sapphire Chameleon Ultra II laser ( Coherent ) tuned to 800 nm .", "Imaging in unanesthetized mice began within 30–60 min of stopping the flow of isoflurane after the last OGB injection .", "Images were acquired using ScanImage software ( Pologruto et al . , 2004 ) written in MATLAB ( MathWorks; RRID:SCR_001622 ) .", "Whole-field images were collected using a 20 × 0 . 95 NA objective ( Olympus ) at an acquisition speed of 3 . 9 Hz ( 512 × 128 pixels ) .", "Several 3 min movies were concatenated and brief segments of motion artifacts were removed ( always <10 s total ) .", "Data were corrected for x–y drift .", "Cell contours were automatically detected and the average ΔF/F signal of each cell body was calculated at each time point .", "The baseline F for each cell was calculated as the mean ROI fluorescence across the entire 3 min timeseries .", "Neuronal and neuropil signals were analyzed separately and astrocytic signals were excluded from analysis .", "Each ΔF/F trace was low-pass filtered using a Butterworth filter ( coefficient of 0 . 16 ) and deconvolved with a 2 s single-exponential kernel ( Yaksi and Friedrich , 2006 ) .", "To remove baseline noise , the standard deviation of all points below zero in each deconvolved trace was calculated , multiplied by two , and set as the positive threshold level below which all points in the deconvolved trace were set to zero .", "Estimated firing rates of the neurons , ri ( t ) , were then obtained by multiplying the deconvolved trace by a factor of 78 . 4 , previously derived empirically from cell-attached recordings in vivo ( Golshani et al . , 2009 ) .", "Data analysis and logistic model calculations were done using MATLAB ( Mathworks; RRID:SCR_001622 ) .", "All simulation codes are available online at https://github . com/cianodonnell/ODonnelletal_2017_imbalances ( copy archived at https://github . com/elifesciences-publications/ODonnelletal_2017_imbalances ) , and the population tracking model code ( O'Donnell et al . , 2017 ) is available at https://github . com/cianodonnell/PopulationTracking .", "( copy archived at https://github . com/elifesciences-publications/PopulationTracking )", "Layer 2/3 model simulations ( Figures 1 and", "2 ) were implemented with the Python-based simulator Brian 2 ( http://briansimulator . org; RRID:SCR_002998 ) ( Goodman and Brette , 2009 ) , and results analyzed with MATLAB ( Mathworks; RRID:SCR_001622 ) .", "The model consisted of four populations of reciprocally connected leaky integrate-and-fire neurons representing a L2/3 somatosensory barrel circuit: 1700 excitatory neurons , 70 PV inhibitory neurons , 115 5HT3AR inhibitory neurons , and 45 SOM inhibitory neurons , driven by a separate population of 1500 excitatory spike sources representing input from L4 .", "Cell numbers were estimated by combining layer-specific excitatory and inhibitory cell count information from ( Lefort et al . , 2009 ) with the approximate percentages of the three inhibitory cell groups given by ( Petersen and Crochet , 2013 ) .", "The voltage V of each neuron evolved asdVdt = ( Rin ( ge ( Erev , e - V ) + gi ( Erev , i - V ) ) - ( V-Vrest ) ) /τm where Rin is the input resistance , Erev , eandErev , i are the excitatory and inhibitory synaptic reversal potentials respectively , τm is the membrane time constant , and geandgi are the summed excitatory and inhibitory synaptic input conductances , respectively .", "Between input events the total excitatory synaptic condunctance ge evolved in time according to the equationdgedt=-ge/τsyn , e where τsyn , e is the excitatory synaptic time constant .", "Similar equations governed the inhibitory conductances .", "When a spike arrived at a synapse , a Bernoulli random number was drawn with release probability set according to the particular synaptic connection type .", "If this number was equal to one , then the total synaptic conductance for that neuron was instantaneously incremented by the specific amplitude of the chosen conductance for that individual synapse , indexed j: ge→ge+g-j .", "All synaptic connections were formed probabilistically by drawing independent random Bernoulli variables with connection type-specific probabilities .", "Synaptic PSP amplitudes were drawn independently for each synapse from a log-normal distribution constrained by the experimentally reported mean and median values for each particular connection type .", "The maximum post-synaptic potential amplitude was set to 8 mV .", "Synapses in the model were conductance-based , but since synaptic strengths reported in the literature were typically in terms of EPSP/IPSP amplitude , in accordance with how the experiments were performed ( Avermann et al . , 2012 ) , we set each maximal synaptic conductance as the value needed to generate a PSP of the desired amplitude when the target neuron started at resting potential in the case of EPSPs or −55 mV in the case of IPSPs , which we computed analytically .", "Refractory periods were calculated as the inter-spike-interval corresponding to the maximal experimentally reported firing rate .", "Release probability and synaptic strength values for unconnected neurons are excluded from Table 1 .", "Excitatory synaptic time constants were set at 2 ms , which is typical for the fast component of AMPA receptor responses , but could not be estimated from the PSP statistics in ( Avermann et al . , 2012 ) because of masking by the slower membrane time constant .", "The mathematical form of our model meant that inhibitory synaptic time constants needed to be equal for all incoming inhibitory synapses to a neuron .", "We set these to 40 ms for E , 5HT3AR and SOM neurons and 16 ms for PV neurons , which were the typical values of the IPSP decay time constants in the ( Avermann et al . , 2012 ) dataset .", "Due to lack of direct data for this circuit , connection probabilities for synapses from L4 E neurons to E , PV and SOM L2/3 neurons was set to a reasonable cortical value of 0 . 15 , while 5HT3AR neurons did not receive any input from L4 ( Gentet et al . , 2012 ) .", "Similarly due to a lack of direct data , we set synaptic release probabilities for connections from L4 to L2/3 neurons to a typical cortical value of 0 . 25 , while mean and median L4 excitatory PSP amplitudes onto L2/3 PV and SOM were set to 0 . 8 and 0 . 48 mV , respectively , to match reported data for L4 EPSP amplitudes onto L2/3 E neurons ( Lefort et al . , 2009 ) .", "The differential equations were solved using the forward Euler method with an integration timestep of 0 . 01 ms . Each simulation run was 50 ms long , during which we recorded whether or not each neuron responded .", "In the rare cases where a neuron spiked more than once , we disregarded the extra spikes .", "L4 neuron dynamics were not explicitly simulated , but instead modeled only as a set of output spike trains .", "After selecting the subset of active L4 neurons , spike times were drawn randomly from a Gaussian distribution with standard deviation of 2 ms . We repeated the simulations 10 times for this identical input pattern to average over the noise due to probabilistic vesicle release .", "We repeated this procedure further 10 times for different random allocations of the ‘ON’ inputs .", "Then , a neuron’s ON probability was defined as the fraction of these 10×10=100 simulations for which it responded with one or more spikes .", "Finally , we repeated the entire procedure for varying levels of L4 input sparsity .", "For the simulations presented in Figure 3 we varied only 76 model parameters , which is 24 less than the total number of 100 model parameters listed in Table 1 .", "We excluded the four neuronal refractory periods ( because in almost all simulations each neuron spiked a maximum of once , making the refractory period irrelevant ) , and the six connection probabilities that were fixed at zero .", "Finally , we grouped together the mean and median PSP amplitudes for each of the fourteen non-zero synaptic connections , so that both parameters were increased or decreased by the same fraction in tandem .", "Together these choices reduced the number of test parameters from 100 to 76 .", "For all parameters that naturally range from 0 upwards , such as the number of neurons or release probability , we increased or decreased their values during testing in the most intuitive way , by adding ±20% of the baseline value .", "However , this method was less useful for other parameters , such as cell resting voltage , for which we reasoned it made more sense to scale relative to another parameter , such as spike threshold .", "As a result , we varied ( 1 ) resting voltage relative to its difference from spike threshold; ( 2 ) spike threshold relative to its difference with resting voltage; ( 3 ) excitatory synaptic reversal potentials relative to resting voltage; ( 4 ) inhibitory synaptic reversal potentials relative to spike threshold .", "Source [1] is ( Lefort et al . , 2009 ) , [2] is ( Avermann et al . , 2012 ) , [3] is ( Fanselow et al . , 2008 ) , [4] is ( Kinnischtzke et al . , 2012 ) , [5] is ( Fino and Yuste , 2011 ) .", "N is number of neurons , Vrest is resting potential , Vth is spike voltage threshold , Rin is input resistance , tref is refractory period , τm is the membrane time constant , τsyn is the synaptic time constant with the first subscript indicating the postsynaptic neuron type and the second subscript the neurotransmitter type of the presynaptic neuron ( e or i ) , Erev is the synaptic reversal potential , pcon is the synaptic connection probability , prel is the synaptic release probability , w is the mean or median post-synaptic potential amplitude as indicated .", "For all neuronal parameters , the subscript indicates the neuron type: E is L2/3 excitatory neurons , Ipv is PV neurons , I5ht is 5HT3AR neurons , Isom is SOM neurons , and EL4 is L4 excitatory neurons .", "For synaptic parameters , the first and second subscripts indicate the pre- and post-synaptic neuron types , respectively .", "An important caveat is that although this model may be considered detailed by some measures , it also simplifies many aspects of L2/3 circuit .", "For example , we assumed that all 5HT3AR cells were homogeneous , even though they likely separate into different subclasses with type-specific connectivity ( Gentet , 2012; Petersen and Crochet , 2013 ) .", "Layer 2 and layer 3 may also consist of distinct cell populations ( Petersen and Crochet , 2013 ) .", "Not all likely connections were included in the model ( Dalezios et al . , 2002; Pfeffer et al . , 2013 ) , and connectivity was assumed to be random , even though it is likely non-random ( Tomm et al . , 2014 ) .", "Although these choices will likely not affect the conclusions of the current study , they may be important to consider for future work that seeks to understand the biological function of the L2/3 somatosensory microcircuit .", "From the L2/3 circuit model simulations , we numerically estimated the probability q that each neuron in the model fires a spike as a function of the fraction of L4 inputs that were active , f .", "We then used the generalized linear model regression tool ‘glmfit’ in MATLAB to find the best fit of the two logistic model parameters for each neuron:q ( f ) =11+exp⁡ ( -β ( f-f1/2 ) ) where the parameter β represents the slope , and the parameter f1/2 represents the fraction of active L4 neurons at which the response probability q=0 . 5 .", "For clarity of presentation , in the main text we converted this f1/2 parameter to what we termed the ‘threshold’ , fthresh , which we defined as the fraction of L4 neurons needed to reach a specified spike probability , qthresh .", "Throughout the study , we fixed qthresh=0 . 01 .", "The threshold is related to qthresh via the inverse of the logistic functionfthresh=f1/2+log⁡qthresh1-qthresh/β We computed firing rates and pairwise correlations from the logistic model ( Figures 4–5 ) in the following way .", "First , we assumed that the fraction of active L4 neurons is described by a normally distributed random variable with zero mean and unit variance:pf=exp⁡ ( -f2/2 ) 2π=𝒩 ( 0 , 1 ) We defined the β and f1/2 parameters relative to the mean and standard deviation of the input distribution .", "Since q is a monotonically increasing function of f , the probability distribution for q ispq=p ( fq ) dfdq where f ( q ) is the inverse of the logistic function q ( f ) and dfdq=exp⁡-βf-f1/2+12βexp⁡ ( -β ( f-f1/2 ) ) .", "We calculate a neuron’s mean firing rate μ as the expectation of q , μ=E[q]=∫0 1[q×p ( q ) ]dq=∫0 1[q×p ( f ( q ) ) |dfdq|]dq We calculate the pairwise covariance of two homogeneous neurons driven by a common input f as cov=E[q2]− ( E[q] ) 2=E[q2]−μ2=∫0 1[q2×p ( f ( q ) ) |dfdq|]dq−μ2 , then find the pairwise correlation by normalizing the covariance by the neurons’ shared variance , var=μ ( 1-μ ) .", "For fitting the logistic model to the recorded neural firing rates and correlations ( Figure 5 ) , we considered a population model where the joint probability distribution across threshold and slope was specified by a 2D Gaussian , which has five parameters: threshold mean and s . d . , slope mean and s . d . , and slope-threshold correlation .", "The three constraint statistics we considered from the neural population data were the mean neural ON probability , the s . d . of neural ON probabilities , and the mean pairwise correlations .", "We found the best-fit model parameters for each dataset using stochastic gradient descent ( code available at https://github . com/cianodonnell/ODonnelletal_2017_imbalances ) .", "Briefly , the fitting procedure followed: ( 1 ) initialize the parameters at a starting guess points , ( 2 ) compute the predicted three output firing statistics via numerical integration over the model’s probability distributions , ( 3 ) compute the fitting error as the summed squared difference between the model output predictions and the target values , ( 4 ) generate a new set of parameter values by adding a small perturbation of a zero-mean Gaussian random number to each parameter , ( 5 ) compute the new output statistics , ( 5 ) recompute the fitting error , ( 6 ) if the new error is smaller than the old error , accept the updated parameter values , otherwise reject them and revert to the old parameters , ( 7 ) return to step 4 unless the error is lower than the desired tolerance .", "We checked for fit convergence by sampling a large number of logistic model parameters from the fitted 2D Gaussian , drawing binary samples from these logistic model neurons and computing the ON probability mean and s . d . , and mean pairwise correlation from the synthetic binary samples , and comparing the computed statistical values to the original data statistics ( Figure 5 – figure supplement 1 ) .", "For the sensitivity analysis presented in Figure 6 , we numerically computed the partial derivative in mean firing rate and pairwise correlation with respect to the mean slope and mean threshold parameters in the population logistical model , using standard finite difference methods .", "To avoid parametric assumptions , all statistical tests were done using standard bootstrapping methods with custom-written MATLAB scripts .", "For example , when assessing the observed difference between two group means Δμobs we performed the following procedure to calculate a p-value .", "First , we pool the data points from the two groups to create a null set Snull .", "We then construct two hypothetical groups of samples S1 and S2 from this by randomly drawing n1 and n2 samples with replacement from Snull , where n1 and n2 are the number of data points in the original groups 1 and 2 respectively .", "We take the mean of both hypothetical sets Δμ1 and Δμ2 and calculate their difference Δμnull=Δμ1−Δμ2 .", "We then repeat the entire procedure 107 times to build up a histogram of Δμnull .", "This distribution is always centered at zero .", "After normalizing , this can be interpreted as the probability distribution f ( Δμnull ) for observing a group mean difference of Δμnull purely by chance if the data were actually sampled from the same null distribution .", "Then the final p-value for the probability of finding a group difference of at least Δμobs in either direction is given by p=∫-∞-ΔμobsfΔμnulldΔμnull+∫Δμobs∞fΔμnulldΔμnull .", "For Figure 5C , we estimated two-dimensional 95% confidence ellipses for the shift in mean slope-threshold parameters between Fmr1 KO and WT by computing the sample error variances and covariance through bootstrapping .", "Then , the 95% confidence ellipse can be computed using the Chi-squared distribution .", "We plotted the confidence interval ellipse using the MATLAB function error_ellipse . m , downloaded from https://www . mathworks . com/matlabcentral/fileexchange/4705-error-ellipse .", "For the Ca2+ imaging data , we began with estimated firing rate time series ri ( t ) for each neuron i recorded as part of a population of N neurons .", "For later parts of the analysis we needed to convert these firing rates to binary ON/OFF values .", "This conversion involves a choice .", "One option would be to simply threshold the data , but this would throw away information about the magnitude of the firing rate .", "We instead take a probabilistic approach where rather than deciding definitively whether a given neuron was ON or OFF in a given time bin , we calculate the probability that the neuron was ON or OFF by assuming that neurons fire action potentials according to an inhomogeneous Poisson process with rate ri ( t ) .", "The mean number of spikes λi ( t ) expected in a time bin of width Δt is λi ( t ) =ri ( t ) Δt .", "We choose Δt = 1 s .", "Under the Poisson model the actual number of spikes m in a particular time bin is a random variable that follows the Poisson distribution P ( m = k ) = λk exp ( -λ ) /k !", ".", "We considered a neuron active ( ON ) if it is firing one or more spikes in a given time bin .", "Hence , the probability that a neuron is ON is pon ( t ) = 1- P ( m=0 ) = 1-exp ( λ ) .", "This approach has two advantages over thresholding: ( 1 ) it preserves some information about the magnitude of firing rates and ( 2 ) it acts to regularize the probability distribution for the number of neurons active by essentially smoothing nearby values together .", "Entropy was estimated by fitting a statistical model we recently developed , called the population tracking model ( O'Donnell , 2017a ) , to the binarized Ca2+ imaging data .", "Briefly , the population tracking model fits two aspects of the data: the probability distribution for the number of neurons synchronously active in the population , and also the conditional firing probability that each individual neuron is active given the population count .", "Hence , the model captures both some aggregate statistics of the population activity , and some aspects of the heterogeneity across neurons .", "See O'Donnell , 2017b ) for complete details and validation of the method .", "Code for fitting the model to data is available at https://github . com/cianodonnell/PopulationTracking .", "The entropy/neuron generally decreased with the number of neurons considered as result of the population correlations ( Figure 7B ) , so we needed to control for neural population size when comparing data from different experimental groups .", "On the one hand , we would like to study as large a number of neurons as possible , because we expect the effects of collective network dynamics to be stronger for large population sizes and this may be the regime where differences between the groups emerge .", "On the other hand , our recording methods allowed us to sample only typically around ~100 neurons at a time , and as few as 40 neurons in some animals .", "Hence , we proceeded by first estimating the entropy/neuron in each animal by calculating the entropy of random subsets of neurons of varying size from 10 to 100 ( if possible ) in steps of 10 .", "For each population size we sampled a large number of independent subsets , calculated the entropy of each .", "Finally , for each dataset we fit a double exponential function to the estimated entropy/neuron as a function of the number of neurons: H/N = A*exp ( -b*N ) +C*exp ( -d*N ) +e , and used this fit to estimate H/N for 100 neurons ." ] ]

[ "A leading theory holds that neurodevelopmental brain disorders arise from imbalances in excitatory and inhibitory ( E/I ) brain circuitry .", "However , it is unclear whether this one-dimensional model is rich enough to capture the multiple neural circuit alterations underlying brain disorders .", "Here , we combined computational simulations with analysis of in vivo two-photon Ca2+ imaging data from somatosensory cortex of Fmr1 knock-out ( KO ) mice , a model of Fragile-X Syndrome , to test the E/I imbalance theory .", "We found that: ( 1 ) The E/I imbalance model cannot account for joint alterations in the observed neural firing rates and correlations; ( 2 ) Neural circuit function is vastly more sensitive to changes in some cellular components over others; ( 3 ) The direction of circuit alterations in Fmr1 KO mice changes across development .", "These findings suggest that the basic E/I imbalance model should be updated to higher dimensional models that can better capture the multidimensional computational functions of neural circuits ." ]

[ "In many brain disorders , from autism to schizophrenia , the anatomy of the brain appears remarkably unchanged .", "This implies that the problem may reside in how neurons communicate with one another .", "Unfortunately , neuroscientists know little about how brain activity might differ from normal in these disorders , or how specific changes in activity give rise to symptoms .", "One leading theory , first proposed over a decade ago , is that these disorders reflect an imbalance in the activity of excitatory and inhibitory neurons .", "Excitatory neurons activate their targets , whereas inhibitory neurons suppress or silence them .", "While studies in mice have lent support to this theory , they have not yet culminated in new treatments for brain disorders .", "One limitation of the excitation-inhibition imbalance theory is that it is one-dimensional .", "It assumes that there is an optimal balance of excitation and inhibition , and that brain disorders can be arranged in an imaginary line on either side of this optimum .", "Disorders to the right of the optimum , such as epilepsy and some forms of autism , feature too much excitation .", "Disorders to the left , such as the developmental disorder Rett syndrome , feature too much inhibition .", "But can diverse brain disorders really be classified on the basis of a single property , or do scientists need to consider other factors ?", "To find out , O’Donnell et al . analyzed recordings of brain activity from genetically modified mice with the mutation that causes fragile X syndrome , the most common form of inherited learning disability and autism .", "The mice showed changes in their overall brain activity compared to control animals .", "Their neurons also tended to fire in a more synchronized manner .", "A computer simulation revealed that an imbalance in excitation and inhibition alone could not explain these changes .", "Yet , a more complex simulation incorporating extra properties of neural circuits did a better job of explaining the altered neural activity seen in the mice .", "O’Donnell et al . propose that this more advanced multi-dimensional model of changes in neural circuits could be used to screen candidate drugs before testing them in patients .", "In principle , the model could even help with designing drugs or other interventions by making it easier for researchers to target more precisely the changes in neural circuits that occur in brain disorders ." ]

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

[ "epidemiology and global health" ]

[ [ "Malaria remains a major public health challenge , with more than 7% of deaths among children under 5 years in the world attributable to the disease ( Liu et al . , 2012 ) .", "Many of these cases arise in regions with high universal immunization coverage .", "In sub-Saharan Africa alone where 80% of malaria cases occur ( World Health Organization , 2012 ) , district coverage rates are estimated at over 70% for the third dose of diphtheria–tetanus–pertussis ( DTP ) vaccines and 75% for measles-containing vaccines ( World Health Organization and UNICEF , 2013 ) .", "Several studies have suggested that childhood immunization may confer non-specific health effects beyond targeted diseases , but the impact of standard vaccination and vitamin A supplementation on malaria infection is unclear .", "In particular , early evidence from murine models indicates that injection with live strains of Bacille Calmette Guerin ( BCG ) vaccine may confer sterilizing protection against Plasmodium infection ( Clark et al . , 1976 ) .", "However , this effect seems to depend on several factors including route of administration ( intradermal , subcutaneous , intramuscular vs intravenous ) , type of immunogen used ( killed vs live attenuated ) , vaccine schedule in relation to malaria exposure ( before vs after infection ) , mouse strain , sex , and Plasmodium species tested ( Clark et al . , 1976; Smrkovski and Strickland , 1978 , Smrkovski , 1981 , 1982; Murphy , 1981 , Matsumoto et al . , 2000 ) .", "Conversely , the risk of malaria infection has been thought to increase during downregulation in cellular immunity ( including interleukin-12 , interferon-gamma , T cells ) and T helper ( Th ) 2 cell upregulation following measles infection and/or immunization ( Desai et al . , 2005 ) .", "Epidemiological evidence to support this theory in humans remains unclear ( Whittle et al . , 1980; Rooth and Bjorkman , 1992 ) .", "Multiple clinical trials in infants and young children have also identified protective effects from vitamin A supplementation against illness ( number and time to first clinical episode , risk of febrile illness , spleen enlargement , and mean parasite density ) ( Shankar et al . , 1999; Zeba et al . , 2008 ) and death caused by malaria ( Fawzi et al . , 1999; Varandas et al . , 2001; Mwanga-Amumpaire et al . , 2012 ) .", "Vitamin A is thought to act as a regulator of pro-inflammatory response genes ( e . g . tumor necrosis factor alpha ) and phagocytotic clearance ( e . g . cluster of differentiation 36 [CD36] ) of Plasmodium falciparum-infected erythrocytes , by way of its active metabolite , retinoic acid ( SanJoaquin and Molyneux , 2009 ) .", "Less certain is its impact on malaria infection .", "While vitamin A did not appear to modulate the risk of parasitemia in a clinical trial study of Ghanaian children ( Binka et al . , 1995 ) and Tanzanian children previously hospitalized with pneumonia ( Villamor et al . , 2003 ) , a cluster randomized intervention trial for breastfeeding in Uganda observed a sixfold decrease in the adjusted risk of malaria infection among children receiving vitamin A ( Nankabirwa et al . , 2011 ) .", "On the other hand , concern has been raised by an apparent increase in levels of parasitemia within mouse models , when combining vitamin A supplements with DTP vaccination; an effect thought to be stronger among female mice ( Jorgensen et al . , 2011 ) .", "The underlying mechanisms of these effects are unclear , although it has been theorized that vitamin A may act as adjuvant to DTP , which is postulated to cause detrimental effects ( Jorgensen et al . , 2011 ) .", "Based on this evidence , the purpose of this study was to determine the risk of Plasmodium parasitemia and P . falciparum-specific antigenemia following vitamin A supplementation and standard vaccination in children under 5 years of age in sub-Saharan Africa ." ], [ "Of 20 , 984 children who presented health cards during survey interviews , 18 , 413 were eligible for blood screening from which 12 , 058 provided capillary blood for malaria testing ( Figure 1 ) .", "From these , 8672 ( 72% ) were tested using both thick blood films and rapid P . falciparum histidine rich protein-2 ( PƒHRP-2 ) tests , 3356 ( 28% ) were tested only with blood films , and 30 ( 0 . 2% ) were tested with only rapid PƒHRP-2 tests .", "Among those tested , we identified 3544 ( 30% ) with positive blood films for Plasmodium spp .", "and 3131 ( 36% ) with positive PƒHRP-2 antigenemia .", "Results from both tests were 87% concordant .", "Complete confounder information was available for 8390 ( 70% ) subjects who were tested for parasitemia and 6121 ( 70% ) tested for antigenemia .", "Table 1 shows BCG , DTP , and poliomyelitis vaccines were used most often .", "Vitamin A was least used .", "Supplementary file 3 shows that immunization schedules were similar across all countries ( World Health Organization and UNICEF , 2007 ) .", "Malaria was most common among subjects from Burkina Faso and least common among those from Rwanda .", "Although Rwanda had the longest rainy season , they also had the greatest ownership of bed nets , and were least likely to have recently used antimalarials or had a mother who used antimalarials during pregnancy .", "Although HIV testing was not conducted among children , the mothers of 2540 subjects were tested for HIV .", "Twenty-four ( 0 . 94% ) were seropositive , suggesting that the potential for vertical transmission might be low .", "There were no significant differences in seropositivity across countries . 10 . 7554/eLife . 03925 . 003Figure 1 . Flow chart of subjects . DOI: http://dx . doi . org/10 . 7554/eLife . 03925 . 00310 . 7554/eLife . 03925 . 004Table 1 . Baseline characteristics of 8390 children tested for malaria using blood film , by survey locationDOI: http://dx . doi . org/10 . 7554/eLife . 03925 . 004CharacteristicLocation of surveyOverall ( n=8390 ) Burkina Faso ( n=2821 ) Mozambique ( n=2266 ) Rwanda ( n=2085 ) Senegal ( n=1218 ) Communities surveyed , n5376074903712005", "( a ) Children tested for malaria , n ( % ) Parasitemia2821 ( 99 ) 2266 ( 100 ) 2085 ( 99 ) 1218 ( 99 ) 8390 ( 99 ) Positive result among children tested for parasitemia 1696 ( 60 ) 578 ( 25 ) 16 ( 0 . 8 ) 22 ( 1 . 8 ) 2312 ( 28 ) Pƒ-HRP-22821 ( 100 ) na2060 ( 99 ) 1217 ( 99 ) 6098 ( 99 ) * Positive results among children tested for Pƒ-HRP-2 2051 ( 73 ) na 35 ( 1 . 7 ) 27 ( 2 . 2 ) 2113 ( 35 )", "( b ) Type of immunization received , n ( % ) Bacille Calmette Guerin ( BCG ) 2799 ( 99 ) 2004 ( 96 ) 2050 ( 100 ) 1160 ( 98 ) 8013 ( 98 ) Diphtheria–tetanus–pertussis ( DTP ) 2721 ( 97 ) 2107 ( 97 ) 2055 ( 98 ) 1161 ( 98 ) 8044 ( 98 ) Measles2225 ( 80 ) 1668 ( 78 ) 1727 ( 86 ) 830 ( 73 ) 6450 ( 80 ) Poliomyelitis2810 ( 100 ) 2156 ( 99 ) 2061 ( 100 ) 1204 ( 100 ) 8231 ( 99 ) Vitamin A87 ( 9 . 3 ) 1554 ( 87 ) 351 ( 72 ) 190 ( 59 ) 2182 ( 62 )", "( c ) Children's characteristics Age in months , median ( IQR ) 22 ( 13–32 ) 20 ( 13–31 ) 24 ( 14–36 ) 19 ( 12–29 ) 22 ( 13–32 ) Girls , n ( % ) 1347 ( 48 ) 1151 ( 51 ) 1020 ( 49 ) 551 ( 45 ) 4069 ( 48 ) Primigravidae , n ( % ) 443 ( 16 ) 477 ( 21 ) 432 ( 21 ) 251 ( 21 ) 1603 ( 19 ) Low birth weight , n ( % ) 867 ( 31 ) 1003 ( 44 ) 672 ( 32 ) 536 ( 44 ) 3078 ( 37 )", "( d ) Malaria-based interventions , n ( % ) Child's family owns bed net2160 ( 77 ) 1528 ( 67 ) 1983 ( 95 ) 1040 ( 85 ) 6711 ( 80 ) Child received antimalarial during past week314 ( 11 ) 117 ( 5 . 2 ) 50 ( 2 . 4 ) 24 ( 2 . 0 ) 505 ( 6 . 0 ) Child's house had indoor insecticide spraying22 ( 0 . 8 ) 541 ( 24 ) na148 ( 12 ) 711 ( 8 . 5 ) † Mother took antimalarial during child's gestational period2616 ( 93 ) 1109 ( 50 ) 337 ( 16 ) 1110 ( 91 ) 5162 ( 62 )", "( e ) Genetic mechanisms of malaria protection , median ( IQR ) Mean predicted HbS allele frequency0 . 06 ( 0 . 05–0 . 06 ) 0 . 03 ( 0 . 01–0 . 03 ) 0 . 03 ( 0 . 03–0 . 03 ) 0 . 07 ( 0 . 06–0 . 07 ) 0 . 04 ( 0 . 03–0 . 06 ) Median predicted G6PDd allele frequency0 . 06 ( 0 . 05–0 . 09 ) 0 . 15 ( 0 . 15–0 . 17 ) 0 . 04 ( 0 . 04–0 . 05 ) 0 . 10 ( 0 . 09–0 . 13 ) 0 . 08 ( 0 . 05–0 . 14 )", "( f ) Climate of communities surveyed , median ( IQR ) Annual range of enhanced vegetation index ( EVI ) 0 . 29 ( 0 . 24–0 . 32 ) 0 . 33 ( 0 . 22–0 . 40 ) 0 . 25 ( 0 . 20–0 . 29 ) 0 . 28 ( 0 . 18–0 . 34 ) 0 . 28 ( 0 . 22–0 . 33 ) Annual mean of EVI for the year0 . 22 ( 0 . 18–0 . 25 ) 0 . 40 ( 0 . 32–0 . 47 ) 0 . 39 ( 0 . 36–0 . 42 ) 0 . 18 ( 0 . 16–0 . 20 ) 0 . 29 ( 0 . 20–0 . 40 ) No .", "of days which EVI above annual mean136 ( 120–160 ) 88 ( 56–128 ) 320 ( 168–384 ) 208 ( 184–264 ) 152 ( 120–200 ) No .", "of days for rainy season ( corresponding to first and last day EVI above annual mean ) 136 ( 120–152 ) 128 ( 72–344 ) 344 ( 296–352 ) 216 ( 152–352 ) 168 ( 120–344 ) *Pƒ-HRP-2 testing not conducted as part of DHS survey protocol for Mozambique .", "†Information on insecticide spraying not collected as part of survey .", "Table 2 shows that while BCG vaccination was not associated with Plasmodium parasitemia , it was linked to an increased risk of PƒHRP-2 antigenemia .", "This effect remained after controlling for patient characteristics associated with BCG use in the inverse probability weighted ( IPW ) model .", "DTP was associated with a lower risk of Plasmodium parasitemia and PƒHRP-2 , but only in the weighted models .", "Measles and poliomyelitis vaccination were not associated with malaria antigenemia or parasitemia .", "Figure 2A shows that the strength of associations between BCG and antigenemia were greater if children were vaccinated during the wet season , among younger children and the more time passed since vaccination . 10 . 7554/eLife . 03925 . 005Table 2 . Relative risk of malaria infection after standard vaccination and vitamin A supplementation among children 6–59 months of ageDOI: http://dx . doi . org/10 . 7554/eLife . 03925 . 005Type of immunizationNo .", "of children vaccinated/total tested ( % ) No .", "of children with positive blood test ( % ) Unadjusted RRAdjusted RR ( 95% CI ) ‡ , §No vaccineVaccineUnweightedWeighted ( IPW )", "( a ) Plasmodium species ( parasitemia ) * Bacille Calmette Guerin ( BCG ) 8013/8140 ( 98 ) 41 ( 32 ) 2227 ( 28 ) 0 . 811 . 25 ( 0 . 81–1 . 91 ) 1 . 24 ( 0 . 76–2 . 05 ) Diphtheria–tetanus–pertussis ( DTP ) 8044/8235 ( 98 ) 83 ( 44 ) 2202 ( 27 ) 0 . 490 . 88 ( 0 . 64–1 . 20 ) 0 . 06 ( 0 . 01–0 . 47 ) Measles6450/8069 ( 80 ) 473 ( 29 ) 1784 ( 28 ) 0 . 931 . 11 ( 0 . 96–1 . 29 ) 1 . 01 ( 0 . 20–5 . 19 ) Poliomyelitis8231/8272 ( 99 ) 14 ( 34 ) 2278 ( 28 ) 0 . 740 . 80 ( 0 . 37–1 . 73 ) 0 . 74 ( 0 . 27–2 . 01 ) Vitamin A supplement2182/3523 ( 62 ) 596 ( 44 ) 438 ( 20 ) 0 . 310 . 46 ( 0 . 39–0 . 54 ) 0 . 43 ( 0 . 36–0 . 52 )", "( b ) Plasmodium falciparum ( antigenemia ) Bacille Calmette Guerin ( BCG ) 6006/6047 ( 99 ) 9 ( 22 ) 2102 ( 35 ) 1 . 914 . 06 ( 2 . 00–8 . 28 ) 3 . 52 ( 1 . 66–7 . 48 ) Diphtheria–tetanus–pertussis ( DTP ) 5933/6054 ( 98 ) 59 ( 49 ) 2049 ( 35 ) 0 . 551 . 34 ( 0 . 88–2 . 02 ) 0 . 06 ( 0 . 01–0 . 38 ) Measles4776/5937 ( 80 ) 410 ( 35 ) 1679 ( 35 ) 0 . 991 . 15 ( 0 . 97–1 . 38 ) 0 . 68 ( 0 . 15–3 . 12 ) Poliomyelitis6 . 072/6084 ( 99 ) 5 ( 42 ) 2111 ( 35 ) 0 . 751 . 39 ( 0 . 55–3 . 49 ) 0 . 93 ( 0 . 37–2 . 35 ) Vitamin A supplement629/1749 ( 36 ) 621 ( 56 ) 75 ( 12 ) 0 . 100 . 23 ( 0 . 17–0 . 29 ) 0 . 22 ( 0 . 16–0 . 29 ) HRP-2: histidine rich protein-2; RR: relative risk; CI: confidence interval; IPW: inverse probability weighted model .", "*Tested in four countries: Burkina Faso , Mozambique , Rwanda and Senegal .", "†Tested in three countries: Burkina Faso , Rwanda and Senegal .", "‡Adjusted for the following factors: age , gender , wealth index score , mother's highest level of education , malaria treatment during previous week , ownership of bed net , proportion of household members under 5 years using bed net during previous night , indoor household insecticide spraying , mother's access to antenatal care during last pregnancy , mother's knowledge regarding vertical HIV transmission , malaria transmission season , and type of community setting ( urban vs rural ) .", "§Inverse probability weighting ( IPW ) based on propensity score model with following factors: age , gender , low birth weight , presence of radio or television , urban versus rural setting , breastfeeding status , wealth index score , mother's age , mother's highest education level , antenatal care during last pregnancy , and mother's tetanus status during last pregnancy . 10 . 7554/eLife . 03925 . 006Figure 2 . Adjusted relative risk of malaria infection according to different features of vitamin A supplementation and BCG vaccination ( Liu et al . , 2012; World Health Organization , 2012 ) .", "( A ) Adjusted for the following factors: age , gender , wealth index score , mother's highest level of education , malaria treatment during previous week , ownership of bed net , proportion of household members under 5 years using bed net during previous night , indoor household insecticide spraying , mother's access to antenatal care during last pregnancy , mother's knowledge regarding vertical HIV transmission , malaria transmission season , and type of community setting ( urban vs rural ) .", "( B ) Covariates ‘Age at vaccination’ and ‘Time since vaccination’ treated as continuous terms when testing for effect modification in the model .", "( C ) Seasonality only available for children vaccinated in 2010 or 2011 calendar year . DOI: http://dx . doi . org/10 . 7554/eLife . 03925 . 006 Vitamin A supplementation appeared protective against Plasmodium parasitemia and PƒHRP-2 antigenemia ( Table 2 ) .", "These effects remained after weighting .", "Figure 2 illustrates that the association between vitamin A and Plasmodium parasitemia was not impacted by season , age or time since supplementation .", "However , for antigenemia , vitamin A was moderately more protective in older children ( ptrend<0 . 001 ) , the more time passed since supplementation ( ptrend<0 . 001 ) or if supplementation was given during the wet season ( ptrend=0 . 04 ) .", "Table 3 reveals that associations between vitamin A and parasitemia were stronger among children 36–59 months of age or if their mother took an antimalarial during pregnancy .", "The relation with PƒHRP-2 antigenemia was stronger if children had a negative malaria blood film , were 36–59 months of age , primigravidae , or had not taken an antimalarial recently .", "Vitamin A was more protective against PƒHRP-2 among children with low birth weight but was less protective against parasitemia .", "The association between vitamin A and PƒHRP-2 was stronger among children tested during the dry season or who lived in communities with a longer rainy season , and in communities with a lower annual mean for enhanced vegetation index ( EVI ) .", "Associations between vitamin A and parasitemia were stronger among communities with a higher mean predicted sickle cell hemoglobin ( HbS ) allele frequency or median predicted glucose-6-phosphate dehydrogenase deficiency ( G6PDd ) allele frequency .", "We had insufficient data to identify cross-interactions between vaccines and vitamin A using an adjusted model , but in unadjusted models we found no significant interactions between vaccination and vitamin A supplementation . 10 . 7554/eLife . 03925 . 007Table 3 . Modifiers of the association between vitamin A supplementation and malaria infection among children 6–59 months of ageDOI: http://dx . doi . org/10 . 7554/eLife . 03925 . 007Characteristics at blood testingLevel in the modelPlasmodium species ( parasitemia ) *Plasmodium falciparum ( antigenemia ) †No .", "of children with positive blood film ( % ) Unadjusted RRAdjusted model3No vitamin AVitamin ARR ( 95% CI ) p Value ( interaction term ) Adjusted RR‡ ( 95%CI ) p Value ( interaction term )", "( a ) Individual levelChildren's characteristics Age at malaria screening6–35 Months484 ( 43 ) 344 ( 20 ) 0 . 330 . 46 ( 0 . 38–0 . 55 ) <0 . 01#0 . 26 ( 0 . 20–0 . 34 ) <0 . 01#36–59 Months112 ( 54 ) 94 ( 21 ) 0 . 230 . 34 ( 0 . 25–0 . 47 ) 0 . 09 ( 0 . 05–0 . 14 ) GenderGirl274 ( 44 ) 226 ( 21 ) 0 . 340 . 54 ( 0 . 44–0 . 67 ) 0 . 290 . 23 ( 0 . 16–0 . 32 ) 0 . 99Boy322 ( 45 ) 212 ( 19 ) 0 . 290 . 40 ( 0 . 32–0 . 49 ) 0 . 23 ( 0 . 17–0 . 30 ) Pregnancy order of childPrimigravidae522 ( 47 ) 366 ( 21 ) 0 . 300 . 45 ( 0 . 38–0 . 53 ) 0 . 270 . 20 ( 0 . 15–0 . 27 ) 0 . 04Multigravidae74 ( 31 ) 72 ( 15 ) 0 . 410 . 51 ( 0 . 39–0 . 66 ) 0 . 33 ( 0 . 22–0 . 48 ) Birth weight2500 mg or greater332 ( 42 ) 204 ( 15 ) 0 . 240 . 39 ( 0 . 32–0 . 48 ) 0 . 010 . 27 ( 0 . 20–0 . 36 ) 0 . 02Less than 2500 mg264 ( 48 ) 234 ( 28 ) 0 . 430 . 53 ( 0 . 43–0 . 66 ) 0 . 13 ( 0 . 09–0 . 18 ) Treatment for intestinal worms during past 6 monthsNot received554 ( 46 ) 212 ( 25 ) 0 . 390 . 50 ( 0 . 40–0 . 61 ) 0 . 080 . 38 ( 0 . 28–0 . 52 ) 0 . 07Received37 ( 30 ) 221 ( 17 ) 0 . 460 . 66 ( 0 . 52–0 . 86 ) 0 . 15 ( 0 . 11–0 . 21 ) Malaria-based interventions Malaria treatment during previous weekNot received550 ( 44 ) 404 ( 19 ) 0 . 310 . 44 ( 0 . 37–0 . 52 ) 0 . 240 . 20 ( 0 . 16–0 . 27 ) 0 . 02Received46 ( 49 ) 34 ( 33 ) 0 . 510 . 88 ( 0 . 62–1 . 26 ) 1 . 01 ( 0 . 60–1 . 68 ) Mother took antimalarial during child's gestational periodNo102 ( 28 ) 226 ( 22 ) 0 . 690 . 78 ( 0 . 59–1 . 03 ) <0 . 0010 . 38 ( 0 . 19–0 . 74 ) 0 . 05Yes491 ( 50 ) 210 ( 19 ) 0 . 230 . 36 ( 0 . 30–0 . 45 ) 0 . 20 ( 0 . 16–0 . 27 ) Family owns bed netDoes not own bed net141 ( 47 ) 123 ( 22 ) 0 . 320 . 43 ( 0 . 34–0 . 56 ) 0 . 420 . 20 ( 0 . 15–0 . 26 ) 0 . 70Owns bed net455 ( 44 ) 315 ( 41 ) 0 . 310 . 48 ( 0 . 40–0 . 58 ) 0 . 24 ( 0 . 18–0 . 32 ) ( B ) Community level ( primary sampling unit ) Type of settingRural518 ( 50 ) 370 ( 25 ) 0 . 350 . 46 ( 0 . 39–0 . 56 ) 0 . 950 . 22 ( 0 . 17–0 . 30 ) 0 . 91Urban78 ( 26 ) 68 ( 9 . 4 ) 0 . 290 . 34 ( 0 . 22–0 . 51 ) 0 . 22 ( 0 . 12–0 . 40 ) Genetic mechanisms of malaria protection Mean predicted HbS allele frequency§Less than 2 . 5%14 ( 20 ) 104 ( 14 ) 0 . 780 . 78 ( 0 . 32–1 . 91 ) <0 . 01#-0 . 26#2 . 5–4 . 9%181 ( 41 ) 269 ( 25 ) 1 . 490 . 95 ( 0 . 64–1 . 42 ) 0 . 96 ( 0 . 24–3 . 91 ) 5% or greater401 ( 48 ) 65 ( 18 ) 0 . 210 . 31 ( 0 . 17–0 . 56 ) 0 . 17 ( 0 . 08–0 . 39 ) Median predicted G6PDd allele frequency§Less than 7 . 5%336 ( 47 ) 47 ( 11 ) 0 . 045 . 42 ( 2 . 01–14 . 6 ) <0 . 001#1 . 43 ( 0 . 28–7 . 21 ) 0 . 02#7 . 5–14 . 9%194 ( 43 ) 128 ( 18 ) 0 . 800 . 89 ( 0 . 52–1 . 50 ) 0 . 41 ( 0 . 12–1 . 34 ) 15% or greater66 ( 40 ) 263 ( 25 ) 0 . 650 . 74 ( 0 . 46–1 . 19 ) -Climate of communities surveyed Season of malaria transmissionDry season198 ( 42 ) 220 ( 19 ) 0 . 320 . 40 ( 0 . 31–0 . 52 ) 0 . 450 . 06 ( 0 . 03–0 . 13 ) <0 . 001Wet season398 ( 46 ) 218 ( 22 ) 0 . 320 . 53 ( 0 . 42–0 . 66 ) 0 . 39 ( 0 . 28–0 . 53 ) Length of rainy seasonLess than 120 days168 ( 49 ) 151 ( 21 ) 0 . 270 . 43 ( 0 . 31–0 . 61 ) <0 . 001#0 . 28 ( 0 . 15–0 . 50 ) 0 . 03#120–179 Days334 ( 56 ) 105 ( 26 ) 0 . 270 . 45 ( 0 . 33–0 . 62 ) 0 . 22 ( 0 . 15–0 . 31 ) 180 Days or more94 ( 23 ) 182 ( 17 ) 0 . 700 . 77 ( 0 . 56–1 . 05 ) 0 . 21 ( 0 . 10–0 . 43 ) Length of time enhanced vegetation index above annual meanLess than 120 days105 ( 42 ) 220 ( 21 ) 0 . 380 . 50 ( 0 . 37–0 . 69 ) 0 . 18#0 . 37 ( 0 . 09–1 . 60 ) 0 . 84#120–179 Days486 ( 52 ) 206 ( 27 ) 0 . 340 . 52 ( 0 . 42–0 . 66 ) 0 . 33 ( 0 . 24–0 . 45 ) 180 Days or more5 ( 3 . 3 ) 12 ( 3 . 2 ) 0 . 970 . 43 ( 0 . 12–1 . 62 ) 0 . 66 ( 0 . 16–2 . 85 ) Range of enhanced vegetation index per yearLess than 0 . 2046 ( 25 ) 41 ( 7 . 9 ) 0 . 260 . 38 ( 0 . 22–0 . 63 ) 0 . 18#0 . 24 ( 0 . 13–0 . 47 ) 0 . 13#0 . 20–0 . 29247 ( 44 ) 105 ( 17 ) 0 . 250 . 48 ( 0 . 36–0 . 64 ) 0 . 36 ( 0 . 25–0 . 53 ) 0 . 30 or greater303 ( 51 ) 292 ( 28 ) 0 . 380 . 48 ( 0 . 39–0 . 61 ) 0 . 19 ( 0 . 13–0 . 29 ) Annual mean for enhanced vegetation indexLess than 0 . 20150 ( 37 ) 21 ( 7 . 2 ) 0 . 130 . 17 ( 0 . 11–0 . 28 ) <0 . 01#0 . 16 ( 0 . 10–0 . 25 ) 0 . 01#0 . 20–0 . 29309 ( 59 ) 89 ( 24 ) 0 . 220 . 51 ( 0 . 37–0 . 70 ) 0 . 34 ( 0 . 23–0 . 51 ) 0 . 30 or greater137 ( 33 ) 328 ( 22 ) 0 . 560 . 61 ( 0 . 46–0 . 81 ) 0 . 56 ( 0 . 20–1 . 53 ) HRP-2: histidine rich protein 2; RR: relative risk; CI: confidence interval; na: not applicable: ref: reference category; HbS: hemoglobin S; G6PD: glucose 6-phosphate dehydrogenase deficiency .", "*Tested in four countries: Burkina Faso , Mozambique , Rwanda and Senegal .", "†Tested in three countries: Burkina Faso , Rwanda and Senegal .", "‡Adjusted for the following factors: age , gender , wealth index score , mother's highest level of education , malaria treatment during previous week , ownership of bed net , proportion of household members under 5 years using bed net during previous night , indoor household insecticide spraying , mother's access to antenatal care during last pregnancy , mother's knowledge regarding vertical HIV transmission , malaria transmission season , and type of community setting ( urban vs rural ) .", "§Geographical waypoints were not recorded for 40 communities .", "Subjects from these PSUs were excluded from analysis .", "#Covariate treated as continuous term when testing for effect modification in the model ." ], [ "Our study extends previous randomized clinical trial evidence from Burkina Faso ( Zeba et al . , 2008 ) , Papua New Guinea ( Shankar et al . , 1999 ) , Tanzania ( Villamor et al . , 2002 ) , and Uganda ( Nankabirwa et al . , 2011 ) showing consistent reductions in malaria-induced morbidity and mortality from vitamin A . Still , it contradicts suggestions that when combined with DTP ( Jorgensen et al . , 2011 ) , vitamin A may increase the risk of malaria infection .", "Not only did we identify a protective association between vitamin A and malaria in a study population with high immunization coverage for BCG , DTP , and poliomyelitis , we also found in unadjusted models that DTP did not modify vitamin A's association with malaria .", "We found no indication of an elevated gender effect for either DTP or vitamin A , although the latter seemed to be more protective among children living in communities with higher allele frequencies for X-linked G6PDd .", "It is unclear how vitamin A could interact with G6PD to influence the risk of malaria infection; the only literature discussion regarding vitamin A and G6PD is limited to animal findings on adipose and lipogenic activities ( Higueret et al . , 1989; Arana et al . , 2008 ) .", "However , G6PDd is independently related to risk reductions in malaria , a driving factor in the high prevalence of G6PDd across Africa ( Howes et al . , 2013 ) .", "G6PDd is also expressed more often in boys than girls due to the X-linked nature of the gene ( Howes et al . , 2013 ) .", "All of the epidemiological evidence to support the detrimental gender-based effects from vitamin A and DTP on child mortality comes from Guinea Bissau ( Benn et al . , 2009a , B2009b; Fisker et al . , 2013 ) .", "Whether relatively mild phenotypic differences between G6PDd alleles could explain such different vaccine effects is not clear .", "Differences in the impact of vitamin A on malaria infection by birth weight , and having a mother who used antimalarials during the child's gestational period suggests exposure to placental malaria may play a critical role in vitamin A's protective effect mechanisms later on in childhood .", "Published evidence regarding placental malaria , antenatal vitamin supplementation , and infant health are limited .", "However , Cox et al . ( 2005 ) have identified a lower level of antibodies involved in placental parasite sequestration and risk reductions in active placental malaria infection at delivery in mothers who received vitamin A supplements during pregnancy .", "Additional research may be warranted on the impact of vitamin A supplementation in young children with known prenatal exposure to malaria .", "Results from a previous clinical trial in Guinea Bissau provides no evidence of an association between BCG re-vaccination and malaria parasitemia ( Rodrigues et al . , 2007 ) .", "Although the study had a number of design limitations ( i . e . did not examine the magnitude of the re-vaccination boosting effect on prior immunity from BCG , was statistically underpowered ( potentially causing type I error ) , and based on passive case detection of malaria at health centers/outpatient clinics ) , it does suggest that our inconsistent findings between Plasmodium parasitemia and PƒHRP-2 antigenemia may be due to another factor .", "In 2012 , the tropical diseases research ( TDR ) programme reported false positivity caused by schistosomiasis and Chagas in one lot of Paracheck Pƒ rapid diagnostics ( World Health Organization on behalf of the Special Programme for Research and Training in Tropical Diseases , 2011 ) .", "Infection with Trypanosoma cruzi , schistosomiasis , and other soil-transmitted helminths is inversely associated with anti-mycobacterial responses due to Th1/Th2 polarization ( Malhotra et al . , 1999; Elias et al . , 2005; Brown et al . , 2006; Dauby et al . , 2009 ) , and albendazole increases BCG immune reactivity among children infected with schistosomiasis ( Dauby et al . , 2009 ) .", "Although Chagas is not endemic to the countries we studied ( Morel and Lazdins , 2003 ) , there is growing evidence that schistosomiasis may be an overlooked , endemic disease among infants and young children in sub-Saharan Africa ( Russell Stothard et al . , 2013 ) .", "Our study represents an active population-based surveillance of malaria infection within four countries .", "The breadth and size of the study population enabled us to evaluate the association between malaria and childhood vaccines/vitamin A despite high vaccination coverage rates within most of the communities surveyed ( Desai et al . , 2005 ) .", "Although our models adjusted for factors that could explain study population differences between blood film and antigenemia models , to address this issue further we ran a sensitivity analysis using blood film data including/excluding Mozambique .", "This analysis showed that when Mozambique data were excluded from blood film analysis , the magnitude of protective vitamin A effects in both blood film and antigenemia models were nearly identical .", "Blood films and antigenemia readings are used to identify two different outcomes ( i . e . blood films test for Plasmodium spp . while antigenemia test for one particular type of Plasmodium [P . falciparum] ) .", "However , due to geography , blood films were likely to be detecting P . falciparum due to geography ( Gething et al . , 2011 , 2012 ) .", "This would explain the similar results identified between blood film and antigenemia results when excluding Mozambique data from our analysis .", "We have chosen to present data in this paper that includes Mozambique because these results provide more conservative estimates of vitamin A effects when using blood films .", "A number of study limitations should also be considered when reviewing our results .", "First , while we used propensity scores to address differences in patient characteristics associated with vaccine uptake , the possibility of residual confounding due to unmeasured factors remains .", "Moreover , because of the observational nature of our study design , high-quality randomized clinical trials are needed to confirm the efficacy of vitamin A in preventing malaria , particularly among children exposed to placental malaria .", "This includes further clarification regarding optimal dosages and the time sequence in which vitamin A is administered in relation to other vaccines , as well as more accurate measures with regard to HbS and G6PDd allele frequency .", "In particular , our study only considered information regarding the last recorded dose of vitamin A supplement rather than a child's history of supplementation .", "Given that vitamin A is fat soluble and can be stored in the liver for long periods of time ( Blomhoff , 1994; Tanumihardjo , 2011 ) , additional research is needed to examine whether regular supplementation has an impact on increased protection .", "Second , although the EVI dataset is advantageous for this research due to its spatial and temporal resolutions , several caveats are associated with EVI that affect its utility as a proxy for seasonal rainfall .", "Foremost among these concerns is the time lag between rainfall and vegetation response , as dormant vegetation does not immediately become green following the return of seasonal rains ( Nicholson et al . , 1990; Davenport and Nicholson , 1993 ) .", "This issue will create a temporal offset between EVI and rainfall that may complicate the interpretation of the results of this research .", "However , this concern will be partially mitigated by a similarly lagged response to rainfall in populations of vector species responsible for spreading Plasmodium ( Mbogo et al . , 2003; Galardo et al . , 2009 ) .", "Another concern when using EVI as a proxy for rainfall relates to non-uniform responses of vegetation to rainfall that reflect long-term moisture conditions ( via land cover ) rather than seasonal oscillations ( Townshend and Justice , 1986 ) .", "This issue is most apparent when comparing dry season EVI values in grasslands to those in forests , as forests tend to retain more green vegetation ( and thus have higher EVI values ) even when dry due to deeper root systems , etc .", "A second aspect of EVI linked to land cover differences is the speed at which vegetation responds to rainfall , as plants in drier ecosystems have evolved to respond more quickly to infrequent rain events ( Ogle and Reynolds , 2004 ) .", "For this research , differing responses to land cover are accounted for by deriving unique EVI curves for each survey cluster location , thus creating relative metrics based on only localized ( i . e . per-cell ) EVI values rather than regional summaries that contain responses of multiple land cover types .", "Finally , data on children's HIV status were not available .", "Although the possibility of mother-to-child HIV transmission was low in our study ( less than 1% of mothers were HIV positive ) , HIV is an established risk factor for malaria infection ( Abu-Raddad et al . , 2006 ) .", "To address this issue , we adjusted a subgroup of models for maternal HIV status .", "While the protective association between vitamin A and malaria remained , the low prevalence of maternal HIV in our study population ( and hence low statistical power ) makes it difficult to rule out potential vitamin A–HIV interactions .", "Further research is needed to evaluate potential interactions between vitamin A , HIV and malaria infection ." ], [ "Data were extracted from Macro International Demographic and Health Surveys ( DHS ) ( ICF International , 2013 ) , a database of nationally representative household surveys conducted in low and middle-income countries .", "We focused on standard DHS surveys carried out since January 2010 , which tested children for malaria using blood films ( gold standard malaria test ) and documented children's use of standard vaccines and vitamin A supplements .", "Malaria indicator surveys were excluded from our study due to the absence of data on childhood immunization .", "From 20 country surveys with accessible data ( last checked 15 January 2014 ) , we identified four surveys that fitted these criteria ( i . e . Burkina Faso , Mozambique , Rwanda , and Senegal ) .", "All surveys were conducted between May 2010 and November 2011 , and used a multistage sampling process , by which study participants were identified from randomly selected households within a primary sampling unit ( PSU ) ( e . g . census enumeration tract ) .", "Trained interviewers used standardized questionnaires to collect information from participants during home interviews on a range of issues related to population , health , and nutrition .", "Geographical waypoints ( World Geodetic System 84 datum , latitude , and longitude ) were also recorded at the center of each PSU .", "Eligible subjects for our analysis included children 6–59 months of age who , during survey interviews , provided blood samples for malaria screening and had their history of vaccination and vitamin A supplementation documented based on health records .", "Each DHS had comprehensive ethical approval and written informed consent was collected for each survey participant ( see below ) .", "Additional data were extracted from external sources on malaria seasonality and the population prevalence of malaria protective genes using the geographical coordinates of each PSU ( n=2604 community waypoints ) .", "Information regarding children's vaccination and vitamin A supplementation history was ascertained from health card data recorded during the survey interview .", "This included a child's date of vaccination using BCG , DTP , measles and polio as well as the number of vaccine doses received ( for DTP and polio only ) .", "We used these data then to calculate age in months at the moment of vaccination , and time in months since vaccination .", "Children whose mother/guardian responded in the affirmative to vaccination ( e . g . child vaccinated during campaign ) but did not provide health cards to document this information were excluded from the analysis .", "For vitamin A supplementations , data were recorded regarding the date of last dose received and use of the vitamin supplement during the 6 months prior to the survey interview .", "Primary study outcomes included the microscopic detection of Plasmodium spp .", "to assess parasitemia and the presence of PƒHRP-2 to assess antigenemia .", "The microscopic presence of Plasmodium spp .", "was determined by reading Giemsa-stained thick blood films .", "Species of Plasmodium spp .", "were not recorded .", "Capillary blood was collected during household interviews and tested onsite for malaria antigenemia .", "Pƒ-specific HRP-2 antigens were detected using the rapid diagnostic Paracheck Pf ( Orchid Biomedical Services , Goa , India ) for Burkina Faso and Senegal surveys and first response malaria antigen HRP-2 ( Premier Medical Corporation , Daman , India ) for the Rwanda survey .", "Thick blood smears were prepared by trained technicians for all children , and sent for gold standard testing of malaria to the Centre National de Recherche et de Formation sur le Paludisme ( Burkina Faso ) , Centro de Investigações em Saúde de Manhiça ( Mozambique ) , TRAC/Plus Malaria Unit in Kigali ( Rwanda ) and Department of Parasitology at the Université Cheikh Anta Diop ( Senegal ) .", "Internal quality control was conducted using standard laboratory protocol and procedures , including having slides randomly read by different laboratory technicians or the chief technician/supervisor for internal quality control measures .", "External quality control ( Mozambique only ) included selecting samples by a computerized system developed by ICF Macro and sending them for external quality control testing at the Muraz Center ( Bobo-Dioulasso ) .", "Capillary blood was collected during household interviews from all women aged 15–29 years .", "Samples were collected on filter paper , dried for 24 hr and then sent for testing to the Centre Regional de Transfusion Sanguine de Ouagadougou ( Burkina Faso ) , National Reference Laboratory ( Rwanda ) and National Reference Laboratory of Bacteriology and Virology at A Le Dantec Hospital ( Senegal ) .", "Viral antibodies were detected using the enzyme-linked immunosorbent assay ( ELISA ) -based assay VironostikaÒ HIV Uni-Form II plus O ( Biomériux , Marcy l'Etoile , France ) .", "Positive samples and a portion of negative samples were tested using the ELISA assay EnzygnostÒ Anti-HIV ½ plus ( Siemens AG , Erlangen , Germany ) .", "Discordant samples were further tested using the Pepti Lav assay ( Bio-Rad Laboratories , Hercules , CA ) or InnoLia ( Burkina Faso and Rwanda ) .", "Internal quality control measures ( Burkina Faso , Rwanda and Senegal ) included using control wells ( positive and negative ) provided with manufacturer's screening kit on each test plate .", "All positive samples and 10% of randomly selected negative samples were also tested using more than one assay , with discordant samples further tested using a third assay .", "External quality control measures ( Burkina Faso only ) consisted of sending all positive samples and 80 randomly selected negative samples to the Center Muraz ( Bobo-Dioulasso ) for additional HIV testing .", "Multivariable logistic regression models were used to estimate associations between malaria and childhood vaccination/supplementation .", "The impact of age at vaccination , time since vaccination , and malaria season on the date of vaccination were also examined .", "To avoid statistical inference errors in our results due to clustering and stratification from the sampling process , we defined country and PSU as strata , and households as clusters using logistic regression models that accounted for the complex survey data structure .", "To test for linear trends , covariates were included as continuous terms in the regression model .", "We adjusted models for continuous variables of age and wealth index score , and categorical variables of gender ( boy vs girl ) , malaria treatment during previous week ( yes vs no ) , ownership of bed net ( yes vs no ) , proportion of household members under 5 years old using bed net during previous night ( none , all , some , or no bed net ) , indoor household insecticide spraying ( yes vs no ) , malaria transmission season ( dry vs wet ) , community setting ( urban vs rural ) , mother's highest educational level ( none , incomplete primary , complete primary , incomplete secondary , complete secondary , post-secondary ) , and access to antenatal care during last pregnancy ( yes vs no ) .", "Complete subject analysis was used .", "Supplementary file 1 presents children's characteristics for missing ( excluded ) and non-missing ( included ) groups .", "Effect modifiers for each vaccine/vitamin model were identified by individually testing cross-product terms between type of immunization/supplement received and individual-based characteristics including age , gender , pregnancy order of child , birth weight , receipt of antimalarial during previous week , receipt of intestinal worm treatment during previous 6 months , if mother took antimalarial during child's gestational period , family owns bed net , or type of community setting; climate-related factors including malaria transmission season at time of immunization , length of rainy season , length of time that EVI was above annual mean , range of EVI per year , or annual mean for EVI; and malaria genetic factors including the predicted allele frequency in a community for HbS ( mean estimate ) and G6PDd ( median estimate ) .", "To account for uncertainty in spatial prediction values for HbS and G6PDd , we conducted a sensitivity analysis by excluding from models any prediction value with degrees of uncertainty ( i . e . interquartile range ) greater than 20% .", "Prior to enrolling in the survey , the parent/adult responsible for the child had to provide written informed consent to participate , along with additional informed consent for blood sample collection and testing .", "If a child tested positive during the survey using malaria RDT , their parent/guardian was informed of results and the child was immediately given treatment according to the current treatment guidelines .", "HIV tests for adults were anonymous , with survey data only being linked to test results after respondent identifiers were deleted from the database .", "Although HIV test results were not available to study participants , patients were offered cards enabling them to obtain free HIV testing and counselling at Voluntary Testing Centers .", "The Institutional Review Board of ICF Macro reviewed and approved the MEASURE Demographic and Health Surveys Project Phase III , in compliance with United States ( U . S . ) Department of Health and Human Services ( DHHS ) regulation 45 Code of Federal Regulations ( CFR ) 46 for ‘Protection of Human Subjects’ research .", "Protocols for blood specimen collection , HIV and malaria testing were also approved by ICF Macro International Institutional Review Board ( IRB ) [all countries] in compliance with U . S . DHHS regulation 45 CFR 46 for ‘Protection of Human Subjects’ research , National Ethics Committees [Burkina Faso , Rwanda and Senegal] and U . S . Centers for Disease Control [Rwanda only] .", "For Senegal , supervisory visits were also organized by the National Ethics Committee to ensure field compliance with ethical regulations .", "Geographical waypoints were randomly displaced to ensure confidentiality [Urban PSU: positional error of 0–2 km; and , Rural PSU: positional error of 0–5 km ( 99% of clusters ) , or 0–10 km error ( 1% of clusters ) ] .", "DHS provided anonymized data to study authors .", "As a secondary analysis of publically available de-identified data , the study was determined exempt from ethics review by Johns Hopkins Bloomberg School of Public Health IRB Office according to U . S . DHHS regulation 45 CFR 46 . 102 for non-human subject research ." ] ]

[ "Recent studies , partly based on murine models , suggest childhood immunization and vitamin A supplements may confer protection against malaria infection , although strong evidence to support these theories in humans has so far been lacking .", "We analyzed national survey data from children aged 6–59 months in four sub-Saharan African countries over an 18-month time period , to determine the risk of Plasmodium spp .", "parasitemia ( n=8390 ) and Plasmodium falciparum HRP-2 ( PfHRP-2 ) -related antigenemia ( n=6121 ) following vitamin A supplementation and standard vaccination .", "Bacille Calmette Guerin-vaccinated children were more likely to be PfHRP-2 positive ( relative risk [RR]=4 . 06 , 95% confidence interval [CI]=2 . 00–8 . 28 ) .", "No association was identified with parasitemia .", "Measles and polio vaccination were not associated with malaria .", "Children receiving vitamin A were less likely to present with parasitemia ( RR=0 . 46 , 95% CI=0 . 39–0 . 54 ) and antigenemia ( RR=0 . 23 , 95% CI=0 . 17–0 . 29 ) .", "Future studies focusing on climate seasonality , placental malaria and HIV are needed to characterize better the association between vitamin A and malaria infection in different settings ." ]

[ "More than half of the world's population is at risk of malaria , with an estimated 198 million clinical cases each year .", "A vaccine that fully prevents it has not yet been discovered .", "Most cases of malaria occur among children living in sub-Saharan Africa , a region where many receive routine vaccinations designed to prevent other diseases; for example , 75% of children in sub-Saharan Africa receive measles vaccines .", "Many also receive vitamin A supplements , which have been linked not only to the protection of a child's vision , but also to a lower risk of death and an improved ability to fight off infections .", "Some researchers have suggested that vitamin A supplements and routine childhood vaccinations for other diseases may also provide some protection against malaria .", "For example , some studies performed in mice have shown that a commonly used tuberculosis vaccine may eliminate Plasmodium parasites that cause malaria infections .", "However , this effect depended on several factors , including how the vaccine was administered and whether the vaccination was given before or after the mouse developed malaria .", "It is less clear whether vaccines or vitamin A have antimalarial effects in humans .", "To address this , Hollm-Delgado et al . analyzed national survey data collected from thousands of children aged between 6 months and 5 years old who lived in four different countries in sub-Saharan Africa .", "The surveys contained information about the vaccines and supplements the children received , and whether their blood showed signs of infection with malaria-causing Plasmodium parasites .", "Hollm-Delgado et al . found that routine vaccinations did not affect the likelihood of malaria parasites being detected in the child's blood .", "However , children vaccinated against tuberculosis were more likely to have a specific type of protein released when malaria infects the blood .", "Hollm-Delgado et al . suspect that the tests may actually have inadvertently detected other parasitic infections in the children , such as Schistosoma , producing false-positive results for malaria .", "In contrast , Hollm-Delgado et al . found that children who received vitamin A supplements were less likely to become infected with malaria .", "The benefits of the supplements appeared to be affected by several conditions , including the time of year when the children received their supplements or when they were tested for malaria , and whether their mother had malaria when pregnant .", "Clinical trials are now needed to confirm these results and investigate how effectively vitamin A prevents malaria ." ]

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

[ "epidemiology and global health", "microbiology and infectious disease" ]

[ [ "Varicella-zoster virus ( VZV ) causes the itching , erythematous vesicular disease called varicella or chickenpox , mainly during childhood , and remains latent in neural ganglia afterwards .", "Latency can then be interrupted by episodes of ( primarily subclinical ) reactivation of VZV as shown by the detection of VZV in saliva or blood from otherwise healthy individuals ( Schünemann et al . , 1998; Nagel et al . , 2011 ) .", "VZV reactivations may also cause herpes zoster ( HZ ) , which presents clinically as a painful dermatomal rash .", "HZ occurs most frequently in individuals with a drastically declined cellular immune status ( Dolin et al . , 1978 ) , but ageing itself is often assumed to substantially reduce resilience of the VZV-specific immune response ( Miller , 1980; Berger et al . , 1981; Levin et al . , 2003 , 2008 ) .", "Recent research supports the hypothesis that waning of VZV cellular mediated immunity ( CMI ) by age is also influenced by cytomegalovirus ( CMV ) infection ( Ogunjimi et al . , 2014 ) .", "Effective and safe pediatric vaccines against varicella exist and have been universally implemented in some countries including the US , Australia , Greece , Germany , Japan and Taiwan ( Marin et al . , 2008 ) .", "However , in many other countries policy makers have been hesitant to introduce childhood VZV vaccination due to the general population's perception of varicella as a relatively mild disease , as well as the so-called exogenous boosting hypothesis ( Hope-Simpson , 1965; Ogunjimi et al . , 2013 ) .", "This hypothesis is based on the concept of the secondary immune response and assumes that boosting of VZV-CMI occurs upon re-exposure to varicella .", "Boosted VZV-specific cellular immunity would subsequently reduce the risk of VZV reactivation and thence HZ .", "The introduction of widespread childhood VZV vaccination would reduce opportunities for varicella re-exposure and could therefore increase HZ incidence , as shown in model-based projections ( Schuette and Hethcote , 1999; Brisson et al . , 2000; Bilcke et al . , 2013 ) .", "Although current data on HZ-incidence post introduction of childhood VZV vaccination has caused controversy , a systematic review rating the quality of the evidence , concluded that exogenous boosting exists , but that its population-wide effect after widespread childhood VZV vaccination remains highly uncertain ( Ogunjimi et al . , 2013 ) .", "One of the main points of criticism on current predictions by deterministic VZV simulation models is the entanglement of exogenous boosting , waning of immunity , immunosenescence and reactivation undermining the possibility of estimating the magnitude and duration of exogenous boosting accurately .", "A concern is that these entangled parameters can be chosen to fit these models well to observed HZ incidence data , but that they are too artificial to allow real and verifiable biological interpretations .", "This leads to currently unverifiable and potentially poor predictions of VZV dynamics beyond the fitted equilibrium states .", "An additional , hitherto ignored uncertainty , is the potential occurrence of endogenous boosting by subclinical VZV reactivation .", "Indeed , some studies have shown VZV to reactivate subclinically both in healthy individuals , immunocompromised and stressed individuals ( Schünemann et al . , 1997; Mehta et al . , 2003; Nagel et al . , 2011 ) .", "However , the effect on VZV-CMI has not yet been quantified .", "In the current paper , we describe the first individual-based VZV model , explicitly combining within and between-host dynamics .", "This model is based on experimental viro-immunological data and allows an accurate estimation of the exogenous boosting characteristics and explicit insertion or validation of experimental data ." ], [ "Using a step-wise algorithm we initially found eight unique parameter sets leading to a reasonable fit of Belgian HZ incidence data ( see Table 1 and Figure 1 ) .", "All best-fitting parameter sets were based on boosting scenario 3 ( predefined exponential loss of boosted VZV-CMI ) .", "Seven of these models include exogenous boosting ( defined as an exponential decay ) with a peak value of 1 . 3 , a boosting duration ranging between 2 and 15 years , no endogenous boosting , a weekly VZV reactivation probability and an annual VZV-CMI loss ( = waning ) estimated to be 1–1 . 5% and 1–2% , respectively .", "We note that although the fits are excellent for the most relevant age groups , 25 years and older , they are less suited to predict HZ incidence for younger ages .", "One model predicted a peak boosting value of 2 . 5 , a boosting duration of only 1 year and no endogenous boosting .", "Although this model was less suited to fit to HZ data for older age groups , it fitted much better to HZ data for younger age groups .", "In additional analyses relaxing on some parameter constraints , we obtained final parameter sets that led to excellent predictions across all age groups ( see Table 1 and Figure 2 ) .", "The two best fitting parameter sets predicted peak boosting values between 2 . 8 and 4 ( maximum allowable value ) , a duration of boosting limited to 1 or 2 years and , as before , no endogenous boosting .", "Averaged VZV-specific CMI levels are shown in Figure 3 . 10 . 7554/eLife . 07116 . 003Table 1 . Best fitting parameter setsDOI: http://dx . doi . org/10 . 7554/eLife . 07116 . 003Parameter setDeviance*Annual waning rate ( % ) Boosting scenarioDuration of boosting ( years ) Peak fold increase following exogenous boostingVZV weekly reactivation probability ( % ) Distribution threshold VZV-CMI for HZPeak fold increase following endogenous boostingOriginal Search ( obtained after Step 2 in Table 1 ) 19262 . 03101 . 31 . 541 29391 . 5331 . 31 . 541 39492 . 0371 . 31 . 541 49512 . 03121 . 31 . 541 59682 . 0371 . 31 . 041 69701 . 0321 . 31 . 041 79692 . 03151 . 31 . 541 89341 . 0312 . 55 . 041Border search 97511 . 0312 . 85 . 041 107991 . 0313 . 15 . 041 119651 . 5323 . 45 . 041 128041 . 5323 . 75 . 041 137221 . 5324 . 05 . 041*Results shown are averaged results per parameter set . VZV , varicella-zoster virus; HZ , herpes zoster . 10 . 7554/eLife . 07116 . 004Figure 1 . Observed ( open circles ) and simulated ( continuous lines ) Belgian herpes zoster ( HZ ) incidence data by age . DOI: http://dx . doi . org/10 . 7554/eLife . 07116 . 00410 . 7554/eLife . 07116 . 005Figure 1—source data 1 . Observed Belgian HZ incidence per age group and per person-year . DOI: http://dx . doi . org/10 . 7554/eLife . 07116 . 00510 . 7554/eLife . 07116 . 006Figure 2 . Observed ( open circles ) and simulated ( continuous lines ) Belgian HZ incidence data by age . DOI: http://dx . doi . org/10 . 7554/eLife . 07116 . 00610 . 7554/eLife . 07116 . 007Figure 2—figure supplement 1 . Observed ( open circles ) Belgian HZ incidence data by age and simulated HZ incidence data ( continuous lines ) for the 13 best parameter sets with a sensitivity analysis for the HZ infectiousness parameter ( values: 0 . 03 , 0 . 10 , 0 . 17 , 0 . 24 , 0 . 31 , 0 . 38 and 0 . 45 ) and three runs per parameter set . DOI: http://dx . doi . org/10 . 7554/eLife . 07116 . 00710 . 7554/eLife . 07116 . 008Figure 3 . Normalized varicella-zoster virus ( VZV ) -specific CMI averaged over 80 simulation years and over all individuals for the two best parameter sets . Caption: note that this figure shows average dynamics although some individuals will have VZV-specific CMI values below 1 ( making them susceptible to HZ ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 07116 . 008 Using our best fitting parameter sets ( set 9 and 13 from Table 1 ) we simulated the effects of a 100% permanently effective varicella vaccine given to all children aged 1 year ( 100% coverage ) .", "Our predictions ( averaged per parameter set ) showed a net increase in HZ incidence during approximately 50 years ( see Figure 4 ) .", "The peak increase in HZ incidence was reached approximately 31 years after programme initiation and was 1 . 75 ( 1 . 64–1 . 87 ) ( 100% CI ) times higher than the HZ incidence prior to varicella vaccination .", "The CI is constructed by using three runs per parameter set and normalizing each run by means of the run-specific averaged equilibrium HZ incidence between 100 and 300 years since the beginning of the simulation .", "This means that per 1 , 000 , 000 person-years the total number of HZ cases would increase on average from 3219 to 5313 ( set 9 ) or from 3209 to 5955 ( set 13 ) .", "Figure 5 shows that the relative contribution of different age groups to HZ incidence changes in the time period after introduction of varicella vaccination .", "Noteworthy is the relative dominance of the 31–40 year old age group between 10–50 years after introduction of varicella vaccination . 10 . 7554/eLife . 07116 . 009Figure 4 . Predicted HZ incidence ( aggregated for all ages ) over time with a CP vaccine for 1 year olds using the best-fitting parameter sets . The red line indicates the moment of CP vaccine introduction , which is assumed to be 100% effective . DOI: http://dx . doi . org/10 . 7554/eLife . 07116 . 00910 . 7554/eLife . 07116 . 010Figure 5 . Time-evolution of the relative contribution to HZ incidence per age group before and after introduction of 100% effective varicella vaccination for 1 year olds . DOI: http://dx . doi . org/10 . 7554/eLife . 07116 . 010" ], [ "It is hypothesized that exogenous re-exposure to varicella increases VZV-specific cellular immunity ( VZV-CMI ) .", "This natural consequence of the secondary immune response will reduce an individual's risk for HZ .", "When the probability of contact per unit-time between currently infectious and previously recovered varicella patients reduces , through a reduction in varicella incidence , it can be expected that HZ incidence temporarily increases due to a lack of exogenous boosting ( Hope-Simpson , 1965; Ogunjimi et al . , 2013 ) .", "To our knowledge , this study is the first to use an individual-based dynamic transmission model for VZV .", "This model allowed us to combine immunological and virological data to estimate key parameters in VZV population dynamics , such as the peak CMI response following re-exposure , duration of boosting and VZV subclinical reactivation characteristics .", "This means that in contrast to the deterministic models where abstract and ad hoc compartments are created to define the transition between different epidemiological states ( for example the transition of a varicella recovered state to a zoster susceptible state ) , we can actually work with true biological , and verifiable , concepts such as VZV-CMI .", "Our best fitting parameter sets for Belgium suggest the effective duration of exogenous boosting to last only between 1 and 2 years .", "These predictions are significantly lower than those from the highly cited deterministic VZV model by Brisson et al . , in which the average duration was predicted to be 20 years ( [7–41 years] 95%CI ) ( Brisson et al . , 2002 ) .", "Our peak immunological response was estimated to be 2 . 8–4 . 0 times larger than the pre-re-exposure value .", "These predictions are consistent with those experimentally found in adults re-exposed to VZV by varicella contacts in the household ( Arvin et al . , 1983; Vossen et al . , 2004 ) or by vaccination ( Levin et al . , 2008 ) .", "A possible limitation of our study was that all close contacts with varicella patients were assumed to exert an equal ‘average’ boosting effect on the exposed individual .", "Future studies could assess how important it would be to incorporate variability in the impact of an exposure through direct contact with a varicella case , based on characteristics of both the exposed and the infectious person ( e . g . , age , comorbidity ) .", "The VZV reactivation probability was estimated to be 5% per week .", "Observed data on VZV reactivation probability are rather sparse and highly divergent regarding study design , sampling site ( saliva , blood , cerebrospinal fluid ) and results ( Schünemann et al . , 1997; Mehta et al . , 2003; Engelmann et al . , 2008; Birlea et al . , 2011; Nagel et al . , 2011; van Velzen et al . , 2013 ) .", "The observed weekly VZV reactivation probability is a topic of discussion in the literature and varies between 0 and 71% .", "As such it is difficult to compare our estimates with the range of observed values .", "We also note that VZV reactivation in our model might only be detectable at the neural ganglia at not ( always ) necessarily in peripheral tissues .", "In order for HZ to occur , we assumed that VZV-CMI should be below a relative threshold ( following a specific distribution ) during VZV reactivation .", "None of our best fitting parameter sets predicted a significant effect of VZV reactivation on VZV-CMI , implying that endogenous boosting most likely does not have an impact on the occurrence of HZ .", "This finding is important since the existence of endogenous boosting has been proposed to have an effect in reducing the negative effects of varicella vaccination on HZ incidence .", "Future experimental studies should focus on confirming our predicted VZV reactivation probability and the lack of endogenous boosting .", "The annual decline of VZV-CMI was predicted to be between 1 and 1 . 5% .", "This result is lower than the 2 . 7–3 . 9% experimentally observed by Levin et al . for individuals older than 60 years ( Levin et al . , 2008 ) .", "The twofold difference in waning rate can be explained by the explicit disentanglement of waning and boosting ( with younger age groups having—on average—higher probabilities of being boosted recently thereby actually increasing the observed waning rate ) .", "A limitation in our study is the use of a fixed waning rate for all ages .", "Our results might be interpreted as an averaged result of lower waning rates for younger age groups and higher waning rates for older age groups ( as documented by Levin et al . ( 2008 ) ) .", "A higher waning rate in older age groups could for example be caused by chronic CMV infection ( Ogunjimi et al . , 2014 ) .", "Although different types of waning ( both in model specification and age dependency ) can be used in this kind of simulation models , we believe that further experimental data documenting VZV specific cellular memory as a function of age is needed so that new waning models can be appropriately formulated .", "One future avenue of research could be the fitting of our predicted VZV-CMI to observed VZV-CMI data , as this will be a mixture of waning , immunosenescence and boosting .", "Better VZV-CMI datasets than those currently available are needed to be able to do this .", "These datasets could and should contain immune responses against different VZV peptides and could differentiate between the different cellular immunity compartments ( CD4 vs CD8 and central vs effector memory cells ) .", "The use of our VZV IBM could help us identify which VZV-CMI compartment is of importance in controlling HZ .", "We used our best fitting models to analyze the effects of a 100% effective varicella vaccine implemented for all 1-year-old children .", "We predicted a net increase in HZ incidence during 50 years and a 1 . 75 peak fold increase 31 years after introduction of the vaccination program .", "This delay in the HZ peak incidence is caused by cohorts born close to the time varicella vaccination was introduced experiencing less repeated boosting instances during their childhood than previously born cohorts ( a mechanism that is similar to the progressive immunity model proposed in the deterministic VZV model by Guzzetta et al . ( 2013 ) ) .", "Although increases in HZ incidences following universal childhood varicella vaccination have been noticed , some authors have attributed these increases to a background evolution that was already present prior to CP vaccination ( see discussion in Ogunjimi et al . ( 2013 ) ) .", "However , our analysis shows that proper documentation of significant increases in HZ incidence might not be possible during the first 10 years , even if a 100% effective vaccine would be used .", "For instance , during the first 10 years of the US program , both uptake and efficacy with the initial single dose vaccination programme were far below 100% .", "Although our model predicts a much lower duration of boosting than used hitherto in compartmental models ( Ogunjimi et al . , 2013 ) , some of our overall HZ projections are qualitatively similar .", "However , in contrast to earlier model estimates our VZV IBM predicts that 31–40 year olds contribute the most to the peak in HZ incidence following varicella vaccination .", "Some observational studies found no effect of varicella vaccination on HZ incidence for those aged 60 years and older ( Hales et al . , 2013 ) .", "This could be compatible with an overall HZ incidence increase due to rising HZ incidence among 31–40 year olds .", "Importantly , younger adults have been shown to be less likely to develop post-herpetic neuralgia ( Opstelten et al . , 2002; Drolet et al . , 2010 ) .", "Thus although our aggregated predictions regarding the increase in HZ incidence following varicella vaccination may appear similar to those published using deterministic models , cost-effectiveness analyses using our VZV IBM would be more in favor of universal childhood varicella vaccination .", "A major benefit of our modeling approach is the possibility to verify our best–fit parameter values via experimental studies , or vice versa , to adapt parameter values when empiricism delivers new insights .", "For example , if a new experimental study would prove the existence of endogenous boosting , this could be readily implemented in our VZV IBM so that parameter sets could be estimated , conditional on the existence of endogenous boosting .", "Although our IBM has given us valuable insights into between-host and within-host VZV dynamics , other influential factors could be introduced in future versions .", "These factors could relate to CMV-immunosenescence ( Ogunjimi et al . , 2014 ) , maternal antibodies , reduced VZV-CMI induction if infected during the first year of life as this might improve the prediction of the teenage group ( Terada et al . , 1994 ) , co-infection with other viruses ( Ogunjimi et al . , 2015 ) , risk groups ( for e . g . , immunosuppressed individuals ) and HLA types ( Meysman et al . , 2015 ) .", "Although current deterministic compartmental models should be able to partially account for these effects , a VZV IBM seems better suited to directly model the influence of these immunity-perturbing factors .", "Future VZV IBM should also explore more realistic vaccination scenarios as well as inter-country variability ( Poletti et al . , 2013 ) .", "We conclude that our VZV individual-based model has explicitly estimated the duration of exogenous boosting to be limited to only 1 or 2 years and that there was no significant effect from endogenous boosting ." ], [ "We present an individual-based model in which the individual's risk of HZ is determined by the individual's VZV-CMI vs the so defined ‘Force of Reactivation ( FoR ) ’ that represents the strength of VZV reactivation ( as detailed in the Modeling VZV reactivation paragraph ) .", "In contrast to classical deterministic epidemiological models , our model is not explicitly compartmentalized by means of ad hoc defined epidemiological groups .", "Instead the main ‘flow diagram’ represents the evolution of VZV-CMI with time and under several events ( for more details see the next paragraphs ) .", "Briefly , Figure 6 illustrates that individuals are born susceptible and that VZV-CMI is equal to zero during this time period .", "After exposure to chickenpox or HZ , VZV-CMI receives an initial value .", "Next , VZV-CMI is expected to undergo waning and several events can occur .", "The first event depicted ( but the reader should realize that the timing of the events are interchangeable ) is exogenous boosting after re-exposure to either chickenpox or HZ .", "Exogenous boosting is assumed to increase VZV-CMI temporary , after which decay towards ‘the original trajectory of VZV-CMI’ is assumed to occur .", "Next , we depicted unsuccessful reactivation of VZV that is assumed to cause endogenous boosting of VZV-CMI ( again followed by a decay ) .", "However , if the FoR is relatively higher than VZV-CMI , VZV reactivation will be successful and will cause HZ ( followed by a reinstatement of VZV-CMI ) . 10 . 7554/eLife . 07116 . 011Figure 6 . Simplified dynamics of VZV-CMI , VZV reactivation and boosting events as modeled . The sequence of exogenous boosting and VZV reactivation can be switched . DOI: http://dx . doi . org/10 . 7554/eLife . 07116 . 011 The dynamics of the synthetic population of the individual-based model are based on Belgian population and mortality data normalized to a fixed total population of 998 , 400 individuals ( Eurostat , 2012 ) .", "The chosen population size is the result of a trade-off between ensuring sufficient heterogeneity and a manageable computational burden .", "Natural deaths are selected based on the age-dependent mortality rate .", "Per time step , the number of newborns equals the number of deaths to obtain a constant population size .", "To conduct predictions over many years , a stationary demographic structure is required throughout the simulated period .", "The Belgian population from 2012 has an overrepresentation of people from age 40 to 60 years ( see Figure 7 ) .", "Applying a fixed age-dependent mortality rate in combination with the replacement by newborns would results in an oscillating population distribution over time with respect to age .", "Therefore , the initial age distribution is not based on the Belgian population count from 2012 but on the survivor function estimated from data on the number of deaths per capita .", "The survivor function by age is estimated from data on the number of deaths per capita by m ( a ) = exp ( −∫0aμ ( t ) dt ) with age-dependent mortality rate μ using a thin plate regression spline model with the gam-function in the R-package mgvc ( Wood , 2011; Hens , 2012 ) .", "Figure 7 illustrates the survivor function by age together with the Belgian population and mortality rates in 2012 . 10 . 7554/eLife . 07116 . 012Figure 7 . VZV IBM demography . DOI: http://dx . doi . org/10 . 7554/eLife . 07116 . 012 The model runs in time steps of 1 week and people with week-age 53 move to the next age class .", "To obtain homogeneous age transitions throughout the simulated period , initial week-ages are randomly assigned between 1 and 52 .", "People from the last age class with 53 weeks are removed from the population so that the demographic structure remains stable throughout the simulated period .", "At the start of the simulation , 30 individuals between ages 1 and 3 are randomly infected with CP .", "The weekly probability λi , t for a susceptible individual to become infected with VZV ( after contact with at least one infectious individual ) is calculated by λi , t=1−∏n=180 ( 1−w ( i , n ) ) ∑CPIn· ( 1−m·w ( i , n ) ) ∑HZIn with w ( i , n ) the weekly effective contact probability for an individual from group i with a random individual from group n , m the relative HZ infectiousness ( empirically estimated to be 0 . 17 [cf . paragraph ‘Modeling VZV endogenous reactivation’] ) and CPIn and HZIn the total number of infectious individuals per age class for CP and HZ , respectively .", "This formula is thus constructed by the complement of the probability that an individual did not have a successful contact with any of the chickenpox or HZ patients .", "The VZV infection probability , w ( i , n ) , is based on empirical social contact data as described elsewhere ( Ogunjimi et al . , 2009 ) .", "Here w ( i , n ) equals the number of close contacts per week lasting longer than 15 min between two random individuals in age classes i and n multiplied by the best fitting proportionality parameter q = 0 . 181 based on Belgian social contact and VZV seroprevalence data ( Ogunjimi et al . , 2009 ) .", "Individuals infected for the first time with VZV are infectious for 1 week after an incubation period of 2 weeks .", "Next , they are CP recovered and receive a normalized CMI value of 1 ± a randomly distributed factor ( normal distribution with variance 0 . 1 as suggested by Terada et al . ( 1994 ) ) .", "Once arrived in the CP recovered state , VZV-CMI starts waning at a weekly rate via the multiplication with ( 1—waning-rate ) .", "The waning-rate ( see Table 2 ) is informed by the annual decline of 2 . 7–3 . 9% per age-year as noted by baseline VZV-CMI values by Levin et al . ( 2008 ) .", "In all model steps , waning is applied to all variables .", "Note that in our model waning and ageing are indistinguishable . 10 . 7554/eLife . 07116 . 013Table 2 . Initial parameter setsDOI: http://dx . doi . org/10 . 7554/eLife . 07116 . 013ParametersStep 1Step 2Annual waning rate ( % ) 2 . 00 . 53 . 01 . 04 . 01 . 5–2 . 0–2 . 5Boosting scenario132–3–Duration of boosting ( years ) 11224374125–7–10–12–15Peak fold increase following exogenous boosting11 . 31 . 61 . 62 . 21 . 9–2 . 2–2 . 5VZV weekly reactivation probability ( % ) 0 . 010 . 0010 . 10 . 050 . 30 . 010 . 50 . 015–0 . 1–0 . 2–0 . 3–0 . 4Distribution threshold VZV-CMI for HZ1122344–Peak fold increase following endogenous boosting111 . 41 . 21 . 8–2 . 2– At each weekly update , the CP recovered individuals have a probability λi , t to receive a boost of VZV-specific immunity .", "Although HZ is less infectious than CP , we assume that if boosting has occurred , the magnitude of VZV-CMI boosting will be similar for both CP and HZ .", "To analyze the effect of boosting , we retain a CMI value for each individual with and without boosting .", "In the first 6 weeks after exogenous boosting , VZV-CMI is assumed to increase linearly up to ‘Peak fold increase’ times the pre-boosting value ( Levin et al . , 2008 ) , but is limited to a maximum of 4 times the VZV-CMI value without re-exposure .", "The 6 week duration between the boosting event and the peak has been influenced by the Levin et al . ( 2008 ) data .", "Limiting the effect of boosting to a factor 4 is based on the recent finding that pediatricians , highly exposed to CP , have T-cell values that are on average 3–4 times higher than controls , but not higher ( Ogunjimi et al . , 2014 ) .", "Next , VZV-CMI will decrease following one of three different boosting scenarios ( as shown in Figure 8 ) until it reaches the pre-boosting value again ( after a ‘duration of boosting’ ) :Exponential decrease from peak to pre-boosting value based on VZV ELISPOT CMI data after Zostavax vaccination as published by Levin et al . ( 2008 ) .", "Levin et al . ( 2008 ) presented ELISPOT data on Zostavax vs placebo-vaccinated individuals and the relative increases were ( visual reference to figure in the Levin et al . paper ) : 120% ( 6 weeks ) , 60% ( 1 year ) , 50% ( 2 years ) and 40% ( 3 years ) .", "Using these average values we performed a regression analysis to estimate the VZV-CMI exponential decay rate per week . Exponential decrease from peak to 60% of pre-boosting value ( [Levin et al . , 2008] , based on simulation from peak to year 1 , cf . supra ) , followed by a constant value for x years with x defined by the parameter set , but ≥3 years .", "After x years , VZV-CMI returns to the pre-boosting value .", "This scenario is compatible with the assumption that memory cells have a predefined and fixed lifespan as implied by Westera et al . ( 2013 ) .", "Exponential decrease from peak to pre-boosting value based on the boosting period of x years as defined by the parameter set .", "The exponential decay is defined by the peak and duration of boosting: Y ( t ) =P⋅Y0⋅e−ln ( P ) x⋅t with Y ( t ) = VZV-CMI ( disregarding age ) , P = peak fold increase , Y0 = VZV-CMI prior to entering boosting sequence ( = ‘original VZV-CMI’ ) and x = duration of boosting .", "This function is constructed by the boundary conditions that Y ( t = x ) = Y0 and Y ( t = 0 ) = P*Y0 . 10 . 7554/eLife . 07116 . 014Figure 8 . Three different boosting scenarios .", "( A ) Illustrates the exponential decline parameterized by a peak ( +120% ) at 6 weeks , ( +60% ) 1 year later , ( 50% ) 2 years later and ( +40% ) 3 years later as presented by the Zostavax vaccine trial by Levin et al . ( B ) Illustrates the exponential decline from peak ( +120% ) to ( +60% ) 1 year later and constant for x years ( as defined by the parameter set ) after wards , as a modified interpretation of the results of the Zostavax vaccine trial by Levin et al . ( C ) Illustrates the increase to a peak value as defined by the parameter set that is followed by an exponential decline so that the pre-boosting value is reached after x years . DOI: http://dx . doi . org/10 . 7554/eLife . 07116 . 014 It is important to clarify that if a new boosting event occurs during an ongoing boosting sequence , the VZV-CMI value attained right before the new boosting event occurs , is assumed to be the baseline ‘pre-boosting’ reference for the new boosting sequence .", "This means that for scenario 3 in case of a new boosting event 6 weeks ( and mutatis mutandis for the other situations ) after the first boost VZV-CMI evolves as Y ( t ) =P⋅Y1⋅e−ln ( P ) x⋅ ( t−t1 ) =P⋅P⋅Y0⋅e−ln ( P ) x⋅t1︸Y1⋅e−ln ( P ) x⋅ ( t−t1 ) =P2⋅Y0⋅e−ln ( P ) x⋅t , with the subscript 1 referring to the situation when the second boosting event occurs .", "This shows that boosting during an ongoing boosting episode prolongs the time before the ‘original VZV-CMI ( Y ( t ) = Y0 ) ’ is reached again .", "After primary infection , VZV is assumed to remain latent , but capable of reactivation .", "The frequencies of VZV reactivation used in the parameter sets were informed by observed VZV reactivation frequencies in random samples from healthy individuals ( 2% in blood [Schünemann et al . , 1997]; 0 out of 112 saliva samples [Mehta et al . , 2003]; 2 . 5% in saliva [Nagel et al . , 2011] ) , immunosuppressed patients ( 8 . 1% from various sites [Engelmann et al . , 2008] ) , individuals with malignancies ( 7 . 5% in blood [Malavige et al . , 2010] ) and HIV patients ( 9% in saliva [van Velzen et al . , 2013]; 16% in cerebrospinal fluid [Birlea et al . , 2011] ) .", "The consequence of reactivation can either be endogenous boosting or clinical reactivation ( HZ ) and this is defined by the difference between VZV-CMI and the FoR .", "The FoR defines the VZV-CMI needed to resist clinical reactivation and if VZV-CMI < FoR , reactivation will lead to HZ .", "The reader should imagine the FoR to represent independent reactivation behavior of VZV and whether this reactivation will lead to HZ or endogenous boosting will depend on the value of VZV-CMI .", "The FoR deviates per time step and individual by means of a gamma probability density function .", "This gamma function is chosen as it represents the summation of unknown biological phenomena that are assumed to have an exponential distribution .", "The parameter set includes four different gamma distributions ( see Table 2 and Figure 9 ) .", "HZ individuals are assumed to be infectious for 1 week and receive a VZV-CMI reset to 1 ± random factor ( normally distributed , cf . primary infection ) .", "There exists no data on the relative infectiousness of HZ patients nor is it likely that this can be estimated through experiments .", "We chose to approximate the relative infectiousness of HZ patients by the relative infectiousness of breakthrough CP patients ( Bernstein et al . , 1993 ) .", "This has previously been estimated at 0 . 17 ( Brisson et al . , 2002 ) .", "Although HZ infectiousness was needed to maintain circulation of VZV in our model , the relative effects on overall VZV transmission are—compared to CP—marginal .", "Figure 2—figure supplement 1 shows the results of a sensitivity analysis in which we varied HZ infectiousness from 0 . 03 to 0 . 45 for the 13 best fitting parameter .", "As can be seen in Figure 2—figure supplement 1 our results are quite robust .", "Reinstallation of VZV-specific immunity after HZ is based on experimental data showing that a second case of HZ only occurs in about 5% of individuals ( Yawn et al . , 2011 ) and that VZV-CMI is higher in recovered HZ patients than in age-matched controls ( Weinberg et al . , 2009 ) . 10 . 7554/eLife . 07116 . 015Figure 9 . Different cumulative distribution functions ( CDF ) for Force of Reactivation ( FoR ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 07116 . 015 If VZV-CMI ≥ FoR , endogenous boosting occurs followed by an exponential decrease according to one of the scenarios described in the previous section .", "The peak following endogenous boosting , however , is restricted to be at most equal to the peak following exogenous boosting .", "In addition , we assume that an endogenous boosting sequence will always be overruled by a new successful exogenous re-exposure boosting episode .", "Simulations were performed using Matlab 2012b on the Flemish VSC supercomputer .", "Simulations ran for 320 years and model output was obtained by averaging the age-specific results over the last 80 years .", "The main outputs were CP incidence , HZ incidence , VZV serology and VZV-CMI .", "In order to optimize the fitting procedure , we performed a two-step parameter set analysis .", "The following 7 parameters were estimated by means of the fitting procedure: VZV-CMI waning rate , type of boosting scenario , duration of exogenous boosting , peak fold increase following exogenous boosting , VZV reactivation probability , FoR distribution and the peak fold increase following endogenous boosting .", "In the first step we ran each parameter set three times .", "We calculated per set the Binomial likelihood by fitting the HZ age-specific output data to Belgian HZ incidence data ( Bilcke et al . , 2012 ) .", "Next , we selected the parameter set leading to the lowest mean deviance ( = −2*loglikelihood ) based on the three repetitions with different stochastic seed and all other parameter sets with deviance +5% at most in order to account for model selection uncertainty ( Castro Sanchez et al . , 2013 ) .", "In order to broaden the parameter selection , we also selected the most prevalent ( marginal ) parameter values in the lowest mean deviance 2 . 5% percentile ( see Table 3 ) . 10 . 7554/eLife . 07116 . 016Table 3 . Step 2 parameter set selectionDOI: http://dx . doi . org/10 . 7554/eLife . 07116 . 016ParametersBest parameter sets + deviance +5%Most prevalent parameters in Q2 . 5Annual waning rate ( % ) 2 . 02 . 0Boosting scenario33Duration of exogenous boosting ( years ) 1142–4–7–12Peak fold increase following exogenous boosting1 . 61 . 6–2 . 2VZV weekly reactivation probability ( % ) 0 . 010 . 010 . 10 . 3Distribution threshold VZV-CMI for HZ2142Peak fold increase following endogenous boosting11 In the second step we adapted our parameter ranges and intervals according to the best fitting values to obtain new parameter combinations ( see Table 3 ) .", "Again , we ran each parameter set three times and calculated the mean deviance .", "The best parameter sets were defined by those parameter sets that had at least one run with a deviance within the 5% range of the deviance of the best fitting parameter set .", "Given the fact that some selected parameter values were on an unexplored border of the parameter grid , we studied whether more extreme values for the border parameters led to better results ( and continuing if deviance was within the second step best deviance +5% ) .", "Doing this , we allowed the other parameters to vary for one unit in both parameter directions .", "We used our best parameter sets and introduced a simplistic hypothetical single dose 100% effective CP vaccine ( without waning of vaccine-induced immunity ) for all children ageing between 1 and 2 years .", "Vaccinated individuals were assumed not to be susceptible to HZ ." ] ]

[ "Varicella-zoster virus ( VZV ) causes chickenpox and reactivation of latent VZV causes herpes zoster ( HZ ) .", "VZV reactivation is subject to the opposing mechanisms of declining and boosted VZV-specific cellular mediated immunity ( CMI ) .", "A reduction in exogenous re-exposure ‘opportunities’ through universal chickenpox vaccination could therefore lead to an increase in HZ incidence .", "We present the first individual-based model that integrates within-host data on VZV-CMI and between-host transmission data to simulate HZ incidence .", "This model allows estimating currently unknown pivotal biomedical parameters , including the duration of exogenous boosting at 2 years , with a peak threefold to fourfold increase of VZV-CMI; the VZV weekly reactivation probability at 5% and VZV subclinical reactivation having no effect on VZV-CMI .", "A 100% effective chickenpox vaccine given to 1 year olds would cause a 1 . 75 times peak increase in HZ 31 years after implementation .", "This increase is predicted to occur mainly in younger age groups than is currently assumed ." ]

[ "The itchy-scratchy misery of a chickenpox was until recently a rite of passage for children around the world .", "The varicella-zoster virus causes chickenpox infections .", "This virus persists in small numbers in nerve cells for many years after infection , and can reactivate from these cells .", "Often this reactivation causes no symptoms , but sometimes it results in a painful skin condition called shingles ( or herpes zoster ) , especially in older adults .", "Some countries—including the United States , Australia , Taiwan and Greece—have virtually wiped out childhood cases of chickenpox by requiring that children be vaccinated against the varicella-zoster virus .", "But some countries have hesitated .", "One reason for this hesitation is that exposure to individuals with a chickenpox infection helps boost the immunity of individuals who have previously been infected .", "This may help reduce the likelihood of these people developing shingles later in life .", "So , some countries have worried that chickenpox vaccinations might inadvertently increase the number of shingles cases .", "To assess this risk , many scientists have created computer models , but the models have some limitations .", "Now , Ogunjimi et al . report a new individual-based model to assess the effect of childhood varicella vaccination on shingles cases that factors in the immune responses to varicella infection .", "The model suggests that re-exposure to the varicella virus through contact with infected people would only provide extra protection for about two years; this is much shorter than previous predictions that suggested it might last 20 years .", "The model also predicts that implementing a varicella vaccination program for children would almost double the number of shingles cases 31 years later .", "But this increase would be temporary .", "The predicted increase in shingles cases is likely to disproportionately occur among 31- to 40-year-olds .", "This is unexpected because most previous models predict that older age groups would bear the brunt of a rise in shingles , but this younger population would be less likely to develop lasting complications of shingles .", "Together , these findings may allay some fears about implementing childhood varicella vaccination programs by showing that the benefits of re-exposure are limited ." ]

[ "Introduction", "Results", "Discussion", "Experimental procedures" ]

[ "developmental biology", "neuroscience" ]

[ [ "Axonal growth cones are guided to their targets by molecular cues laid down at discrete decision steps along their routes .", "This information is transduced inside growth cones via dedicated intracellular signaling pathways , eventually modulating cytoskeletal dynamics .", "How multiple pathways interact and compound the limited molecular diversity of axon guidance cues to produce the rich complexity of nerve trajectories is unclear ( Dickson , 2002; Kolodkin and Tessier-Lavigne , 2011; Tessier-Lavigne and Goodman , 1996 ) .", "Multiple modes of axon guidance signaling pathway interaction have been documented .", "Axon guidance ligand co-incidence can result in emergent growth cone behaviors that are qualitatively different from those induced by either cue alone ( Bielle et al . , 2011; Kuwajima et al . , 2012 ) .", "The exposure to one cue can also silence the response to another , as Slit silences Netrin-1 attraction in commissural axons , allowing them to continue their journey past the nervous system midline ( Stein , 2001 ) .", "Contrasting these is signal co-operation , where co-activation of two pathways changes the magnitude of growth cone responses , but not their quality .", "Co-operative steering of a growth cone in the same direction could entail two co-localized congruent cues , either both repellent or attractants , or two opposing cues , where a repellent reinforces the decision to grow towards an attractant cue ( Dudanova et al . , 2010; Schmitt et al . , 2006 ) .", "At the molecular level , co-operating axon guidance signaling pathways can intersect in additive or synergistic manners .", "Additive integration involves the summation of responses to individual cues implying parallel signaling pathways intersecting at the level of actin stability , as observed in additive phenotypes of mutations affecting motor axon guidance and muscle target selection in Drosophila and in vertebrates ( Kramer et al . , 2006; Winberg et al . , 1998 ) .", "Synergistic integration , that is , whose magnitude , when the signals are coincident , is greater than the sum of the effects of separate signals , implies signaling pathways intersecting at an intermediate step .", "It has been observed for congruent cues , such as bone morphogenetic proteins , whose repulsive signal integration occurs at the level of their receptors ( Butler and Dodd , 2003; Yamauchi et al . , 2008 ) , and for Sonic Hedgehog and Ntn1 ( Sloan et al . , 2015 ) .", "Despite this growing list of signal intersection modalities ( Dudanova and Klein , 2013 ) , their precise molecular mechanisms at a simple axon guidance waypoint are still unclear .", "One such point is traversed by spinal lateral motor column ( LMC ) axons as they enter the vertebrate limb where lateral and medial LMC axons diverge to form , respectively , dorsal and ventral limb nerves ( Lance-Jones and Landmesser , 1981; Tosney and Landmesser , 1985 ) .", "A molecular model of this event implicates limb mesenchyme ephrin signaling to LMC growth cone tyrosine kinase Eph receptors .", "Lateral LMC growth cone expression of EphA receptors and medial LMC growth cone expression of EphB receptors results in their repulsion from cognate ephrin-A and ephrin-B ligands present in the ventral and dorsal limb mesenchyme , respectively ( Eberhart et al . , 2000; Helmbacher et al . , 2000; Kania and Jessell , 2003; Luria et al . , 2008 ) .", "Additional mechanisms , such as reverse signaling from Eph receptors or semaphorins and glial cell-derived neurotrophic factor ( GDNF ) in the limb , co-operate with forward ephrin-A:EphA signaling to contribute to the fidelity of LMC axon trajectory choice ( Bonanomi et al . , 2012; Chai et al . , 2014; Dudanova et al . , 2010; Huber et al . , 2005; Kao and Kania , 2011; Kramer et al . , 2006; Marquardt et al . , 2005 ) .", "These observations imply a fragmented molecular logic of spinal motor axon guidance – ephrin:Eph forward signaling as a common effector of all LMC axon guidance , integrating with molecularly diverse LMC-subpopulation specific cues .", "Netrins and their receptors have been implicated in diverse axon guidance events including motor axon exit from the spinal cord ( Bai et al . , 2011; Hedgecock et al . , 1990; Keino-Masu et al . , 1996; Keleman and Dickson , 2001; Kennedy et al . , 1994; Lai Wing Sun et al . , 2011; Serafini et al . , 1994 ) .", "The cellular response to Netrins is dictated by its receptors: deleted in colorectal cancer ( DCC ) and perhaps Neogenin enable attraction to Netrin-1 ( Keino-Masu et al . , 1996; Palmesino et al . , 2012; Xu et al . , 2014 ) , while Unc5 proteins , in some cases in conjunction with DCC , elicit repulsion from Netrin-1 ( Hong et al . , 1999; Leonardo et al . , 1997 ) .", "Modulatory signals such as Slit and transforming growth factor β ( TGF-β ) act at the level of DCC to change Netrin-1 responses ( Bai et al . , 2011; Leyva-Diaz et al . , 2014; Stein , 2001 ) or to set the sensitivity of repulsive Netrin receptors ( MacNeil et al . , 2009 ) .", "The extensive modulation of Netrin-1 signaling raises the question of its relationship with ephrin signaling that is prominently involved in axon guidance decisions in the developing nervous system .", "In this study , we provide genetic in vivo and in vitro evidence that limb mesenchyme Netrin-1 is a bifunctional effector , attracting lateral LMC and repelling medial LMC axons .", "Using in vitro growth preference assays , we demonstrate synergistic integration of congruent and opposing Netrin and ephrin signals .", "We also show that repulsive Netrin and ephrin receptors co-localize in a complex whose formation is modulated by ephrin-B2 signaling and EphB2 kinase activity , and that Src family kinase is a node for integration of Netrin-ephrin synergistic growth cone responses ." ], [ "Netrin-1 ( Ntn1 ) mRNA expression in developing limb buds ( Krawchuk and Kania , 2008 ) , prompted us to analyze the expression of Netrin ligands and their receptors at the time of LMC axon growth into the limb mesenchyme , between the embryonic day ( e ) 10 . 5 and e11 . 5 in mouse and between Hamburger-Hamilton stage ( HH st . ) 25 and 27 in chick ( Hamburger and Hamilton , 1951; Kania et al . , 2000; Tosney and Landmesser , 1985 ) .", "Comparing Netrin family ligand mRNA expression with that of Lmx1b , a dorsal limb marker ( Riddle et al . , 1995 ) , revealed that at the base of the mouse forelimb and hindlimb , where LMC axons select a dorsal or ventral limb trajectory , Netrin-1 mRNA and protein were enriched in the dorsal limb mesenchyme ( Figure 1A–D ) .", "Expression of other Netrin ligands was either absent or not localized in a manner consistent with a role in LMC axon guidance at this choice point ( Figure 1—figure supplement 1 ) .", "In addition , the expression of β-galactosidase from an Ntn1 gene trap allele ( Ntn1Gt ) recapitulated Netrin-1 mRNA and protein distribution ( Figure 1E , F; Serafini et al . , 1996 ) .", "In HH st . 25 chickens , Ntn1 mRNA was also confined to the dorsal limb ( Figure 1G , H ) . 10 . 7554/eLife . 10841 . 003Figure 1 . Expression of Netrin-1 in the limb mesenchyme and of Netrin receptors in medial and lateral LMC neurons . Netrin-1 mRNA and protein detected by in situ hybridization and immunohistochemistry compared with the dorsal limb marker Lmx1b in mouse and chick limbs at the time of LMC axon limb ingrowth .", "( A , B )", "In situ hybridization for Ntn1 ( A ) and the dorsal limb marker Lmx1b ( B ) mRNAs in consecutive sections of e11 . 5 wild-type mouse forelimb .", "( C , D )", "Immunostaining for Netrin-1 , neurofilament , and Lmx1b in e11 . 5 mouse forelimb .", "( E , F )", "Immunostaining for β-galactosidase and neurofilament in e11 . 5 Ntn1Gt/+ mouse forelimb .", "β-galactosidase is expressed at the dorsal limb mesenchyme domain abutting dorsally projecting LMC axons .", "( G , H )", "In situ hybridization for Ntn1 mRNA ( G ) and Lmx1b mRNA ( H ) in consecutive sections of HH st . 27 chick hindlimbs .", "Unc5c and DCC ( or Neogenin in chick ) mRNA and protein expression was compared with LMC divisional markers in e11 . 5 mice and HH st . 27 chick spinal cord or to EphA4 in lateral LMC axons .", "( I , J )", "Selective expression of Unc5c in medial LMC motor neurons .", "In situ detection of Unc5c mRNA and immunodetection of Isl1/2 in e11 . 5 lumbar mouse spinal cord .", "The green signal in ( J ) represents the pseudocolor image shown in ( I ) .", "M and L indicate the position of medial and lateral LMC , respectively , as assessed by Isl1/2 ( red ) or Lhx1 immunostaining ( not shown ) .", "( K , L )", "DCC is expressed at both LMC divisions .", "In situ hybridization of Dcc mRNA and immunodetection of Isl1/2 in e11 . 5 mouse lumbar spinal cord .", "The green signal represents the pseudocolor image .", "Note the higher level of Dcc in lateral ( Isl1- ) vs . medial ( Isl1+ ) LMC motor neurons .", "( M–R )", "Localization of Unc5c and DCC proteins in dorsal and ventral axon branches entering the hindlimb in e11 . 5 embryos .", "( M–O )", "Double immunostaining for Unc5c and DCC .", "Note the high expression level of Unc5c in ventral nerves and DCC in both dorsal and ventral nerves .", "( P–R )", "Double immunostaining for Unc5c and EphA4 .", "( S–V )", "Expression of Netrin-1 receptors in chick LMC neurons .", "In situ hybridization for Unc5c , Neo1 , Isl1 , and Lhx1 in consecutive sections of HH St . 27 chick lumbar spinal cord .", "Note the presence of in medial ( Isl1+ ) LMC neurons and of Neo1 in both LMC divisions .", "( W ) Summary of Netrin-1 , DCC , and Unc5c expression in limb mesenchyme and LMC neurons .", "LMC , lateral motor column; Ntn1 , Netrin-1; NF , neurofilament; M , medial; L , lateral; d , dorsal; v , ventral .", "Scale bars: ( A–H ) 250 μm , ( I–L ) 80 μm , ( M–V ) 50 μm . DOI: http://dx . doi . org/10 . 7554/eLife . 10841 . 00310 . 7554/eLife . 10841 . 004Figure 1—figure supplement 1 . Expression of Netrin family ligands and receptors in mouse and chick hindlimbs .", "( A and B )", "Analysis of mRNA expression by in situ hybridization of Netrin family ligands in mouse e11 . 5 and chick HH st . 27 hindlimbs .", "Note the absence of expression of mNtn3 , mNtn4 , cNtn2-like , and cNtn4 and the expression of mNtnG2 , mRgma , mRgmb , cNtnG1 , and cNtnG2 in patterns that do not suggest a clear LMC axon dorsoventral limb nerve selection function .", "( C ) Analysis of mRNA expression by in situ hybridization of Netrin family receptors in e11 . 5 mouse hindlimbs .", "Note the absence of expression of these receptors in hindlimbs with the exception of Unc5c that is present in ventral limb mesenchyme .", "( D and E )", "Specificities of Unc5c and DCC antibodies .", "( D ) Immunostaining for Unc5c and neurofilament in e 11 . 5 mice lumbar spinal cords ( left ) or peripheral axons ( right ) of wild-type and Unc5c-/- mice .", "( E ) Immunostaining for DCC in spinal cords of wild-type or Dcc-/- mice .", "( F ) Immunostaining for Unc5c and DCC in sensory and motor axons .", "Note the absence of DCC from axons exiting the DRG and the lower expression of Unc5c in the sensory-only branch compared with the motor branch .", "( G ) Analysis of mRNA expression by in situ hybridization for genes encoding Netrin receptors in e11 . 5 mouse and HH St . 27 chick spinal cords .", "Expression of mouse Neo1 , Dscam , Unc5a , and Unc5d is observed in both medial and lateral LMCs , while mouse Unc5b is not expressed in LMC neurons .", "In chick , both Unc5a and Unc5b are expressed in medial LMC while Unc5d is absent from LMC neurons .", "SpC , spinal cord .", "Scale bars: ( A–C ) 300 μm , ( D ) 40 μm , ( E–G ) 80 μm . DOI: http://dx . doi . org/10 . 7554/eLife . 10841 . 004 To determine whether Netrin-1 receptors are present in developing LMC neurons , we first compared their mRNA expression in mouse spinal cord sections with that of medial and lateral LMC neuron markers , Isl1 and Lhx1 , respectively , between e10 . 5 and e11 . 5 ( Tsuchida et al . , 1994 ) .", "Unc5c , a repulsive Netrin-1 receptor gene , was expressed in Isl1+ medial LMC neurons ( Figure 1I , J ) , while Dcc and Neogenin1 ( Neo1 ) , encoding attractive Netrin-1 receptors , were expressed by both LMC divisions at hindlimb and forelimb levels ( Figure 1K , L; Figure 1—figure supplement 1 ) .", "Detection of Unc5c protein using an antibody raised against its C terminus , and Isl1 and Lhx1 , confirmed that Unc5c is present in medial LMC neurons , but not in lateral LMC neurons ( Figure 1—figure supplement 1; data not shown ) .", "Co-detection of DCC and Isl1 or Lhx1 proteins indicated that DCC is expressed by both LMC divisions ( Figure 1—figure supplement 1; data not shown ) .", "Examination of motor and sensory nerves near their spinal cord and dorsal root ganglion exit , before they merge into mixed sensory-motor nerves , revealed that DCC is present exclusively on motor axons , while Unc5c is expressed at high levels in motor axons with some expression in sensory axons ( Figure 1—figure supplement 1 ) .", "In the limb , high levels of Unc5c protein were present in ventral limb nerves ( Figure 1M–O ) , consistent with its medial LMC expression , while DCC was detected in both dorsal and ventral nerves ( Figure 1N–O ) , consistent with its pan-LMC expression .", "Additional analysis revealed that mouse Dscam and Unc5a were expressed in all LMC neurons , while Unc5b and Unc5d were not detected in this population ( Figure 1—figure supplement 1 ) .", "Chick Unc5b and Unc5c were expressed in medial LMC neurons while Unc5b and Neo1 mRNAs were found in all LMC neurons ( Figure 1S–V , Figure 1—figure supplement 1 ) .", "Altogether , these studies suggested that dorsal limb Ntn1 may play a dual role in LMC axon guidance , attracting lateral LMC axons that do not express Unc5c into the dorsal limb , and repelling Unc5c-expressing medial LMC axons into the ventral limb ( Figure 1W ) .", "We next determined whether loss of Netrin-1 or its receptors affects LMC axon pathfinding , by analyzing the limb trajectories of medial and lateral LMC axons in e11 . 5 and e12 . 5 wild-type , Ntn1Gt/Gt , Dcc-/- , Neo1Gt/Gt , Unc5c-/- , and Unc5a-/- mutant mice ( Bae et al . , 2009; Boyer and Kozak , 1991; Burgess et al . , 2006; Fazeli et al . , 1997; Williams et al . , 2006 ) .", "In all strains examined , the specification of LMC neurons , their axon outgrowth , and Eph and ephrin expression was normal ( Figure 2—figure supplement 2; data not shown ) .", "To examine LMC axon trajectory selection , we injected the retrograde tracer horseradish peroxidase ( HRP ) into the ventral or dorsal limb muscles of Ntn1Gt/Gt and Netrin receptor mutant embryos and determined the divisional identity of labeled LMC neurons using the marker Foxp1 ( Dasen et al . , 2008; Rousso et al . , 2008 ) , and the medial LMC marker Isl1 ( Luria et al . , 2008 ) .", "All our retrograde tracing observations were confirmed using an alkaline phosphatase medial LMC reporter transgene or Unc5c and EphA4 axonal staining ( Figure 2—figure supplement 1 ) .", "When HRP was injected into the ventral limb to detect aberrant lateral LMC axons , in Dcc mutants 9 . 7% of HRP-labeled neurons were medial LMC , compared with 4 . 4% in controls , 4 . 7% in Ntn1Gt/Gt and 5 . 6% in Unc5c-/- mice ( Figure 2A–H; p=0 . 164 , 1 , and 0 . 748 for Dcc-/- , Ntn1Gt/Gt , and Unc5c-/- vs . controls; all values listed in Supplementary file 1B ) .", "We also injected HRP into the ventral limbs of Neo1Gt/Gt or Dscam-/- single or Dcc-/-; Neo1Gt/Gt double mutants and assessed the divisional identity of labeled LMC neurons ( Figure 2—figure supplement 2 ) .", "Neither single nor double mutant lateral LMC axons were found in the ventral limb at an incidence greater than that in control embryos ( Figure 2—figure supplement 2; p=0 . 105 , 0 . 537 and 0 . 537 for Neo1Gt/Gt , Dscam-/- and Dcc-/-; Neo1Gt/Gt vs . wild-type controls ) .", "Thus , extensive redundancy notwithstanding , Dcc , Neogenin , or Dscam are not required for in vivo lateral LMC axon guidance . 10 . 7554/eLife . 10841 . 005Figure 2 . The requirement of Netrin-1 and its receptors for the fidelity of LMC axon trajectory selection . Lateral and medial LMC axon projections were analyzed by injecting the retrograde tracer HRP into dorsal or ventral limb muscles followed by the assessment of LMC divisional identity of backfilled LMC neurons .", "( A–H )", "Analysis of lateral LMC motor axon projections in wild-type ( A and B ) , Dcc-/- ( C and D ) , Ntn1Gt/Gt ( E and F ) and Unc5c-/- ( G and H ) mutant mice .", "A retrograde tracer ( HRP , red ) was injected in the ventral forelimb of e12 . 5 wild-type or mutant mice followed by detection of Isl1 ( green , A , C , E , G ) and Foxp1 ( blue , B , D , F , H ) to identify medial ( Isl1+ , Foxp1+ ) and lateral ( Isl1- , Foxp1+ ) LMC neurons .", "Insets in A , C , E and G show examples of magnified HRP+ backfilled cells that are Isl1- ( Dcc-/- mice ) or Isl1+ ( wild-type , Ntn1Gt/Gt or Unc5c-/- mice ) .", "( I ) Quantification of retrogradely labeled lateral LMC axon projections .", "The graph depicts the mean percentage ± SD of HRP+ backfilled motor neurons that express the medial LMC marker Isl1 after a dorsal limb injection .", "n = 5 ( wild-type ) , 4 ( Ntn1Gt/Gt ) , 3 ( Unc5c-/- ) , 5 ( Dcc-/- ) embryos .", "( J ) Summary of analysis of lateral LMC projections in different Netrin signaling mutant mice .", "See Figure 2—figure supplement 2 for results of additional mutant analyses .", "( K–R )", "Analysis of medial LMC motor axon projections in wild-type ( K and L ) , Dcc-/- ( M and N ) , Ntn1Gt/Gt ( O and P ) , and Unc5c-/- ( Q and R ) mutant mice .", "HRP ( red ) was injected in the dorsal forelimb of e12 . 5 wild-type or mutant mice followed by immunostaining of spinal cord sections for Isl1 ( green , K , M , O , Q ) and Foxp1 ( blue , L , N , P , R ) to identify medial ( Isl1+ , Foxp1+ ) and lateral ( Isl1- , Foxp1+ ) LMC neurons .", "Arrowheads indicate examples of HRP+ cells that are Isl1- ( wild-type and Dcc-/- mice ) or Isl1+ ( Ntn1Gt/Gt or Unc5c-/- mice ) and are magnified in the insets .", "( S ) Quantification of retrogradely labeled medial LMC axon projections .", "The graph depicts the mean percentage ± SD of HRP+ backfilled motor neurons that express the medial LMC marker Isl1 after a dorsal limb injection .", "n = 3 ( wild-type ) , 3 ( Ntn1Gt/Gt ) , 5 ( Unc5c-/- ) , 3 ( Dcc-/- ) embryos .", "( T ) Summary scheme of medial LMC projections in wild-type , Ntn1Gt/Gt , Unc5c-/- , and Dcc-/- mice .", "In Ntn1Gt/Gt and Unc5c-/- mice some medial LMC axons project to the dorsal limb .", "HRP , horseradish peroxidase; wt , wild-type; Ntn1 , Netrin-1; error bars = SD; *** = p<0 . 001; * = p<0 . 05; statistical significance computed using Fisher’s exact test .", "All values ( mean ± SD ) can be found in Supplementary file 1B; scale bar: 20 μm . DOI: http://dx . doi . org/10 . 7554/eLife . 10841 . 00510 . 7554/eLife . 10841 . 006Figure 2—figure supplement 1 . Analysis of medial LMC axon projections in mutant mice . Medial LMC axons were identified by genetic labeling with placental alkaline phosphatase ( PLAP ) ( A–H ) or identified with immunostaining for NF and EphA4 ( J ) or NF and Unc5c ( K ) .", "( A–H ) hCrest/Isl1-PLAP mice were crossed to wild-type , Dcc-/- , Ntn1Gt/Gt , and Unc5c-/- mice .", "Developing for alkaline phosophatase allowed the selective visualization of medial LMC axon projections .", "Immunostaining for NF in combination with alkaline phosphatase reaction in wild-type ( A and B ) , Ntn1Gt/Gt ( C and D ) , Unc5c-/- ( E and F ) , and Dcc-/- ( G and H ) mice .", "( I ) Quantification of the percentage of PLAP signal present in dorsal or ventral nerve branches .", "( J ) Immunostaining for NF and EphA4 in e11 . 5 lumbar spinal cords of wild-type ( top panels ) and Unc5c-/- mutants ( bottom panels ) .", "Note the overlap of NF and EphA4 in the dorsal branch of wild-type mice and the presence of a large cohort of EphA4-negative axons in the dorsal nerve of Unc5c-/- mice .", "( K ) Analysis of Unc5c-expressing medial LMC axon trajectories in Ntn1Gt/Gt , Dcc-/- , Neo1Gt/Gt or double Neo1Gt/Gt , Dcc-/- mice .", "Limb sections were immunostained for NF and Unc5c ( at a dilution in which motor but not sensory axons can be detected ) in the different mutant mice as indicated .", "Note the presence of Unc5c-positive axons in the dorsal branch of Ntn1Gt/Gt mice .", "LMC , lateral motor column; NF , neurofilament; wt , wild-type; error bars = SD; *** = p<0 . 001; ** = p<0 . 01; n . s . = not significant; statistical significance computed using Student’s unpaired t test; all values are mean ± SD .", "Scale bars: ( A–H , K ) 80 μm; ( J ) 25 μm . DOI: http://dx . doi . org/10 . 7554/eLife . 10841 . 00610 . 7554/eLife . 10841 . 007Figure 2—figure supplement 2 . Normal specification of LMC neurons in Unc5c , Dcc , and Ntn1Gt mutants and summary of LMC axon trajectory analysis in mutant mice .", "( A–C )", "Normal specification of LMC neurons in Unc5c-/- , Dcc-/- , and Ntn1Gt/Gt mice .", "( A ) The number of medial and lateral LMC neurons was determined in e13 . 5 cervical and lumbar spinal cord sections from different mutant mice immunostained for Isl1 and Foxp1 .", "( B ) Quantification of medial and lateral LMC neurons in wt , Unc5-/- , Dcc-/- , and Ntn1Gt/Gt mice .", "( C ) Normal expression of EphB1 and EphA4 in Unc5c-/- , Dcc-/- , and Ntn1Gt/Gt mice .", "E13 . 5 lumbar spinal cord sections from different mice were immunostained for Isl1 and EphA4 ( C1-2 , C5-6 , C9-10 ) or analyzed by in situ hybridization for Isl1 and Ephb1 mRNA in consecutive sections ( C3-4 , C7-8 , C11-12 ) .", "( D and E )", "Summary of analysis of lateral ( ventral labeling ) and medial ( dorsal labeling ) LMC trajectories by backfill experiments in mice forelimbs of Neo1Gt/Gt double Dcc-/-;Neo1Gt/Gt , Unc5a-/- , double Unc5a-/-; Unc5c-/- , and Dscam-/- mice .", "Only the proportion of HRP+ LMC expressing Isl1 in dorsally filled double Unc5a-/-; Unc5c-/- embryos is significantly different from that of wt embryos ( p<0 . 001 ) .", "Number of embryos quantified for ventral fill: n = 5 ( wt ) , 4 ( Neo1Gt/Gt ) , 3 ( Dcc-/- and Neo1Gt/Gt ) , 4 ( Unc5a-/- ) , 4 ( Unc5c-/- and Unc5a-/- ) , 4 ( Dscam-/- ) .", "Number of embryos quantified for dorsal fill: n = 3 ( wt ) , 4 , ( Neo1Gt/Gt ) , 4 ( Dcc-/-; Neo1Gt/Gt ) , 4 ( Unc5a-/- ) , 3 ( Unc5c-/-; Unc5a-/- ) , 3 ( Dscam-/- ) .", "( F ) Summary of analysis of lateral ( ventral labeling ) and medial ( dorsal labeling ) LMC trajectories by backfill experiments in e12 . 5 mice hindlimbs of different Netrin mutants as indicated .", "The proportions of HRP+ LMC expressing Isl1 in dorsally filled Unc5c-/-and Unc5a-/-; Unc5c-/- embryos are significantly different from that of wt embryos ( p<0 . 001 for both groups ) .", "Number of embryos quantified for ventral fill: n = 7 ( wt ) , 3 , ( Neo1Gt/Gt ) , 4 ( Dcc-/-; Neo1Gt/Gt ) , 4 ( Unc5a-/- ) , 3 ( Unc5c-/-; Unc5a-/- ) , 4 ( Dscam-/- ) .", "Number of embryos quantified for dorsal fill: 4 ( wt ) , 4 , ( Neo1Gt/Gt ) , 3 ( Dcc-/- and Neo1Gt/Gt ) , 4 ( Unc5a-/- ) , 3 ( Unc5c-/-; Unc5a-/- ) , 3 ( Dscam-/- ) .", "HRP , horseradish peroxidase; LMC , lateral motor column; wt , wild-type; error bars = SD; *** = p<0 . 001; statistical significance computed using Student’s unpaired t test ( B ) or Fisher’s exact test on raw numbers ( E , F ) ; all values are mean ± SD .", "Quantification details in supplemental file 1C .", "Scale bars: ( A ) 50 μm; ( C ) 80 μm; ( D ) 40 μm . DOI: http://dx . doi . org/10 . 7554/eLife . 10841 . 007 Next , to detect aberrant medial LMC axon projections , we injected HRP into the dorsal limb of control and mutant mice and the proportion of labeled medial motor neurons determined as a percentage of all labeled motor neurons .", "In wild-type mice , 3 . 9% of tracer-labeled LMC neurons were medial LMC ( Figure 2K , L , S ) , whereas in Unc5c mutants , 40 . 4% of labeled LMC neurons were medial LMC , corresponding to essentially random limb nerve selection ( Figure 2Q , R , S; p<0 . 001 wild-type vs . Unc5c-/- ) .", "In Ntn1Gt/Gt mice , 12 . 6% of labeled LMC neurons were medial LMC , representing a significant projection error ( Figure 2O , P , S; p<0 . 05 vs . wild-type ) .", "Similar incidence of misprojections in Ntn1Gt/Gt and Unc5c mutants were also observed using a medial LMC-specific transgene ( Figure 2—figure supplement 1 ) , suggesting that Ntn1 in the dorsal limb repels medial LMC axons through Unc5c .", "As Unc5c-DCC heterodimers have been proposed to mediate repulsion from Ntn1 ( Hong et al . , 1999 ) , we asked whether DCC is required to specify the limb trajectory of medial LMC axons .", "In Dcc-/- mice with dorsal limb tracer injection , medial LMC neurons represented 3 . 3% of labeled LMC neurons , a proportion not significantly different from the 3 . 9% observed in controls ( Figure 2M , N , S , p=1 vs . wild-type controls ) .", "To determine whether Neo1 might act with DCC to mediate repulsion from Ntn1 , we investigated the fidelity of medial LMC axon guidance in Neo1Gt/Gt single or Dcc-/-; Neo1Gt/Gt double mutants .", "Medial LMC projections in Neo1Gt/Gt or in Dcc-/-; Neo1Gt/Gt double mutants were not different from controls , and neither were they affected in Dscam-/- mice ( Figure 2—figure supplement 2; p=0 . 537 , 0 . 748 , 0 . 537 for Neo1Gt/Gt , for Dcc-/-; Neo1Gt/Gt , and for Dscam-/-vs . wild-type controls , respectively ) , suggesting that attractive Ntn1 receptors are not required for medial LMC axon guidance .", "We next assessed whether Netrin-1 can directly modulate the behavior of LMC axons by monitoring the response of medial and lateral LMC axons to Netrin-1 protein in an in vitro stripe assay ( Kao and Kania , 2011 ) .", "We exposed chick LMC axons to protein carpets in alternating stripes of either Fc protein and Cy3-labeled secondary antibody mixed with Fc ( Fc/Fc ) or Fc protein and Cy3-labeled secondary antibody mixed with Netrin-1 ( Fc/N ) .", "In all stripe assay experiments , the total amount of LMC axon outgrowth was similar ( data not shown ) .", "Lateral LMC axons , identified by EphA4 expression , did not show significant stripe preference when grown on Fc/Fc stripes , with similar proportion of axons growing over either stripe ( Figure 3A , 48% on Cy3-Fc vs . 52% on Fc ) .", "However , when confronted with Fc/N stripes , lateral LMC axons preferentially grew on Netrin-1stripes ( Figure 3B; 75% on N , 25% on Fc stripes p<0 . 001 vs . Fc/Fc controls ) . 10 . 7554/eLife . 10841 . 008Figure 3 . Opposing responses of medial and lateral LMC axons to Netrin-1 require Neogenin and Unc5c function . Growth preference on protein stripes exhibited by medial and lateral LMC axons .", "Each experiment is composed of three panels ( left , middle , right ) and a quantification .", "( A–E )", "Left panels: explanted lateral ( EphA4+ ) LMC neurites on Fc/Fc ( A ) or Netrin-1 ( N ) /Fc stripes without ( B ) or with ( C ) the addition of anti-neogenin antibody .", "Lateral ( GFP+ EphA4+ ) LMC neurites of DccΔICD and GFP ( D ) or Dcc and GFP ( E ) co-electroporated explants treated with anti-neogenin antibody .", "Middle panels: inverted images where EphA4 signal is dark pixels overlaid on substrate stripes .", "Right panels: superimposed images of five representative explants from each experimental group highlighting the distribution of lateral LMC neurites .", "Quantification of lateral ( EphA4+ ) LMC neurites on first ( pink ) and second ( pale ) stripes expressed as a percentage of total EphA4 signals .", "Number of neurites: 77 .", "Minimal number of explants: 11 .", "( F–I )", "Left panels: detection of medial ( GFP+ ) LMC neurites of explants on Fc/Fc ( F ) or N/Fc stripes ( G ) , [Unc5c]siRNA co-electroporated explants on N/Fc stripes ( H ) and [Unc5c]siRNA + Unc5c co-electroporation rescue experiment ( I ) .", "Middle panels: inverted images where GFP signal is dark pixels overlaid on substrate stripes .", "Right panels: superimposed images of five representative explants from each experimental group highlighting the distribution of medial LMC neurites ( right panels ) .", "Quantification of medial ( GFP+ ) LMC neurites on first ( pink ) and second ( pale ) stripes expressed as a percentage of total GFP signals .", "Minimal number of neurites: 83 .", "Minimal number of explants: 12 .", "( J–K )", "Left panels: explanted lateral ( EphA4+ ) LMC neurites on N/Fc stripes .", "Lateral ( GFP+ EphA4+ ) LMC neurites of Unc5c and GFP ( J ) or Unc5c and Csk-GFP ( K ) co-electroporated explants .", "Middle panels: inverted images where EphA4 signal is dark pixels overlaid on substrate stripes .", "Right panels: superimposed images of five representative explants from each experimental group highlighting the distribution of lateral LMC neurites .", "Quantification of lateral ( EphA4+ ) LMC neurites on first ( pink ) and second ( pale ) stripes expressed as a percentage of total EphA4 signals .", "Minimal number of neurites: 90 .", "Minimal number of explants: 11 .", "( L ) Left panels: detection of medial ( GFP+ ) LMC neurites of explants on N/Fc stripes with anti-neogenin antibody addition .", "Middle panels: inverted images where GFP signal is dark pixels overlaid on substrate stripes .", "Right panels: superimposed images of five representative explants from each experimental group highlighting the distribution of medial LMC neurites ( right panels ) .", "Quantification of medial ( GFP+ ) LMC neurites on first ( pink ) and second ( pale ) stripes expressed as a percentage of total GFP signals .", "Minimal number of neurites: 83 .", "Minimal number of explants: 12 .", "LMC , lateral motor column; N , netrin-1; error bars = SD; *** = p<0 . 001; ** = p<0 . 01; n . s . = not significant; statistical significance computed using Mann-Whitney U test; all values ( mean ± SD ) can be found in Supplementary file 1B; scale bar: 50 µm . DOI: http://dx . doi . org/10 . 7554/eLife . 10841 . 00810 . 7554/eLife . 10841 . 009Figure 3—figure supplement 1 . Functional blocking of Neogenin has no effect on ephrin-mediated lateral LMC repulsion , quantification of Unc5c-expressing medial LMC neurons in mice and chick .", "( A–C )", "Characterization of Netrin-1/Fc stripes .", "Detection of Netrin-1 and Fc-Cy3 in alternating stripes by immunostaining .", "( D–E )", "Detection of lateral ( EphA4+ ) LMC neurites of explants on C100 eA5-Fc/Fc stripes without ( D ) or with ( E ) anti-neogenin antibody treatment .", "Middle panels: inverted images where EphA4 signal is dark pixels overlaid on substrate stripes .", "Right panels: superimposed images of five representative explants from each experimental group highlighting the distribution of lateral LMC neurites .", "Quantification of lateral ( EphA4+ ) LMC neurites on first ( pink ) and second ( pale ) stripes expressed as a percentage of total EphA4 signals .", "( F ) In situ hybridization for Unc5c in conjunction with immunostaining for Isl1 in e11 . 5 mice lumbar spinal cord .", "Bottom: quantification of the number of LMC neurons ( Isl1+ ) which are also Unc5c+ .", "12 sections , n=581 neurons .", "( G ) Double in situ hybridization for Unc5c and Isl1 in HH stage 27 chick lumbar spinal cord .", "Bottom: quantification of the number of LMC neurons ( Isl1+ ) which are also Unc5c+ .", "6 sections , n=229 neurons .", "( H ) Double in situ hybridization for Unc5c and Ephb1 in HH stage 27 chick lumbar spinal cord .", "Bottom: quantification of the number of Ephb1+ LMC neurons which also are Unc5c+ .", "3 sections , n=119 neurons .", "LMC , lateral motor column; N , Netrin-1; eA5 , ephrin-A5-Fc; error bars = SD; n . s . = not significant; statistical significance computed using Mann-Whitney U test .", "Scale bars: ( A–E ) 100 μm , ( F–H ) 20 μm . DOI: http://dx . doi . org/10 . 7554/eLife . 10841 . 009 In chick , the attractive Netrin receptor function has been proposed to be carried out by Neogenin , since there is no Dcc gene in this species ( Phan et al . , 2011 ) .", "To address this possibility , we performed an Fc/N stripe preference assay incubating LMC axons in the presence of a function-blocking antibody raised against the extracellular domain of Neo1 .", "In such experiments , LMC axon preference for Ntn1 stripes was abolished ( Figure 3C , 53% on N , 47% on Fc stripes p<0 . 001 vs . untreated N/Fc; [Palmesino et al . , 2012] ) , but could be rescued by electroporation of rat Dcc expression plasmid ( Figure 3E; 66% on N , 34% on Fc stripes ) but not with a plasmid encoding a truncated DCC ( DccΔICD; Figure 3D; 48% on N , 52% on Fc stripes; p<0 . 001 vs . Dcc overexpression ) .", "Lateral LMC axons incubated with anti-Neo1 antibodies did not lose their sensitivity to ephrin-A5 ( Figure 3—figure supplement 1 ) .", "These experiments show that Neo1 mediates lateral LMC neurite growth preference on Netrin-1 , and that DCC is functionally equivalent .", "To study the behavior of medial LMC axons in response to Netrin-1 , we explanted green fluorescent protein ( GFP ) -expressing LMC neurons from chick embryos electroporated with the medial LMC-specific expression plasmid e[Isl1]::GFP ( Kao et al . , 2009 ) .", "While GFP-expressing medial LMC axons did not favor either control stripe ( Figure 3F; 51% on Cy3-Fc , 49% on Fc stripes ) , when challenged with Fc/Netrin-1 stripes , medial LMC axons avoided Netrin-1 in favor of Fc ( Figure 3G; 24% on N , 76% on Fc stripes , p<0 . 001 ) .", "This repulsive effect was abolished in medial LMC neurons electroporated with the siRNA targeting Unc5c , an effect that was rescued by expression of mouse Unc5c ( Figure 3H , I; 48% on N , 52% on Fc stripes , p<0 . 01; 28% on N , 72% on Fc stripes , p=0 . 281 ) .", "Inclusion of a function-blocking Neogenin antibody in the stripe assays did not block the repulsion of medial LMC neurites from Netrin-1 ( Figure 3L; 27% on N , 73% on Fc stripes , p=0 . 113 ) .", "Thus , Netrin-1 causes repulsion of medial LMC axons through Unc5c , independently of Neogenin , providing direct evidence that Netrin-1 is a bifunctional axon guidance cue for LMC axons .", "Finally , we asked whether expression of Unc5c is sufficient to evoke LMC axon repulsion from Netrin-1 .", "Explanted lateral LMC neurons electroporated with an Unc5c expression plasmid avoided Netrin-1 stripes .", "UNC5-mediated repulsion from Netrin-1 has been shown to depend on the Src kinase ( Williams et al . , 2006 ) , and indeed the repulsion of Unc5c-expressing LMC axons from Netrin-1 stripes was abolished by the expression of the Src-inhibiting kinase Csk ( Figure 3J , K; 30% on N , 70% on Fc stripes vs . 59% on N , 41% on Fc stripes , p<0 . 001; [Imamoto and Soriano , 1993] ) .", "Given the prominent function of ephrins in motor axon guidance , we next investigated the possibility that Netrin and ephrin signals co-operate in LMC neurons .", "To do this we focused on the function of Unc5c and EphB2 , an ephrin-B receptor guiding medial LMC axons in vivo ( Luria et al . , 2008 ) .", "We first tested whether chicken Unc5c is required in vivo by reducing its expression through [Unc5c]siRNA and GFP plasmid electroporation of presumptive LMC neurons at HH st . 17–19 .", "Analysis of the proportion of electroporated LMC axons entering the dorsal versus ventral limb nerves at HH st . 26 showed that in embryos electroporated with [Unc5c]siRNA , 72% of GFP+ axons were present in the dorsal limb nerve , compared with 52% in GFP electroporated controls ( Figure 4A , B; Figure 4—figure supplement 1; p<0 . 01 ) , demonstrating that chicken Unc5c is required for the fidelity of LMC trajectory choice . 10 . 7554/eLife . 10841 . 010Figure 4 . Co-operation between Unc5c and EphB2 receptors in LMC trajectory selection .", "( A–E )", "GFP and neurofilament detection in the limb nerve branches in the crural plexus of embryos electroporated with the following expression plasmids and siRNAs: GFP ( A ) , [Unc5c]siRNA and GFP ( B ) , Unc5c and GFP ( C ) , EphB2-GFP ( D ) , or EphB2-GFP and Unc5c ( E ) .", "Quantification of GFP signals in all electroporation experiments expressed as , respectively , percentage in dorsal and ventral limb nerves ( GFP Fluo [%] ) .", "n = 5 embryos .", "( F–I )", "GFP and neurofilament detection in the limb nerve branches in the crural plexus of embryos electroporated with the following expression plasmids with 20% of normal concentration: low GFP ( F ) , low Unc5c and low GFP ( G ) , low EphB2-GFP ( H ) , or low EphB2-GFP and low Unc5c ( I ) .", "Quantification of GFP signals in all electroporation experiments expressed as , respectively , percentage in dorsal and ventral limb nerves ( GFP Fluo [%] ) .", "n = 5 embryos .", "See text for detailed description .", "d , dorsal; v , ventral; error bars = SD; *** = p<0 . 001; ** = p<0 . 01; * = p<0 . 05; n . s . = non significant; statistical significance computed using Mann-Whitney U test; All values ( mean ± SD ) can be found in Supplementary file 1B .", "Scale bar: 150 µm . DOI: http://dx . doi . org/10 . 7554/eLife . 10841 . 01010 . 7554/eLife . 10841 . 011Figure 4—figure supplement 1 . Characterization of siRNA-mediated Unc5c knockdown and Unc5c overexpression in LMC neurons in chicken .", "( A ) Detection of Isl1 , Foxp1 , GFP , and Unc5c in chick embryos electroporated with [Unc5c]siRNA and GFP .", "( B ) Quantification of the reduction in expression levels of Unc5c in LMC neurons after in ovo electroporation of [Unc5c]siRNA .", "Note a significant decrease ( p<0 . 001 ) in Unc5c expression levels by 50% upon [Unc5c]siRNA and GFP but not GFP-only electroporation .", "Number of embryos quantified: n = 3 for both groups .", "( C , D )", "The total number of LMC neurons and the proportion of medial and lateral LMC are normal after [Unc5]siRNA electroporation .", "Number of embryos quantified: n = 4 for all groups .", "( E ) Equal proportion of medial and lateral LMC neurons electroporated with either GFP only or [Unc5c]siRNA and GFP .", "Number of embryos quantified: n = 4 for all groups .", "( F ) The expression of EphA4 and EphB1 is not altered after [Unc5]siRNA electroporation .", "( G ) Detection of Isl1 , Foxp1 , GFP , and mouse Unc5c in chick embryos expressing mUnc5c and GFP .", "( H , I )", "The total number of LMC neurons and the proportion of medial and lateral LMC are normal after mUnc5c and GFP electroporation .", "Number of embryos quantified: n = 4 for all groups .", "( J ) Equal proportion of medial and lateral LMC neurons electroporated with either GFP only or mUnc5c and GFP .", "Number of embryos quantified: n = 4 for all groups .", "( K ) The expression of EphA4 and EphB1 is not altered after mUnc5c and GFP electroporation .", "Number of embryos quantified: n = 4 for all groups .", "( L ) Detection of GFP , mUnc5c , mEphb2 , and Foxp1 in chick embryos expressing normal ( top ) or low ( 1/5th ) ( bottom ) concentrations of Ephb2-GFP and Unc5c plasmids .", "( M ) Quantification of the ratio of the ectopic expression levels of low and normal concentration of GFP , Unc5c , and EphB2 in chick embryos .", "Number of embryos quantified: n = 6 for all groups .", "( N ) Analysis of lateral LMC motor axon projections in chick embryos electroporated with Unc5c , Ephb2 , Unc5c and Ephb2 , or GFP expression plasmids ( all at low concentrations ) .", "A retrograde tracer ( HRP , blue ) was injected in the ventral forelimb of HH st . 29/30 chick embryos followed by detection of Lhx1 ( red ) to identify lateral LMC neurons .", "Insets show examples of magnified HRP+ backfilled cells that are Lhx1+ ( in low Unc5c + low Ephb2 co-electroporation ) or Lhx1- ( in all other conditions ) .", "( O ) Quantification of retrogradely labeled lateral LMC axon projections .", "The graph depicts the mean percentage ± SD of electroporated ( GFP+ ) and HRP+ backfilled motor neurons that express the lateral LMC marker Lhx1 after a ventral limb injection .", "N ≥ 3 embryos .", "HRP , horseradish peroxidase; LMC , lateral motor column; error bars = SD; *** = p<0 . 001; n . s . = not significant; statistical significance computed using Mann-Whitney U test ( B–E , H–J , M ) , or Fisher’s exact test on raw numbers ( O ) ; all values are mean ± SD .", "Scale bars: ( A , G , L ) 56 μm; ( F , K ) 145 μm; ( N ) 40 μm . DOI: http://dx . doi . org/10 . 7554/eLife . 10841 . 011 To test whether Unc5c and EphB2 co-operate to direct LMC axons into the ventral limb , we compared the limb trajectory of LMC axons overexpressing GFP , Unc5c and GFP , EphB2 fused to GFP ( EphB2-GFP ) or Unc5c together with EphB2-GFP .", "On their own , Unc5c and EphB2-GFP caused a significantly greater fraction of GFP+ axons to select the ventral limb nerve when compared with GFP-only controls ( Figure 4A , C , D; ventral limb: 48% in GFP , 57% in Unc5c/GFP , p<0 . 05 vs . GFP; 64% in Ephb2-GFP , p<0 . 01 vs . GFP ) .", "However , when co-expressed , Unc5c and EphB2-GFP caused significantly more GFP+ axons to select the ventral limb nerve branch ( Figure 4E; ventral branch: 80% in Unc5c and Ephb2-GFP , p<0 . 01 vs . Unc5c/GFP; p<0 . 01 vs . Ephb2-GFP ) , suggesting that EphB2 and Unc5c can co-operate to guide LMC axon guidance .", "We next assessed whether electroporation of combined Unc5c and EphB2 at low concentrations elicits an effect not observed when each receptor expression plasmid is electroporated alone .", "To do this , we electroporated low concentrations of Ephb2-GFP , Unc5c , and GFP , or Ephb2-GFP and Unc5c plasmids into LMC neurons , and compared spinal cord section Unc5c , GFP , and EphB2 protein and mRNA levels against standard DNA level electroporations .", "We estimated that EphB2 and Unc5c proteins produced from the plasmids electroporated at low concentrations were approximately 30% of that induced by electroporating high plasmid concentrations ( Figure 4—figure supplement 1 ) .", "The proportion of LMC axons entering the ventral limb nerve in such Unc5c/GFP or Ephb-GFP embryos was not significantly different from controls ( Figure 4F–H; both p>0 . 107 ) .", "However , LMC axons co-expressing low levels of Ephb2-GFP and Unc5c entered the ventral limb nerve with an incidence of 74% , a proportion significantly different from controls and confirmed by retrograde tracer injection into the ventral limb ( Figure 4I; p<0 . 001; Figure 4—figure supplement 1 ) .", "Together , these results suggest in vivo LMC co-operative responses to ephrin-B and Ntn1 could be synergistic in nature .", "To directly determine how ephrin and Netrin-1 signals co-operate in LMC axons , we took advantage of e[Isl1]::GFP-electroporated medial LMC axons’ preferential growth on Fc stripes when confronted with Fc/ephrin-B2 ( eB2 ) stripes , generated by incubation with a standard eB2 concentration ( C100 eB2=10 μg/ml; Figure 5A; 79% on Fc , 22% on eB2; [Kao and Kania , 2011] ) .", "This preference was significantly increased when medial LMC neurites were confronted with Fc/eB2+Netrin-1 ( each at C100 ) stripes mimicking the restriction of eB2 and Netrin-1 to the dorsal limb mesenchyme ( Figure 5B; 90% on Fc , 10% on eB2+Netrin-1 , p<0 . 05 vs . eB2; C100 Netrin-1 = 100 ng/ml ) .", "The increased LMC axon repulsion from ephrins in the presence of Netrin-1 could be due to Netrin and Eph signaling acting simultaneously at the level of single growth cones , or due to recruitment of non-overlapping subpopulations of LMC neurons that only express one of the receptor families .", "The vast majority of LMC neurons co-express both types of receptors ( Kania and Jessell , 2003; Luria et al . , 2008; Figure 1I–R; Figure 3—figure supplement 1; data not shown ) , arguing that ephrins and Ntn1 can co-operatively act in individual LMC axons . 10 . 7554/eLife . 10841 . 012Figure 5 . Congruent and opposing modes of Netrin and ephrin synergy in cultured LMC axons .", "( A–J )", "Left panels: explanted medial ( GFP+ ) LMC neurites of e[Isl1]::GFP-electroporated explants on C100 eB2-Fc/Fc ( A ) , C100 eB2-Fc + C100 N/Fc ( B ) , C10 N/Fc ( C ) , C10 eB2-Fc/Fc ( D ) , C10 eB2-Fc + C10 N/Fc ( E ) stripes and explanted lateral ( EphA4+ ) LMC neurites on C100 eA5-Fc/Fc ( F ) , C100 eA5-Fc/ C100 N stripes ( G ) , C10 N/Fc ( H ) , C10 eA5-Fc/Fc ( I ) , or C10 eA5-Fc/C10 N ( J ) stripes .", "Middle panels: inverted images where GFP ( A–E ) or EphA4 ( F–J ) signal is dark pixels overlaid on substrate stripes .", "Right panels: superimposed images of five representative explants from each experimental group highlighting the distribution of LMC neurites .", "Quantification of medial ( GFP+ ) or lateral ( EphA4+ ) LMC neurites on first ( pink ) and second ( pale ) stripes expressed as a percentage of total GFP ( A–E ) or EphA4 ( F–J ) signals .", "C100 stripes were generated by incubating with ephrins at 10 μg/ml and/or Netrin-1 at 100 ng/ml .", "Experiments with intervening concentrations ( C50 and C25 ) are shown in Figure 5—figure supplement", "1 . Minimal number of neurites: 72 .", "Minimal number of explants: 11 .", "( K , L )", "Plots of relative concentrations ( x axis ) over the fidelity index ( y axis ) .", "Medial ( K ) or lateral ( L ) LMC neurites were challenged with one of five concentrations ( C100 , C50 , C25 , C10 , 0 ) of ephrin or Netrin-1 to test for preferential LMC neurite growth .", "The fidelity index is the absolute value of: ( percent growth on 2nd stripes – 50% ) /50% .", "Index of 1 represents the complete repulsion or attraction of LMC neurites from the 1st stripes , and 0 represents no preference .", "Note that stripes at C10 concentrations induced little or no preferential LMC neurite growth when only ephrin ( middle plots of K and L ) or Netrin-1 ( left plots of K and L ) was presented , but allowed a strong preferential growth of LMC neurites when both cues were present ( right plots of K and L ) .", "LMC , lateral motor column; N , Netrin-1; eA5 , ephrin-A5-Fc; eB2 , ephrin-B2-Fc; error bars = SD; *** = p<0 . 001; * = p<0 . 05; statistical significance computed using Mann-Whitney U test; All values ( mean ± SD ) can be found in Supplementary file 1B .", "Scale bar: 50 µm . DOI: http://dx . doi . org/10 . 7554/eLife . 10841 . 01210 . 7554/eLife . 10841 . 013Figure 5—figure supplement 1 . Netrin and ephrin synergy in LMC axon guidance .", "( A–L )", "Left panels: explanted lateral ( EphA4+ ) LMC neurites on C50 N/Fc ( A ) C50 eA5-Fc/Fc ( B ) C50 eA5-Fc/N stripes ( C ) C25 N/Fc ( D ) C25 eA5-Fc/Fc ( E ) or C25 eA5-Fc/N ( F ) stripes , and medial ( GFP+ ) LMC neurites of e[Isl1]::GFP-electroporated explants on C50 N/Fc ( G ) C50 eB2-Fc/Fc ( H ) C50 eB2-Fc + C50 N/Fc ( I ) C25 N/Fc ( J ) C25 eB2-Fc/Fc ( J ) C25 eB2-Fc + C25 N/Fc ( L ) stripes .", "Middle panels: inverted images where EphA4 ( A–F ) or GFP ( G–L ) signal is dark pixels overlaid on substrate stripes .", "Right panels: superimposed images of five representative explants from each experimental group highlighting the distribution of LMC neurites .", "Quantification of lateral ( EphA4+ ) or medial ( GFP+ ) LMC neurites on first ( pink ) and second ( pale ) stripes expressed as a percentage of total EphA4 ( A–F ) or GFP ( G–L ) signals .", "Minimal number of neurites: 72 .", "Minimal number of explants: 11 .", "LMC , lateral motor column; N , Netrin-1; eA5 , ephrin-A5-Fc; eB2 , ephrin-B2-Fc; error bars = SD; n . s . = not significant; *** = p<0 . 001; statistical significance computed using Mann-Whitney U test; all values are mean ± SD; scale bar: ( A–L ) 50 µm . DOI: http://dx . doi . org/10 . 7554/eLife . 10841 . 013 To address whether Netrin-1-ephrin co-operation is additive or synergistic in nature we performed ligand titration experiments .", "We considered the possibility that simultaneous application of Netrin-1 and ephrin-B2 at concentrations that do not elicit a stripe preference on their own could result in their summation exceeding a threshold required for a preference response .", "Thus , signals at subthreshold concentrations could elicit a growth cone response when presented together , through simple additive means .", "However , we reasoned that a stripe preference evoked by simultaneous exposure to Netrin-1 and eB2 concentrations that are each half or less of those subthreshold concentrations not eliciting stripe preference when alone , would indicate synergistic co-operation between Netrin and ephrin signaling since simple arithmetic additivity could not account for the crossing of a threshold .", "Thus , we tested whether a reduction to 50% , 25% , and 10% of our standard ephrin-B2 and Netrin-1 concentrations ( C50 , C25 , and C10 , respectively ) is sufficient to abolish the stripe assay response by LMC growth cones to individual cues , and whether , at these concentrations , their co-incidence rescues stripe preference .", "Medial LMC neurons challenged with Fc stripes versus stripes with Netrin-1 at C50 , C25 , or C10 displayed no preference ( Figure 5C; Figure 5—figure supplement 1; at C10 48% neurites on Netrin-1; p=0 . 081 ) , whereas ephrin-B2 exposure resulted in reduced preference at C50 , and no detectable preference at C25 , and C10 ( Figure 5D; Figure 5—figure supplement 1; at C10 45% neurites on eB2; p=0 . 081 ) .", "However , when Netrin-1 and eB2 were presented together in the same stripes , alternating with Fc stripes , we observed a striking repulsion of medial LMC axons from Netrin-1+eB2-containing stripes towards Fc stripes , at all suboptimal concentrations studied ( Figure 5E; 21% on eB2+Netrin-1 stripes at C10; p<0 . 001 vs . Netrin-1/Fc or eB2/Fc; Figure 5—figure supplement 1 ) , revealing that medial LMC axons synergistically integrate repulsion from Netrin-1 and ephrin-B2 ( Figure 5K ) .", "To determine the mode by which lateral LMC neurons integrate repulsive and attractive signals we studied their in vitro response to simultaneous exposure to ephrin-A5 ( eA5 ) and Netrin-1 .", "To do this , we quantified the preference of lateral LMC neurites for growth over Fc/eA5 stripes versus Fc stripes ( eA5/Fc ) or alternating stripes containing eA5 and Netrin-1 ( eA5/Netrin-1 ) , mimicking their in vivo distribution in ventral and dorsal limb mesenchyme , respectively .", "When challenged with eA5/Fc stripes , lateral LMC axons preferentially grew on Fc stripes ( Figure 5F; 77% on Fc , 23% on eA5 ) , but when facing eA5/Netrin-1 stripes , the preference of lateral LMC axons for non-eA5 stripes was significantly increased ( Figure 5G; 87% on Fc , 13% on eA5 + Ntn1 , p<0 . 05 vs . eA5 only ) .", "Next , we asked whether lateral LMC neurons integrate Netrin-1 and eA5 signaling synergistically by performing ligand titration experiments as above .", "Lateral LMC neurons challenged with Fc control stripes next to stripes with either Netrin-1 or eA5 at C50 displayed a reduced preference , which was abolished at C25 , and C10 ( Figure 5H , I; Figure 5—figure supplement 1; at C10 , 52% on Netrin-1 vs . 46% on eA5; p=0 . 065 vs . Netrin-1/Fc or eA5/Fc ) .", "However , when Netrin-1 and eA5 were presented together in alternating stripes , each at C50 , C25 , and C10 , the repulsion of lateral LMC axons from eA5 stripes was dramatically rescued ( Figure 5J; at C10 25% on eA5 and 75% on Netrin-1; p<0 . 001 vs . Netrin-1/Fc or eA5/Fc ) .", "These data are summarized in a fidelity index plot in Figure 5L and reveal that lateral LMC axons integrate attractive and repulsive responses to Ntn1 and eA5 , respectively , in a synergistic manner .", "Together , these experiments reveal that lateral and medial LMC growth cones synergistically integrate opposing and congruent Netrin and ephrin signals .", "To begin to characterize the molecular mechanisms by which Ephrin-B2-Netrin-1 synergize , we assessed whether LMC neurons integrate Netrin-1 and ephrin signals on a short timescale , using a growth cone collapse paradigm .", "Explanted HH st . 24–25 chicken LMC neurons electroporated with the medial LMC marker e[Isl1]::GFP were exposed to Fc , eB2 , Netrin-1 , and eB2 and Netrin-1 for 30 min and the extent of growth cone collapse determined .", "A high concentration of eB2 ( 10 μg/ml ) elicited a significant increase in collapse over controls ( Figure 6A–C; 62% vs . 18% Fc-treated; p=0 . 008 ) , and exposure to lower eB2 ( 1 μg/ml ) or Netrin-1 ( 300 ng/ml ) concentrations resulted in negligible collapse ( p=0 . 194 and p=0 . 412 , respectively , vs . Fc ) .", "Exposure of medial LMC growth cones to a mix of eB2 and Netrin-1 at low concentrations resulted in 73% collapse , significantly different from either ligand alone , or Fc controls ( Figure 6B; p< 0 . 029 ) , suggesting that the molecular mechanisms underlying ephrin-B2- Netrin-1 synergy can operate over a relatively short timescale . 10 . 7554/eLife . 10841 . 014Figure 6 . Ligand-dependent and signal-dependent EphB2-Unc5c complex formation and EphB2 phosphorylation .", "( A ) Medial LMC neuron explant growth cone collapse assay scheme .", "( B ) Percentage of collapsed e[Isl1]::GFP medial LMC growth cones following 30 min exposure to Fc ( 10 μg/ml ) , eB2-Fc ( high: 10 μg/ml; low: 1 μg/ml ) , Netrin-1 ( 300 ng/ml ) or Netrin-1 and eB2-Fc ( 300 ng/ml and 1 μg/ml ) .", "Significance computed using Fisher’s exact test .", "( C ) Examples of growth cones labeled with Tuj1 ( green ) and phalloidin ( red ) .", "( D–I )", "Unc5c and EphB2 protein localization in non-permeabilized LMC growth cones treated with Fc or eB2 and Netrin-1 for 15 min .", "Individual channels are inverted .", "All treatments result in same receptor protein signal levels ( eB2 and Netrin-1 images are in Figure 6—figure supplement 1 ) .", "Receptor clusters are depicted in insets , arrowheads: Unc5c and EphB2 co-localization .", "( J ) Pearson's R value as a measure of surface Unc5c and EphB2 co-localization in LMC growth cones .", "Co-localization levels are higher than expected by chance , as demonstrated by Costes’ shuffled image P-value calculations ( Figure 6—figure supplement 1 ) .", "Ligand treatment does not increase the levels of receptor co-localization observed ( p=0 . 7940 , one-way analysis of variance ( ANOVA ) and Tukey's multiple comparisons test; N = 3; n ≥ 33 growth cones per treatment ) .", "( K ) Unc5c and EphB2 receptor interactions .", "Unc5c-Myc was co-immunoprecipitated with EphB2-GFP but not with EphA3-GFP in transfected HEK-293 cells .", "All samples shown were run in same gel .", "( L ) Unc5c-EphB2 interaction is selectively enhanced by 15 min incubation with eB2-Fc ( 1 . 5 μg/ml ) or eB2-Fc+Netrin-1 ( 1 . 5 μg/ml +250ng/ml ) but not with Netrin-1 ( 250 ng/ml ) , Fc ( 1 . 5 μg/ml ) , or ephrin-A3-Fc ( 1 . 5 μg/ml ) prior to lysates preparation .", "Fc fusion proteins were pre-clustered by incubating them with anti-human or anti-mouse Ig for 1 hr at 4°C .", "For quantifications see Figure 6—figure supplement 1N .", "( M ) Comparison of Unc5c interactions with wild-type or kinase-dead EphB2 .", "Unc5c-Myc/EphB2-GFP interaction is blocked when a single point mutation is introduced in EphB2-GFP abolishing its kinase function ( EphB2-KD-GFP , Dalva et al . , 2000 ) .", "For quantifications see Figure 6—figure supplement 1O .", "All samples shown were run in same gel .", "( N ) P-EphB2 levels are increased upon stimulation with Netrin-1+eB2-Fc compared with eB2-Fc alone .", "p-EphB2 was developed first , followed by stripping of the membrane and re-blotting with anti-EphB2 antibody .", "Two replicate comparisons are shown; one sample t-test; p<0 . 02 , N=10 comparisons , 4 experiments .", "eB2 , ephrin-B2-Fc; ip , immunoprecipitation; LMC , lateral motor column; tr , transfection .", "All error bars = SD; *** = p<0 . 001; n . s . = not significant; scale bars: ( C ) 10 μm; ( D–I ) 2 μm .", "All values ( mean ± SD ) can be found in Supplementary file 1B . DOI: http://dx . doi . org/10 . 7554/eLife . 10841 . 01410 . 7554/eLife . 10841 . 015Figure 6—figure supplement 1 . Unc5c–EphB2 receptor association .", "( A ) Unc5c and EphB2 protein localization in permeabilized LMC growth cones treated for 15 min with Fc , Netrin-1 , eB2 , Netrin-1+eB2 .", "Individual channels are inverted .", "( B ) Growth cone size does not change following Fc , eB2 , Netrin-1 , or Netrin-1+eB2 treatment for 15 min ( p=0 . 8161 using one-way ANOVA; N = 3; n ≥ 36 growth cones per treatment ) .", "All treatments result in same receptor protein signal levels .", "( C ) Quantification of Unc5c protein signal in LMC growth cones as fraction of growth cone area above threshold ( p=0 . 3643 , one-way ANOVA and Tukey's multiple comparisons test; N = 3; n ≥ 36 growth cones per treatment ) .", "( D ) Quantification of EphB2 protein signal in LMC growth cones as fraction of growth cone area above threshold ( p=0 . 3862 , one-way ANOVA and Tukey's multiple comparisons test; N = 3; n ≥ 36 growth cones per treatment ) .", "( E ) Unc5c and EphB2 protein localization in non-permeabilized LMC growth cones treated with eB2 or Netrin-1 for 15 min .", "Individual channels are inverted .", "Receptor clusters are depicted in insets , arrowheads: Unc5c and EphB2 co-localization .", "( F ) Costes’ P-value analysis for Fc-treated , eB2-treated , Netrin-1-treated , and Netrin-1+eB2-treated non-permeabilized growth cones , calculated by automatically shuffling appropriately sized chunks of one of the channels of an image and running co-localization analysis .", "This was done 100 times per image .", "A value of 1 signifies that 100% of the shuffled results had a Pearson's R value lower than the one calculated for the original image , i . e . observed co-localization is higher than expected by chance ( N = 3; n ≥ 33 growth cones per treatment ) .", "( G ) Quantification of growth cone size for non-permeabilized growth experiments ( p=0 . 1675 using one-way ANOVA , N = 3; n ≥ 33 growth cones per treatment ) .", "( H ) Quantification of Unc5c protein signal in LMC growth cones as fraction of growth cone area above threshold ( no significant differences , except between permeabilized and non-permeabilized , p<0 . 05; one-way ANOVA followed by Tukey's multiple comparisons test; N = 3; n ≥ 33 growth cones per treatment; detailed values in supplemental file 1C ) .", "( I ) Quantification of EphB2 protein signal in LMC growth cones as fraction of growth cone area above threshold ( no significant differences , except between permeabilized and non-permeabilized , p<0 . 05; one-way ANOVA followed by Tukey's multiple comparisons test; N = 3; n ≥ 33 growth cones per treatment ) .", "( J ) Quantification of phalloidin signal reflecting successful non-permeabilized staining , expressed as fraction of growth cone area above threshold ( no significant differences , except between permeabilized and non-permeabilized , p<0 . 05; one-way ANOVA followed by Tukey's multiple comparisons test; N = 3; n ≥ 33 growth cones per treatment ) .", "( K ) Permeabilized versus non-permeabilized signal for fluorescent-conjugated phalloidin and early endosome marker 1 ( EEA1 ) , showing successful staining with minimal membrane permeabilization .", "n . s . = not significant; error bars = SD .", "( L ) Specificity of antibodies in immunoblot detection .", "HEK293 cells were transfected with Unc5c-Myc or EphB2-GFP and anti-Myc and anti-GFP antibodies were used in western blots to detect expressed proteins from total lysates .", "( M ) Co-immunoprecipitation of EphB2 by Unc5c .", "EphB2-GFP was immunoprecipitated by Myc antibody from lysates of Unc5c-Myc-transfected and Ephb2-GFP-transfected HEK293 cells .", "( N ) Co-immunoprecipitated Unc5c fold changes .", "Pixel intensity and area of unsaturated western blot bands were measured in inverted images in Photoshop and total intensity calculated .", "For fold changes calculations , values of co-immunoprecipitated Unc5c in each treatment were normalized to immunoprecipitated EphB2-GFP and compared with Fc condition .", "One sample t-test , * = p<0 . 05; N=4 in each condition .", "( O ) Reduction in co-immunoprecipitated Unc5c by EphB2-KD-GFP when compared with EphB2-GFP interactions .", "Calculation of band intensities were done as described in ( N ) .", "Values of co-immunoprecipitated Unc5c-Myc were normalized to immunoprecipitated EphB2-GFP or EphB2-KD-GFP and the fold change between EphB2-KD-GFP and EphB2-GFP calculated .", "One sample t-test , * = p<0 . 05; N=4 in each condition .", "LMC , lateral motor column; scale bars: ( A , E , K ) 2 μm . DOI: http://dx . doi . org/10 . 7554/eLife . 10841 . 015 The synergistic behavior of growth cones exposed to Netrin and ephrin stripes implies a cross-talk that could occur at the receptor level and/or through downstream signaling effectors .", "We first tested whether exposure to ephrin-B2 and/or Netrin-1 increases the levels of EphB or Unc5c receptor on the surface of LMC growth cones by treating LMC neurites with Fc , eB2 , Netrin-1 , and both eB2 and Netrin-1 and visualizing EphB and Unc5c receptors under permeabilizing and non-permeabilizing fixation conditions .", "In all cases , EphB and Unc5c expression levels remained essentially unchanged in LMC growth cones ( Figure 6—figure supplement 1 ) .", "However , immunodetection of Unc5c and EphB2 revealed a high incidence of Unc5c- and EphB-containing membrane patches , whose number and size were constant under all conditions ( Figure 6D–J , p>0 . 05; Figure 6—figure supplement 1 , data not shown [Costes et al . , 2004] ) , suggesting the existence of ephrin-Netrin receptor complexes in discrete cell membrane domains of the growth cone .", "To test whether EphB2 and Unc5c can form a molecular complex , we performed co-immunoprecipitation experiments with lysates of HEK293 cells co-transfected with", "1 . Epha3-GFP , Unc5c-Myc;", "2 . Ephb2-GFP , Unc5c-Myc;", "3 . Unc5c-Myc fusion protein expression plasmids .", "In western blot analysis of cell lysates , anti-epitope tag antibodies selectively recognized their respective fusion proteins ( Figure 6—figure supplement 1L ) .", "We found that the anti-GFP antibody precipitated Unc5c when Unc5c-Myc was co-expressed with EphB2-GFP but not with EphA3-GFP or in the absence of EphB2-GFP ( Figure 6K ) .", "We estimated the amount of co-precipitated Unc5c-Myc to be ~15% of the amount precipitated by Myc antibodies ( not shown ) .", "The Unc5c-Myc–EphB2-GFP interaction was also observed in reciprocal co-immunoprecipitation experiments ( Figure 6—figure supplement 1M ) .", "We next determined whether the association between Unc5c and EphB2 was ligand-dependent .", "Anti-GFP antibodies were used to precipitate lysates of Ephb2-GFP and Unc5c-Myc expression plasmid-transfected HEK293 cells that had been exposed to either Netrin-1 , eB2-Fc , Netrin-1 and eB2-Fc , Fc or ephrin-A3-Fc ( eA3-Fc ) .", "We found that the association between EphB2-GFP and Unc5c-Myc was increased ~22 times after pre-incubation with eB2-Fc ( Figure 6L and Figure 6—figure supplement 1N , p<0 . 01 ) .", "This association was induced in a ligand-specific manner , since incubations with Netrin-1 , eA3-Fc or Fc did not stimulate Netrin-ephrin receptor interactions ( Figure 6L ) .", "Exposure to combined Netrin-1 and eB2-Fc increased EphB2-GFP–Unc5c interactions to a similar extent as eB2-Fc alone ( Figure 6L and Figure 6—figure supplement 1N ) .", "Thus , a basal association between EphB2 and Unc5c is increased by ephrin-B2 .", "Finally , since many EphB receptor functions depend on its tyrosine kinase activity ( Kullander et al . , 2001; Soskis et al . , 2012 ) , we addressed whether it might also be required for the Unc5c–EphB2 association .", "To do this , we compared the amount of Unc5c interacting with wild-type EphB2 or kinase dead point mutant EphB2 ( EphB2-KD-GFP; Dalva et al . , 2000 ) , upon ephrin-B2 stimulation .", "We found that the loss of kinase function reduced the extent of interaction by ~67% when compared with the wild-type EphB2-GFP ( Figure 6M , Figure 6—figure supplement 1O , p<0 . 01 ) .", "Eph receptor tyrosine phosphorylation induced by ephrin binding is a requirement and a correlate of their cellular activity ( Zisch et al . , 1998 ) , leading us to examine whether following EphB2-Unc5c complex induction by ephrin-B2 , the presence of Netrin-1 might potentiate EphB2 tyrosine phosphorylation .", "To this effect , from lysates of a cell line stably expressing EphB2 and transfected with Unc5c-Myc , we pulled down EphB2 using eB2-Fc in the presence or absence of Netrin-1 and used specific antibodies to detect tyrosine-phosphorylated EphB2 ( p-EphB2; Figure 6N; ( Dalva et al . , 2000; Holland et al . , 1997; Takasu , 2002 ) .", "Exposure to both eB2 and Netrin-1 generated a significant increase of ~23% in tyrosine phosphorylation of EphB2 when compared with ephrin-B2 alone ( Figure 6N , p<0 . 02 ) .", "Together , our biochemical experiments indicate that concomitant stimulation with ephrin-B2 and Netrin-1 induces the formation of an EphB2-Unc5c receptor complex and increases EphB2 tyrosine phosphorylation levels , commensurate with elevated biological activity .", "Src family kinase ( SFK ) activation is critical for the intracellular relay of Eph receptor signaling , growth cone collapse , and medial LMC axon guidance in vivo ( Kao et al . , 2009; Knoll , 2004; Zisch et al . , 1998 ) .", "Additionally , since some studies and our experiments point to a role for SFKs in Netrin-1:Unc5 signaling ( Williams et al . , 2006 ) , we considered SFK involvement in the integration of Netrin-ephrin activities .", "First , we observed that SFKs were not required for the formation of the Unc5c-EphB2 complex ( data not shown ) .", "Second , we considered whether eB2 and Netrin-1 co-incidence could result in SFK activation being higher than that induced by either ligand alone .", "We examined the levels of SFK-activating phosphorylation ( pSFK; Boggon and Eck , 2004 ) , in LMC growth cones treated with Fc , eB2 , Netrin-1 , and both eB2 and Netrin-1 .", "After 15 min , following ligand application but before completion of growth cone collapse , increasing doses of eB2 generated increased LMC growth cone pSFK signal that coincided with EphB receptor clusters , while Netrin-1 alone failed to induce pSFK , when compared with Fc treatment ( Figure 7—figure supplement 1; data not shown ) .", "After 30 min of simultaneous exposure to eB2 and Netrin-1 , significantly higher levels of pSFK were seen in collapsing LMC growth cones when compared with those exposed to either ligand alone or Fc ( Figure 7A–F , G; p<0 . 01 ) .", "Together , these experiments suggest that simultaneous exposure to eB2 and Netrin-1 might result in prolonged elevation of pSFK levels . 10 . 7554/eLife . 10841 . 016Figure 7 . Src family kinases ( SFKs ) are required for synergistic repulsion from ephrin-B2 and Netrin-1 . ( A–F ) Detection of pSFKs ( green ) and Tuj1 ( blue ) in collapsed growth cones after 30’ treatment with Fc ( 10 μg/ml ) , low Netrin-1 ( 0 . 3 μg/ml ) , high Netrin-1 ( 1 μg/ml ) , low eB2 ( 1 μg/ml ) , high eB2 ( 10 μg/ml ) , or low eB2 and low Netrin-1 ( 1 μg/ml and 0 . 3 μg/ml ) .", "Bottom panels show inverted images of the pSFK channel .", "( G ) Quantification of pSFK detected in collapsed growth cones treated as above .", "In the presence of low Netrin-1 and eB2 concentrations , pSFK signal is increased when compared with low eB2 or low Netrin-1 alone .", "Statistical significance computed using one-way ANOVA and Tukey's multiple comparisons test; N = 3 , n ≥ 10 growth cones per condition per experiment .", "( H ) LMC neuron explant growth cone collapse assay and SFK inhibition scheme .", "( I ) Percentage of collapsed LMC growth cones following 30 min exposure to Fc ( 10 μg/ml ) , or Netrin-1 and eB2-Fc ( 0 . 3 μg/ml and 1 μg/ml; 1/10th of this concentration; or 1/30th of this concentration ) , in the presence or absence of 0 . 1 μM SFK inhibitor SU6656 .", "N ≥ 3 , significance computed using Fisher’s exact test with n > 400 growth cones for each treatment .", "( J–Q )", "Quantification of medial ( GFP+ ) LMC neurites of e[Isl1]::GFP-electroporated explants on C100 eB2/Fc ( J ) , C100 N/Fc ( L ) , C100 eB2+N/Fc ( N ) , and C10 eB2+N/Fc ( P ) , and Csk and e[Isl1]::GFP-co-electroporated explants on C100 eB2/Fc ( K ) , C100 N/Fc ( M ) , C100 eB2+N/Fc ( O ) , and C10 eB2+N/Fc ( Q ) .", "Quantification of neurites on first ( pink ) and second ( gray ) stripes expressed as a percentage of total GFP signals .", "Minimal number of neurites: 80 .", "Minimal number of explants: 12 .", "Statistical significance computed using Mann-Whitney U test .", "eB2 , ephrin-B2-Fc; LMC , lateral motor column; N , Netrin-1; All error bars = SD; n . s . : not significant; *: p<0 . 05; **: p<0 . 01; ***: p<0 . 001; scale bar: 2 μm .", "All values ( mean ± SD ) can be found in Supplementary file 1B . DOI: http://dx . doi . org/10 . 7554/eLife . 10841 . 01610 . 7554/eLife . 10841 . 017Figure 7—figure supplement 1 . pSFK controls and Csk-electroporated axons in stripe assays .", "( A–F )", "Detection of pSFK in LMC growth cones treated for 15 min . ( G–I ) Co-localization of pSFK and EphB1 receptor clusters in LMC growth cones .", "( J ) pSFK signal increases with increasing dose of ephrin-B2 ligand .", "( K ) A 15-min .", "treatment with 1 μg/ml ephrin-B2 activates pSFK to the same extent as 1 μg/ml ephrin-B2 + 0 . 3 μg/ml Netrin-1 , but not as much as 10 μg/ml ephrin-B2 .", "Statistical significance computed using one-way ANOVA and Sidak’s multiple comparisons test; N ≥ 3 , n ≥ 10 growth cones per condition per experiment .", "( L ) Area of collapsed growth cones analyzed for pSFK quantification .", "Statistical significance computed using one-way ANOVA .", "( M ) Medial LMC growth cones of e[Isl1]::GFP-electroporated explants collapse in response to a high dose of ephrin-B2 ( 10 μg/ml ) , and this can be blocked by including 0 . 1 μM SU6656 with the ligand .", "Statistical significance computed using Fisher’s exact test; N = 3 , n > 190 growth cones per condition .", "( N–U )", "Left panels: medial ( GFP+ ) LMC neurites of e[Isl1]::GFP-electroporated explants on C100 eB2-Fc/Fc ( N ) , C100 N/Fc ( P ) , C100 eB2-Fc+N/Fc ( R ) , and C10 eB2-Fc+N/Fc ( T ) , and Csk and e[Isl1]::GFP-co-electroporated explants on C100 eB2-Fc/Fc ( O ) , C100 N/Fc ( Q ) , C100 eB2-Fc+N/Fc ( S ) , and C10 eB2-Fc+N/Fc ( U ) .", "Middle panels: inverted images where GFP signal is dark pixels overlaid on substrate stripes .", "Right panels: superimposed images of five representative explants from each experimental group highlighting the distribution of LMC neurites .", "Images correspond to the experiments in Figure 7 .", "eB2 , ephrin-B2-Fc; LMC , lateral motor column; N , Netrin-1; SFK , Src family kinase; All error bars = SD; n . s . = not significant; ***: p<0 . 001; *: p<0 . 05; scale bars: ( A–F ) 8 μm; ( G–I ) 6 μm; ( N–U ) 50 μm . DOI: http://dx . doi . org/10 . 7554/eLife . 10841 . 017 Finally , to assess SFK role in Netrin-ephrin responses , we performed LMC growth cone collapse and stripe assays in the presence of SFK blockers .", "SU6656 , an SFK inhibitor , completely blocked the collapse of LMC growth cones evoked by ephrin-B2 but only attenuated by 8% the collapse caused by simultaneous eB2 and Netrin-1 application ( Figure 7H–I; p<0 . 05; Figure 7—figure supplement 1; data not shown; Blake et al . , 2000 ) .", "The combination of SU6656 with eB2 and Netrin-1 at 1/10th and 1/30th of the above concentrations , resulted in a 19% and 29% attenuation of synergistic collapse , respectively ( Figure 7I , p<0 . 001 ) .", "In stripe assays , medial LMC neuron over-expression of Csk , an inhibitory kinase of Src ( Imamoto and Soriano , 1993 ) , completely blocked responses to Fc/N and Fc/eB2 stripes at C100 ( Figure 7J–M , p<0 . 01 ) , while the preference elicited by Fc/N+eB2 stripes at C100 concentration was attenuated by 57% ( Figure 7N–O , p<0 . 01 ) and it was reduced by 87% when challenged with Fc/N+eB2 stripes at C10 ( Figure 7P–Q; p<0 . 001; detailed quantification: Supplementary file 1B ) .", "Together , these results indicate that SFK activation is a key step in the integration of ephrin-B2 and Netrin-1 signaling that leads to elevated repulsion of LMC growth cones ." ], [ "Our results argue for a model in which Netrin-1 present in the dorsal limb mesenchyme concomitantly directs lateral LMC axons into dorsal limb nerves , and medial LMC axons into ventral limb nerves .", "Our most direct evidence of this is the contrasting response of cultured medial and lateral LMC axons to Netrin-1 protein , which depends on ( 1 ) the selective expression of Unc5c in medial LMC neurons , and ( 2 ) the expression of attractive Netrin receptors in LMC neurons .", "Unc5c loss results in decreased repulsion from Netrin-1 in vitro and entry of medial LMC axons into the dorsal limb in vivo while inhibition of attractive Netrin receptors leads to a loss of lateral LMC axon preference for growth on Netrin-1 .", "In line with previous data ( Hong et al . , 1999 ) , Unc5c signaling dominates over attraction to Netrin-1 , since over-expression of Unc5c in LMC neurons results in more LMC axons entering the ventral limb .", "Following this logic , the growth of Unc5c mutant medial LMC axons into the dorsal limb of mice might be caused by the loss of the dominant repulsive receptor uncovering Netrin attraction .", "However , since in vitro loss of Unc5c function in LMC neurons does not uncover an increased preference for Netrin-1 stripes , it is plausible that attractive Netrin-1 receptors in medial LMC axons are not functional in the context of our assays .", "Following dorsoventral limb trajectory selection , LMC axons are constrained to the permissive axonal pathways in the limb where they make subsequent trajectory selections that bring them to their muscle target ( Tosney and Landmesser , 1985 ) .", "During these stages , Netrin receptor expression in LMC neurons is dynamic and Netrin-1 is expressed in the distal limb mesenchyme , suggesting that it is involved in the selection of muscle nerve trajectory by LMC axons as implied by muscle nerve trajectory defects in Unc5c mutants ( S . P . and Thomas Jessell; unpublished observation ) .", "Thus , along with its regulation of motor axon exit from the nervous system ( Bai et al . , 2011; Varela-Echavarria et al . , 1997 ) , and in contrast to other signals that apparently guide subpopulations of LMC axons ( Chai et al . , 2014; Huber et al . , 2005; Kramer et al . , 2006 ) , Netrin-1 appears to be a pervasive signal controlling motor axon guidance in vertebrates and non-vertebrates ( Winberg et al . , 1998 ) .", "Limb rotation experiments predict that a localized axon guidance signal constrains LMC axon trajectory choice at the base of the limb ( Ferns and Hollyday , 1993 ) , thus excluding Netrin-1’s long-range diffusible axon guidance activity as a dorsoventral nerve selection signal .", "However , Netrin-1 can also act as a short range , non-diffusible signal ( Brankatschk and Dickson , 2006; Timofeev et al . , 2012 ) and in our in vitro assays , medial and lateral LMC axons exhibit robust and specific responses to immobilized Netrin-1 .", "Additionally , since Netrin-1 protein localization in the limb matches closely that of its mRNA , Netrin-1 likely functions as a contact-dependent axon guidance cue for LMC growth cones at the limb nerve bifurcation abutting the Netrin-1 expression domain .", "One corollary of this , based on non-vertebrate models of Netrin repulsion ( Keleman and Dickson , 2001 ) , would be the lack of requirement for attractive Netrin receptors for short range Unc5c-mediated LMC axon guidance , in agreement with our genetic experiments showing that DCC is dispensable for Unc5c-mediated medial LMC guidance .", "Unc5c mutation causes more severe medial LMC guidance defects than Ntn1 mutation .", "One possible explanation is that other Unc5 ligands such as FLRTs might contribute to Unc5c repulsion of LMC axons ( Yamagishi et al . , 2011 ) .", "In addition , the hypomorphic nature of the Ntn1Gt mutation ( Serafini et al . , 1996 ) , with residual Netrin-1 protein guiding some medial LMC axons in Ntn1 mutants , could account for the weaker misprojection phenotype .", "Neogenin has been long proposed to be a Netrin-1 receptor; however , most direct functional evidence of its function in axon guidance implicates it as a repulsive guidance molecule ( RGM ) receptor ( Rajagopalan et al . , 2004; Xu et al . , 2014 ) .", "Our in vitro antibody blocking experiments directly demonstrate that Neo1 function is required for Netrin-1-mediated axon guidance and the functional equivalence of DCC and Neo1 .", "The rescue of Neo1 antibody block by DCC expression also implies that the intracellular effectors of Netrin-1:DCC attraction signaling are present in chicken lateral LMC neurons .", "Contrary to our in vitro experiments where Neo1 loss of function results in loss of growth preference on Netrin-1 , genetic loss of DCC and nearly all of Neo1 , or Dscam , does not lead to any lateral LMC guidance defects , suggesting that in vivo , attractive Netrin-1 receptors are dispensable for the fidelity of lateral LMC axon guidance .", "How can the in vitro and in vivo data be reconciled ?", "One possibility is that non-Netrin-1 lateral LMC guidance cues such as ephrin-As in the ventral limb are operational even when Netrin-1 attractive receptors are removed , preserving the high fidelity of lateral LMC axon trajectory choice .", "It is also possible that the 10% of wild-type Neo1 protein levels produced from the Neo1 hypomorphic allele ( Bae et al . , 2009 ) are sufficient to elicit normal attraction to Netrin-1 in lateral LMC axons and synergize with other axon guidance cues in the limb .", "Our present experiments show that coincidence of Netrin-1 and ephrin is integrated in a synergistic manner by spinal motor neurons leading to robust preference responses .", "Our argument against simple additivity of ephrin and Netrin-1-evoked responses is based on the quantitative analysis of neurite growth preferences .", "Thus , observable effects from the combination of ligands , each at concentrations of less than half of a sub-threshold concentration ( without effect on its own ) , implies synergy and not additive mechanisms .", "Our stripe assays exhibited similar overall outgrowth in all conditions , and detected a robust axon guidance response to ephrin and Netrin-1 even at 1/10th of concentrations insufficient for ephrin or Netrin-1 to elicit an effect on their own .", "Moreover , these responses reflect the bifunctional nature of Netrin-1’s chemotropic activity: Netrin-1 attraction synergizes with ephrin-A repulsion of lateral LMC neurons while Netrin-1 repulsion synergizes with ephrin-B repulsion of medial LMC axons .", "Thus , contrasting modes of synergistic integration occur in related populations of spinal motor neurons , possibly reflecting the evolutionary advantage of axon guidance synergy .", "Furthermore , it is likely that the molecular mechanisms that integrate two repulsive cues , and an attractive cue with a repulsive cue are fundamentally different , even if the cues are molecularly related as is the case for ephrin-As and ephrin-Bs .", "Synergy implies a cross-talk between Netrin and ephrin signaling , which could be initiated at the receptor level or at downstream signaling nodes .", "Our experiments suggest a two-step model of congruent ephrin-Netrin synergy , likely starting at the receptor level ( Figure 8 ) .", "As a first step of our model , independent of ligands , a considerable fraction of EphB and Unc5c receptors on the surface of medial LMC growth cones is apparently found in the same membrane compartment , contrasting the non-overlapping distribution of some ephrins and Eph receptors ( Marquardt et al . , 2005 ) .", "Such membrane compartment co-existence might facilitate the formation of EphB2-Unc5c complexes following the addition of ephrin-B2 , as detected in our biochemical assays .", "Importantly , this effect is ligand-specific , and signaling through the EphB2 receptor is important since tyrosine kinase point mutation inhibits complex formation .", "The second step involves the action of Ntn1 alone or in conjunction with ephrin-B2 , through EphB2-Unc5c complexes , resulting perhaps in the activation of novel downstream signaling pathways , or increased activation of common intracellular effectors .", "In support of the second possibility , Netrin-1 presence increased EphB2 tyrosine-phosphorylation , a correlate of Eph receptor activation required for many of its functions ( Zisch et al . , 1998 ) .", "We also found that the coincidence of ephrin-B2 and Netrin-1 resulted in prolonged activating phosphorylation of SFKs when compared with that induced by ephrin-B2 alone , and that SFK function is required in LMC neurons for ephrin-B2-Ntn1 synergistic responses .", "Thus , since Src mutation abolishes the fidelity of medial LMC limb nerve selection ( Kao et al . , 2009; Liu et al . , 2004 ) , the second step might entail increased EphB2 and Src kinase activity leading to more intense activation of common signaling pathways .", "The fact that SFK inhibitors could not totally block repulsive growth cone responses upon Netrin-1-ephrin-B2 stimulation raises the interesting possibility that additional , non-SFK mechanisms integrating Netrin-ephrin signaling exist .", "Furthermore , the genetic observation that in EphB1 and EphB3 mutants many medial LMC axons can be labeled from dorsal limb muscles ( Luria et al . , 2008 ) , raises the question of whether the EphB2-Unc5c interaction is a general property of B-class Eph receptors . 10 . 7554/eLife . 10841 . 018Figure 8 . Model summarizing EphB2 interactions with Unc5c .", "( A ) Under non-stimulated conditions there is a low level interaction between EphB2 and Unc5c ( depicted by dotted two-directional arrow ) .", "( B , C )", "Upon ephrin-B2 stimulation , signaling through EphB2 kinase activity induces direct or indirect association ( arrow ) with Unc5c .", "( D ) Netrin-1 signals through the novel receptor complex resulting in elevated EphB2 phosphorylation and , together with EphB2 , in SFK potentiation .", "SFK , Src family kinase; TK , tyrosine kinase domain .", "Y-416 , tyrosine-416 of Src , whose phosphorylation positively correlates with Src kinase activity . DOI: http://dx . doi . org/10 . 7554/eLife . 10841 . 018 Our experiments also shed light on the significance of synergistic axon guidance .", "With additive signals , blocking one completely would have only partial effect on the overall fidelity of LMC trajectory selection .", "However , in the case of synergistic signals , taking out most of a signal might still leave enough of it to synergize with its partner pathway since this effect can occur at suboptimal signal levels .", "Completely inactivating a synergizing signal will lead to extreme effects where its partner might not function efficiently enough to maintain LMC axon pathway selection fidelity .", "In this view limb-expressed GDNF and ephrin-A appear as additive signals , such that deleting one of them results only in a partial loss of LMC trajectory choice fidelity ( Kramer et al . , 2006 ) .", "However , genetic perturbation of ephrin and Netrin signaling in medial LMC axons suggest a synergistic interaction: loss of EphB signaling leads to severe medial LMC axon guidance phenotypes ( Luria et al . , 2008 ) , as does the loss of Unc5c in the present study , arguing against redundancy between the two systems , and together with our in vitro data , for synergy .", "Synergistic integration of axon guidance cues might endow neurons with a powerful means of preventing potential axon guidance errors caused by fluctuations in expression levels of guidance receptors and their ligands ." ], [ "Mouse lines were as previously described: Unc5crcmTg ( Ucp ) 1 . 23Kz ( Ackerman et al . , 1997; Burgess et al . , 2006 ) ; Dcctm1WBG ( Fazeli et al . , 1997 ) ; Ntn1Gt ( pGT . 8TM ) 629Wcs ( Serafini et al . , 1996 ) ; Neo1Gt ( Bae et al . , 2009 ) ; Unc5a ( Williams et al . , 2006 ) ; Dscam ( Amano et al . , 2009 ) .", "Timed mating vaginal plug was designated as e0 . 5 .", "Fertilized chicken eggs ( Couvoir Simetin ) were incubated at 38°C and staged according to Hamburger and Hamilton , 1951 .", "Chick spinal cord electroporation of expression plasmids or siRNAs was performed at HH st . 18/19 as described ( Croteau and Kania , 2011 ) .", "siRNA sequences used ( sense strand ) : [Unc5c]siRNA , 1:1:1 mixture of GAACCCGAAGAAGCTTACATCGTGA , CAGCAACTGTGATTGTCTATGTGAA , and CACCGTGACTTTGAGTCAGATATTA .", "Dcc and DccΔICD expression plasmids were described previously ( Hong et al . , 1999 ) and were co-electroporated with the GFP expression plasmid at a 1:10 molar ratio .", "Retrograde labeling of mouse or chick motor neurons using HRP ( Roche ) or conjugated dextran ( Invitrogen ) as tracers was performed as described ( Luria et al . , 2008 ) .", "Protein carpets were produced using silicon matrices ( Knöll et al . , 2007 ) .", "Carpets contained an alternating stripe pattern of ephrin-Fc , Netrin-1 , or Fc only as controls: the first stripe was labeled with Fc-specific Cy3-conjugated antibody ( 4:1 weight ratio ) while the second stripe contained unconjugated Fc-specific antibody .", "Dissection of E5 chick spinal motor column and dissociated culture was as described ( Kao and Kania , 2011 ) .", "In situ mRNA detection , immunofluorescence and live-cell staining were performed as described ( Kao and Kania , 2011 ) or using standard methods .", "Probes are available upon request .", "A rabbit polyclonal anti-Unc5c antiserum was raised against a c-terminal peptide of mouse Unc5c: CGRHETVVSLAAEGQY .", "The antiphospho-Tyr416-Src antiserum ( Life Technologies ) also reacts with Yes and Fyn .", "See Supplementary file 1A for all antisera .", "For non-permeabilized assays , tissue was exposed to ligands for 15 min and then placed on ice , and a 5-min pre-blocking step was performed by replacing half the media with phosphate-buffered saline ( PBS ) containing 2% bovine serum albumin ( BSA ) ( final , 1% on tissue ) and incubating at 4°C .", "Half of the media was then replaced with motor neuron media containing primary antibodies against Unc5c ( final dilution of 1 in 1000 ) , EphB2 ( 1 in 1000 ) , and EEA1 ( 1 in 500 ) as control ( fluorescent-conjugated phalloidin ( 1 in 400 ) was also used as control in some experiments due to species cross-reaction issues with the EEA1 antibody ) and tissue was incubated 30 min at 4°C .", "Tissue was then fixed with a mixture of ⅕th 30% sucrose and ⅘ths 4% PFA for 15 min at 4°C .", "Three quick washes with PBS were followed by replacing half the PBS with PBS containing secondary antibodies ( final , 1 in 1000 ) and a 1-hr incubation at 4°C .", "Finally , three quick washes were followed by mounting in Mowiol .", "For the permeabilized control , fixation occurred after ligand incubation and before primary antibody staining , primaries were added in motor neuron media with added triton ( 0 . 3% ) , and secondaries in PBS with added triton ( 0 . 3% ) .", "Otherwise , all concentrations , incubation times , and temperatures were identical .", "GFP and PLAP-labeled axonal projection , protein , and mRNA expressions , and motor neuron numbers of limb section images were quantified using Photoshop ( Adobe ) or ImageJ ( NIH ) as described ( Kao et al . , 2009 ) .", "Stripe assays were quantified as previously described ( Kao and Kania , 2011 ) .", "Data from the experimental replicates were evaluated using Microsoft Excel .", "Experimental measurements were compared using statistical tests as indicated in figure legends , with 0 . 05 a significance threshold .", "HEK293 cells ( ATCC CRL 1573 ) were transfected using the calcium phosphate method , combining the following plasmids as described in results: Ephb2-GFP ( Kao et al . , 2009 ) , mUnc5c-Myc ( gift of Franck Polleux ) , mDcc-HA ( Stein , 2001 ) , Epha3-GFP ( Gift of Dimitar Nikolov ) and Ephb2-KD-GFP ( Dalva et al . , 2000 ) .", "In experiments with ligand incubations , after ~40 hr the media was replaced and cells were starved for 2 hr in OPTIMEM ( Gibco ) , followed by incubation in Fc ( 1 . 5 μg/ml; R&D ) , Netrin-1 ( 250 ng/ml , R&D ) , eB2 ( 1 . 5 μg/ml , R&D ) , eA3 ( 1 . 5 μg/ml , R&D ) or ligand combinations for 15 min at 37°C .", "Fc and Fc-fusion ligands were pre-clustered by mixing them with anti-Fc antibody ( human for Fc and eB2 and mouse for eA3 ) for 1hr at 4°C ) .", "After a wash in PBS , cell lysates were prepared in lysis buffer containing 10mM Tris-Cl pH 8 , 137 mM NaCl , 2mM EDTA , 1% NP40 and proteinase inhibitors ( cOmplete ULTRA Tablets , Mini , EDTA-free , EASYpack , Roche ) .", "Immunoprecipitations were performed overnight by binding pre-cleared lysates ( centrifuged two times at 14 , 000 rpm for 15 min and 5 min , respectively ) to ProteinA/G agarose that were previously incubated for 2 hr at 4°C with the corresponding antibody .", "After two washes in 0 . 1% NP40 in PBS and one in PBS , samples were separated on 4–12% gradient Bis-Tris polyacrylamide gels ( Nupage Novex , Life Technologies ) .", "Western blots were incubated overnight with the indicated antibodies diluted in 3% milk and developed with ECL reagent ( Pierce ) .", "For detection of p-EphB2 , Ephb2-stably transfected HEK293 cells ( Poliakov et al . , 2008; gift from Tony Pawson , cell line identity not authenticated ) were transfected with Unc5c-Myc and starved overnight in OPTIMEM ( Gibco ) prior to treatment with eB2-Fc ( 0 . 15 μg/ml ) or eB2-Fc and Netrin-1 ( 0 . 15 μg/ml + 0 . 6 μg/ml; [Holland et al . , 1997 ) ] ) for 15 min .", "Cell lysates were prepared in buffer , pre-cleared as described and bound overnight to ProteinA/G agarose .", "Samples were run in 4–12% gradient Bis-Tris polyacrylamide gels .", "Detection of p-EphB2 ( Dalva et al . , 2000; Holland et al . , 1997 ) was followed by membrane stripping ( Thermo Scientific ) for 20 min at 37°C , blocked in 3% BSA and re-blotted with a goat anti-EphB2 antiserum ( R&D systems ) .", "Pixel intensity and area of western blot unsaturated bands were measured in Photoshop and total intensity calculated .", "P-EphB2 signals were normalized to EphB2 and Netrin-1+ eB2-Fc vs . eB2-Fc ratios calculated .", "In the case of immunoprecipitation experiments , the amount of Unc5c was normalized to immunoprecipitated EphB2-GFP .", "Statistics were performed with a one-sample t-test , with the null hypothesis mean=1 ." ] ]

[ "During neural circuit assembly , axonal growth cones are exposed to multiple guidance signals at trajectory choice points .", "While axonal responses to individual guidance cues have been extensively studied , less is known about responses to combination of signals and underlying molecular mechanisms .", "Here , we studied the convergence of signals directing trajectory selection of spinal motor axons entering the limb .", "We first demonstrate that Netrin-1 attracts and repels distinct motor axon populations , according to their expression of Netrin receptors .", "Quantitative in vitro assays demonstrate that motor axons synergistically integrate both attractive or repulsive Netrin-1 signals together with repulsive ephrin signals .", "Our investigations of the mechanism of ephrin-B2 and Netrin-1 integration demonstrate that the Netrin receptor Unc5c and the ephrin receptor EphB2 can form a complex in a ligand-dependent manner and that Netrin–ephrin synergistic growth cones responses involve the potentiation of Src family kinase signaling , a common effector of both pathways ." ]

[ "The ability of animals to walk and perform skilled movements depends on particular groups of muscles contracting in a coordinated manner .", "Muscles are activated by nerve cells called motor neurons found in the spinal cord .", "The connections between the motor neurons and muscles are established in the developing embryo .", "Each motor neuron produces a long projection called an axon whose growth is guided towards the target muscle by signal proteins .", "The motor neurons are exposed to many such signal proteins at the same time and it is not clear how they integrate all this information so that their axons target the correct muscles .", "Poliak , Morales et al . used a variety of genetic and biochemical approaches to study the formation of motor neuron and muscle connections in the limbs of mice and chicks .", "The experiments show that a signal protein called Netrin-1 is produced in the limbs of developing embryos and attracts the axons of some types of motor neurons and repels others .", "This is due to the motor neurons producing different types of receptor proteins to detect Netrin-1 .", "Further experiments show that individual axons can combine information from attractive or repulsive Netrin-1 signals together with repulsive signals from another family of proteins called ephrins in a 'synergistic' manner .", "That is , the combined effect of both cues is stronger than their individual effects added together .", "This synergy involves ligand-dependent interactions between the Netrin-1 and ephrin receptor proteins , and the activation of a common enzyme .", "Poliak , Morales et al . ’s findings reveal a new role for Netrin-1 in guiding the development of motor neurons in the limb .", "Future work will focus on further understanding the mechanism of synergy between Netrin-1 and ephrins .", "Netrin-1 and ephrins are also involved in the formation of blood vessels and many other developmental processes , so understanding how they work together would have a wide-reaching impact on research into human health and disease ." ]

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

[ "neuroscience" ]

[ [ "Animals execute neural control over their motor systems to generate a multitude of behaviors such as grooming , courtship or foraging .", "These behavioral strategies often require very distinct types of motor patterns while employing the same muscle groups .", "For example , different modes of locomotion serve the optimal strategy for food finding .", "Under the assumption that food is nearby , a large variety of invertebrate and vertebrate species including mammals perform local or area-restricted search ( ARS ) consisting of short moves and frequent high-angled turns .", "Alternatively , in order to localize more distant food sources , animals disperse via long-distance travelling ( LDT ) by moving along straight paths ( Bell , 1990; Fryxell et al . , 2008; Hills , 2006 ) .", "Understanding how nervous systems operate these different strategies of foraging behavior requires a detailed description of the underlying motor patterns as well as mechanistic insights into the neural circuits coordinating them .", "We addressed these problems through investigations of the nematode C . elegans , which is a tractable genetic model organism with an anatomically mapped small nervous system and which employs a variety of strategies to navigate through its environment .", "Nematodes advance via undulatory crawling movements ( Gray , 1953 ) , which can be precisely quantified ( Brown et al . , 2012; Stephens et al . , 2008; Yemini et al . , 2013 ) .", "The animal’s posture is tightly coupled to ventral and dorsal body wall muscle activity and therefore a reliable proxy to characterize the motor patterns underlying gait production ( Butler et al . , 2014 ) .", "When removed from food C . elegans performs frequent reorientation maneuvers , which consist of brief periods of backward crawling ( reversals ) followed by sharp turning ( omega turns ) ; however , when no food is detected for a prolonged time C . elegans maintains forward crawling and only infrequently reorients .", "These two locomotion strategies have been explicitly characterized as ARS after removal from food and LDT ( Calhoun et al . , 2015; Gray et al . , 2005; Hills et al . , 2004; Peliti et al . , 2013; Tsalik and Hobert , 2003; Wakabayashi et al . , 2004 ) .", "Also , two major locomotion strategies for goal-directed chemotaxis have been observed: the biased random walk , which is reminiscent of bacterial chemotaxis ( Berg and Brown , 1972 ) , where animals modulate the probability of reorientation depending on sensory history ( Pierce-Shimomura et al . , 1999 ) , and weathervaning , where animals directly steer toward the favored direction by introducing a subtle bias to their path ( Iino and Yoshida , 2009 ) .", "The above-mentioned studies have quantified locomotion by the frequency and duration of forward runs , reversals and omega turns; however , quantitative descriptions of the motor patterns underlying the different locomotion strategies of C . elegans , or those of any animal species in general , have been missing .", "Oxygen ( O2 ) chemotactic responses of C . elegans are an effective experimental paradigm to investigate the neural control of locomotion .", "In nature , C . elegans is found in association with anthropogenic soil habitats constituting complex environments rich in olfactory cues ( Félix and Braendle , 2010; Félix and Duveau , 2012 ) , in which local O2 concentrations vary and might serve to indicate the presence of bacterial food , pathogens or predators ( Sexstone et al . , 1985 ) .", "In the laboratory , worms are fed on E . coli lawns , which generate steep O2 gradients ranging from atmospheric 21% down to 12% O2 ( Gray et al . , 2004 ) .", "C . elegans possesses sophisticated sensory systems to detect O2 ( Busch et al . , 2012; Gray et al . , 2004; Zimmer et al . , 2009 ) .", "A sudden decline in O2 levels activates BAG class O2 sensory neurons ( Zimmer et al . , 2009 ) , and during navigation of spatial O2 gradients , animals accumulate at intermediate concentrations ( Cheung et al . , 2005; Gray et al . , 2004; Zimmer et al . , 2009 ) .", "Interneuron circuits that are involved in locomotory responses to changes in ambient O2 and that communicate with O2 sensory neurons have been partially described ( Busch et al . , 2012; Kato et al . , 2015; Laurent et al . , 2015 ) .", "However , the navigational strategies used during O2 chemotaxis are uncharacterized .", "In the present study we use O2 chemotactic responses of food-deprived C . elegans as a paradigm to experimentally control a shift from LDT to ARS-like behavior .", "We show that the worm’s undulatory motions can be decomposed into two modes corresponding to undulation and turning motions .", "By controlling the coordination of these modes animals can gradually adjust their locomotion strategy on a behavioral spectrum ranging from LDT to O2-induced ARS .", "The interneurons AVK and DVA releasing the neuropeptides FLP-1 and NLP-12 , respectively , are required for the control over this fine adjustment in response to acute sensory stimulation as well as during spatial navigation in O2 gradients .", "In summary , this work shows how neuromodulatory circuit elements control elementary motor components to flexibly adjust the strategy of locomotion ." ], [ "We analyzed the locomotion behavior of 1 hr food-deprived worm populations filmed while freely crawling on agarose in a behavioral flow arena , which permitted tight control over ambient O2 concentrations ( Zimmer et al . , 2009 ) .", "An immediate drop from atmospheric 21% to 10% O2 ( O2 downshift ) evoked a variety of behavioral changes , including transient reduction of locomotion speed and concomitant up-regulation of reversals and omega turns as previously described ( Zimmer et al . , 2009 ) ( Figure 1A , Figure 1—figure supplement 1 ) .", "In addition , we observed up-regulation of shallow turning maneuvers during forward movement , evident as increased curvature of the animals’ locomotion trajectories ( track curvature ) ( Figure 1B ) .", "The compound effect of these behavioral changes was a drastic reduction in the spatial displacement of worms during the first three minutes upon O2 downshift ( Figure 1C ) .", "Thus , a sudden drop in environmental O2 concentrations evoked a change in the locomotion strategy switching from predominantly forward crawling to a mixture of reduced crawling and increased reorientations , i . e . from LDT to O2-induced ARS ( see also Video 1 and Video 2 upper left panel ) .", "This interpretation is in accordance with previous literature classifying similar behavioral strategy changes in the context of removal from food ( Calhoun et al . , 2015; Gray et al . , 2005; Hills et al . , 2004; Peliti et al . , 2013; Tsalik and Hobert , 2003; Wakabayashi et al . , 2004 ) .", "The changes in behavioral parameters all depended on an O2 chemosensory neuron pair , termed BAG , which is activated upon O2 downshift ( Zimmer et al . , 2009 ) ( Figure 1—figure supplement 1C ) .", "We first qualitatively evaluated these behaviors by examining single worms ( see Video 1 ) .", "Upon O2 downshift , animals tended to stop forward locomotion and adopted complex and variable postures , unlike the more regular sinusoid-like postures associated with LDT ( Figure 1D , E ) .", "Some of these postures resembled omega turns , while others were associated with shallow turns and pauses ( Figure 1D ) .", "In order to obtain quantifiable time-series of animal postures , we skeletonized the worm images and calculated 24 inter-segment angles for each worm and movie frame ( Figure 1F ) ; an example is shown by the kymograph in Figure 1G .", "LDT was associated with long stretches of regular and coherent body posture patterns , while O2-induced ARS after O2 downshift exhibited variability in the amplitude , direction and frequency of body postural changes ( Figure 1G ) .", "Note that shallow turns , slowing bouts , reversals and omega turns also occurred sparsely during LDT; however , during O2-induced ARS these behaviors were up-regulated 2-4-fold ( compare pre- with post- stimulus period in all panels of Figure 1 , Figure 1—figure supplement 1 ) . 10 . 7554/eLife . 14116 . 003Figure 1 . Worms transit from LDT to ARS after oxygen-sensory stimulation .", "( A–C )", "Locomotion parameters from population assays of wild type ( N2 ) worms stimulated with O2 downshifts from 21% to 10% .", "Traces show mean , shadings show SEM .", "n = 36 populations assays ( ~25 animals per assay ) .", "Time is relative to O2 downshift ( A ) Forward movement speed based on centroid coordinates ( 1 s binning ) .", "( B ) Curvature of forward moving trajectories ( 1 s binning ) .", "( C ) Centroid displacement between 30 s intervals .", "( D–G )", "Representative example of a wild type ( N2 ) worm .", "t1-4 indicate corresponding time points .", "Time is relative to O2 downshift .", "( D ) Single video frames: ( t1 ) forward run at 21% O2 , ( t2 ) pause after O2 downshift , ( t3 ) shallow and ( t4 ) deep turning maneuver .", "( E ) Trajectory of centroid position for 35 s at 21% and 70 s at 10% O2 , respectively .", "Color indicates crawling speed; reverse movement is shown as negative speed .", "( F ) Worm image overlaid with a skeleton .", "The inset shows how inter-segment angles were calculated .", "( G ) Top: Body posture kymograph of 24 inter-segment angles from head ( 1 ) to tail ( 24 ) .", "Middle: time course of representative head ( #2 ) and mid-body angle ( #11 ) .", "Bottom: translational speed of the worm’s centroid .", "Reverse movement is indicated by color . DOI: http://dx . doi . org/10 . 7554/eLife . 14116 . 00310 . 7554/eLife . 14116 . 004Figure 1—figure supplement 1 . Behavioral transition from LDT to ARS after oxygen downshift depends on oxygen-sensory neurons BAG .", "( A–B )", "Locomotion parameters from population assays of wild type ( N2 ) worms stimulated with O2 downshifts from 21% to 10% .", "Traces show mean , shadings show SEM .", "Data bins are 10 s .", "n = 36 populations assays ( ~25 animals per assay ) .", "Time is relative to O2 downshift .", "( A ) Omega turns and ( B ) reversals .", "( C ) Quantifications of behavioral parameters presented in ( A , B ) and Figure 1A–C .", "Means ± SEM of wild type N2 ( n = 36 assays ) and BAG- worms ( genetic cell ablation , n = 6 assays ) .", "Significance values are determined by paired t-tests ( ****p≤0 . 0001 , *0 . 01<p≤0 . 05 , ns p>0 . 05 ) .", "Note that BAG-ablated worms ( strain CX11697 ) additionally lack the ASG sensory neurons ( Roger Pocock , pers . communication ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 14116 . 00410 . 7554/eLife . 14116 . 005Video 1 . Wild type N2 C . elegans behavioral responses to O2 downshift . Exemplary wild type ( N2 ) worms from population behavioral assays at 21% ambient O2 and after a sudden shift at 10% O2 .", "Time is relative to the O2 downshift .", "Note how the animals slow down their locomotory rate and up-regulate more complex and flexible postures .", "Time lapse 2x ( 20fps ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 14116 . 00510 . 7554/eLife . 14116 . 006Video 2 . Animated undulation and turning mode shapes of a wild type N2 worm . Reconstructed worm shapes of the decomposed undulation and turning modes and their superpositions from an exemplary wild type ( N2 ) worm ( same as in Figures 1D–G , 2C , D ) .", "Time is relative to the O2 downshift from 21% to 10% .", "Upper left panel shows original movie frame captures .", "The right panels show worm shapes reconstructed from undulation mode , turning mode and body posture time series shown in the kymographs below .", "The undulation mode corresponds to regular sinusoid-like worm shapes , whereas the turning mode corresponds to more strongly bent postures during shallow turns and omega turns .", "The diverse and complex postures observed are obtained by simple linear addition of undulation and turning shapes .", "Time lapse 1x ( 10fps ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 14116 . 006 In summary , LDT is associated with extended periods of coherent movement across all body segments leading to efficient relocation , which are only sparsely interspersed by reorientation maneuvers , .", "Contrarily , O2-induced ARS is associated with slow locomotion and frequent reorientation maneuvers mediated through more complex postures .", "Having observed different postural characteristics underlying LDT versus O2-induced ARS , we aimed for a more detailed and precise description thereof .", "C . elegans uses 95 body wall muscles to move .", "However , the coordinated motions of the muscle-associated body segments can be quantified with far fewer than 95 parameters , leading to a reduced but comprehensive description of body posture ( Stephens et al . , 2008 ) .", "Briefly , in this approach , principal components analysis ( PCA ) ( Jolliffe , 2002 ) calculates the eigenvectors ( termed eigenworms , EWs ) of the covariance matrix of inter-segment angles observed over many posture time series ( Figure 2A ) .", "EW’s are a set of representative worm postures ( as defined by a vector of inter-segment angles ) , in descending order of explanatory power; i . e . each EW contributes a certain amount of postural variance and linear combinations of the first four EWs account for the gross majority ( >85% ) of the observed postural variance ( Figure 2B ) .", "For example , the first two eigenworms ( EW1-2 ) capture the dominant correlations between segment body angles as reflected in Figure 2A , and subsequent EWs capture secondary correlational structure between segment body angles .", "We found that despite the strong effect of O2 downshift on the animals’ postures , the respective underlying EWs were nearly identical ( Figure 2A ) .", "However , the relative contributions of each EW changed: the percentage of total variance explained by EW1 and EW2 decreased whereas the percentage of total variance explained by lower-order EWs increased ( e . g . EW3-6 , Figure 2B ) .", "Thus , the different postures observed during LDT versus O2-induced ARS can be described by linear combinations of the same elementary shapes , the relative weights of which are modulated upon stimulation . 10 . 7554/eLife . 14116 . 007Figure 2 . Decomposition of the body posture into undulation and turning motor patterns .", "( A ) The panels show the top six of 24 eigenworms ( EWs ) calculated from pooled segment angle time series of wild type ( N2 ) worms .", "Black and red EWs are calculated from 60s intervals pre ( n = 312 ) or post- ( n = 469 ) O2 downshift respectively .", "( B ) Bars show the variance explained by the EWs shown in A . Traces show cumulative values .", "The p-value was determined by a random resampling approach ( see Materials and methods ) and estimates an upper boundary of the probability that the difference between the cumulative variance distributions would occur by chance .", "( C ) Example kymographs from the same worm shown in Figure 1D–G .", "Time is relative to O2 downshift .", "Arrowheads indicate time points t1-4 as in Figure 1 .", "Top: body posture as in Figure 1G .", "Middle: undulation mode reconstructed from EW 1–2 .", "Bottom: turning mode reconstructed from EW 3–24 .", "( D ) Worm shapes graphically reconstructed from body posture , undulation mode and turning mode at time points t1-4 .", "Note that the worm’s posture is a linear sum of undulation and turning mode . DOI: http://dx . doi . org/10 . 7554/eLife . 14116 . 00710 . 7554/eLife . 14116 . 008Figure 2—figure supplement 1 . Characterization of undulation and turning modes by signal cross-correlation analysis . Undulation and turning modes during forward movement from wild type N2 time series ( n = 36 assays , ~25 animals per assay , yielding to n = 9358 time series > = 20 s duration ) were analyzed by cross-correlation .", "Traces display mean ± standard deviation .", "Maximum absolute correlation and corresponding lag times are indicated .", "( A–D )", "Intra-mode cross-correlation between segment angles of head ( segment angle #2 ) and mid-body ( segment angle #11 ) ( A , B ) or head and posterior body ( segment angle #19 ) ( C , D ) , for undulation ( A , C ) or turning mode ( B , D ) .", "( E , F )", "Inter-mode cross-correlation between undulation and turning mode for ( E ) head and ( F ) mid-body segment angle . DOI: http://dx . doi . org/10 . 7554/eLife . 14116 . 008 Next , we employed the eigenworm approach to obtain a more intuitive , yet quantitative , description of the worm postures characterizing LDT vs . O2-induced ARS .", "Here and in all subsequent analyses throughout this manuscript we used canonical EWs calculated from the full wild type N2 time series ( n = 36 population assays , ~25 animals per assay ) .", "The combination of EW1 and 2 corresponds to the regular undulatory movements along the worm’s body , which generates forward and backward crawling ( Stephens et al . , 2008 ) .", "We thus reconstructed posture time series based only on EWs 1–2 ( see Materials and methods for details ) .", "This led to a regular wave-like pattern corresponding to the undulatory worm movements; we termed this pattern the ‘undulation mode’ ( see Figure 2C for an example ) .", "Subtracting the undulation mode from the full body posture time series , which is equivalent to reconstructing posture time series based on all remaining EWs 3–24 , revealed a strikingly organized residual mode .", "Strong transients in the residual time series coincided with shallow turns as well as with highly bent omega turns ( see Figure 2C for examples ) ; we thus termed this time series the ‘turning mode’ .", "The turning mode was qualitatively different in comparison to the undulation mode: the head moved in anti-phase with the tail and in phase with the mid-body whereas the undulation mode was generated by anti-phase undulations of head and mid-body and in phase undulation of head and tail .", "We quantitatively assessed these differences by signal cross-correlation analysis ( Figure 2—figure supplement 1A–D ) .", "The undulation mode and turning mode were not independent as they were often phase-locked: signal cross-correlation analysis showed that the onset of turning movements lagged on average ~0 . 6 s behind the undulation mode ( Figure 2—figure supplement 1E , F ) .", "We graphically synthesized worm shapes from the undulation mode and turning mode separately , which illustrated that the undulation mode corresponded to regular sinusoid-like worm shapes , whereas the turning mode corresponded to more strongly bent postures during shallow turns and omega turns .", "The diverse and complex postures observed in worms were obtained by simple linear addition of undulation and turning shapes ( Figure 2D; Video 2 ) .", "In conclusion , apparently complex postures of C . elegans can be linearly composed out of simpler elementary shapes , which constitute behaviorally relevant motor patterns of the worm: the undulation mode describes regular forward and backward crawling , onto which the turning mode imposes turning movements .", "We used the eigenworm decomposition method to calculate three instantaneous ( i . e . for each movie frame ) parameters of worm locomotion: ( 1 ) undulation amplitude , a measure of the curvature across the body contributing to undulation movements , calculated as the sum of the absolute values of all inter-segment angles of the undulation mode , ( 2 ) undulation frequency of the undulation movements , determined from the projection amplitude time series of EW1 and EW2 ( Stephens et al . , 2008 ) ( see Materials and methods for details ) , and ( 3 ) turning amplitude , a measure of how strong the animals bend their bodies in order to turn , calculated as the sum of the absolute values of all inter-segment angles of the turning mode .", "Biomechanical calculations indicate that both body bending amplitude and frequency synergistically generate thrust against the substrate thereby contributing to the animals’ locomotion speed ( Gray , 1953 ) .", "The sum of the absolute values of all inter-segment angles of the body posture is a measure of the total curvature ( or tortuousness ) of the animals’ posture , which we term body amplitude .", "To obtain separate measures of worm locomotion during LDT and O2-induced ARS , we performed analyses on the pre- and post-stimulus periods .", "High signals in turning amplitude consistently coincided with regions of high track curvature , i . e . events when animals adjusted their heading direction ( see Figure 3A for examples ) ; track curvature was a graded function of turning amplitude ( Figure 3B ) .", "Turning amplitude and undulation amplitude exhibited a graded inverse relationship to each other ( Figure 3C ) .", "As expected , undulation frequency exhibited a tight linear relationship with translational locomotion speed ( Figure 3D ) ( See also reference [Stephens et al . , 2008] ) .", "We found that directional changes during forward movement were associated with slow locomotion: there was an inverse relationship between undulation frequency and centroid track curvature ( Figure 3E ) .", "High undulation amplitude was associated with fast locomotion ( high undulation frequency ) whereas high turning amplitude was associated with slow , low-frequency locomotion ( Figures 3F ) .", "All relationships described were preserved during both pre- and post- stimulus periods ( Figure 3B–F ) . 10 . 7554/eLife . 14116 . 009Figure 3 . Turning amplitude is reciprocally regulated with undulation amplitude and undulation frequency .", "( A ) Exemplary worm trajectories during the 10% O2 interval aligned at their starting points with color code indicating turning amplitude .", "( B–F )", "The graphs show interdependencies of the indicated locomotion parameters during forward movement .", "Solid lines show the medians and dashed lines show interquartile ranges on the y–axis .", "Data are binned by the corresponding values on the x-axis .", "Tick marks indicate bin medians of the x-axis .", "Data from wild type N2 worms ( n = 36 assays , ~25 animals per assay ) during 3 min intervals pre- ( gray ) and post- ( black or color ) O2 downshift were analyzed .", "Very high values on the x-axis are omitted due to data sparseness .", "( B ) Track curvature versus turning amplitude .", "Bin width = 0 . 2 rad .", "( C ) Turning amplitude versus undulation amplitude .", "Bin width = 0 . 1 rad .", "( D ) Centroid speed versus undulation frequency .", "Bin width = 0 . 015 cycles/s .", "Pearson correlation coefficients are indicated .", "( E ) Track curvature versus undulation frequency .", "Bin width = 0 . 015 cycles/s .", "( F ) Turning amplitude ( left ) or undulation amplitude ( right ) versus undulation frequency .", "Bin width = 0 . 015 cycles/s . DOI: http://dx . doi . org/10 . 7554/eLife . 14116 . 009 In conclusion , turning amplitude , undulation amplitude and undulation frequency are coupled: during turning maneuvers , animals reduce the amplitude and speed of undulation motions , leading to slow locomotion .", "Contrarily during periods of fast locomotion turning maneuvers are suppressed .", "Under our experimental conditions , the coupling of these motor parameters is a robust property of worm locomotion during both spontaneous and stimulus-evoked behaviors .", "We next analyzed the averaged time courses of the turning and undulation amplitudes in response to the stimulus .", "Upon stimulus onset , the mean amplitudes were abruptly up- or down-regulated , respectively , and gradually returned to baseline within three minutes ( Figure 4A ) .", "For comparison , we calculated the time course of total body amplitude , which changed upon stimulation with a biphasic response profile , i . e . initial suppression followed by transient up-regulation ( Figure 4A ) . 10 . 7554/eLife . 14116 . 010Figure 4 . Undulation amplitude and turning amplitude are inversely and gradually regulated in response to O2 stimulation .", "( A , B )", "Behavioral responses of wild type N2 worms during forward movement evoked by decreasing O2 concentrations .", "Traces show mean amplitude of body posture ( black ) , undulation mode ( blue ) and turning mode ( green ) ; shadings indicate SEM .", "Time is relative to onset of O2 concentration change .", "( A ) O2 downshift from 21% to 10% O2 ( n = 36 assays , ~25 animals each ) and ( B ) gradual O2 ramp from 21 to 4% O2 ( n=15 assays , ~25 animals each ) .", "Some O2 concentrations during the ramp are indicated on top .", "Colors correspond to ( C ) .", "( C ) Trial-mean ( ± SEM ) fractional distribution functions of turning amplitude from the same data shown in ( B ) .", "All distributions are calculated from consecutive 15 s intervals during the ramp with amplitude bin width of 0 . 1 rad .", "The legend indicates the median O2 concentration during each interval .", "Color code corresponds to ( B ) .", "( D ) 2D density map showing the fractional distribution of forward undulation frequency versus turning amplitude from all worms ( n ≈ 375 ) during the ramp assays shown in ( B , C ) .", "Data from -60 s to 180 s were included .", "Bin width are 0 . 002 cycles/s and 0 . 025 rad .", "The y- and x-axes omit very high values due to data sparseness . DOI: http://dx . doi . org/10 . 7554/eLife . 14116 . 010 Based on the graded relationships exhibited by turning and undulation parameters ( Figure 3B–F ) we hypothesized that animals were able to gradually control the contributions of undulation and turning motions .", "To test this hypothesis , we stimulated the animals with a temporal oxygen ramp ranging from atmospheric 21% to 4% O2 ( -0 . 01%/s ) .", "This protocol caused a subtle change in mean total body amplitude but stronger and gradual reciprocal changes in mean undulation and turning amplitudes ( Figure 4B ) .", "We then plotted the fraction of postures with a given turning amplitude as distribution functions during subsequent time segments of the ramp .", "We did not observe bimodality but instead that the skew of these distributions gradually increased towards higher levels ( Figure 4C ) .", "Thus , animals were able to gradually adjust turning and undulation motions within a wide continuous range , as further revealed in a two-dimensional density plot of turning amplitude against undulation frequency ( Figure 4D ) .", "In summary , oxygen stimulation results in a shift in the contributions of undulation and turning motor patterns .", "Animals display control of the strength of undulation and turning motions in response to both abruptly and gradually changing environments .", "LDT and O2-induced ARS behaviors appear to lie on a continuum of behavioral mode compositions .", "We hypothesized that undulation and turning modes are under control of some of the animals’ pre-motor interneuronal circuits .", "To identify such circuits we analyzed candidate neuropeptide modulators that were reported to affect body posture and found roles for FLP-1 and NLP-12 neuropeptides: flp-1 mutants have been reported to exhibit elevated body curvature and locomotion speed ( Chang et al . , 2015; Nelson et al . , 1998 ) .", "NLP-12 neuropeptides have been reported to be exclusively released from the proprioceptive interneuron DVA; interfering with the DVA/NLP-12 system leads to decreased body curvature and locomotion speed ( Bhattacharya et al . , 2014; Garrison et al . , 2012; Hu et al . , 2011; Janssen et al . , 2008; Li et al . , 2006 ) .", "We first confirmed these previously reported results in our 1 hr food deprivation paradigm .", "We assayed flp-1 and nlp-12 mutants , DVA ablated ( DVA- ) animals , as well as transgenic nlp-12 rescue strains in DVA , and found that all phenotypes could be recapitulated in 1 hr fasted worms ( Figure 5A , B; Figure 6A ) .", "Next , we aimed to identify the relevant site of FLP-1 release .", "flp-1 is expressed in various interneurons of the worm’s head and ventral ganglia ( Nelson et al . , 1998 ) .", "We found that flp-1 expression in the single ventral ganglion interneuron class AVK , which we observed to be the neuron class with strongest expression , is sufficient to restore a wild type phenotype ( Figure 6A ) .", "We generated AVK- animals , which caused enhanced body posture amplitude similar to flp-1 mutants ( Figure 5A , B ) .", "flp-1;nlp-12 double mutants as well as AVK-;DVA- animals exhibited intermediate total body amplitude similar to wild type levels , indicating an antagonism between both systems ( Figure 5A , B ) .", "For comparison across different genetic backgrounds , we calculated undulation and turning modes for all genetic backgrounds using the wild type N2 eigenworms as a common basis .", "The decomposition revealed that a large proportion of altered body posture across all phenotypes could be explained by a corresponding effect on undulation amplitude .", "Interfering with AVK/FLP-1 function increased undulation amplitude , while interfering with DVA/NLP-12 decreased undulation amplitude .", "flp-1;nlp-12 double mutants exhibited intermediate phenotypes ( Figure 5C ) .", "In addition , all genotypes except flp-1;nlp-12 double mutants and DVA- animals showed an increase in turning amplitude , which was especially strong for flp-1 mutants and animals lacking AVK ( Figure 5D ) .", "A comparison of 2D probability density distributions of undulation vs . turning mode revealed that to a large extent the postures of mutants and manipulated strains reflected postures that were exhibited , however less frequently , by wild type animals ( Figure 5—figure supplement 1 ) .", "Besides their effects on worm postures at constant atmospheric O2 levels , all analyzed mutant and cell ablation strains were compromised in regulating undulation and turning mode in response to O2 downshift .", "The strongest defects in stimulus-evoked postural changes were seen in strains lacking AVK neurons , as well as in flp-1;nlp-12 double mutants ( Figure 5E–G ) .", "The DVA/NLP-12 and AVK/FLP-1 systems were both implicated in controlling locomotion speed ( Figure 5—figure supplement 2 ) .", "Cell-specific expression of nlp-12 in DVA and flp-1 in AVK partially restored the respective phenotypes , confirming these cells as important release sites for the respective neuropeptides ( Figure 6A–F; Figure 5—figure supplement 2C , E ) . 10 . 7554/eLife . 14116 . 011Figure 5 . Regulation of undulation mode and turning mode through antagonistic peptidergic interneurons .", "( A ) Top: Anatomical sketch of interneurons AVK and DVA .", "All other panels show representative video frames during forward movement at 21% O2 of worms with indicated genotypes .", "flp-1 and nlp-12 are loss of function mutations .", "AVK- and DVA- denote transgenes driving caspase expression leading to cell death in the respective neuron .", "Note the effects of genotypes on body posture .", "Heads are pointing upwards .", "( B–D )", "Boxplots of amplitudes during forward movement measured during a 4 min interval at 21% O2 of the ( B ) body posture , ( C ) undulation mode and ( D ) turning mode .", "( E ) Time profiles of mean undulation amplitude ( blue ) and mean turning amplitude ( green ) .", "Shadings show SEM .", "Dashed lines indicate onset of O2 downshift .", "The traces are vertically aligned to emphasize differences in behavioral responses .", "The vertical bar indicates amplitude; the horizontal bar indicates time axis .", "( F , G )", "Boxplots showing changes of ( F ) undulation amplitude and ( G ) turning amplitude in response to the O2 downshift .", "Changes were calculated by subtracting the means of 60s intervals post-downshift from the means of 60s pre-downshift intervals .", "Boxplots in all panels display trial-median and interquartile range with 5–95 percentile whiskers .", "Asterisks on top of box plots indicate significance levels compared to wild type N2 and asterisks on top of brackets indicate significance levels for selected comparisons , using one-way-ANOVA with Sidak’s correction for multiple comparisons ( ****p≤0 . 0001 , ***0 . 0001<p≤0 . 001 , **0 . 001<p≤0 . 01 , *0 . 01<p≤0 . 05 , ns p>0 . 05 ) .", "Number of experiments ( ~25 animals per assay ) : N2 n = 36 , flp-1 n = 29 , AVK- n = 21 , nlp-12 n = 21 , DVA- n = 13 , flp-1;nlp-12 n = 12 and AVK-;DVA- n = 10 . DOI: http://dx . doi . org/10 . 7554/eLife . 14116 . 01110 . 7554/eLife . 14116 . 012Figure 5—figure supplement 1 . Postures of manipulated animals with mis-regulated motor patterns largely lie within the wild type N2 posture space . 2D density maps of the indicated strains showing the fractional distribution of forward undulation amplitude versus turning amplitude from all worms during the shift assays shown in Figure 5E .", "Data from -60 s to 60 s respective to the shift are included .", "Bin widths are 0 . 05 rad .", "White boundary encompasses 99% of wild type N2 fractional data .", "The fractions of data that lie within this boundary are indicated .", "Except for flp-1 mutants , >90% of the posture data are within the 99% posture space of wild type animals .", "This indicates that the postures of mutants and manipulated strains largely reflect postures that are exhibited , however less frequently , by wild type animals .", "Postures of flp-1 mutants that are outside the wild type profile exhibit mostly an increase in undulation amplitude . DOI: http://dx . doi . org/10 . 7554/eLife . 14116 . 01210 . 7554/eLife . 14116 . 013Figure 5—figure supplement 2 . Regulation of locomotion speed through antagonistic peptidergic interneurons .", "( A ) Time profiles of mean forward locomotion speed .", "Shadings show SEM .", "Dashed lines indicate onset of O2 downshift .", "The horizontal bar indicates time axis .", "( B–E )", "Boxplots of forward locomotion speed ( B , C ) measured during a 4 min interval at 21% oxygen and ( C , D ) of the percent change in response to the oxygen downshift .", "Changes were calculated by subtracting the means of 60 s intervals post-downshift from the means of 60 s pre-downshift intervals divided by the means of the interval pre-downshift .", "Genotypes indicate transgenic rescue using cell specific promoters ( pDVA , pAVK ) .", "Boxplots display trial-median , interquartile range and 5–95 percentile whiskers .", "Asterisks on top of box plots indicate significance levels compared to wild type N2 and asterisks on top of brackets indicate significance levels for comparisons with the respective mutant or selected genotypes , using one-way-ANOVA with Sidak’s correction for multiple comparisons ( ****p≤0 . 0001 , ***0 . 0001<p≤0 . 001 , **0 . 001<p≤0 . 01 , *0 . 01<p≤0 . 05 , ns p>0 . 05 ) .", "Number of experiments ( each ~25 animals ) : ( B , D ) N2 n = 36 , flp-1 n = 29 , AVK- n = 21 , nlp-12 n = 21 , DVA- n = 13 , flp-1;nlp-12 n = 12 and AVK-;DVA- n = 10 .", "( C , E )", "N2 n = 10 , nlp-12 n = 6 , pDVA: nlp-12 rescue under Pnlp-12 n = 11 , flp-1 n = 11 , pAVK: flp-1 rescue under Pflp-1 fragment n = 9 , N2 n = 11 , flp-1;nlp-12 n = 11 , pAVK;pDVA: flp-1 rescue under Pflp-1 fragment & nlp-12 rescue under Pnlp-12 n = 11 . DOI: http://dx . doi . org/10 . 7554/eLife . 14116 . 01310 . 7554/eLife . 14116 . 014Figure 5—figure supplement 3 . Gradual behavioral transitions depend on flp-1 and nlp-12 neuropeptide genes .", "( A ) Behavioral responses of flp-1; nlp-12 mutant worms during forward movement evoked by a gradual O2 ramp from 21 to 4% ( n = 15 assays ~25 animals each ) .", "Traces show mean amplitude of body posture ( black ) , undulation mode ( blue ) and turning mode ( green ) ; shadings indicate SEM .", "Time is relative to onset of O2 ramp .", "Some O2 concentrations during the ramp are indicated on top .", "Colors correspond to ( B ) .", "( B ) Trial-mean ( ± SEM ) of fractional distribution functions of turning amplitude from the same data shown in ( A ) .", "All distributions are calculated from consecutive 15 s intervals during the ramp with amplitude bin width of 0 . 1 rad .", "The legend indicates the median O2 concentration during each interval .", "Color code corresponds to ( A ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 14116 . 01410 . 7554/eLife . 14116 . 015Figure 6 . Peptidergic regulation of undulation and turning mode by flp-1 and nlp-12 through interneurons AVK and DVA .", "( A–C )", "Boxplots of amplitude during forward movement measured during a 4 min interval at 21% O2 of the ( A ) body posture ( B ) undulation mode and ( C ) turning mode .", "( D ) Time profiles of mean undulation amplitude ( blue ) and mean turning amplitude ( green ) .", "Shadings show SEM .", "Dashed lines indicate onset of O2 downshift .", "The traces are vertically aligned to emphasize differences in behavioral responses .", "The vertical bar indicates amplitude; the horizontal bar indicates time axis .", "( E–H )", "Boxplots showing changes of undulation amplitude ( E , G ) and turning amplitude ( F , H ) in response to the O2 downshift ( E , F ) or to an O2 ramp ( G , H ) .", "Changes were calculated by subtracting the means of 60 s intervals post-downshift from the means of 60 s pre-downshift intervals ( E , F ) or by subtracting the means of 60 s intervals at the end of the ramp from the means of 60 s intervals before ramp onset ( G , H ) .", "Genotypes indicate transgenic rescue using cell specific promoters ( pDVA , pAVK ) .", "Boxplots in all panels display trial-median , interquartile range and 5–95 percentile whiskers .", "Asterisks on top of box plots indicate significance levels compared to wild type N2 and asterisks on top of brackets indicate significance levels for comparisons with the respective mutant , using one-way-ANOVA with Sidak’s correction for multiple comparisons ( ****p≤0 . 0001 , ***0 . 0001<p≤0 . 001 , **0 . 001<p≤0 . 01 , *0 . 01<p≤0 . 05 , ns p>0 . 05 ) .", "Number of experiments ( ~25 animals per assay ) : N2 n = 10 , nlp-12 n = 6 , pDVA: nlp-12 rescue under Pnlp-12 n = 11 , flp-1 n = 11 , pAVK: flp-1 rescue under Pflp-1 fragment n = 9 , N2 n = 11 , flp-1;nlp-12 n = 11 , pAVK;pDVA: flp-1 rescue under Pflp-1 fragment & nlp-12 rescue under Pnlp-12 n = 11 . DOI: http://dx . doi . org/10 . 7554/eLife . 14116 . 015 Next we tested whether the DVA/NLP-12 and AVK/FLP-1 systems were required for fine control of behavior in the O2 ramp stimulus paradigm .", "We focused on flp-1;nlp-12 double mutants , since their baseline behavioral parameters during LDT were similar to wild type N2 animals ( Figure 5B–D ) .", "However , we hypothesized that if the neuropeptides were part of a control system of worm postures , the double mutants should lack control over the composition of behavioral modes .", "Indeed , in the ramp paradigm the mutant animals’ modulation of undulation and turning amplitudes was largely compromised ( Figure 5—figure supplement 3 ) .", "This phenotype could be rescued by cell-specific expression of nlp-12 in DVA and flp-1 in AVK ( Figure 6G–H ) .", "In conclusion , control of the composition of undulation and turning modes can be partially dissected at the genetic and single neuron level .", "The DVA/NLP-12 and AVK/FLP-1 systems are required for normal body posture during LDT in constant environmental conditions as well as for the differential fine control of undulation and turning motions in response to both abruptly and smoothly changing environments .", "Our data suggest that decomposition of posture into undulation and turning modes is not only a useful quantitative description of behavior , but that these motor patterns are under differential neural control .", "In order to gain more mechanistic insight into the action of DVA and AVK neurons , we investigated the relationship between their activity and behavior .", "We performed high-magnification ( 40x ) fluorescence Ca2+ imaging , using the indicator GCaMP5K , simultaneously with lower magnification infrared behavioral recording of worms freely moving on agarose .", "A closed-loop tracking system kept neurons of interest in the field of view of the imaging objective ( Faumont et al . , 2011; Kato et al . , 2015 ) and oxygen levels were controlled via a custom-fabricated oxygen flow arena mounted on the microscope stage ( Kato et al . , 2015 ) .", "An example recording of AVK activity is shown in Figure 7A .", "We observed frequent Ca2+ transients of varying magnitude , which were associated with changes in the animal’s locomotion speed .", "Consistent with this observation , AVK Ca2+ signals in average decreased upon O2 downshift ( Figure 7B–C ) .", "When measuring AVK activity in immobilized animals , we did not detect O2 downshift-evoked activity changes ( Figure 7C ) .", "In freely moving animals , we found that AVK activity positively correlated with locomotion speed ( Figure 7D ) , but unlike previously reported speed-encoding interneurons , which exclusively encode either forward or reverse locomotion speed ( Kato et al . , 2015; Li et al . , 2014 ) , AVK correlated with speed during both forward and reverse movement ( Figure 7E ) .", "Consistent with our findings that speed and turning motions were reciprocally regulated , AVK activity negatively correlated with turning amplitude during forward movement ( Figure 7F ) .", "The relationship of AVK activity with behavioral output was unchanged when comparing pre- and post-stimulus periods ( Figure 7G–H ) .", "Our activity recordings were corrected for motion artifacts by co-expressing mCherry and calculating GCaMP/mCherry fluorescence ratios; additional control experiments using Ca2+-insensitive GFP confirmed that our measurements were GCaMP-specific and thus not derived from possible motion artifacts ( Figure 7—figure supplement 1 ) . 10 . 7554/eLife . 14116 . 016Figure 7 . Neural activity of interneuron AVK reflects locomotion speed and suppression of turning motions .", "( A–H )", "Neural activity of AVK neurons was measured in freely moving worms by calcium imaging and is displayed as normalized ratio of GCaMP5K/ mCherry fluorescence .", "( A ) Exemplary recording of neural activity ( top ) and locomotion speed ( bottom ) .", "Time is relative to O2 downshift .", "Periods of reverse movement are labeled in orange .", "( B ) Mean AVK activity ( left ) and mean locomotion speed ( right ) .", "Shadings indicate SEM .", "Time is relative to O2 downshift .", "n = 51 worms .", "( C ) AVK neural activity changes in response to O2 downshift compared between freely moving worms and worms immobilized in a microfluidic device .", "Quantifications of mean activity during 60 s intervals pre- and post- O2 downshift indicated by dashed lines in ( B ) .", "Values from single recordings are shown in gray and population means ± SEM are shown in magenta .", "AVK activity significantly decreased in freely moving animals , but did not change in immobilized worms ( ****p≤0 . 0001; ns , p=0 . 34 , paired t-test ) .", "( D ) Median ( solid line ) and interquartile range ( dashed lines ) of AVK activity versus binned locomotion speed ( bin size = 0 . 01 mm/s ) .", "( E ) Box plots of linear correlation coefficients between forward or reverse movement speed and AVK activity calculated for each recording ( n = 51 ) .", "Unpaired t-test shows no significant difference ( ns , p=0 . 13 ) .", "( F ) Median ( solid line ) and interquartile range ( dashed lines ) of AVK activity during forward movement versus binned turning amplitude ( bin size = 0 . 2 rad ) .", "( G , H )", "Box plots of linear correlation coefficients calculated from time intervals pre- and post- O2 downshift ( n = 51 each ) .", "Paired t-tests show no significant differences .", "( G ) Locomotion speed versus AVK activity; 2 min intervals ( ns , p = 0 . 89 ) .", "( H ) Turning amplitude during forward movement versus AVK activity , 4 min intervals ( ns , p=0 . 56 ) .", "All boxplots display median , interquartile range and 5–95 percentile whiskers . DOI: http://dx . doi . org/10 . 7554/eLife . 14116 . 01610 . 7554/eLife . 14116 . 017Figure 7—figure supplement 1 . Neural activity changes of interneuron AVK are not derived from motion artifacts .", "( A–F )", "Neural activity of AVK neurons was measured by calcium imaging and is displayed as normalized ratio of GCaMP5K/mCherry fluorescence or for control calcium-insensitive GFP/mCherry .", "( A ) Mean AVK ratio ( top ) and mean locomotion speed ( bottom ) from GFP-expressing animals .", "Shadings indicate SEM .", "Time is relative to O2 downshift .", "n = 28 worms .", "( B ) Boxplots showing changes of AVK ratio in response to the O2 downshift compared between freely moving worms expressing GCaMP or GFP and worms immobilized in a microfluidic device .", "Changes were calculated by subtracting the means of 60 s intervals post-downshift from the means of 60 s pre-downshift intervals , indicated by dashed lines in ( A ) .", "Significantly smaller ratio changes of immobilized worms ( *p = 0 . 015 ) or control GFP-expressing worms ( ****p<0 . 0001 , one-way-ANOVA with Dunnett’s correction for multiple comparisons ) compared to GCaMP-expressing freely moving worms .", "( C ) Median ( solid line ) and interquartile range ( dashed lines ) of AVK GFP/mCherry ratio versus binned locomotion speed ( bin size = 0 . 01 mm/s ) .", "( D ) Box plots of linear correlation coefficients between speed and AVK ratio calculated for each recording ( n as indicated ) .", "Total recordings of GCaMP-expressing worms show significantly stronger correlation than GFP-expressing worms ( unpaired t-test ****p<0 . 0001 ) .", "( E ) Median ( solid line ) and interquartile range ( dashed lines ) of AVK ratio versus binned turning amplitude ( bin size = 0 . 2 rad ) during forward locomotion .", "( F ) Box plots of linear correlation coefficients between turning amplitude and AVK ratio during forward movement calculated for each recording .", "GCaMP-expressing worms have significantly stronger correlation than GFP-expressing worms ( unpaired t-test ****p<0 . 0001 ) .", "Boxplots display median , interquartile range and 5–95 percentile whiskers . DOI: http://dx . doi . org/10 . 7554/eLife . 14116 . 017 In summary , elevated AVK neuronal activity reflects high locomotion speed and low turning amplitude , thus corresponding to aspects of motion rather than to sensory inputs; similar observations have been made for many interneurons in C . elegans ( Kato et al . , 2015; Larsch et al . , 2013; Laurent et al . , 2015; Li et al . , 2014; Luo et al . , 2014b ) .", "The neural activity is consistent with the behavioral phenotypes of AVK- animals , which exhibit reduced locomotion speed ( Figure 5—figure supplement 2A , B ) and strong up-regulation of turning maneuvers ( Figure 5D ) .", "Taken together , these results suggest that AVK activity promotes locomotion speed and suppresses turning .", "However , since AVK activity changes are low in immobilized worms we propose that AVK can influence these functions only in the context of feedback from the motor system .", "Next we tested the neural response of the DVA neuron in respect to behavioral output .", "Figure 8A shows an example trace of DVA neuronal activity in a freely moving worm .", "DVA Ca2+ signals increased on average upon O2 downshift ( Figure 8B , C upper panel , see Figure 8—figure supplement 1A , B for motion artifact controls ) .", "We found that DVA activity correlated with multiple aspects of locomotory behavior: DVA activity rose on average at the transition from forward to backward directed movement ( Figure 8—figure supplement 1C–E ) and DVA was strongly activated during periods of paused locomotion ( Figure 8—figure supplement 1F , G ) , a behavior that was observed more frequently upon O2 downshift ( Figure 8—figure supplement 1H ) .", "After restricting the analysis exclusively to periods of forward locomotion , i . e . removing reversal and pausing periods from the data , we did not detect any O2 downshift-evoked change in the residual DVA activity ( Figure 8C , lower panel ) .", "In contrast to AVK , a global correlation analysis did not detect a relationship between DVA activity and forward locomotion speed , turning amplitude or undulation amplitude ( R = -0 . 04 , R = 0 . 09 , R = 0 . 06 , respectively ) .", "However , discernable DVA Ca2+ transients were evident during periods of forward locomotion ( Figure 8A ) .", "To test whether these signals had a behavioral correlate , we calculated Ca2+ peak-triggered averages of undulation amplitude , turning amplitude and body posture amplitude .", "This approach showed that DVA Ca2+ signals were on average associated with transient increases in undulation amplitude and transient decreases in turning amplitude ( Figure 8D ) .", "Triggering body posture amplitude to DVA activity peaks did not reveal any discernible signal ( Figure 8—figure supplement 1I ) ; these findings highlight the strength of the eigenworm decomposition approach for decoding behavior from neural activity . 10 . 7554/eLife . 14116 . 018Figure 8 . Neural activity of interneuron DVA is associated with the amplitude of undulation motions .", "( A–D )", "Neural activity of DVA neurons was measured by calcium imaging in freely moving animals and is displayed as normalized ratio of GCaMP5K/mCherry fluorescence .", "( A ) Exemplary recording of neural activity ( top ) and locomotion speed ( bottom ) .", "Time is relative to O2 downshift .", "Periods of reverse movement are labeled in orange .", "Peaks in DVA calcium transient during forward displacement are marked by blue dots .", "( B ) Mean DVA activity ( top ) and mean locomotion speed ( bottom ) .", "Shadings indicate SEM .", "Time is relative to O2 downshift .", "n = 46 worms .", "( C ) DVA neural activity changes in response to O2 downshift using all data ( top ) or only during forward displacement , i . e . excluding reverse movement and pause phases ( bottom ) .", "Quantifications of mean activity during 60s intervals pre- and post- O2 downshift indicated by dashed lines in ( B ) .", "Values from single recordings are shown in gray and population median ± interquartile range are shown in magenta .", "DVA activity significantly increases upon O2 downshift , but not when animals are moving forward ( ****p<0 . 0001; ns , p=0 . 12 , Wilcoxon matched-pairs signed rank test ) .", "( D ) Traces show mean undulation amplitude and turning amplitude triggered to DVA activity peaks ( t = 0", "s ) during forward displacement ( see blue dots in ( A ) for example events ) .", "Shadings show SEM .", "Number of events and worms is indicated .", "Wilcoxon matched-pairs signed rank tests indicate significant changes ( ****p≤0 . 0001 ) between 1 s or 2 s intervals pre- and post-event as indicated . DOI: http://dx . doi . org/10 . 7554/eLife . 14116 . 01810 . 7554/eLife . 14116 . 019Figure 8—figure supplement 1 . Neural activity of interneuron DVA is associated with reversals and pausing phases .", "( A–I )", "Neural activity of DVA neurons was measured by calcium imaging and is displayed as normalized ratio of GCaMP5K/mCherry fluorescence or for control calcium-insensitive GFP/mCherry .", "( A ) Mean DVA ratio ( left ) and mean locomotion speed ( right ) from GFP-expressing worms .", "Shadings indicate SEM .", "Time is relative to O2 downshift .", "n = 14 worms .", "( B ) Boxplots showing changes of DVA ratio in response to the O2 downshift compared between GCaMP- and GFP- expressing worms .", "Changes were calculated by subtracting the means of 60s intervals post-downshift from the means of 60s pre-downshift intervals , indicated by dashed lines in ( A ) .", "Mann-Whitney test shows significantly different changes ( **p = 0 . 003 ) .", "( C ) Mean DVA activity of GCaMP-expressing worms triggered to reversal starts ( t = 0 ) occurring during a 4 min interval at 21% oxygen .", "Shadings show SEM .", "Wilcoxon matched-pairs signed rank test compares DVA activity during 5s intervals before and after reversal start as indicated ( ****p<0 . 0001 ) .", "( D ) Boxplots showing DVA ratio change after reversal start of GCaMP- and control GFP- expressing animals .", "Mann-Whitney test shows significant difference ( ****p<0 . 0001 ) .", "Numbers of events and worms are indicated .", "( E ) DVA neural activity changes in response to O2 downshift using data only during reverse movement .", "Quantifications of mean activity during 60s intervals pre- and post- oxygen downshift .", "Values from single recordings are shown in gray and population median + interquartile range are shown in magenta .", "DVA activity significantly increases in GCaMP-expressing animals after the stimulus ( ***p=0 . 0002 , Wilcoxon matched-pairs signed rank test ) .", "( F ) Exemplary recording of neural activity ( top ) and locomotion speed ( bottom ) .", "Time is relative to O2 downshift .", "Periods of reverse movement are labeled in orange .", "Phases when the animal paused are marked by blue shadings .", "( G ) Quantifications of mean DVA neural activity during moving and pausing phases from worms that were pausing at least 60s of the recording time .", "Values from single recordings are shown in gray .", "Number of worms is indicated .", "DVA activity significantly differs in GCaMP-expressing animals ( **p<0 . 0059 ) but not in GFP-expressing animals ( ns p = 0 . 0625 , Wilcoxon matched-pairs signed rank tests ) .", "( H ) Fraction of GCaMP-expressing worms in pause phase .", "Time is relative to O2 downshift .", "n = 46 worms .", "( I ) Mean amplitude of body posture triggered to DVA activity peaks ( t = 0s ) during forward displacement ( same events as in Figure 8D ) .", "Shadings show SEM .", "Number of events and worms is indicated .", "Wilcoxon matched-pairs signed rank tests indicates no significant changes between 2 s intervals pre- and post-event as indicated ( ns p = 0 . 086 ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 14116 . 019 In summary DVA activity correlates to multiple aspects of behavior including the transition from forward to reverse movement and prolonged pausing .", "Consistent with the role of DVA/NLP-12 in promoting undulation amplitude ( Figure 5C ) DVA neural activity peaks co-occur with undulation amplitude increases and reciprocal turning amplitude decreases .", "In the previous sections we have described that worms coordinate a variety of locomotion parameters when responding to rapidly and gradually changing ambient O2 concentrations .", "In the following sections we address whether animals make use of this behavioral repertoire in a navigational chemotaxis paradigm .", "Previous work has shown that C . elegans navigates O2 gradients towards preferred intermediate O2 concentrations avoiding both hypoxia and atmospheric O2 levels ( Cheung et al . , 2005; Gray et al . , 2004 ) .", "O2 sensing by BAG sensory neurons is required for these behaviors under fasted conditions ( Zimmer et al . , 2009 ) .", "We thus analyzed the distribution of animal populations exposed to linear gas phase O2 gradients ranging from 4% to 21% ( 0 . 6% O2 /mm ) generated in a previously reported microdevice ( Gray et al . , 2004 ) ( Figure 9A ) .", "The conditions used in the present study excluded the hypoxia regime ( <4% O2 ) since the neuronal mechanisms of acute hypoxia avoidance in C . elegans have not been identified yet ( Gray et al . , 2004; Zimmer et al . , 2009 ) .", "The average distribution profile suggested that 1–1 . 5 hr food-deprived animals strongly avoided low O2 concentrations , accumulated at intermediate O2 concentrations around a bin center of 16 . 4% and slightly avoided atmospheric levels of 21% O2 ( Figure 9B ) .", "For each experiment a paired control experiment was performed ( 21% O2 flow from both microdevice inlets ) , during which animals tended to slightly accumulate in the middle part of the arena ( Figure 9B ) . 10 . 7554/eLife . 14116 . 020Figure 9 . O2 chemotaxis involves regulation of turning , reversals and locomotion speed .", "( A ) O2 chemotaxis assay .", "Top: video frame capture showing worms in O2 gradient arena .", "Arrows indicate gas inlets and outlets .", "Bottom: Measured O2 gradient in the device .", "( B ) Distributions ( trial-means ± SEM ) of wild type N2 worms along the length of the arena .", "Data binning on the x-axis is 1 . 3% O2 .", "For gradient ( solid line ) or control ( dashed line ) assays either 21% and 4% , or 21% and 21% oxygen were applied at the inlets .", "Dashed line at 16 . 4% marks the bin center of the average peak accumulation during the gradient .", "n = 53 assays , 30-40 animals/assay .", "( C , D )", "Analysis of weathervaning chemotaxis .", "( C ) Example trajectories of wild type N2 worms in the oxygen gradient .", "Color code indicates heading orientation ( bearing ) with respect to the O2 gradient: bearing towards or away from 16% O2 is defined as 0° or 180° , respectively .", "Gray trajectory sections were excluded from the analysis .", "Inset: example trajectory with color-code indicating the change of bearing ΔB .", "( D ) Trial-mean curving bias ΔB per displacement ( ± SEM ) under control ( left ) and gradient ( middle ) conditions as a function of bearing ( 20° binning ) .", "Right: quantification of curving bias averaged across a bearing range of 40°–150° .", "( E ) Left: Trial-mean reversal frequency ( ± SEM ) under control ( dashed line ) and gradient ( solid line ) conditions as a function of bearing ( 20° binning ) .", "Right: quantification of difference in mean reversal frequency below versus above 90° bearing .", "( F ) Left: Trial-mean forward centroid speed ( ± SEM ) under control ( dashed line ) and gradient ( solid line ) conditions as a function of bearing ( 20° binning ) .", "Right: quantification of difference in mean forward crawling speed below versus above 90° bearing .", "( G ) Trial-mean mid-body ( segment angle #11 ) signal amplitude of individual turning events under control ( light gray ) and gradient ( dark gray ) conditions as a function of bearing ( 5° binning ) .", "The linear fit and the correlation coefficients", "( r ) are indicated; corresponding p-values indicate significance of correlation .", "The indicated sample sizes are n = number of experiments .", "All boxplots display trial-median , interquartile range and 5–95 percentile whiskers .", "Quantifications of control and gradient experiments are significantly different by Mann-Whitney test ( ****p<0 . 0001 ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 14116 . 020 Previous studies showed that C . elegans navigation in chemical and temperature gradients involves various navigational strategies such as modulation of reorientation rate , modulation of speed and finely controlled steering ( Iino and Yoshida , 2009; Luo et al . , 2007; Luo et al . , 2014a; Pierce-Shimomura et al . , 1999; Schild and Glauser , 2013 ) .", "Since it was unknown which strategies contribute to O2 chemotaxis , we performed a quantitative characterization thereof .", "Here and in the subsequent section , we focused on the low oxygen avoidance condition ( 16% O2–4% O2 ) .", "Finely controlled steering during navigation of chemical gradients was previously described as a chemotaxis strategy termed weathervaning: animals introduce a curving bias towards preferred conditions depending on their heading direction with respect to the gradient direction ( bearing ) ( Iino and Yoshida , 2009 ) .", "Heading straight towards 16% or 4% O2 was defined as 0° or 180° bearing respectively while heading perpendicular to the gradient was defined as 90° bearing ( Figure 9C ) .", "We then calculated the animals’ curving bias , which is the average change in the bearing trajectory ΔB/Δx as a function of bearing ( Figure 9C–D ) .", "In comparison to control experiments lacking a gradient , animals exhibited a curving bias towards preferred O2 concentrations , strongest at bearing orientations around 90° ( Figure 9D ) .", "Next , we investigated the modulation of reorientation rate and found that reversal rates were suppressed or increased at low or high bearing angles , respectively ( Figure 9E ) .", "We also observed a gradual reduction of locomotion speed as a function of increased bearing angles ( Figure 9F ) .", "Finally , we detected individual shallow turning events based on finding local peaks of turning mode signal of the mid-body during forward locomotion , excluding omega turns .", "On average , turning increased gradually as a function of bearing angles during O2 chemotaxis but not under control conditions ( Figure 9G ) .", "In summary , we found that wild type N2 animals when fasted for 1–1 . 5 hr perform O2 chemotaxis to approach intermediate O2 concentrations centered around 16 . 4% .", "The low O2 avoidance mechanisms employ multiple navigational strategies: when heading along directions around perpendicular to the gradient animals steer towards preferred O2 concentrations using weathervaning , and when animals are heading away from preferred O2 concentrations ( high bearing angles ) the random biased walk mechanism of chemotaxis is prevalent as indicated by an up-regulation of reversal rates .", "In addition , animals exhibit enhanced ARS-like behavior indicated by a gradual reduction of locomotion speed and up-regulation of turning dependent on their heading direction .", "Based on our data , we hypothesized a role of the AVK/FLP-1 and DVA/NLP-12 systems for turning maneuvers during O2 chemotaxis .", "flp-1;nlp-12 double mutants showed a defect in their ability to distribute in response to the O2 gradient ( Figure 10A ) .", "We subtracted the fractional animal distributions during controls from the distributions during gradient application in order to calculate O2 chemotaxis indices for low O2 concentration ranges .", "This revealed a compromised ability of flp-1;nlp-12 double mutants to avoid low O2 concentrations ( Figure 10B ) .", "A similar defect was observed in nlp-12 and flp-1 single mutants but not in AVK- animals ( Figure 10—figure supplement 1A–D ) .", "However , flp-1 single mutants as well as AVK- animals showed a stronger tendency to accumulate in the center of the assay arena during control experiments ( Figure 10—figure supplement 1B , C ) .", "This effect appeared similar to crowding behavior animals perform under conditions of high population density and food scarcity ( unpublished observation ) .", "Since this effect could potentially mask O2 chemotaxis behaviors and thus might impede the interpretability of results we focused our further in-depth analysis on flp-1;nlp-12 double mutants ( Figure 10 ) and nlp-12 single mutants ( Figure 10—figure supplement 1E–H ) .", "nlp-12 mutants as well as flp-1;nlp-12 double mutants showed reduced weathervaning O2 chemotaxis ( Figure 10C; Figure 10—figure supplement 1E ) but were able to modulate reversal rates ( Figure 10D; Figure 10—figure supplement 1F ) despite lower reversal rates in nlp-12 single mutants ( Figure 10—figure supplement 1F ) .", "flp-1;nlp-12 double mutants did not modulate locomotion speed and turning mode as a function of bearing ( Figure 10E , F ) ; both parameters were modulated in nlp-12 single mutants despite low and high basal rates in speed and body turning signal , respectively ( Figure 10—figure supplement 1G , H ) . 10 . 7554/eLife . 14116 . 021Figure 10 . FLP1 and NLP-12 neuropeptides are implicated in the regulation of turning and locomotion speed during O2 chemotaxis .", "( A ) Distributions ( trial-means ± SEM ) of flp-1;nlp-12 mutants along the length of the arena .", "Data binning on the x-axis is 1 . 3% O2 .", "For gradient ( solid line ) or control ( dashed line ) assays either 21% and 4% , or 21% and 21% oxygen were applied at the inlets .", "Dashed line at 16 . 4% marks the bin center of the average peak accumulation of wild type animals during the gradient .", "n = 45 assays , 30-40 animals/assay .", "( B ) Left: Indices ( trial-means ± SEM ) of worm distributions along the length of the arena of wild type N2 ( gray ) and flp-1;nlp-12 mutant ( green ) animals , calculated by subtracting in every bin the fraction of the paired control experiment from the fraction of the respective gradient experiment .", "Dashed line at 13 . 8% O2 marks equal average accumulation during control and gradient conditions .", "Right: Cumulative indices ( summed indices of all bins below 13 . 8% ) of mutant and wild type N2 are compared by one-way-ANOVA with Dunnett’s correction ( *p=0 . 035 , ***p=0 . 0003 ) .", "All strains shown in Figure 10—figure supplement 1D were included in the statistical analysis .", "( C ) Mean curving bias ( ± SEM ) of flp-1;nlp-12 mutants under control ( left ) and gradient ( middle ) conditions as a function of bearing ( 20° binning ) .", "Right: quantification of curving bias averaged across a bearing range of 40°–150° .", "( D ) Left: Trial-mean reversal frequency ( ± SEM ) of flp-1;nlp-12 mutants under control ( dashed line ) and gradient ( solid line ) conditions as a function of bearing ( 20° binning ) .", "Right: quantification of difference in mean reversal frequency below versus above 90° bearing .", "( E ) Left: Trial-mean forward centroid speed ( ± SEM ) of flp-1;nlp-12 mutants under control ( dashed line ) and gradient ( solid line ) conditions as a function of bearing ( 20° binning ) .", "Right: quantification of difference in mean forward crawling speed below versus above 90° bearing .", "( F ) Trial-mean mid-body ( segment angle #11 ) signal of individual turning events of flp-1;nlp-12 mutants under control ( light gray ) and gradient ( dark gray ) conditions as a function of bearing ( 5° binning ) .", "The linear fit and the correlation coefficients", "( r ) are indicated; corresponding p-values indicate significance of correlation .", "The indicated sample sizes are n = number of experiments .", "All boxplots display trial-median , interquartile range and 5–95 percentile whiskers .", "Quantifications of control and gradient experiments per strain are compared by Mann-Whitney test .", "Comparisons between flp-1;nlp-12 mutant and N2 are performed with Kruskal-Wallis test with Dunn’s correction ( ****p≤0 . 0001 , ***0 . 0001<p≤0 . 001 , **0 . 001<p≤0 . 01 , *0 . 01<p≤0 . 05 , ns p>0 . 05 ) .", "Results of wild type N2 worms ( shown in gray ) used for comparison to mutants rely on the same data shown in Figure 9 . DOI: http://dx . doi . org/10 . 7554/eLife . 14116 . 02110 . 7554/eLife . 14116 . 022Figure 10—figure supplement 1 . Regulation of turning during O2 chemotaxis depends on NLP-12 neuropeptides .", "( A–C )", "Distribution ( trial-means ± SEM ) of manipulated worms along the length of the arena .", "Data binning on the x-axis is 1 . 3% O2 .", "For gradient ( solid line ) or control ( dashed line ) assays either 21% and 4% , or 21% and 21% oxygen were applied at the inlets .", "Dashed line at 16 . 4% marks the bin center of the average peak accumulation of wild type animals during the gradient .", "Numbers of assays are indicated , 30-40animals/assay .", "( A ) nlp-12 mutants , ( B ) flp-1 mutants , ( C ) AVK cell-ablated worms .", "( D ) Indices were calculated by subtracting in every bin the fraction of the paired control experiment from the fraction of the respective gradient experiment .", "Cumulative indices ( summed indices of all bins below 13 . 8% ) of mutant strains are separately compared to wild type N2 by one-way-ANOVA with Dunnet’s correction ( ****p≤0 . 0001 , ***0 . 0001<p≤0 . 001 , **0 . 001<p≤0 . 01 , *0 . 01<p≤0 . 05 , ns p>0 . 05 ) .", "Wild type ( N2 ) and flp-1;nlp-12 data are from the same experiments shown in Figure 9B or 10A , B . ( E ) Trial-mean curving bias ( ± SEM ) of nlp-12 mutants under control ( left ) and gradient ( middle ) conditions as a function of bearing ( 20° binning ) .", "Right: quantification of curving bias averaged across a bearing range of 40°–150° .", "( F ) Left: Trial-mean reversal frequency ( ± SEM ) of nlp-12 mutants under control ( dashed line ) and gradient ( solid line ) conditions as a function of bearing ( 20° binning ) .", "Right: quantification of difference in mean reversal frequency below versus above 90° bearing .", "( G ) Left: Trial-mean forward centroid speed ( ± SEM ) of nlp-12 mutants under control ( dashed line ) and gradient ( solid line ) conditions as a function of bearing ( 20° binning ) .", "Right: quantification of difference in mean forward crawling speed below versus above 90° bearing .", "( H ) Trial-mean mid-body ( segment angle #11 ) signal amplitude of individual turning events of nlp-12 mutants under control ( light gray ) and gradient ( dark gray ) conditions as a function of bearing ( 5° binning ) .", "The linear fit and the correlation coefficients", "( r ) are indicated; corresponding p-values indicate significance of correlation .", "The indicated sample sizes are n = number of experiments .", "All boxplots display trial-median , interquartile range and 5–95 percentile whiskers .", "Quantifications of control and gradient experiments per strain are compared by Mann-Whitney test .", "Comparisons between nlp-12 mutant and N2 are performed with Kruskal-Wallis test with Dunn’s correction ( ****p≤0 . 0001 , ***0 . 0001<p≤0 . 001 , **0 . 001<p≤0 . 01 , * 0 . 01<p≤0 . 05 , ns p>0 . 05 ) .", "Results of wild type N2 worms ( shown in gray ) used for comparison to mutants rely on the same data shown in Figure 9 . DOI: http://dx . doi . org/10 . 7554/eLife . 14116 . 022 Taken together , these results show that when both FLP-1 and NLP-12 signaling is abolished , animals exhibit specific defects in navigating O2 gradients: both weathervaning at intermediate bearing angles and up-regulation of ARS-like behaviors , i . e . slowing and increased turning , at high bearing angles are compromised; however , modulation of reversal rates as a function of bearing angle is unaffected ." ], [ "A detailed description of motor patterns underlying animal locomotion during LDT and ARS contributes toward understanding the neural operations that execute the animals’ strategy to navigate in the environment .", "Here we report on a posture control system in C . elegans that coordinates two elementary motor modes to drive efficient dispersal or local search behaviors , respectively .", "This coordination is required for foraging and goal-directed chemotaxis .", "We show that a sudden decrease in environmental O2 , detected by BAG O2 sensory neurons , evokes an array of behavioral changes in food-deprived animals ( Figures 1A–E; Figure 1—figure supplement 1 ) .", "Unlike previously described brief escape reflexes ( Goodman , 2006; Hilliard et al . , 2002 ) , these behaviors are sustained for several minutes and can be described as a switch from LDT to O2-induced ARS , in accordance to previous literature studying similar behavior strategies after removal from food ( Calhoun et al . , 2015; Gray et al . , 2005; Hills et al . , 2004; Peliti et al . , 2013; Tsalik and Hobert , 2003; Wakabayashi et al . , 2004 ) .", "The reduction in ambient oxygen could indicate the proximity of a food source and therefore evoke similar ARS behavior as observed after removal from food .", "In both paradigms the search strategy is thus likely built on the vicinity of a potential food source .", "However , whether the same interneuron circuits are controlling the two ARS behaviors has not been focus of this study and remains to be investigated .", "Yet , we know that NLP-12 neuropeptides play a role in both O2-induced ARS ( this study ) and ARS after removal from food ( Bhattacharya et al . , 2014 ) indicating some potential overlap between both circuits .", "The bending of each body segment of C . elegans is tightly coupled to the activity of the underlying body wall muscles .", "Therefore , posture time series are an indirect readout of muscle activity patterns ( Butler et al . , 2014 ) .", "Here we show that strikingly different movement strategies of C . elegans can be described by projecting posture time series to a common basis of EWs ( Figure 2A ) .", "We reconstruct two motor modes from these projections: reconstructing posture time series from EW1-2 reveals the regular undulation mode of the animal consisting of sinusoid-like postures ( Figure 2C , D ) .", "The residual movement ( turning mode ) is a set of motor patterns with distinct characteristics ( Figure 2—figure supplement 1 ) , capturing the animals’ curving and turning movements .", "Strikingly , complex postural time series can be additively composed out of these two simpler modes ( Figure 2D , Video 2 ) : The eigenworm approach decomposes the biphasic temporal profile of animal posture observed upon sensory stimulation into two reciprocally regulated , simpler , monophasic components ( Figure 4A ) .", "This indicates that the undulation and turning modes may explicitly map to neural control mechanisms .", "We speculate that summation of distinct neural activity patterns might be an elegant solution for a neural system to generate complex movements .", "In this view the turning and undulation modes could have specific neural correlates at the level of interneurons and/or motor neurons , which are summed at the respective downstream postsynaptic targets .", "This hypothesis bears analogy to the proposal of muscle synergies producing coordinated movements in vertebrates ( Tresch and Jarc , 2009 ) .", "Further imaging studies simultaneously recording the activity of muscles , motor neurons and interneurons are required to test this hypothesis .", "We describe three basic parameters of C . elegans locomotion: frequency and amplitude of the undulation mode contributing to the animals’ locomotion speed and the turning amplitude contributing to changes in the direction of locomotion .", "All three parameters are in a lawful relationship: high undulation frequency is associated with high undulation amplitude , and both parameters are counter-regulated with turning amplitude ( Figure 3C , F ) .", "This finding demonstrates an important tradeoff between flexibility and efficiency of movement: explorative behaviors with frequent directional changes require flexible postures and slower locomotion while fast undulation for efficient displacement requires movements coherent across all body parts .", "Therefore , undulation and turning motor modes are counter-regulated; however , they are not mutually exclusive as their contributions to movement are graded ( Figure 3–4 ) .", "Such counter-regulation might not be surprising , yet it has not been thoroughly described how animals in general coordinate underlying motor patterns for their purposes .", "Furthermore , counter-examples with co-regulation of those motor patterns exist in animal locomotion: e . g . hares sharply turn corners during fast escape runs , which suggests the need of neural coordination mechanisms of motor patterns .", "Based on our findings we propose that LDT and ARS are not discrete alternative behavioral states , between which the animals switch , but rather represent extremes of a behavioral spectrum reflecting the animals’ ability to flexibly adapt locomotion to their instantaneous needs .", "This contrasts with descriptions of C . elegans’ roaming and dwelling behaviors in the presence of food , which have been characterized as discrete alternative behavioral states maintained for seconds to minutes , which is regulated by the neuromodulators serotonin ( 5-HT ) and PDF neuropeptides ( Ben Arous et al . , 2009; Flavell et al . , 2013 ) .", "However , a different study has reported behavioral intermediates between roaming and dwelling ( Gallagher et al . , 2013 ) .", "Nevertheless , roaming and dwelling characterized by ARS-related behavioral features on food might be different from O2-induced ARS and LDT of food-deprived animals; it remains to be tested whether neural mechanisms , such as 5-HT and PDF signaling in roaming-dwelling decisions or FLP-1 and NLP-12 signaling in O2-induced ARS , are shared between or exclusive for the two paradigms .", "Our data show that undulation and turning modes can be dissected at the genetic and cellular level .", "We identified peptidergic neurons and neuropeptide genes , the genetic manipulations of which strongly affect the animals’ undulation and turning motions .", "Our data support the following model: FLP-1 neuropeptides released from AVK interneurons promote a shallow posture by constitutively restricting turning and undulation amplitude .", "AVK neuronal activity promotes undulation speed and suppresses turning ( Figure 5 , 7 and Figure 5—figure supplement 2 ) .", "Unlike other speed encoding interneurons in C . elegans that selectively encode the speed of forward or reverse movement ( Kato et al . , 2015; Li et al . , 2014 ) AVK encodes speed during the execution of both gaits .", "The decreased activity after stimulation is consistent with an increase of turning amplitude during ARS .", "Changes of AVK activity after sensory stimulation are evident only in unrestrained animals .", "Therefore , AVK might function as part of a locomotor feedback system to monitor speed changes and to inversely couple turning motions to these changes .", "In addition , it is likely that AVK neurons receive input from their presynaptic partners , the RIG interneurons ( White et al . , 1986 ) , which are among the major postsynaptic partners of BAG sensory neurons and candidate transducers of O2 stimuli ( Kato et al . , 2015 ) , which could be a pathway for sensory-evoked postural control .", "The DVA interneuron and NLP-12 neuropeptides secreted by it have differential effects on both motor patterns: our data support a model in which DVA activity/NLP-12 during forward locomotion promote undulation movements and locomotion speed while counteracting turning movements ( Figures 5 , 8 and Figure 5—figure supplement 2 ) .", "In freely moving animals , we discovered various behavioral correlates of DVA neural activity , measured by Ca2+-imaging , including pausing , reversing as well as transient reciprocal changes in undulation and turning amplitude ( Figures 8 and Figure 8—figure supplement 1 ) .", "In our recordings DVA activity does not seem to encode sensory stimulus as it corresponds best to the execution of the various behaviors , the frequency of which changes upon stimulation .", "So far no candidate sensory to motor pathways from BAG to DVA have been characterized .", "DVA has been described as a proprioceptive neuron activated by body flexure in semi-restrained worms ( Li et al . , 2006 ) .", "In our data obtained in freely moving worms we did not find a relationship between total body flexure and DVA activity .", "It therefore remains to be shown to what extent proprioception contributes to DVA activity in unrestrained animals .", "When both characterized regulatory systems are compromised simultaneously , either in flp-1;nlp-12 double mutants or in AVK-;DVA- animals , most locomotion parameters during LDT are either unaffected or only marginally affected ( Figure 5 and Figure 5—figure supplement 2 ) .", "These results suggest that both signaling systems act in parallel but antagonistically .", "Yet , the ability of these animals to control locomotion speed , undulation amplitude and turning amplitude in response to stimulation is drastically affected ( Figure 5—figure supplement 2 , Figure 5E–G , Figure 5—figure supplement 3 ) .", "These data indicate the importance of a counter-regulatory system including AVK/FLP-1 and DVA/NLP-12 for controlling undulation and turning motions in response to environmental changes .", "We hypothesize that NLP-12 and FLP-1 exert their effects on undulation and turning motions by modulating neurotransmission at neuromuscular junctions: NLP-12 peptides have been shown to enhance synaptic transmission at cholinergic motorneuron synapses ( Bhattacharya et al . , 2014; Hu et al . , 2011; Hu et al . , 2015 ) .", "A similar effect on GABAergic motor neuron synapses has been suggested for FLP-1 peptides ( Stawicki et al . , 2013 ) .", "We show here that 1 hr food-deprived wild type N2 animals , unlike well-fed N2 animals ( Gray et al . , 2004 ) , navigate in a linear O2 gradient towards a concentration range centered around 16% ( Figure 9A , B ) .", "This is a laboratory condition associated with abundant bacterial food ( Gray et al . , 2004 ) , which suggests that fasted animals utilize information about ambient O2 concentrations in order to search for food .", "We show that C . elegans employs multiple strategies during O2 chemotaxis , the choice of which depends on how the animals are oriented along the gradient: when their orientation is close to perpendicular to the gradient direction , the weathervaning strategy prevails ( Figure 9D ) .", "When heading straight up or down the gradient , they employ a biased random walk strategy by down- or up-regulating reorientation rate , respectively ( Figure 9E ) .", "Similar observations have been made in other chemotaxis paradigms in C . elegans ( Iino and Yoshida , 2009 ) .", "In addition , we found that heading away from preferred O2 levels causes the animals to slow down and up-regulate turning movements , i . e . they engage in an ARS-like behavior ( Figure 9F , G ) .", "flp-1;nlp-12 double mutants exhibit specific defects in weathervaning , speed modulation and up-regulation of turning movements , while the random biased walk mechanism remains intact ( Figure 10C–F ) .", "The overall consequence is a decrease in chemotaxis performance ( Figure 10A , B ) .", "These data indicate that the fine-tuned control of undulation and turning motions is essential for normal navigation of spatial gradients; nevertheless , the biased random walk strategy can partially compensate for these deficits .", "In bacteria the biased random walk strategy is the only reported mechanism supporting the organism to reach its goal ( Berg and Brown , 1972 ) .", "It will be interesting to find out whether similar counter-regulation of motor patterns underlies the chemotactic strategies of other animals , e . g . Drosophila larvae .", "These move via whole-body propulsions and are also capable of controlling run directions ( = weathervaning ) besides regulating timing and direction of whole-body turns in response to odorant gradients ( Gomez-Marin et al . , 2011; Gomez-Marin and Louis , 2014 ) .", "In conclusion , we propose that combination of elementary motor patterns enables animals to flexibly adjust their locomotion strategy .", "The undulation mode dominates LDT while turning movements shape O2-induced ARS .", "However , these behaviors lie on a continuum .", "Neuromodulatory peptidergic circuits gradually adjust the underlying movement parameters while enforcing lawful reciprocal relationships between them .", "In this way , movement can be finely controlled according to the animals’ instantaneous needs , allowing for a rapid sensory-evoked shift from LDT to O2-induced ARS as well as for subtle movement changes during navigation .", "Our approach is thus complementary with others that describe different behaviors as discrete and maintained behavioral states between which animals switch ( Ben Arous et al . , 2009; Brown et al . , 2012; Dankert et al . , 2009; Kain et al . , 2013; Wiltschko et al . , 2015 ) .", "The coordination of motor patterns for foraging accounts for a tradeoff that animals have to make in order to effectively achieve the goals of each strategy: fast undulation requires a coherent coordination of all body parts , while slow search movements require flexibility in gait and posture .", "In this work we provide experimental support for this concept obtained from C . elegans and speculate that analogous control schemes may govern locomotion in higher animals ." ], [ "Behavioral studies of C . elegans populations were done as described previously ( Zimmer et al . , 2009 ) with some modifications for increased image resolution and improved stimulus delivery: For each assay ~25 adult animals grown on OP50 seeded food plates ( 1 day post L4 larval stage ) were transferred ( via manual picking ) without food onto a plane food-free nematode growth medium ( NGM ) agar surface in a 14 cm petri dish ( NGM assay plate ) .", "Animals were starved for one hour on the NGM assay plate prior to examination .", "A 36 mm x 36 mm area was cut out of Whatman filter paper soaked in 20 mM of repelling CuCl2 to prevent animals from leaving the assay arena .", "A custom-made transparent plexiglass device with a flow arena of 39 mm x 39 mm x 0 . 7 mm was placed onto the assay arena and animals were exposed to a gas flow of 25 ml/ min containing 21% ( v/ v ) O2 for six minutes , followed by a switch to 10% O2 for six minutes ( downshift shift assays ) or followed by a temporal ramp from 21% to 4% O2 lasting three minutes ( step size 0 . 094%/s ) .", "All gas mixtures were balanced with N2 .", "Gases were mixed with a static mixing element connected to mass flow controllers ( Vögtlin Instruments , Aesch , Switzerland ) that were operated by custom written LabVIEW ( National Instruments , Austin , TX ) software .", "The temporal O2 shifts and ramp were confirmed by measuring O2 concentrations in the device with an O2-sensitive fluorescent spot sensor ( PreSens , Regensburg , Germany ) .", "We measured that O2 shifts equilibrate the arena within 12 s .", "The O2 ramps were found to be linear as expected .", "Recordings of freely behaving animals illuminated with 200 mm x 200 mm flat red LED lights were made at 10 fps on 4–5 megapixel CCD cameras ( JAI , Copenhagen , Denmark ) using Streampix software ( Norpix , Montreal , Canada ) .", "The pixel resolution was 0 . 0129 mm/ pixel .", "Movies were analyzed with a custom written image processing and tracking code in MATLAB ( MathWorks , Natick , Massachusetts ) .", "It is build upon a previously reported script ( Ramot et al . , 2008; Tsunozaki et al . , 2008 ) , available at http://med . stanford . edu/wormsense/tracker/ .", "Briefly , worms were detected by gray level thresholding .", "Worm trajectories were generated by connecting nearby centroid coordinates in adjacent frames and each trajectory coordinate was assigned with recorded binary images , centroid coordinates and shape parameters; the resulting data-structures are termed here worm tracks .", "Trajectories are terminated when worms collide with each other or with the boundaries of the arena .", "Each worm track therefore represents a fragment of each worm’s complete behavior during the recording time .", "Worm tracks with a length of less than 200 ( 20 s duration ) frames were discarded .", "Each worm is typically represented by multiple worm tracks of varying length .", "This is important to keep in mind when displaying and quantifying the data ( see below ) .", "The resulting trajectories were smoothed and used to calculate instantaneous translational speed of the worm’s centroid , which is measured along the axis of progression .", "Periods of backward locomotion were detected based on changes in angular velocity and building on the fact that animals were moving most of the time in forward direction , which was confirmed also for all mutants used in this study .", "Reversal frequency was calculated in 10 s bins .", "Reversals were usually excluded ( data set to NaN ) from the population behavioral assay analyses to obtain forward locomotion only .", "Time periods during which the animals were within 80pixels ( = 1 . 0 mm ) distance to the Whatman paper were also excluded .", "Deep , so-called omega turns were detected based on characteristic changes in object eccentricity and angular speed , and their frequency was calculated in 10 s bins .", "Track curvature was determined from smoothed centroid trajectories for every frame as absolute change of heading angle between adjacent frames .", "Therefore the heading angle per frame i was extracted as the four-quadrant inverse tangent of the x/y-coordinates resulting from the differences between the centroid x/y-positions in frame i-5 and frame i+5 ( 1 s intervals ) .", "The result ( change of heading angle per frame ) was divided by the mean crossed distance per frame during the same 1 s interval .", "This yielded change of heading angle over distance .", "Data during which animals were barely moving ( speed < 0 . 03 mm/s ) had to be excluded as centroid 'wobbling' due to tracking issues at very low speeds caused falsely high curvature levels .", "Artificially high values ( >31 rad/mm = 99 . 5 percentile ) were removed .", "Data during reverse movement were excluded .", "Displacement was calculated in 30 s bins .", "Distance of the centroid positions between start and end point for every bin were determined for each worm track .", "For displacement , movement in reverse direction was not excluded .", "After thresholding , the binary worm images were processed ( by dilation , erosion , edge-smoothing , bridging unconnected points , hole-filling ) before they were eventually skeletonized ( including branch trimming for optimization ) to obtain one-dimensional splines tracing the midline of the worms from head to posterior end .", "The pointy tail tips were omitted due to thresholding issues .", "The extracted splines were smoothed and divided into 25 equally spaced body segments .", "Artificially short skeletons were excluded and the data of these frames set to NaN .", "Images of worms forming coil-like postures could not be skeletonized .", "This typically occurred during deep bended omega turns when the head touches the tail .", "However , the most highly curved posture typically happened slightly before so that body posture , undulation mode and turning mode amplitude maxima during omega turns were still included in most of the data .", "Therefore , time points during those turning events before and after the moments of head-body touching could be skeletonized .", "Head positions were determined based on direction of movement and taking into account when the animals moved backward .", "Then skeletons were reordered accordingly .", "Head-tail flips were further prevented due to the fact that the head position could only change by limited distance in-between adjacent frames ( 0 . 1 s ) : both skeleton end points were compared to the head position of the previous frame and the end point with smaller distance was usually ( except for identified deep omega turns , when head and tail position were very close ) taken as the new head position .", "Manually proof checking confirmed the reliability of this method .", "We calculated 24 inter-segment angles between the adjacent segments from head ( segment angle #1 ) to tail ( segment angle #24 ) .", "Segment angle time series were added to the worm tracks structures .", "They were termed body posture .", "Previous studies had used at least 48 ( up to 100 ) segments ( Brown et al . , 2012; Stephens et al . , 2008; Yemini et al . , 2013 ) .", "In this paper , we can recapitulate similar eigenworm shapes with 24 segment angles .", "The advantage of choosing ( in comparison to these studies ) lower resolution was being able to record from multiple animals simultaneously and to acquire data with the parallel worm tracker described above .", "This approach provides higher sample power required for trial averaging and statistics when assaying behavioral responses that naturally exhibit high single trial animal-to-animal variability .", "For calculation of eigenworms ( except for Figure 2A , B ) , all wild type N2 segment angle time series ( containing forward and backward locomotion ) were concatenated .", "For Figure 2A , B , only wild type segment angle time series that were spanning defined 60 s intervals directly pre- or post- O2 shift , respectively , were concatenated .", "Then eigenworms ( EW ) ( and eigenvalues for Figure 2A , B ) were derived by standard principal components analysis ( PCA ) on the concatenated angle time series using MATLAB .", "Variance contribution per eigenworm ( Figure 2A , B ) was calculated as the relative fraction each eigenvalue contributed to the sum of all eigenvalues .", "The difference between the cumulative variance distributions of the two 60 s intervals was evaluated for significance by a random resampling approach: the same angle time series were shuffled and then randomly split into two groups with n numbers matching the original counts of time series pre- or post-shift , respectively .", "Then PCA was performed on the concatenated angle time series per group ( as before ) and the cumulative difference between the two cumulative variance contributions was calculated .", "These steps were iterated one million times during which an equal or higher value than the one obtained from the original non-shuffled groups never occurred .", "This procedure thus yielded an estimate of an upper bound of 10–6 for a p-value .", "Segment angle time series of all individual worm tracks from all strains were projected onto these wild type-derived eigenworms .", "The time series mean of each angle was subtracted from each angle .", "Then , undulation mode or turning mode was generated by firstly calculating the cross-product of the 24 segment angles with a matrix made of eigenworms 1–2 or remaining eigenworms 3–24 , respectively .", "This step yielded a description of the body posture in terms of 2 or 22 projection amplitudes along the respective eigenworms .", "Secondly , the cross product of the result with the respective eigenworm matrix was calculated and the previously subtracted mean was added .", "This step retrieved the description in terms of 24 segment angles . undulationmode=[EW1:EW2]× ( [EW1:EW2]T×[angles−mean ( angles ) ] ) +mean ( angles ) turningmode=[EW3:EW24]× ( [EW3:EW24]T×[angles−mean ( angles ) ] ) +mean ( angles ) This procedure decomposed the body posture on a frame-by-frame basis into undulation mode and turning mode .", "As PCA is a linear decomposition method , the total body posture is the sum of every corresponding undulation and turning mode:bodyposture=undulationmode+turningmode Cross correlation functions in Figure 2—figure supplement 1 were calculated from selected segment angles ( as indicated ) of all wild type N2 undulation or turning mode time series during forward locomotion .", "This method was robust with respect to the varying lengths ( at least 20 s ) of worm tracks .", "Phase velocity ( = undulation frequency ) of undulation mode was derived by calculating the cross-product of the 24 segment angles time series ( after subtracting the time series mean of each angle ) with the eigenworms 1–2 .", "The obtained two projection amplitudes a1 and a2 oscillate in quadrature ( Stephens et al . , 2008 ) .", "They were gently smoothed and the phase angle for each frame was derived from the cross-product of vectors in the ( a1 , a2 ) space from adjacent frames; then the phase velocity was calculated from the obtained angles as cycles ( 360˚ angle ) per time ( cycles/second ) .", "We calculated the amplitude of body posture , undulation and turning mode by summing the absolutes of all 24 segment angles per time point .", "In order to account only for forward locomotion , periods of backward locomotion were excluded from population data .", "Worm shapes in Figure 2D and in Video 2 were reconstructed from total body posture , undulation mode and turning mode: synthetic worm silhouettes were calculated at each point in a time series as follows .", "From the 24 segment angles and a list of 25 fixed end-to-end segment lengths , the position of 26 2D points along the central line of the worm body from head to tail were calculated .", "Combining these 26 central line points with a fixed list of 26 cross sectional widths from head to tail , a filled 2D polygonal model of the worm consisting of 50 quadrilaterals and 2 triangles , one for the head-most segment and one for the tail-most segment , was created .", "The overall orientation for these reconstructed worms was determined at each time point by matching the centroid-to-nose-tip vector to a corresponding vector extracted from real movies .", "The filled 2D polygonal model was then rendered into an image using a standard triangle-filling pixel scan-line algorithm .", "For averaging , means and standard errors of the mean ( SEM ) of behavioral time courses were determined on a frame-by-frame basis from all worm tracks of all experiments available in each frame , for centroid speed , amplitudes and track curvature .", "These averages were finally binned ( by 10 frames = 1 s intervals ) for display reasons .", "Averages of reversal and omega turn frequency or displacement rate ( Figure 1—figure supplement 1A , B or Figure 1C ) were determined by averaging worm track data derived from 10 s or 30 s bins , respectively .", "Distributions of behavioral features were derived as follows .", "For relative distributions of turning amplitude during short time intervals ( Figure 4C ) , all data from the indicated time interval were pooled per experiment and normalized histograms with 0 . 1 rad bin size were derived and then averaged over all experiments .", "For analyzing two behavioral features against each other , all worm tracks ( binned by 5 frames = 0 . 5 s ) within the respective indicated time interval of all experiments were pooled .", "For density maps displaying 2D distributions of undulation frequency against turning amplitude ( Figure 4D ) , normalized 2D histograms were derived with the bin sizes of 0 . 002 cycles/s for undulation frequency and 0 . 025 rad for turning amplitude .", "Very high values of undulation frequency ( >0 . 6 cycles/s ) and amplitude ( >4 rad ) were not shown due to data sparseness .", "For density maps displaying 2D distributions of undulation amplitude against turning amplitude ( Figure 5—figure supplement 1 ) , normalized 2D histograms were derived with bin sizes of 0 . 05 rad .", "A boundary encompassing the 99% most abundant data of wild type N2 was calculated .", "The per cent of data of manipulated strains lying within this wild type N2 boundary was determined .", "For sorting one behavioral feature Y over another feature X ( Figure 3 ) , feature X was divided into bins of equal length ( undulation frequency 0 . 015 cycles/s; turning amplitude 0 . 2 rad [against track curvature]; undulation amplitude 0 . 1 rad [against turning amplitude] ) .", "Medians and inter-quartile ranges of feature Y from all worm track data within each bin of X were calculated and plotted over the medians of bins from X . Linear correlation coefficients were calculated via a MATLAB standard function .", "Example trajectories in Figure 3A were chosen during the 10% O2 phase .", "Positions and turning amplitude were binned by 5 frames ( 0 . 5 s ) and the trajectories’ start points were aligned .", "For quantifications and statistics we chose to account for experiment-to-experiment variability .", "Therefore the population mean for each individual experiment was calculated from respective indicated time intervals .", "For changes after O2 downshift or through the O2 ramp , the means of two equally sized intervals were subtracted per experiment .", "Relative changes of locomotion speed were determined by normalizing the change over the level obtained during the interval pre-downshift for each experiment .", "Sample sizes were the number of experiments .", "All data of mutants were compared to the wild type N2 dataset , or between selected strains as indicated , by one-way-ANOVA with Sidak correction .", "For comparing neuropeptide mutants and cell ablated lines to wild type ( Figure 5 ) , all acquired data per strain were used , because the flp-1 mutant displayed relatively increased behavioral variability between different sets of experiments .", "In order to get the best picture of its phenotype all data were taken into account .", "For analyzing neuropeptide rescues strains ( Figure 6 ) , only the subset of mutant and the subset of wild type experiments performed in parallel to the rescue strains were used .", "Only for the rescue of flp-1;nlp-12 mutants ( Figure 6 ) , exclusive datasets of double rescue , mutant and wild type strains were acquired .", "For comparing intervals pre- and post-shift per strain ( Figure 1—figure supplement 1 ) , paired t-tests were performed .", "For O2 chemotaxis assays we used a previously reported O2 gradient device made of polydimethylsiloxane ( PDMS ) ( Gray et al . , 2004 ) .", "We chose different experimental conditions than in previous studies ( Chang and Bargmann , 2008; Chang et al . , 2006; Gray et al . , 2004; Zimmer et al . , 2009 ) in order to optimally match all conditions across behavioral experiments in this study .", "The important parameters were: low population density , 1 hr food deprivation and no hypoxia conditions in the device ( 21-4% instead of 21-0% gradients ) .", "Hence different preferred O2 levels are reported here ( 16–17% in this study versus 7-10% ) .", "Animals were starved for 1 hr on an NGM agar plate without E . coli feeding bacteria before the aerotaxis assays .", "In each assay , 30–40 adult ( 1 day post L4 larval stage ) animals were transferred onto a new NGM agar plate and the PDMS device was placed over them .", "This device comprised an arena of 33 × 15 mm .", "Gas in- and outlets were included within 3 mm wide stretches on either side of the arena ( Figure 9A ) .", "The gas was producing a linear gradient by diffusion .", "21% or 4% ( v/v ) O2 , balanced with nitrogen , was delivered with a flow rate of 0 . 75 ml/min from gas-tight syringes using a syringe pump ( PHD2000 , Harvard Apparatus ) .", "Each gradient experiment was accompanied by a preceding control experiment performed with application of 21% O2 on both sides .", "After performing control experiments for 30 min , gradients were established and animals were recorded for another time course of 30 min .", "The O2 gradient establishment was confirmed in separate measurements with the VisiSens O2 imaging system ( PreSens ) by ratiometric fluorescence imaging of an O2-sensitive foil covered with an agarose film and the gradient device .", "These experiments revealed that a nearly linear gradient is established within 10 min .", "Recordings of freely behaving animals illuminated with flat red LED lights were made at 3 , 10 or 15 fps on a 4 megapixel CCD camera ( Jai ) at a pixel resolution of 0 . 0276 or 0 . 0155 mm/ pixel using Streampix software ( Norpix ) .", "For worm population profiles of gradient and control experiments , one video frame at 30 min of the recording was evaluated .", "Animals within 13 equally spaced 2-mm bins of the assay arena were manually counted and the fraction of animals per bin was calculated for every experiment .", "An index was calculated by subtracting per bin the fraction of the respective pre-run control experiment from the fraction of each gradient experiment .", "Means and SEM were calculated from all experiment replicates .", "Sample sizes were the number of experiments with available paired controls .", "For quantification , cumulative indices were determined per experiment by summing the index from all bins below , respectively , the bin that displayed a mean index of zero for wild type animals ( 13 . 8% O2 at its center ) .", "Cumulative indices of mutant strains were compared to wild type indices by one-way-ANOVA with Dunnett’s correction in separate tests .", "For further analyses , movies were analyzed as described above for worm population behavioral assays .", "Bearing ( B , measured in degree° ) was quantified as the instantaneous orientation of the animals’ smoothed centroid heading relative to the optimal O2 concentration isoline .", "We defined the metric range from 0° ( orientation exactly towards the isoline ) to 180° ( orientation away from the isoline ) .", "Left or right orientation was neglected , as the gradient in the arena is one-dimensional .", "We excluded sections of the trajectories that were within 20 pixels ( =0 . 55 mm ) distance to the arena edges .", "Our weathervaning analysis was restricted to the area of the gradient with concentrations lower than 16% and 2 min after O2 flow onset .", "Curving bias was calculated as the instantaneous change of bearing over distance travelled dB/dX ( rad/mm ) for each frame .", "The analysis was restricted to continuous forward run periods: Omega turns , reversals , forward runs of very short duration ( <3 s ) or forward runs with a total displacement of less than 1 mm were excluded .", "A negative curving bias indicates a change of orientation towards the optimum isoline .", "Curving bias , reversal frequency and speed of forward runs relative to bearing were obtained by calculating histograms using bins of 20° bearing .", "For quantification and statistical comparisons curving biases were averaged across a bearing range of 40°–150° .", "Relative reversal and speed change was calculated as the percent change of reversal frequency or speed , respectively , for animals with a bearing >90° relative to the reversal frequency or speed of all animals with bearing <90° .", "These parameters were calculated independently for each experiment .", "Kruskal-Wallis test with Dunn’s correction was used for comparing mutant strains against wild type N2 and Mann-Whitney test for comparing gradient vs . control for each strain .", "Sample size equals number of experiments ( successfully worm-tracked control and gradient runs counted spearately ) .", "For calculating mean body turning with respect to the animals’ bearing we used the high pixel resolution datasets ( hence the lower n numbers in Figure 9G , 10F ) , skeletonized worms and performed eigenworm analysis as above .", "Peak values of the mid-body turning mode ( segment angle #11 ) only during forward movement and excluding omega turns were detected with a peak-finding algorithm and averaged over bins of 5° for corresponding bearing values .", "Standard MatLab functions were used for the linear fit and calculation of linear correlation coefficients .", "p values are calculated based on a Student's t distribution using the corr function in MatLab .", "Calcium imaging recordings were made using an automatic re-centering system previously described ( Faumont et al . , 2011 ) .", "Adult ( 1 day post L4 larval stage ) worms expressing both mCherry and GCaMP5K ( or GFP ) in the neuron of interest were placed on food-free nematode growth medium ( NGM ) agarose pads and sealed in a custom built airtight chamber with inlet and outlet connectors for gas delivery .", "Animals were starved for 1 hr prior the imaging experiment on a food-free normal NGM plate .", "21% ( v/ v ) O2 , balanced with nitrogen , was applied for 4 min with a gas flow of 50 ml/ min , followed by a switch to 10% O2 for 4 min .", "Gases were mixed with a static mixing element connected to mass flow controllers ( Vögtlin Instruments ) .", "To avoid out of focus movement it was critical to carefully prepare plane agarose pads .", "For this , freshly made NGM agarose mix was melted and poured into a ring 2 . 45 mm thick and 50 mm in diameter and enclosed with glass on both sides to harden .", "Another ring of 38 mm diameter was pressed onto the agarose in order to make an indentation into which 20 mM copper chloride was pipetted to restrict the worm to the center of the pad .", "The pad was then placed inside the chamber , which was sealed shut and covered with a glass slide ( 0 . 55 mm thickness ) 0 . 7 mm from the agarose surface .", "The chamber was then placed onto a motorized stage with associated controller ( MS-2000-PhotoTrack , Applied Scientific Instrumentation ) .", "Images were acquired on an inverted compound microscope ( Zeiss Axio Observer . Z1 ) using two Charge-Coupled Device cameras ( Evolve 512 , Photometrics ) .", "Dual wavelength excitation light ( 470 and 585 nm ) was provided by a CoolLED pE-2 excitation system using an ET-EGFP/mCherry filter set ( 59022x , Chroma ) and dichroic ( 59022bs , Chroma ) .", "A long-distance 63x objective ( Zeiss LD Plan-Neofluar 63x , 0 . 75 NA ) was used to stream unbinned images at 33 ms exposure time ( 30 Hz ) with Visiview software ( Visitron Systems GmbH , Germany ) .", "A dichroic mirror ( 620 spxr , Chroma ) directed high wavelength mCherry emission to a four-quadrant photomultiplier tube ( Hamamatsu ) for recentering .", "The remaining emission was split by a DualCam DC2 cube ( 565 lpxr , Photometrics ) to each of the two CCD cameras , one for mCherry emission ( 641/75 nm , Brightline ) and one for GCaMP emission ( 520/35 nm , Brightline ) .", "mCherry emission was further reduced to 50% by a neutral density filter to prevent signal saturation .", "Simultaneous behavior recordings under infrared LED illumination ( 780 nm ) were made using an IR-sensitive CCD camera ( Manta Prosilica GigE , Applied Vision Technologies ) at 4x magnification with 100 ms exposure time ( 10 Hz ) and StreamPix software ( Norpix ) .", "The pixel resolution was 1 . 6 mm/ px .", "We extracted fluorescence intensity values for GCaMP and mCherry with a custom-made MATLAB tracking script .", "We determined a threshold intensity value for the mCherry channel-derived images capturing the neuron of interest during all recording frames and then measured the sum of pixels from a connected region of at least 50-pixel size above threshold for each frame .", "For the GCaMP channel-derived images we measured the sum of pixels from the region matching the mCherry thresholded area .", "Both datasets were background-corrected by subtracting the average background pixel value ( obtained from the respective first image frame capturing unspecific tissue signal ) multiplied by the number of pixels thresholded in that frame .", "The ratio GCaMP5K/ mCherry was calculated per frame to correct for artificial GCaMP fluorescence changes due to motion artifacts or out-of-focus movements of the cell body .", "Very strong out-of-focus movements were automatically excluded , as there was no connected region >50 pixels above threshold in these frames .", "In that case data were set to NaN .", "We further corrected for strong out-of-focus movements , not fully adjusted through the ratio calculation , by determining strong drops in mCherry fluorescence and occasionally unreasonably large drops in GCaMP fluorescence values .", "Normalized ratio dR/R ( % ) was calculated as the difference of R extracted from each frame to the mean R of the total recording and divided by mean R [ ( R-mean ( R ) ) /mean ( R ) ] .", "Further signal artifacts were determined and excluded by determining extremely high ratio values ( absolute and relative to a finer local mean ) and drops ( relative ) of the normalized ratio , which had been carefully evaluated for each cell .", "All excluded data were set to NaN .", "GFP control data were extracted and processed exactly the same way as GCaMP data .", "Worm skeleton extraction from infrared videos was performed as described ( Yemini et al . , 2013 ) , save for a few minor modifications .", "The source code is available at https://github . com/openworm/SegWorm .", "Videos were down-sampled to 520 × 519 pixels .", "The stereotypy of the images permitted choosing a fixed manual threshold ( an 8-bit grayscale value of 127 ) for every recording to separate worm from background .", "The thresholded worm was dilated by 3 pixels so as to smooth any imperfections , and then eroded by 8 pixels to achieve an accurate representation of the worm .", "The original algorithm was too stringent in rejecting poorly segmented worm shapes .", "Therefore , all thresholds for worm-shape rejection were relaxed by 80% without compromising the effectiveness of this step .", "Head/tail classification was a matter of determining which end of the worm was more central .", "Therefore , within video chunks of contiguous worm segmentations , individual skeletons were oriented relative to each other as previously described , then head and tail were determined by taking the mean distance of both worm ends from the video center .", "Thereafter , all steps to skeletonize the worm are as formerly described , save for a reduction in skeleton size to 26 worm points .", "Precise positions of the motorized tracking stage and thus the tracked neural target were recorded for every frame ( 30 fps ) via the VisiView software and extracted using a MetaMorph ( Universal Imaging ) custom-made script .", "Obtained stage positions were further analyzed with MATLAB-based custom-made processing scripts .", "Locomotion speed was calculated with a step-size of 30 frames ( =1 s ) , i . e . speed of frame i was the distance between positions of frame i-15 and frame i+15 divided by the passed time .", "Artificially high values ( >0 . 6 mm/s ) , when the tracking of the cell was lost , were excluded .", "Angular velocity was calculated with a step-size of 5 frames after extracting heading angles from changes in x/y-coordinates during 30-frame bins of smoothed ( 30 frames ) trajectories .", "Then , periods of backward locomotion were detected based on characteristic changes of angular velocity , the speed derivative and speed , and by considering a maximum length of reversals per strain .", "The splines encoded by the extracted 26 worm skeleton points were smoothed per frame and 24 inter-segment angles were calculated for each frame .", "When no or artificial ( too long or short due to image segmentation issues ) skeletons were retrieved , segment angles were set to NaN .", "Small gaps ( <0 . 5 s ) in segment angle time series were filled by cubic interpolation and angles were smoothed over time ( 15 frames = 1 . 5 s ) .", "In order to match calcium imaging and segment angle data , the segment angle time series were cubically interpolated from their original acquisition rate of 10 Hz to the 30 Hz-frame rate of the calcium imaging recordings .", "Single traces of normalized ratio or amplitude were smoothed by 15 frames ( 0 . 5 s ) prior trial-averaging .", "Then means and SEMs from time courses of normalized ratio dR/R or behavior parameters were calculated from all recordings and averages were binned ( by 30 frames = 1 s intervals ) for display reasons .", "Fraction of animals pausing were calculated and binned into 5 s bins .", "When indicated , only data during certain locomotion phases e . g . forward movement were used to calculate averages by setting ratio or behavior data e . g . during reversals ( and pause phases ) to NaN .", "For statistics , means of normalized ratio or behavior parameters were calculated per worm from respective indicated time intervals .", "The means pre- and post- O2 downshift were compared with paired tests ( paired t-test or Wilcoxon matched-pairs signed rank test ) as indicated to evaluate the changes .", "For comparing these O2-evoked changes between different worm strains ( GCaMP- vs . GFP-expressing worms ) or conditions ( freely moving vs . immobilized ) the means of the two equally sized intervals ( pre and post ) were subtracted per worm and evaluated by one-way-ANOVA with Dunnett’s correction or Mann Whitney test , as indicated .", "Single worm traces for illustration were smoothed by 5 frames and every 10th point was plotted for display reasons .", "Eigenworms of all pooled segment angle time series ( containing forward and backward locomotion ) of wild type GCaMP-expressing N2 worms from calcium-imaging experiments were concatenated ( separately for AVK and DVA imaging lines ) .", "Then eigenworms ( EW ) were derived by standard principal components analysis ( PCA ) on the concatenated angles using MATLAB .", "The individual segment angle time series of all recorded worms were projected onto these wild type-derived ( respective AVK- or DVA-GCaMP ) eigenworms exactly the same way , as done for the population behavioral assays to retrieve undulation and turning modes .", "Further the amplitude of each mode was calculated accordingly by summing the absolutes of all 24 segment angles per time point .", "For further analyses all data time series were smoothed by usually 5 frames , or 15 frames when peak detection was involved .", "For analyzing the relation of the neural activity ratio to selected behavior features , data points per worm were binned by 15 frames ( = 0 . 5 s ) and then pooled from all recordings .", "When indicated only data within respective time intervals or data during specific locomotion phases were taken into account .", "The ratio data were then sorted over bins of equal length of the behavior feature ( speed 0 . 01 mm/s , amplitude 0 . 2 rad ) .", "Medians and inter-quartile ranges of the ratio data within each bin were calculated and plotted over the medians of the behavior bins .", "Very high values of speed ( >0 . 35 mm/s ) and amplitude ( >4 rad ) were not shown due to data sparseness .", "Linear correlation coefficients were calculated per worm with a MATLAB standard function and compared between intervals , locomotion phases or between strains , using paired or unpaired t-test as indicated .", "Boxplots displaye median , interquartile range and 5–95 percentile whiskers .", "Before analyzing the relation of AVK ratio and locomotion speed , ratio data were shifted relative to speed data by a lag time of 1 . 67 s , which had been derived from the peak r value of a performed cross-correlation of the two signals .", "For event-triggered averages , a peak-detection algorithm was used to determine DVA ratio peaks during forward moving phases ( from identified ‘moving phases’ and excluding reversals ) .", "Further reversal start time points derived from the stage position were used .", "The respective traces of amplitude plus surrounding 6–10 s on either side were aligned to these events and means and SEMs per frame of all events were calculated and plotted .", "Wilcoxon matched-pairs signed rank tests compared means calculated per event of short intervals ( length as indicated ) pre- and post- event .", "The means of the pre- and post- reversal start intervals were subtracted per event and the changes were evaluated by Mann-Whitney test comparing different GFP-expressing worms to GCaMP-expressing worms .", "Moving and pausing phases were classified as phases of at least 2 s duration above or below a speed threshold of 0 . 05 mm/s , tolerating gaps of maximum 1 s .", "Means of normalized dR /R during moving or pausing phases , from worms that were pausing at least 60 s of the recording time ( with interruptions ) , were compared via Wilcoxon matched-pairs signed rank tests .", "Microfluidic two-layer PDMS devices were constructed as previously described ( Chronis et al . , 2007; Zimmer et al . , 2009 ) .", "The worm channel was connected to a reservoir containing S-Basal buffer .", "All components were connected with Tygon tubing ( 0 . 02 in ID , 0 . 06 in OD; Norton ) or polyethylene tubing ( 0 . 066 in ID , 0 . 095 in OD; Intramedic ) using 23G Luer-stub adapters ( Intramedic ) .", "21% ( v/v ) O2 , balanced with nitrogen , was applied with a gas flow of 50 ml/ min for 4 min , followed by a switch to 10% O2 for 4 min .", "Gases were mixed with a static mixing element connected to mass flow controllers ( Vögtlin Instruments ) .", "After 1-hr starvation on a food-free NGM plate , single adult ( 1 day post L4 larval stage ) worms expressing both mCherry and GCaMP5K in AVK were loaded into the worm channel: Animals were transferred into a drop of S-Basal on the NGM plate .", "By applying a short vacuum to the worm outlet , we sucked animals up into Tygon tubing , which was afterwards connected again to the worm inlet to position the worm in the channel .", "We used an epifluorescence microscope equipped with a CoolLED pE-2 excitation system to provide dual wavelength excitation light using an ET-EGFP/mCherry filter set ( 59022x , Chroma ) and dichroic ( 59022 bs , Chroma ) , and an Optosplit II ( Cairns ) image splitter ( filter set used: 580 nm beam splitter and 520/35 nm and 641/75 nm bandpass emission filters ) .", "Split imaging data were acquired with an Andor iXon 397 EMCCD camera with 100 ms exposure time , streaming images at 10 Hz acquisition rate to MetaMorph software ( Universal Imaging ) .", "Fluorescence values were measured with a custom-made tracking script written in MetaMorph software .", "The region of bright mCherry signal is detected by thresholding and robustly tracked using the built-in track object function .", "Measurement regions for GCaMP signal as well as nearby regions for measuring background values were manually selected for the first image frame .", "Their positions were updated according to the frame-to-frame displacement of the mCherry-tracking region .", "Normalized ratio dR/R ( % ) was calculated in the same way as for the freely moving calcium-imaging data .", "Worms were maintained at 20˚C on plates of agar nematode growth medium ( NGM ) seeded with OP50 Escherichia coli bacteria as a food source .", "Wild-type was C . elegans Bristol strain N2 .", "Mutant strains used in this study were: ZIM144 , flp-1 ( ok2811 ) IV , 6x outcrossed to N2 ZIM550 , nlp-12 ( ok335 ) I , 6x outcrossed to N2 ZIM551 , flp-1 ( ok2811 ) IV;nlp-12 ( ok335 ) I , derived from crossing ZIM144 with ZIM550 .", "We verified the flp-1 ( ok2811 ) allele by cDNA sequencing: it deletes the last two bases of exon 1 and the entire exon 2 of the flp-1 coding region resulting in an artificial exon 1 ( made up of remaining unspliced parts of intron 2 ) with a predicted premature stop codon; further this causes a frame shift starting from exon 3 including the whole sequence section encoding the actual neuropeptides .", "Therefore flp-1 ( ok2811 ) is a prospective null allele .", "As opposed to the previously described alleles flp-1 ( yn2 ) and flp-1 ( yn4 ) , flp-1 ( ok2811 ) does not affect the nearby daf-10 coding sequence .", "Mutant worm strains were received from Caenorhabditis Genetics Center ( CGC ) , Liliane Schoofs Laboratory and Chris Li Laboratory .", "Transgenic animals were generated by injecting plasmid mixes into gonads of young adult hermaphrodites and generating heritable extra-chromosomal arrays .", "All injection mixes were prepared to yield 100ng/ul by adding empty pSM plasmids when necessary .", "When indicated , Punc-122::gfp , Punc-122::dsRed ( expressed in so-called coelomocytes ) or Pmyo-3::mCherry ( expressed in body wall muscles ) were used as co-injection markers .", "StrainGenotypeDescriptionZIM466lite-1 ( xu-7 ) X; mzmEx300 [Pflp-1 ( AVK ) ::GCaMP5K; Pflp-1 ( AVK ) ::mCherry]AVK imaging lineZIM626lite-1 ( xu-7 ) X; mzmEx407 [Pflp-1 ( AVK ) ::GFP; Pflp-1 ( AVK ) ::mCherry]AVK gfp control imaging lineZIM563lite-1 ( xu-7 ) X; mzmEx365 [Pnlp12::GCaMP5K; Pnlp-12::wCherry]DVA imaging lineTRL144lite-1 ( xu-7 ) X; [Pnlp12::GFP; Pnlp-12::wCherry]DVA gfp control imaging lineZIM319flp-1 ( ok2811 ) IV; mzmEx142 [Pflp-1 ( AVK ) ::flp-1::SL2::gfp; Pmyo-3::mCherry]flp-1 rescue in AVKZIM837nlp-12 ( ok335 ) I; mzmEx505 [Pnlp12::nlp12::SL2::gfp; Pflp-17::gfp]nlp-12 rescue under endogenous promoterZIM1255flp-1 ( ok2811 ) IV;nlp-12 ( ok335 ) I; mzmEx505 [Pnlp12::nlp12::SL2::gfp; Pflp-17::gfp]; mzmEx142 [Pflp-1 ( AVK ) ::flp-1::SL2::gfp; Pmyo-3::mCherry]flp-1 rescue in AVK & nlp-12 rescue under endogenous promoterZIM367mzmEx249 [Pflp-1 ( AVK ) ::egl-1::SL2::gfp; Pflp-1::mCherry; Punc-122::dsRed]AVK-ZIM625mzmEx406 [Pnlp-12::p12::SL2::mCherry; Pnlp-12::p17::SL2::mCherry; Pnlp-12::mCherry; Punc-122::gfp]DVA-ZIM627mzmEx249 [Pflp-1 ( AVK ) ::egl1::SL2::gfp; Pflp-1::mCherry; Punc-122::dsRed]; mzmEx406 [Pnlp-12::p12::SL2::mCherry; Pnlp-12::p17::SL2::mCherry; Pnlp-12::mCherry; Punc-122::gfp]AVK-; DVA-CX11697kyIs536 [Pflp-17::p17::SL2::gfp elt-2::gfp]; kyIs538 [Pglb-5::p12::SL2::gfp; elt-2::mCherry]BAG- ( also ASG- , Roger Pocock , pers . comm . ) Pro-apoptotic genes used for cellular ablation were egl-1 ( Conradt and Horvitz , 1998 ) , or p12 and p17 , which encode domains from split caspase 3 ( ced-3 ) ( Chelur and Chalfie , 2007 ) .", "GCaMP5k in mzmEx365 is codon-optimized for C . elegans ( GenScript ) .", "wCherry is codon-optimized mCherry and was kindly donated by Mei Zhen laboratory .", "PCR-amplified DNA fragments of interest flanked by restriction sites were cloned into pSM vectors .", "Promoters were inserted via FseI and AscI sites while coding regions were usually inserted via NheI and Acc651 sites .", "Pflp-1 ( AVK ) : a 505bp fragment of flp-1 promoter which extends from position −513 to −9 relative to the flp-1 start codon , expressed in AVK only ( Altun-Gultekin et al . , 2001; Nelson et al . , 1998 ) .", "Pnlp-12: 383bp fragment , directly upstream of the ATG start codon of the nlp-12 gene , reported to be solely expressed in DVA ( Hu et al . , 2011 ) and kindly provided by the Josh Kaplan laboratory .", "flp-1 genomic region including the whole coding regions: 1414bp fragment beginning at start codon and including 127bp of 3’UTR .", "nlp-12 genomic region including the whole coding regions: 437bp fragment from start to stop codon ." ] ]

[ "In animal locomotion a tradeoff exists between stereotypy and flexibility: fast long-distance travelling ( LDT ) requires coherent regular motions , while local sampling and area-restricted search ( ARS ) rely on flexible movements .", "We report here on a posture control system in C . elegans that coordinates these needs .", "Using quantitative posture analysis we explain worm locomotion as a composite of two modes: regular undulations versus flexible turning .", "Graded reciprocal regulation of both modes allows animals to flexibly adapt their locomotion strategy under sensory stimulation along a spectrum ranging from LDT to ARS .", "Using genetics and functional imaging of neural activity we characterize the counteracting interneurons AVK and DVA that utilize FLP-1 and NLP-12 neuropeptides to control both motor modes .", "Gradual regulation of behaviors via this system is required for spatial navigation during chemotaxis .", "This work shows how a nervous system controls simple elementary features of posture to generate complex movements for goal-directed locomotion strategies ." ]

[ "Animals navigate through their environment using different strategies according to their current needs .", "For example , when the goal is to travel long distances , they move quickly and in an efficient way by employing regular , repetitive movements .", "However , when the aim is to explore the nearby area – to search for food , for example – animals move slowly and make more flexible movements .", "These different types of movement mostly use the same groups of muscles , and so animals must be able to alter how they control their muscles to yield these different strategies .", "These movement strategies have been observed in many animal species , from worms to grazing cows , and researchers have mostly classified them into distinct behavioral states that the animals switch between .", "To date , the patterns of movements that underlie these strategies have not been described in detail .", "The wavelike movement of the roundworm Caenorhabditis elegans has the advantage of being relatively easy to measure .", "By analyzing precise recordings of how the worms change posture as they move , Hums et al . now show that two main patterns of motion underlie worm movement .", "Regular whole-body waves ( undulations ) efficiently drive long-distance travel , while more complex turning motions allow the animals to flexibly change direction and so explore the local environment .", "Furthermore , the worms can fine-tune their movement strategy by gradually transitioning between the two patterns .", "This finding is opposed to the standard view , where animals switch between distinct behavioral states .", "Hums et al . then studied how neuronal regulation in the C . elegans nervous system enables the worms to transition between the different movement strategies .", "In these experiments , neurons were manipulated and their activity was recorded .", "The results suggest that two classes of so called interneurons enable the worms to fine-tune their movements .", "Each class of these interneurons produces a signaling molecule ( or neuropeptide ) that counteracts the activity of the other signal; together both neuropeptides regulate the patterns of movements .", "Further work is now needed to identify and investigate the downstream neurons that work together to represent the different patterns of movements in the roundworm .", "Future studies could also analyze whether other animals – such as swimming animals and limbed animals – use similar principles to change between distinct forms of movement and thus enact a range of behavioral strategies ." ]

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

[ "biochemistry and chemical biology" ]

[ [ "Macro-autophagy ( hereafter autophagy ) is a conserved pathway for the delivery of cytoplasmic material into the lysosomal system for degradation ( Kraft and Martens , 2012 ) .", "Upon induction of autophagy , double membrane organelles termed autophagosomes are formed in a de novo manner .", "Initially , autophagosome precursors appear as small membrane structures referred to as isolation membranes ( or phagophores ) .", "As isolation membranes expand , they gradually enclose cytoplasmic cargo material .", "Upon closure of the isolation membranes , autophagosomes are formed within which the cargo is isolated from the rest of the cytoplasm .", "Autophagosomes subsequently fuse with the lysosomal compartment where the inner membrane and the cargo are eventually degraded .", "When induced by starvation autophagy can be relatively non-selective with regard to the cargo that is sequestered within autophagosomes .", "However , it has become clear that autophagy can be highly selective and even exclusive when induced by the presence of intracellular cargo material ( Zaffagnini and Martens , 2016 ) .", "Substances including aggregated proteins , cytosolic pathogens and damaged or surplus organelles have all been shown to be selectively degraded by autophagy ( Khaminets et al . , 2016 ) .", "Autophagy thereby protects the organism from pathological conditions such as neurodegeneration , cancer and infection ( Levine and Kroemer , 2008; Mizushima and Komatsu , 2011 ) .", "In S . cerevisiae the cytoplasm-to-vacuole-targeting ( Cvt ) pathway mediates the delivery of the oligomeric prApe1 enzyme as well as Ams1 and Ape4 into the vacuole via small autophagosomes that are referred to as Cvt vesicles ( Nakatogawa et al . , 2009 ) .", "Selectivity of autophagic processes is mediated by cargo receptors that link the cargo to isolation membranes due to their ability to simultaneously bind the cargo and Atg8-family proteins on the isolation membrane ( Johansen and Lamark , 2011; Rogov et al . , 2014; Stolz et al . , 2014 ) .", "The interaction of the cargo receptors with Atg8-family proteins is mediated by LC3-interacting regions ( LIRs ) ( Pankiv et al . , 2007;Ichimura et al . , 2008 ) also known as Atg8 interacting motifs ( AIMs ) in the cargo receptors ( Noda et al . , 2010 ) .", "Atg8-family proteins are ubiquitin-like proteins that are conjugated to the headgroup of the membrane lipid phosphatidylethanolamine ( PE ) rendering the otherwise soluble proteins membrane-bound ( Ichimura et al . , 2000 ) .", "This conjugation reaction is also referred to as lipidation .", "The Atg8 conjugation cascade is analogous to the chain of reactions that mediate the conjugation of ubiquitin to its substrates .", "Thus , Atg8 is activated by the E1-like enzyme Atg7 under consumption of ATP and subsequently transferred to the E2-like enzyme Atg3 from which Atg8 is ultimately transferred to the headgroup of PE ( Ichimura et al . , 2000; Klionsky and Schulman , 2014 ) .", "This last step is strongly facilitated by a complex composed of the Atg12~Atg5 protein conjugate and Atg16 .", "The Atg12~Atg5-Atg16 complex acts in an E3-like manner and determines the site of Atg8 conjugation ( Fujita et al . , 2008b; Hanada et al . , 2007 ) .", "The Atg8 conjugation machinery acts in concert with other proteins of the autophagic machinery including the Atg1/ULK1 complex , the class III PI3K complex 1 , Atg9 and the WIPIs to mediate the efficient generation of autophagosomes or Cvt vesicles ( Dooley et al . , 2014; Fujita et al . , 2008a; Juris et al . , 2015; Kishi-Itakura et al . , 2014; Komatsu et al . , 2005; Kraft et al . , 2012; Mizushima et al . , 1998 , 2001; Sou et al . , 2008 ) .", "The precise mechanisms by which the Atg12~Atg5-Atg16 complex and Atg8 aid the formation , elongation or closure of the autophagosomal membranes are unclear .", "Recent work has provided important information about how the presence of an autophagic cargo induces the formation of an isolation membrane .", "In particular , it was shown that the Atg19 cargo receptor recruits the Atg11 scaffold protein to the prApe1 cargo for Atg1 kinase activation ( Kamber et al . , 2015; Torggler et al . , 2016 ) .", "In addition , it was demonstrated that the cargo receptors Optineurin and NDP52 recruit the ULK1 complex to damaged mitochondria ( Lazarou et al . , 2015 ) .", "Furthermore , TRIM proteins were shown to localize the ULK1 , PI3K complexes and ATG16L1 to their cargo in a process referred to as precision autophagy ( Chauhan et al . , 2016; Kimura et al . , 2015 ) .", "A major question is how the presence of an autophagic cargo is coupled to Atg8 conjugation and thus isolation membrane formation in space and time .", "Here we show that the S . cerevisiae Atg19 and Atg34 as well as the human p62 , Optineurin and NDP52 cargo receptors interact with the E3-like Atg12~Atg5-Atg16 complex .", "Employing Atg19 as a model in a fully reconstituted system we show that it is capable of recruiting Atg12~Atg5-Atg16 to the prApe1 cargo .", "This recruitment is mediated by a direct interaction of the AIM motifs in Atg19 with the Atg5 subunit .", "In our in vitro system the recruitment of the Atg12~Atg5-Atg16 complex is sufficient to drive accumulation of lipidated Atg8 at the cargo .", "Since the interaction of the Atg19 cargo receptor with the E3-like Atg12~Atg5-Atg16 complex is outcompeted by Atg8 , the system may have an inherent directionality whereby the final product in form of Atg8~PE could displace the upstream conjugation machinery at the concave side of the isolation membrane ." ], [ "During classical ubiquitination reactions the localization of the E3 ligase determines where ubiquitin is conjugated to its substrates ( Deshaies and Joazeiro , 2009; Komander and Rape , 2012 ) .", "We therefore asked if autophagic cargo receptors could interact with the Atg12~Atg5-Atg16 E3-like complex and thereby recruit it to the cargo .", "Indeed , in pull down experiments GST-Atg19 used as a bait successfully pulled down Atg12~Atg5-Atg16 , demonstrating a direct interaction between these two components ( Figure 1A ) .", "In a complementary approach we imaged the recruitment of Atg12~Atg5-Atg16-mCherry to beads coated with GST-Atg19 under equilibrium condition ( Figure 1B ) .", "Atg12~Atg5-Atg16-mCherry was robustly and specifically recruited to these beads ( Figure 1B ) .", "The α-mannosidase ( Ams1 ) receptor Atg34 was also able to bind the Atg12~Atg5-Atg16 complex ( Figure 1C ) , suggesting that this interaction is a more general property of cargo receptors .", "In order to test if this interaction occurs in cells we performed immunoprecipitation experiments using Atg5-TAP to pull down 6xmyc-Atg19 ( Figure 1D ) .", "Atg19 was specifically pulled down by Atg5-TAP .", "Employing the M-Track assay , which is based on the methylation of the human histone 3 N-terminus by the human SUV39H1 methyltransferase when the two components come into close contact ( Brezovich et al . , 2015; Zuzuarregui et al . , 2012 ) , we confirmed that Atg19 and the Atg12~Atg5-Atg16 complex are in close proximity in living cells ( Figure 1E ) .", "It was previously shown that overexpression of a methyltransferase does not result in unspecific methylation of the histone 3 N-terminus ( Brezovich et al . , 2015 ) .", "Next , we tested if the interaction of cargo receptors with the E3-like complex is conserved .", "To this end , we co-expressed human GFP-ATG5 and mCherry-p62 in HeLa cells in which the endogenous p62 had been knocked-down by RNAi .", "mCherry-p62 was efficiently co-precipitated by GFP-ATG5 ( Figure 1F ) .", "We confirmed this result by using a microscopy-based assay in which we imaged the recruitment of mCherry-p62 to GFP-ATG5 coated beads in HeLa cell lysates ( Figure 1G ) .", "We extended this analysis by investigating other human cargo receptors and found that NDP52 was also pulled down by GFP-ATG5 ( Figure 1H ) .", "In addition , we detected a weak but consistent co-precipitation of Optineurin ( OPTN ) ( Figure 1H ) .", "In summary , the S . cerevisiae Atg19 and Atg34 cargo receptors directly interact with the Atg12~Atg5-Atg16 E3-like enzyme and an interaction with this complex is also detectable for the human cargo receptors p62 , OPTN and NDP52 . 10 . 7554/eLife . 18544 . 003Figure 1 . Cargo receptors interact with the Atg12~Atg5-Atg16 complex in vitro and in vivo .", "( A ) Western blots of GST-pull down experiments using GST-Atg19 as bait and the Atg12~Atg5-Atg16 complex as prey .", "Degradation bands of GST-Atg19 are marked with an asterisk ( * ) .", "( B ) Glutathione Sepharose beads were coated with GST-Atg19 or GST and imaged in the presence of the Atg12~Atg5-Atg16-mCherry complex at equilibrium .", "The quantification shows the relative mCherry signal intensity measured at the bead in percent .", "Three independent experiments were considered for quantification .", "Scale bar: 100 µm .", "( C ) Same assay as shown in ( A ) but using GST-Atg34 as a bait .", "( D ) Western blots of a co-immunoprecipitation experiment using atg19Δ , atg8∆ S . cerevisiae cells with integrated Atg5-TAP and transformed with 6xmyc-Atg19 .", "Atg5-TAP was precipitated using magnetic Epoxy IgG-beads .", "( E ) M-Track assay using Protein A-Histone 3 ( H3 ) -tagged Atg16 and Atg19 fused to 9xmyc and the SUV39H1 methyl-transferase ( HKMT ) .", "Shown is a Western blot with an anti-trimethylation-specific antibody to assess the methylation signal and anti-ProteinA to assess the amount of cleaved protein A-H3 on beads .", "The Atg13 interaction with Atg17 was used as a positive control for the assay .", "( F ) Co-immunoprecipitation experiment using GFP-TRAP beads incubated with lysates from HeLa cells transfected with the indicated expression constructs .", "Endogenous p62 was down regulated by RNAi .", "( G ) Lysates from HeLa cells transfected with GFP and mCherry-p62 or GFP-ATG5 and mCherry-p62 were incubated with GFP-TRAP beads and the recruitment of the proteins to the beads was imaged by spinning disc microscopy .", "The graph shows the average and standard deviation over all beads from one experiment .", "The endogenous p62 was downregulated by RNAi .", "( H ) Western blot analysis of lysates from HeLa cells co-transfected with the indicated constructs and subjected to anti-GFP immunoprecipitation ( GFP-TRAP , Chromotek ) .", "Numbers below each blot indicate the relative band intensity for the particular blots shown .", "The beads/input enrichment factors ( EF ) indicate the fold of enrichment of each mCherry-tagged cargo receptor in the GFP-ATG5 beads fraction over its correspondent GFP control , normalized on the input levels and equalized to the GAPDH blots .", "Representative blots of at least four independent experiments are shown ( left ) .", "The plot shows the average sample/control fold enrichment in the indicated fractions for each cargo receptor .", "The beads/input enrichment factor is defined as above .", "Averages and standard deviations of at least four independent experiments are shown ( right ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 18544 . 00310 . 7554/eLife . 18544 . 004Figure 1—figure supplement 1 . Human ATG5 pulls down p62 from cell lysates . Anti-GFP blot of the GFP-TRAP experiment shown in Figure 1F .", "Shown are input and bead fractions . DOI: http://dx . doi . org/10 . 7554/eLife . 18544 . 004 Further focusing on Atg19 , we tested which of the Atg12~Atg5-Atg16 complex subunits interacts with the Atg19 cargo receptor by using GST-Atg19 as bait to pull down Atg5 stabilized with the N-terminal helix of Atg16 ( Atg5-Atg16 ( 1–46 ) ) , the Atg12~Atg5 conjugate , the Atg5-Atg16 complex and the full Atg12~Atg5-Atg16 complex ( Figure 2A and Figure 2—figure supplement 2A ) .", "Atg12 could not be tested in isolation since we were unable to purify the protein .", "All proteins tested showed interaction with Atg19 suggesting that Atg5 is sufficient for the binding to Atg19 ( Figure 2A , B ) .", "We confirmed this result in size exclusion chromatography experiments using Atg5-Atg16 ( 1–46 ) and Atg19 .", "Indeed , a fraction of Atg5-Atg16 ( 1–46 ) shifted to higher molecular weight fractions in the presence of Atg19 ( Figure 2—figure supplement 1 ) . 10 . 7554/eLife . 18544 . 005Figure 2 . Atg19 directly binds Atg5 via its C-terminal domain and requires its coiled-coil domain to interact with the Atg12~Atg5-Atg16 complex .", "( A ) GST-pull down experiment using GST-Atg19 or GST as bait in the presence of recombinant Atg5-Atg16 ( 1–46 ) , Atg12~Atg5 , Atg5-Atg16 or Atg12~Atg5-Atg16 complexes as preys .", "Input and bead fractions were loaded on a SDS-PAGE gel and subjected to Western blotting .", "Proteins were detected using an anti-Atg5 antibody .", "See also Figure 2—figure supplements 1 and 2 .", "( B ) Quantification of GST-pull down experiments , one of which is shown in ( A ) .", "The amount of pulled down protein for the Atg12~Atg5-Atg16 complex was set to 100% .", "Average values were calculated from three independent experiments and plotted in the histogram together with the standard deviations .", "( C ) GST-pull down experiment using GST-Atg19 or GST as bait and recombinant Atg16-meGFP or the Atg12~Atg5-Atg16-meGFP complex as prey .", "Input and bead samples were loaded on a SDS-PAGE gel and subjected to Western blotting .", "Proteins were detected using an anti-GFP antibody .", "See also Figure 2—figure supplement 2 .", "( D ) Schematic representation of the Atg19 domain organization .", "N-terminal domain ( NTD , residues 1–124 ) , coiled-coil domain ( CC , residues 124–254 ) , Ams1 binding domain ( ABD , residue 254–365 ) and C-terminal domain ( CTD , residues 365–415 ) .", "( E ) The Atg12~Atg5-Atg16 complex and its subunits , Atg5-Atg16 ( 1–46 ) , Atg5-Atg16 and the Atg12~Atg5 conjugate were incubated with either full length GST-Atg19 or truncated versions thereof as baits and the pulled down protein was detected by Western blotting using an anti-Atg5 antibody .", "The bead fractions showing GST-labeled Atg19 and truncated versions thereof are depicted in Figure 2—figure supplement 2 .", "( F ) Glutathione beads coated with full length GST-Atg19 or truncations thereof were incubated with the Atg12~Atg5-Atg16-mCherry complex and its recruitment to the beads was determined by spinning disc microscopy .", "A quantification of three independent experiments is shown to the left .", "The signal measured for binding to GST-Atg19 full length was set to 100% .", "( G ) Glutathione beads were coated with Atg19 full length ( GST-Atg19 ) , Atg19 C-terminal domain ( GST-Atg19 ( 365–415 ) ) or Atg19 lacking the C-terminal canonical AIM motif ( GST-Atg19 ( 1–407 ) ) and incubated with Atg12~Atg5-Atg16-mCherry .", "A quantification of three independent experiments is shown to the left .", "The signal measured for binding to GST-Atg19 full length was set to 100% .", "DOI: http://dx . doi . org/10 . 7554/eLife . 18544 . 00510 . 7554/eLife . 18544 . 006Figure 2—figure supplement 1 . Atg19 and Atg5 interact in solution . Atg19 and Atg5-Atg16 ( 1–46 ) were co-incubated at concentrations of 580 µM and run on a Superose 6 size exclusion column .", "Aliquots of individual fractions were run on SDS-PAGE gels and Coomassie stained ( top panel ) .", "Size exclusion chromatography fractions of individual Atg5-Atg16 ( 1–46 ) ( middle panel ) and Atg19 ( bottom panel ) proteins run at 55 µM concentration . DOI: http://dx . doi . org/10 . 7554/eLife . 18544 . 00610 . 7554/eLife . 18544 . 007Figure 2—figure supplement 2 . Mapping Atg19 interaction with Atg12~Atg5-Atg16 complex .", "( A ) Ponceau staining of input samples of pull down experiment shown in Figure 2A .", "( B ) Ponceau staining of input and bead samples of pull down experiment shown in Figure 2C .", "( C ) Coomassie stained gels of GST-labeled Atg19 and truncated versions thereof used for the pull down experiments shown in Figure 2E .", "Each panel represents final bead fractions after incubation with the indicated prey proteins . DOI: http://dx . doi . org/10 . 7554/eLife . 18544 . 007 The presence of Atg16 had a stimulatory effect on the interaction of Atg5 with Atg19 suggesting that Atg16 could directly interact with Atg19 ( Figure 2A , B and Figure 2—figure supplement 2B ) .", "However , when tested in isolation Atg16 did not show any detectable binding to Atg19 ( Figure 2C ) .", "Full length Atg16 may therefore enhance the interaction by an allosteric effect on Atg5 or by increasing the avidity of the interaction due to its ability to self-associate ( Fujioka et al . , 2010; Kaufmann et al . , 2014 ) .", "In order to identify the regions in Atg19 that are required for the interaction with Atg12~Atg5-Atg16 we tested a series of Atg19 truncation mutants ( Figure 2D ) for their interaction with the entire complex and components thereof ( Figure 2E ) .", "Atg5 and Atg5-Atg16 showed robust interaction with all Atg19 truncations including the C-terminal domain encompassing amino acids 365–415 ( Figure 2E and Figure 2—figure supplement 2C ) .", "The presence of Atg12 , either in context of the Atg12~Atg5 conjugate or the Atg12~Atg5-Atg16 complex , changed the properties of the interaction and required the presence of amino acids 124–254 , which include the cargo binding domain of Atg19 ( Yamasaki et al . , 2016 ) .", "We corroborated the results of the pull down experiments for the full Atg12~Atg5-Atg16 complex in a microscopy-based assay under equilibrium conditions ( Figure 2F ) .", "This assay confirmed that the coiled-coil domain of Atg19 is required for the interaction when Atg5 is conjugated to Atg12 ( Figure 2F ) .", "To interrogate the role of the C-terminal region of Atg19 we performed further microscopy-based interaction experiments ( Figure 2G ) .", "Consistent with the pull down experiments the Atg12~Atg5-Atg16 complex bound strongly to full length Atg19 but not to the isolated C-terminus ( amino acids 365–415 ) ( compare Figure 2E and G ) .", "Intriguingly , deletion of the last eight amino acids containing the canonical AIM motif ( Noda et al . , 2008; Sawa-Makarska et al . , 2014 ) from the C-terminus of Atg19 strongly reduced the interaction ( Figure 3A ) suggesting that this AIM motif contributes to the interaction of Atg19 with the Atg12~Atg5-Atg16 complex .", "Next , we further dissected the contribution of the AIM motifs in the Atg19 C-terminus to the interaction with Atg5 .", "While deletion of the canonical AIM motif in the extreme C-terminus resulted in strong but incomplete reduction in Atg5 binding , further mutation of two additional AIM-like sequences ( Abert et al . , 2016; Sawa-Makarska et al . , 2014 ) completely abolished the interaction ( Figure 3B and Figure 3C Figure 3—figure supplement 1 ) .", "The AIM-dependent association of Atg19 with Atg5 is relevant for the interaction of the two proteins in vivo as the W412A mutation in the canonical AIM motif of Atg19 results in markedly reduced interaction of the two proteins in co-immunoprecipitation experiments ( Figure 3D ) . 10 . 7554/eLife . 18544 . 008Figure 3 . The AIM motifs in the C-terminal domain of Atg19 are required for its interaction with Atg5 and are competitively bound by Atg8 . ( A ) Amino acid sequence of the C-terminal domain ( 365-415 ) of Atg19 containing the canonical AIM motif ( 412WEEL415 ) and two AIM-like motifs ( 376FYSF379 , 384LPEL387 ) .", "( B ) Glutathione beads coated with the indicated proteins were imaged in the presence of Atg5-mCherry-Atg16 ( 1–46 ) complex under equilibrium conditions .", "The mCherry signal is shown in false color ( ImageJ: Fire ) .", "See also Figure 3—figure supplement 1 .", "( C ) Quantification of three independent experiments of the relative mCherry signal intensity measured at the bead .", "Due to optical reasons very low signals at the beads resulted in values lower than the background and thus negative values .", "Error bars represent standard deviations .", "( D ) Co-immunoprecipitation experiment of 6xmyc-Atg19 or 6xmyc-Atg19W412A with Atg5-TAP as bait in S . cerevisiae cell lysates .", "Western blots of bead and lysate fractions are shown .", "Atg12~Atg5-TAP was detected with an anti-TAP and 6xmyc-Atg19 with an anti-Myc antibody .", "A quantification of three independent experiments is shown to the right .", "Shown are averages and standard deviations .", "( E ) Glutathione beads coated with the GST-Atg19 C-terminus ( 365-415 ) or GST were imaged in the presence of Atg5-mCherry-Atg16 ( 1–46 ) complex under equilibrium conditions .", "For the competition experiment recombinant GFP-Atg8 ( or buffer ) was added to the sample at a final concentration corresponding to 1x initial concentration of Atg5-mCherry-Atg16 ( 1–46 ) .", "Purified Atg8 ( or buffer ) was added to a final concentration of 22x the initial concentration of Atg5-mCherry-Atg16 ( 1–46 ) ) .", "Representative microscopy pictures are shown .", "( F ) Quantification of three independent experiments , one of which is shown in ( E ) .", "The mCherry intensity in the ‘+ buffer’ sample were GST-Atg19 ( 365–415 ) was used as bait was set to 100% .", "Due to optical reasons very low signals at the beads resulted in values lower than the background and thus negative values .", "Bars represent standard deviations .", "( G ) Glutathione Sepharose beads were coated with GST-prApe1 ( 1–41 ) and Atg19 or GST and imaged in the presence of the Atg12~Atg5-Atg16-mCherry complex .", "Subsequently , recombinant GFP-Atg8 was added to the sample at a final concentration corresponding to 1x or 10x the initial concentration of the Atg12~Atg5-Atg16-mCherry complex .", "The binding of the complex to the beads in the absence of Atg8 was set to 100% .", "The histogram shows the averaged values of three independent experiments and the error bars represent standard deviations .", "N = 3 for the prApe1 samples .", "See also Figure 3—figure supplement 2 . DOI: http://dx . doi . org/10 . 7554/eLife . 18544 . 00810 . 7554/eLife . 18544 . 009Figure 3—figure supplement 1 . AIM-dependent interaction of the Atg19 C-terminal domain with Atg5 . Western blot of a GST-pull down experiment employing GST fusions of the wild type Atg19 CTD ( 365-415 ) or the indicated mutants as baits and Atg5-Atg16 ( 1–46 ) as prey .", "Bead samples were subjected to Western blotting using an anti-Atg5 antibody for detection ( bead samples , upper panel ) or Coomassie staining ( input samples , lower panel ) , respectively . DOI: http://dx . doi . org/10 . 7554/eLife . 18544 . 00910 . 7554/eLife . 18544 . 010Figure 3—figure supplement 2 . Recruitment of Atg19 and Atg12~Atg5-Atg16 to cargo mimetic beads . Representative pictures and experimental scheme of mCherry-Atg19 recruitment to cargo mimetic beads coated with GST-prApe1 ( 1–45 ) in ( A ) and Atg12~Atg5-Atg16-eGFP to beads in the presence or absence of Atg19 in ( B ) .", "A quantification of three independent experiments is shown in ( B ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 18544 . 010 The dependence of the Atg19 - Atg12~Atg5-Atg16 interaction on the AIM motifs suggested that it is mutually exclusive with the interaction of Atg19 with Atg8 .", "To test this possibility , we immobilized the C-terminus of Atg19 on beads and added Atg5-mCherry-Atg16 ( 1–46 ) .", "Subsequently , we added GFP-Atg8 or Atg8 to the beads and determined the signal of Atg5-mCherry-Atg16 ( 1–46 ) on the beads ( Figure 3E ) .", "Indeed , Atg8 outcompeted the Atg19-bound Atg5-mCherry-Atg16 ( 1–46 ) in a dose dependent manner .", "The loss of the Atg5-mCherry signal correlated with an increased GFP-Atg8 signal at the bead ( Figure 3E , F ) .", "Thus , the interaction of Atg5 with the C-terminus of Atg19 is AIM-dependent and mutually exclusive with Atg8 binding .", "Next , we asked if the interaction of Atg19 with Atg5 is also mutually exclusive with the Atg19 - Atg8 interaction in the context of the entire Atg12~Atg5-Atg16 complex and when Atg19 is bound to the prApe1 cargo .", "First , we generated cargo mimetic beads by attaching the prApe1 propeptide to them via a GST-tag .", "Consistent with previous results Atg19 was robustly recruited to these beads ( Figure 3—figure supplement 2A ) ( Pfaffenwimmer et al . , 2014; Sawa-Makarska et al . , 2014 ) .", "Next we tested if Atg19 recruited the Atg12~Atg5-Atg16 complex to the artificial cargo .", "Indeed , the Atg12~Atg5-Atg16 complex showed a strong Atg19 dependent signal at the beads ( Figure 3—figure supplement 2B ) .", "Employing this experimental setup , we then went on by adding GFP-Atg8 to the cargo mimetic beads ( Figure 3G ) .", "The complex was displaced in a concentration dependent manner confirming that Atg12~Atg5-Atg16 and Atg8 compete for the same binding sites on Atg19 .", "The Atg5 subunit of the Atg12~Atg5-Atg16 complex and the AIM-like motifs in Atg19 are both essential for the binding of these two components .", "Moreover , the interaction of Atg19 with Atg5 and Atg8 is mutually exclusive , strongly suggesting that the AIM motifs in Atg19 directly interact with Atg5 .", "To identify potential binding sites in Atg5 for the AIM motif we employed computational modeling and molecular dynamics simulations .", "To this end , we used the structure of Atg5-Atg16 ( 1–46 ) from S . cerevisiae ( PDB:2DYO ) ( Matsushita et al . , 2007 ) and performed molecular dynamics simulations which allowed us to capture the flexibility of the Atg5-Atg16 ( 1–46 ) complex .", "Subsequently , in silico docking was performed for 10 randomly selected snapshots from the molecular dynamics trajectory using a peptide encompassing the canonical AIM motif ( 411TWEEL415 ) of Atg19 .", "Modelling analysis suggested three possible binding sites for the peptide , two of which mapped to Atg5 ( Figure 4A , B ) and one to a site formed by Atg16 .", "Since our pull down experiments ( Figure 2C ) did not detect any direct interaction of Atg16 with Atg19 the latter site was excluded from further analysis .", "The other two sites were analyzed .", "Both contained residues that were persistently involved in forming salt bridges and/or hydrogen bonds with the TWEEL peptide .", "These were K57 and K137 in the first binding site ( Figure 4A , pose 1 ) and N84 and R208 in the second binding site ( Figure 4A , pose 2 ) .", "These two sites show a similar architecture composed of a hydrophobic pocket surrounded by positively charged residues .", "Specifically , molecular dynamics simulations showed that the hydrophobic pocket serves to dock W412 and L415 of the TWEEL peptide , while lysine , arginine and asparagine residues on the surface of Atg5 contact the peptide via salt bridges and hydrogen bonds with the glutamic acid residues E413 and E414 .", "Structural superposition showed that the two sites are unrelated to the AIM-binding sites of Atg8 ( Figure 4—figure supplement 1 ) . 10 . 7554/eLife . 18544 . 011Figure 4 . Structure of the Atg5-Atg16 ( 1–46 ) complex with the predicted binding sites for the TWEEL peptide .", "( A ) Structure of Atg5 in complex with the N-terminus of Atg16 ( PDB:2DYO , [Matsushita et al . , 2007] ) .", "The backbone of Atg5 is shown in green and Atg16 is shown in orange .", "The position of the predicted TWEEL pentapeptide binding sites on Atg5 are shown in blue ( pose 1 and 2 ) .", "The residues predicted to contact the glutamates of the TWEEL peptide are shown as sticks .", "( B ) Surface representation of the Atg12~Atg5-Atg16 ( 1–46 ) complex ( PDB:3W1S [Noda et al . , 2013] ) .", "The amino acids in the potential TWEEL binding sites predicted to contact the glutamates of the peptide are shown in blue .", "Atg5 is shown in grey , Atg12 is shown in violet and Atg16 is shown in orange .", "( C ) Representative blots of GST-pull down experiments conducted with GST-Atg19 or GST in the presence of Atg5-Atg16 ( 1–46 ) wild type and its single mutant versions ( K57E , N84E , K137E and R208E ) are shown .", "Input and bead samples were subjected to Western blot analysis .", "GST-fusion bait proteins were detected with Ponceau Staining ( lower row ) and prey proteins were detected with an anti-Atg5 antibody ( upper row ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 18544 . 01110 . 7554/eLife . 18544 . 012Figure 4—figure supplement 1 . Structural relationship between the Atg5 and Atg8 AIM binding sites . Shown is the structure of Atg5 ( blue ) in complex with the N-terminus of Atg16 ( yellow ) ( PDB: 2DYO , [Matsushita et al . , 2007] ) .", "The canonical AIM motif of Atg19 ( 412WEEL415 ) ( Noda et al . , 2008 ) is shown in green and is located at the positions it would occupy if the binding mode was analogous to its binding to Atg8 .", "The Atg8 K57 and K137 in pose 1 as well as N84 and R208 in pose 2 are shown in red . DOI: http://dx . doi . org/10 . 7554/eLife . 18544 . 012 In order to validate the predictions from the molecular dynamics simulations we set out to interfere with the interaction .", "To this end , we mutated the predicted binding sites in Atg5 at residues K57 , K137 , N84 or R208 to E . We refrained from mutating the hydrophobic pockets of Atg5 in order to avoid non-specific loss of function effects due to disruption of the hydrophobic core of the protein .", "The mutant proteins were tested for their ability to bind to full length GST-Atg19 in pull down experiments ( Figure 4C ) .", "Atg5 K137E as well as Atg5 R208E still efficiently bound to Atg19 , while the Atg5 K57E and Atg5 N84E mutants showed no detectable binding in this assay .", "We went on to test the effect of the K57E and N84E mutations on the recruitment of the Atg12~Atg5-Atg16 complex to cargo mimetic beads ( Figure 5A–C ) .", "The wild type Atg12~Atg5-Atg16-mCherry complex robustly localized to the beads .", "Introduction of the K57E into Atg5 resulted in a strongly reduced recruitment of the Atg12~Atg5-Atg16 complex , while the N84E mutation rendered the recruitment undetectable ( Figure 5B , C ) .", "When combined , the two mutations also resulted in a loss of Atg12~Atg5-Atg16-mCherry complex recruitment to the beads .", "Thus , introducing negative charge at positions 57 and 84 in the two predicted AIM binding sites interfered with the interaction of the two proteins .", "These residues are therefore likely to be directly involved in the formation of binding sites for the AIM motif .", "Interestingly , in the context of the Atg12~Atg5 conjugate N84 in pose two would be largely covered by Atg12 ( Figure 4B ) .", "This may explain why the C-terminal domain of Atg19 containing the AIM motif is not sufficient for the interaction with Atg12~Atg5 and requires the coiled-coil domain ( Figure 2D–G ) , which may reorient Atg12 away from pose 2 . 10 . 7554/eLife . 18544 . 013Figure 5 . Mutation of the predicted binding sites for Atg19 in Atg5 impairs the recruitment of the Atg12~Atg5-Atg16 complex to cargo mimetic beads and Cvt pathway function but does not affect bulk autophagy .", "( A ) Coomassie stained gels showing the input amounts of the Atg12~Atg5-Atg16-mCherry complex ( upper gel ) and of the GST-prApe1 ( 1–41 ) + Atg19 or GST proteins on the beads ( lower gel ) used for the experiment shown in ( B ) .", "( B ) GST-prApe1 ( 1–41 ) + Atg19 or GST coated glutathione beads imaged in the presence of Atg12~Atg5-Atg16-mCherry complex ( wild type , K57E , N84E or K57E , N84E ) .", "( C ) Quantification of three independent experiments of the relative mCherry signal intensity measured at the beads .", "One experiment used for the quantification is shown in ( B ) .", "The signal measured for the wild type Atg5~Atg12-Atg16-mCherry complex was set to 100% .", "Due to optical reasons very low signals at the beads resulted in values lower than the background and thus negative values .", "Error bars represent standard deviations .", "( D ) Coomassie-stained gel showing the result of a liposome co-sedimentation assay using wild type Atg12~Atg5-Atg16-mCherry and the indicated point mutants thereof .", "Liposome binding allows the protein to be pelleted ( P ) .", "The unbound protein remains in the supernatant ( S ) .", "( E ) Quantification of in vitro Atg8 conjugation assays using the indicated mutants of Atg12~Atg5 .", "The amount of conjugated Atg8 and un-conjugated Atg8 was measured as the band intensity signal on a Coomassie stained gel and set as 100% .", "Amounts of conjugated Atg8 were determined relative to this .", "Averages of these values were calculated from three independent experiments and the final values are plotted together with standard deviations .", "See also Figure 5—figure supplement 1 .", "( F ) Co-immunoprecipitation using Atg5-TAP or Atg5 K57E , N84E-TAP as bait in the presence of 6xmyc-Atg19 and either Atg16 wild type or Atg16 E102A .", "6xmyc-Atg19 pulled down protein in wild type Atg5 and Atg16 expressing samples was set to 100 .", "All the other conditions were quantified in relation to this ( Atg5 wild type and Atg16 E102A , 85% ( ±59 ) p-value = 0 . 63 , n . s . ; Atg5 K57E , N84E and Atg16 wild type , 13% ( ±12 . 9 ) p-value < 0 . 0001; Atg5 K57E , N84E and Atg16 E102A , 2 . 9% ( ±4 . 6 ) p-value < 0 . 0001 ) .", "The p-values were calculated using a two-tailed Student t-test .", "Proteins were detected using anti-TAP and anti-Myc antibodies , anti-Pgk1 was used as loading control .", "Shown is a representative blot of three experiments .", "( G ) prApe1 processing assay using an atg5Δ strain transformed with the indicated expression constructs .", "The lower Ape1 band indicates prApe1 processing and thus its delivery into the vacuole .", "The prApe1 and Ape1 bands were detected with an anti-Ape1 antibody .", "The expression levels of Atg5 were visualised with an anti-Myc antibody .", "The Pgk1 signal served as a loading control .", "The bar graph to the right shows a quantification of six independent experiments .", "The p-values were calculated using a two-tailed Student t-test .", "( H ) prApe1 processing assay using yeast atg16Δ strain with Atg5 wild type-TAP or Atg5 K57E , N84E-TAP stably integrated in the genome and transformed with the indicated Atg16 constructs .", "The blot shows the prApe1 processing in the Atg5 mutants in combination with Atg16 wild type in rich conditions ( the full set of tested Atg16 can be found in Figure 5—figure supplement 2 ) .", "A blot showing the full set of Atg16 mutants after 6 hr nitrogen-starvation is shown in Figure 5—figure supplement 3 .", "The Ape1 bands were detected using an anti-Ape1 antibody .", "The bar graph shows quantification of the prApe1 processing of four independent experiments .", "The p-value was calculated using a two-tailed Student t-test .", "( I ) A representative blot of a GFP-Atg8 cleavage assay is shown .", "( J ) Pho8∆60-activity assay under rich ( black bars ) and 5 hr N-starvation ( white bars ) growing conditions using a pho13∆ , pho8∆60 , atg5Δ strain transformed with the indicated Atg5 expression constructs or an empty vector .", "At least three independent experiments were conducted and the mean values for each conditions were plotted .", "The error bars represent the standard deviation . DOI: http://dx . doi . org/10 . 7554/eLife . 18544 . 01310 . 7554/eLife . 18544 . 014Figure 5—figure supplement 1 . The K57E and N84E mutations do not impair the ability of the Atg12~Atg5 conjugate to promote Atg8 conjugation . In vitro Atg8 lipidation reaction using SUVs and wild type as well the indicated Atg12~Atg5 mutants .", "Shown are Coomassie stained gels of a time series of Atg8 conjugation reactions in the presence of Atg12~Atg5 K57E or N84E ( left panel ) and wild type or K57E , N84E mutant ( right panel ) .", "The Atg12~Atg5 band is not visible on the gel at the concentrations used in this experiment .", "The Atg8~PE is detected as a faster migrating band in a urea containing gel . DOI: http://dx . doi . org/10 . 7554/eLife . 18544 . 01410 . 7554/eLife . 18544 . 015Figure 5—figure supplement 2 . Effect of the disruption of the Atg16 – Atg21 interaction on prApe1 processing . prApe1 processing assay in atg16Δ yeast strain expressing transiently transformed Atg16 D101A or E102A or D101A , E102A-double mutant proteins and Atg5-TAP or Atg5 K57E , N84E-TAP stably integrated in the genome ( complete blot from Figure 5H upper blot ) .", "The Atg5 K57E , N84E-TAP in combination with Atg16 wild type shows reduced prApe1 processing .", "This mutant , in combination with Atg16 D101A , E102A and D101A , E102A-double mutation , shows fully arrested prApe1 processing .", "Atg5 expression levels were checked using anti-TAP-antibody; Pgk1 served as a loading control . DOI: http://dx . doi . org/10 . 7554/eLife . 18544 . 01510 . 7554/eLife . 18544 . 016Figure 5—figure supplement 3 . The Cvt pathway is affected upon disrupting Atg19 – Atg5 interaction .", "( A ) prApe1 processing assay using yeast atg16Δ strain with Atg5-TAP or Atg5 K57E , N84E-TAP stably integrated in the genome and transformed with the indicated Atg16 constructs .", "The blot shows the full set of Atg16 mutants after 6 hr nitrogen-starvation .", "The Ape1 bands were detected using anti-Ape1 antibody .", "The bar graph in ( B ) shows quantification of the prApe1 processing of four independent experiments .", "The p-value was calculated using a two-tailed Student t-test . DOI: http://dx . doi . org/10 . 7554/eLife . 18544 . 01610 . 7554/eLife . 18544 . 017Figure 5—figure supplement 4 . Mutation of the predicted binding sites for Atg19 in Atg5 does not affect bulk autophagy . Pho8∆60-activity assay under rich ( black bars ) and 5 hr N-starvation ( white bars ) growing conditions using a pho13∆ , pho8∆60 , atg16∆ , Atg5-TAP or Atg5 K57E , N84E-TAP strain transformed with the indicated Atg16 expression constructs or an empty vector .", "Three independent experiments were conducted and the mean values for each conditions were plotted .", "The error bars represent the standard deviation .", "No significant difference was observed between the wild type Atg5 and the Atg5 K57E , N84E mutant . DOI: http://dx . doi . org/10 . 7554/eLife . 18544 . 017 The K57 and N84 mutations did not abolish the conjugation of Atg5 to Atg12 ( Figure 5A ) .", "We also did not observe significant defects of the mutant Atg12~Atg5-Atg16 complexes with respect to liposome binding ( Figure 5D ) or of the single and double mutant Atg12~Atg5 conjugates with respect to promoting Atg8 lipidation ( Figure 5E and Figure 5—figure supplement 1 ) .", "We went on to test if the K57E , N84E double mutation would interfere with the Atg5 - Atg19 interaction in vivo by performing co-immunoprecipitations ( Figure 5F ) .", "Consistent with the results shown above ( Figure 3D ) , wild type Atg5 robustly co-precipitated Atg19 .", "The K57E , N84E double mutant Atg5 showed a strongly reduced ability to interact with Atg19 , but the interaction was still detectable ( Figure 5F ) .", "It was previously shown that Atg16 interacts with Atg21 and that this interaction recruits the Atg12~Atg5-Atg16 complex to the pre-autophagosomal structure ( PAS ) ( Juris et al . , 2015 ) .", "We therefore asked if the residual interaction of the K57E , N84E mutant Atg5 with Atg19 could be dependent on the recruitment of the Atg12~Atg5-Atg16 complex to the PAS by Atg21 .", "To this end , we introduced the E102A mutation into Atg16 , which was reported to abolish the Atg16 - Atg21 interaction ( Juris et al . , 2015 ) .", "Indeed , the interaction of the Atg5 K57E , N84E with Atg19 became undetectable in context of the Atg16 E102A mutation ( Figure 5F ) .", "Next , we tested the impact of the single mutations or their combination on the functionality of the Cvt pathway by monitoring prApe1 processing .", "The N84E mutation , which had the stronger effect on the Atg19 - Atg5 interaction also affected prApe1 processing more pronouncedly compared to the K57E mutation while the K57E , N84E double mutation had the strongest effect on prApe1 processing ( Figure 5G , H and Figure 5—figure supplement 2 ) .", "For the cells expressing Atg5-TAP ( Figure 5H and Figure 5—figure supplement 2 ) we consistently noticed a somewhat lower levels of the Atg12~Atg5 for the K57E , N84E double mutant under rich conditions .", "This effect was not seen for the myc-tagged version .", "prApe1 processing was also affected by the K57E , N84E double mutation under starvation condition in the context of the Atg16 D101A , E102A mutation ( Figure 5—figure supplement 3 Figure 5H ) .", "In contrast , starvation-induced bulk autophagy as measured by GFP-Atg8 cleavage and the Pho8∆60 assay was not significantly affected by the Atg5 K57E , N84E double mutation ( Figure 5I , J ) , even when tested in combination with the Atg16 D101A , E102A mutant ( Figure 5—figure supplement 4 ) .", "The data presented so far have shown that the Atg19 cargo receptor can recruit the E3-like Atg12~Atg5-Atg16 complex to the prApe1 cargo and that mutations that abolish this interaction reduce the efficiency of the Cvt pathway .", "The cargo receptor - E3 interaction might therefore be a minimal axis to couple Atg8 conjugation to the cargo .", "To test this hypothesis , we developed a fully reconstituted system to recapitulate these reactions .", "In analogy to the experiment shown in Figure 3—figure supplement 2 , we added the Atg12~Atg5-Atg16 complex to cargo mimetic beads bound by Atg19 .", "The Atg12~Atg5-Atg16 complex directly binds to membranes ( Figure 5D ) ( Romanov et al . , 2012 ) and it may thus be able to link membranes and the cargo .", "To test this , we added small unilamellar vesicles ( SUVs ) labeled by the incorporation of ATTO390-PE or Rhodamine to Atg19-bound cargo mimetic beads in the presence or absence of the Atg12~Atg5-Atg16 complex ( Figure 6A , Figure 6—figure supplement 1 ) .", "The SUVs were recruited to the beads in an Atg12~Atg5-Atg16 complex dependent manner ( Figure 6A , Figure 6—figure supplement 1 ) .", "The complexes containing the Atg19 binding defective mutants of Atg5 were less efficiently recruited to cargo mimetic beads and were also less efficient in membrane recruitment ( Figure 6—figure supplement 2 ) .", "Thus , the complex brings the membrane substrate for Atg8 conjugation in proximity of the cargo .", "Next we analyzed if this minimal system would allow local accumulation of conjugated Atg8 by adding the ubiquitin-like molecule GFP-Atg8 , the E1-like enzyme Atg7 , the E2-like enzyme Atg3 , and the cofactors MgCl2 and ATP to the system .", "Intriguingly , GFP-Atg8 showed increased signal at the cargo in the presence of ATP ( Figure 6A ) .", "We also detected an increased signal for the membrane and the Atg12~Atg5-Atg16 complex ( Figure 6A ) , likely due to its association with Atg8 and the membranes ( Kaufmann et al . , 2014; Romanov et al . , 2012 ) .", "Since the presence of ATP increased the GFP-Atg8 signal at the beads , this effect may be due to lipidation of Atg8 and thus its stable anchoring and concentration on the membranes .", "If so , then the lipidated Atg8 should be more strongly bound by the Atg19 receptor ( Abert et al . , 2016; Sawa-Makarska et al . , 2014 ) .", "To test this , we conducted FRAP experiments on the cargo mimetic beads ( Figure 6B ) .", "Indeed , Atg8 recovered more slowly in the presence of ATP and recovery was slowest for the Atg8 positive puncta , which we interpret as larger Atg8-positive vesicular structures ( Figure 6B ) . 10 . 7554/eLife . 18544 . 018Figure 6 . In vitro reconstitution of cargo-directed Atg8 lipidation .", "( A ) GST-prApe1 ( 1–45 ) + Atg19 ± Atg12~Atg5-Atg16-mCherry coated Sepharose beads were imaged in the presence of ATTO390-containing SUVs and GFP-Atg8∆R117 , Atg7 , Atg3 , MgCl2 with or without ATP .", "Representative pictures and the experimental scheme are shown .", "The images corresponding to the mCherry channel are displayed in false color ( ImageJ: Fire ) .", "See also Figure 6—figure supplement 1 and Figure 6—figure supplement 2 .", "( B ) Representative pictures of a GFP-Atg8 FRAP experiment conducted on the surface of GST-prApe1 ( 1–45 ) + Atg19 + Atg12~Atg5-Atg16-mCherry coated Sepharose beads after the conjugation reaction performed in the presence ( lower row ) or absence ( upper row ) of ATP .", "The graph shows the quantification of the GFP signal measured on the surface of at least two beads per condition ( Cx: Atg12~Atg5-Atg16-mCherry ) .", "( C ) Representative pictures , experimental scheme and quantification of Atg8 lipidation on cargo mimetic beads coated with GST-prApe1 ( 1–45 ) + Atg19 + Atg12~Atg5-Atg16-mCherry + ATTO390-containing SUVs in the presence of the conjugation machinery ( Atg7 , Atg3 , GFP-Atg8∆R117 , MgCl2 , ±ATP ) after removal of SUVs from the solution by washing .", "( D ) Representative pictures of a GFP-Atg8 FRAP experiment conducted on the surface of GST-prApe1 ( 1–45 ) + Atg19 + Atg12~Atg5-Atg16-mCherry + ATTO390-containing SUVs-coated Sepharose beads , after conjugation reaction performed in the presence ( lower row ) or absence ( upper row ) of ATP .", "The graph shows the quantification of the recovered GFP signal on the beads in the presence or absence of ATP , respectively , for at least two beads per condition .", "See also Figure 6—figure supplement 3 . DOI: http://dx . doi . org/10 . 7554/eLife . 18544 . 01810 . 7554/eLife . 18544 . 019Figure 6—figure supplement 1 . Atg19 AIM-dependent recruitment of the Atg12~Atg5-Atg16 complex and vesicles to cargo mimetic beads . Representative pictures and experimental scheme of Rhodamine-SUV recruitment to cargo mimetic beads coated with GST-prApe1 ( 1–45 ) and Atg19 ( wild type or AIM mutant ( F376A , F379A , P385A , E386A , W412A ) ) with or without the Atg12~Atg5-Atg16-meGFP complex . DOI: http://dx . doi . org/10 . 7554/eLife . 18544 . 01910 . 7554/eLife . 18544 . 020Figure 6—figure supplement 2 . Vesicle recruitment by Atg12~Atg5-Atg16 mutants to cargo mimetic beads . Representative pictures and quantification of Atg12~Atg5-Atg16-mCherry and ATTO390-SUVs recruitment to cargo mimetic beads coated with GST-prApe1 ( 1–45 ) and Atg19 .", "The quantification is based on three independent experiments and the bars indicate the standard deviation .", "Signal corresponding to wild type Atg12~Atg5-Atg16 was set to 100% .", "DOI: http://dx . doi . org/10 . 7554/eLife . 18544 . 02010 . 7554/eLife . 18544 . 021Figure 6—figure supplement 3 . De-conjugation of lipidated Atg8 on the beads .", "( A ) Quantification of three independent experiments performed using cargo mimetic beads prepared as in Figure 6A .", "Atg4 was added to cargo mimetic beads after an overnight conjugation reaction .", "The GFP-Atg8∆R117 signal at the beads was measured after 30 min of de-conjugation reaction .", "( B ) Representative images of the Atg4 de-conjugation of Atg8 at the beads at the indicated time points . DOI: http://dx . doi . org/10 . 7554/eLife . 18544 . 021 In the experiments shown in Figure 6A , B the vesicles were also present in solution and we could therefore not exclude the possibility that conjugation occurred remotely from the cargo , followed by attachment of the vesicles harboring lipidated Atg8 .", "For this reason , we recruited vesicles to cargo mimetic beads via the Atg12~Atg5-Atg16 complex and washed away unbound vesicles ( Figure 6C ) .", "Subsequently , we added the Atg8 conjugation machinery .", "In the presence of ATP the bead associated signal was increased , consistent with local lipidation of Atg8 at the cargo ( Figure 6C ) .", "To corroborate this result , we performed FRAP experiments on these beads ( Figure 6D ) .", "Indeed , the recovery of Atg8 was much reduced in the presence of ATP , consistent with its stable attachment to the membrane via lipidation .", "Furthermore , upon addition of the Atg4 protein , which removes Atg8 from PE the GFP-Atg8 signal decreased ( Figure 6—figure supplement 3 ) ." ], [ "Here we have shown that the cargo receptor Atg19 directly interacts with the Atg5 subunit of the E3-like Atg12~Atg5-Atg16 complex and that this interaction is sufficient to direct Atg8 conjugation to the cargo in a reconstituted system .", "The recruitment of the Atg12~Atg5-Atg16 complex requires the AIM motifs of Atg19 and it is likely that these motifs directly bind Atg5 since our modeling analysis and molecular dynamics simulations predicted two binding sites for the C-terminal AIM motif of Atg19 and mutation of these sites abolished Atg19 binding .", "In addition , the interaction of Atg19 with Atg5 is competitive with the interaction with Atg8 , which also directly interacts with the C-terminal AIM motif of Atg19 ( Noda et al . , 2008 ) .", "Additional regulation of the two mutually exclusive binding events may occur due to phosphorylation as it was shown that Atg19 is phosphorylated in its C-terminal domain ( Pfaffenwimmer et al . , 2014; Tanaka et al . , 2014 ) .", "Since Atg19 contains multiple AIM-like sequences , it is possible that both sites in Atg5 contribute to Atg19 binding .", "Mutation of N84 in site two had a more pronounced effect on the interaction with Atg19 .", "In the context of the Atg12~Atg5 conjugate this site would be buried by the Atg12 subunit ( Noda et al . , 2013 ) .", "Since the coiled-coil domain of Atg19 was required for the interaction of Atg19 with Atg5 when conjugated to Atg12 , we hypothesize that the coiled-coil domain is required to expose binding site two by changing the position of Atg12 .", "The interaction of cargo receptors with the Atg12~Atg5-Atg16 complex is conserved since we detected an interaction of the S . cerevisiae Atg34 and human p62 , NDP52 and Optineurin cargo receptors with Atg12~Atg5-Atg16 and ATG5 , respectively , suggesting that the recruitment of the E3-like ligase for ATG8-family members conjugation to the cargo is a more general property of cargo receptors .", "Future work will have to elucidate the biochemical details of the interaction of the human cargo receptors with ATG5 and the ATG12~ATG5-ATG16L complex .", "It is possible that this interaction is at least for p62 in part mediated indirectly by ALFY since it interacts with p62 and ATG5 ( Filimonenko et al . , 2010 ) .", "A similar mechanism has been described for C . elegans where EPG-7 binds both , p62/SQST-1 and ATG12/LGG-3 ( Lin et al . , 2013 ) .", "The results presented in this study suggest the following sequence of events , at least for the Cvt pathway ( Figure 7 ) .", "When bound to their respective cargo , cargo receptors cluster and provide a high-avidity binding platform that recruits the autophagy machinery , including the E3-like Atg12~Atg5-Atg16 complex .", "This machinery is able to bring and keep membranes in close proximity to the cargo and to act catalytically by promoting several rounds of Atg8 conjugation .", "Consistent with the idea of local Atg8 lipidation the E2-like Atg3 enzyme localizes to the site of autophagosome formation ( Ngu et al . , 2015 ) .", "The Atg12~Atg5-Atg16 complex is able to directly bind lipids ( Romanov et al . , 2012 ) and thus it may link the cargo to the membrane .", "However , in vivo additional interactions with PROPPINs/WIPIs will render the system more robust ( Dooley et al . , 2014; Juris et al . , 2015 ) . 10 . 7554/eLife . 18544 . 022Figure 7 . Model for the molecular mechanism of cargo-directed Atg8 lipidation . The Atg19 cargo receptor binds the cargo ( 1 ) and recruits the E3-like Atg12~Atg5-Atg16 complex to the cargo via its AIM motifs ( 2 ) .", "In the presence of a membrane source , Atg8 is locally conjugated ( 3 ) and may eventually outcompete the Atg12~Atg5-Atg16 complex from Atg19 binding .", "At the same time the AIM motifs of Atg19 may keep the Atg8-coated isolation membrane close to the cargo excluding non-cargo material from incorporation into selective autophagosomes ( 4 ) .", "See main text for extended discussion . DOI: http://dx . doi . org/10 . 7554/eLife . 18544 . 022 Due to the action of the Atg8 conjugation machinery Atg8 accumulates at the isolation membrane and may subsequently outcompete the Atg12~Atg5-Atg16 complex on the concave side of the isolation membrane .", "In conflict with this hypothesis is the finding that the signal of the Atg12~Atg5-Atg16 complex at the bead was not decreased upon addition of ATP ( Figure 6C ) .", "We interpret this result with the previously described interactions of the Atg12~Atg5-Atg16 complex with the membrane and lipidated Atg8 on the convex side ( Kaufmann et al . , 2014; Romanov et al . , 2012 ) .", "Consistent with exclusion of Atg12~Atg5-Atg16 from the concave side of the isolation membrane , the Atg12~Atg5-Atg16 complex is excluded from the autophagosomal lumen in vivo ( Mizushima et al . , 2003 ) .", "On the concave side Atg8 may be subsequently bound with high avidity by the cargo receptors , which could result in close apposition of the membrane and the cargo and thus exclusion of non-cargo material from the autophagosome ( Figure 7 ) ( Abert et al . , 2016; Sawa-Makarska et al . , 2014; Wurzer et al . , 2015 ) .", "As a consequence , this mechanism would localize the Atg8 conjugation machinery to the highly curved edge of the membrane where the isolation membrane has not yet formed , which may additionally stimulate Atg8 conjugation ( Nath et al . , 2014 ) .", "Thus , the intricate AIM/LIR-based interplay between the cargo , the cargo receptors , the Atg8 conjugation machinery and Atg8 may serve to confer directionality to the Atg8 lipidation resulting in robust membrane growth exclusively around the cargo .", "In vivo , this may occur at a permissive site such as the vacuole or the endoplasmic reticulum ( Lamb et al . , 2013; Nakatogawa et al . , 2009 ) ." ], [ "Atg3: NP_014404; Atg4: NP_014176 . 2; Atg5: NP_015176 . 1; Atg7: NP_012041 . 1; Atg8 NP_009475 . 1; Atg10: NP_013058 . 1; Atg12: NP_009776 . 1; Atg16: NP_013882 . 1; Atg19: NP_014559 . 1; Atg34: NP_014558 . 1; prApe1: NP_012819; p62/SQSTM1: NP_003891; NDP52: AAA75297 . 1; OPTN: NP_001008212 .", "A list of constructs for protein expression can be found in ( Table", "1 . ) .", "Expression and purification of Atg19 and shortened variants thereof , Atg34 and prApe1 ( 1–45 ) was described previously ( Sawa-Makarska et al . , 2014 ) .", "prApe1 ( 1–41 ) was subcloned into pGEX4T-1 vector , expressed and purified as a GST fusion protein with the same approach as the propeptide prApe1 ( 1–45 ) variant described in ( Sawa-Makarska et al . , 2014 ) .", "The mCherry-Atg19 was purified via a hexahistidine tag as described in ( Sawa-Makarska et al . , 2014 ) .", "Atg12~Atg5-Atg16 , Atg12~Atg5 , Atg16 and Atg16-meGFP as well as all proteins required for Atg8~PE conjugation ( Atg3 , Atg7 , Atg10 , Atg8∆R117 , meGFP-Atg8∆R117 ) were expressed and purified as described previously ( Romanov et al . , 2012 ) .", "Atg5-Atg16 ( 1–46 ) for analytical size exclusion chromatography was produced by co-expressing Atg5 subcloned into pGEX4T-3 and the first 46 amino acids of Atg16 ( Atg16 ( 1–46 ) ) subcloned into pCOLADuet-1 in E . coli Rosetta pLySS .", "The co-transformed cells were grown at 37°C to an OD600 of 0 . 6 , induced with 0 . 1 mM IPTG and further grown over night at 18°C .", "Cells were pelleted and resuspended in a buffer containing 50 mM HEPES pH 7 . 5 , 300 mM NaCl , 1 mM MgCl2 , 2 mM β−mercaptoethanol , complete protease inhibitors ( Roche , Basel , Switzerland ) and DNAse I ( Sigma , USA , Missouri ) .", "Cells were disrupted by freeze-thawing and lysates were cleared by ultracentrifugation ( 140 , 000 g for 30 min at 4°C in a Ti45 rotor Beckman , Brea , CA , USA ) .", "Supernatants were applied to glutathione beads ( GE Healthcare , Buckinghamshire , UK ) for 1 hr at 4°C .", "Beads were washed five times with 50 mM HEPES , 300 mM NaCl , 1 mM DTT and the protein was cleaved from the GST tag by incubation with thrombin protease ( SERVA , Heidelberg , Germany ) overnight at 4°C .", "The supernatant containing Atg5 - Atg16 ( 1–46 ) was concentrated and applied to a Superdex 200 ( 16/60 prep grade , GE Healthcare ) and eluted with a buffer containing 25 mM HEPES at pH 7 . 5 , 150 mM NaCl and 1 mM DTT .", "Fractions containing pure protein were pooled , concentrated , frozen in liquid nitrogen and stored at −80°C .", "For all other experiments , wild type Atg5 as well as Atg5 ( K57E ) , Atg5 ( N84E ) , Atg5 ( K137 ) , Atg5 ( R208 ) and Atg5 ( K57E , N84E ) were tagged with an N-terminal hexahistidine-tag followed by a TEV cleavage site .", "The proteins were co-expressed and purified in complex with Atg16 ( 1–46 ) as described in ( Romanov et al . , 2012 ) .", "Atg16-mCherry with an N-terminal hexahistidine-tag followed by a TEV cleavage site ( pETDuet-1 ) was expressed in E . coli Rosetta pLysS .", "Cells were grown at 37°C to an OD600 of 0 . 6 and induced with 0 . 1 mM IPTG .", "The protein expression continued overnight at 16°C .", "Cells were pelleted and resuspended in a buffer containing 50 mM HEPES pH 7 . 5 , 300 mM NaCl , 10 mM Imidazole , 1 mM MgCl2 , 2 . 5 mM β−mercaptoethanol , complete protease inhibitors ( Roche ) and DNAse I ( Sigma ) .", "Cells were lysed by freeze-thawing followed by 30 s sonication and the lysate was centrifuged at 40000 rpm ( Beckman Ti45 rotor ) for 40 min at 4°C .", "The supernatant was applied to a 5 ml Ni-NTA column ( GE Healthcare ) and eluted via a stepwise imidazole gradient ( 50 , 75 , 100 , 150 , 200 , and 300 mM ) .", "Protein-containing fractions were pooled and subjected to overnight cleavage with TEV protease at 4°C in the dark .", "The cleaved protein was applied to a Superdex 200 column ( 16/60 prep grade , GE Healthcare , Sweden ) and eluted with a buffer containing 25 mM HEPES pH 7 . 5 , 500 mM NaCl and 1 mM dithiothreitol ( DTT ) .", "Fractions containing the purified proteins were pooled , concentrated , frozen in liquid nitrogen , and stored at −80°C .", "For purification of Atg5-Atg16 and Atg5-Atg16-meGFP , Atg5 was expressed as a GST-tagged protein in E . coli Rosetta pLysS from pGEX4T-3 vector .", "The expression and purification followed the same procedure as for Atg5-Atg16 ( 1–46 ) .", "In short , cells were grown at 37°C to an OD600 of 0 . 5 and induced with 100 μM IPTG for 16 hr at 18°C .", "Cells were disrupted by freeze-thawing and the cleared lysate was incubated with glutathione beads ( Glutathione Sepharose 4B , GE Healthcare , Uppsala , Sweden ) .", "The protein was cleaved off from the beads with thrombin protease ( SERVA ) .", "Next , the supernatant containing Atg5 was mixed with purified Atg16 or Atg16-meGFP in a molar ratio 1:1 at 4°C for 30 min , concentrated and the resulting Atg5-Atg16 or Atg5-Atg16-meGFP complex was further purified by size exclusion chromatography on Superdex S200 ( 16/60 prep grade , GE Healthcare ) .", "The wild type and Atg12~Atg5-Atg16 ( untagged and -mCherry tagged ) as well as the point mutants Atg12~Atg5 ( K57E ) -Atg16-mCherry , Atg12~Atg5 ( N84E ) -Atg16-mCherry , Atg12~Atg5 ( K57E , N84E ) -Atg16-mCherry were purified in two steps .", "In the first step , Atg12~Atg5 wild type conjugate or point mutants thereof were generated as described in ( Romanov et al . , 2012 ) .", "Next the conjugates were mixed with purified Atg16 or Atg16-mCherry in a molar ratio 1:1 ratio and incubated on ice for 30 min .", "The resulting Atg12~Atg5-Atg16 or Atg12~Atg5-Atg16-mCherry complexes were further purified by size exclusion chromatography on Superdex S200 ( 16/60 prep grade , GE Healthcare ) .", "The Atg5-mCherry was subcloned into pETDuet-1 vector with N-terminal hexahistidine-tag followed by a TEV cleavage site ( 6xHis-TEV-Atg5-mCherry ) .", "The protein was co-expressed as a complex with the first 46 amino acids of Atg16 ( Atg16 ( 1–46 ) ) subcloned into pCOLADuet-1 .", "The E . coli Rosetta pLysS cells were co-transformed with 6xHis-TEV-Atg5-mCherry and Atg16 ( 1–46 ) and grown at 37°C to an OD600 of 0 . 6 , induced with 1 mM IPTG and further grown overnight at 18°C .", "Cells were pelleted and resuspended in a buffer containing 50 mM HEPES pH 7 . 5 , 300 mM NaCl , 10 mM Imidazole , 1 mM MgCl2 , 2 mM β−mercaptoethanol , complete protease inhibitors ( Roche ) and DNAse I ( Sigma ) .", "Cells were disrupted by freeze-thawing followed by 30 s sonication .", "Lysates were cleared by ultracentrifugation ( 140 , 000 g for 30 min at 4°C in a Beckman Ti45 rotor ) .", "Supernatant was applied to a 5 ml Ni-NTA column ( GE Healthcare , Sweden ) and eluted via a stepwise imidazole gradient ( 50 , 75 , 100 , 150 , 200 , and 300 mM ) .", "Protein-containing fractions were pooled , concentrated , applied onto a Superdex 200 column ( 16/60 prep grade , GE Healthcare ) and eluted with a buffer containing 25 mM HEPES pH 7 . 5 , 150 mM NaCl and 1 mM dithiothreitol ( DTT ) .", "Fractions containing the purified proteins were pooled , concentrated , frozen in liquid nitrogen , and stored at −80°C .", "Atg4 was expressed and purified as described in ( Zens et al . , 2015 ) .", "To probe the direct Atg19 interaction with Atg5-Atg16 ( 1–46 ) in solution the analytical size exclusion chromatography was performed .", "Atg19 and Atg5-Atg16 ( 1–46 ) were premixed at 55 μM , concentrated to 580 μM ( Amicon Ultra-0 . 5 ml Centrifugal Filters 3 kDa MWCO , Millipore , Cork , Ireland ) and subsequently applied onto a Superose 6 gel filtration column ( PC 3 . 2/30 , GE Healthcare ) equilibrated with a buffer containing 25 mM HEPES at pH 7 . 5 , 150 mM NaCl and 1 mM DTT .", "Resulting fractions were subjected to SDS-PAGE and the protein bands were detected with Coomassie Brilliant Blue staining .", "To perform GST pull down binding assays GST or GST-fused Atg19 wild type or shortened variants thereof were used as a bait and Atg12~Atg5-Atg16 or Atg12~Atg5-Atg16-meGFP , Atg12~Atg5 , Atg5-Atg16 ( 1–46 ) , Atg5-Atg16 or Atg16-meGFP were used as a prey .", "The purified GST-fused proteins ( 5 µM for pull downs in Figures 1A , C , 2A and C; 20 µM for pull downs in Figure 2E , Figure 3—figure supplements 1 Figure 4C , ) and purified GST-free proteins ( 5 µM ) as well as glutathione Sepharose 4B beads ( GE Healthcare ) were simultaneously incubated for 1 hr at 4°C on a rotating wheel .", "After washing the beads three times with 25 mM HEPES at pH 7 . 5 , 150 mM NaCl , 1 mM DTT ( and 0 . 1% TritonX100 for pull-downs in Figures 1A , C , 2A and C ) , the glutathione beads together with bound proteins were subjected to SDS-PAGE .", "The protein bands were detected either by Coomassie Brilliant Blue staining or Ponceau or by immunoblotting carried out with a mouse monoclonal anti-Atg5 ( Romanov et al . , 2012 ) , mouse anti-GFP ( Roche , diluted 1:5000 in 0 . 5% Milk in TBST , 1% TritonX100 ) or anti-GST ( diluted 1:1000 in 3% Milk in TBST , 1% TritonX100 ) antiserum used as primary antibodies .", "Secondary antibodies were used as described in ´prApe1 processing assay´ .", "For experiments shown in Figure 3E , glutathione Sepharose 4B beads were incubated with a 30 µM GST or GST-Atg19 ( 365–415 ) solution for 30 min at 4°C on a rotating wheel and afterwards twice washed in buffer containing 25 mM HEPES at pH 7 . 5 , 150 mM NaCl and 1 mM DTT .", "2 µl of these beads were added to a Atg5-mCherry-Atg16 ( 1–46 ) solution pre-pipetted in the wells of a 384-wells glass-bottom plate ( Greiner Bio One , Frickenhausen , Germany ) resulting in a final concentration of 18 µM .", "After at least 20 min of incubation , samples were imaged as described in the ‘Microscopy-based protein-protein interaction assay’ section .", "For the competition experiment GFP-Atg8∆R117 ( or buffer ) was added to the wells at a final concentration of 18 µM ( 1x initial Atg5-mCherry-Atg16 ( 1–46 ) concentration ) and allowed to compete the Atg5-mCherry-Atg16 ( 1–46 ) protein and to bind to the GST-protein for at least 20 min .", "An equivalent volume of empty buffer was added to the control well in order to account for the dilution factor applied to the sample .", "After imaging , Atg8 ( or buffer ) was further added to the same wells at a final concentration of 400 µM ( 22x initial Atg5-mCherry-Atg16 ( 1–46 ) concentration ) and allowed to reach the equilibrium of binding for at least 20 min .", "The samples were then imaged as described in Microscopy-based protein-protein interaction assay´ section .", "For experiments shown in Figure 3G , glutathione Sepharose 4B beads were incubated with a 10 µM GST or GST-prApe1 ( 1–40 ) solution for 30 min at 4°C on a rotating wheel and subsequently washed twice in buffer containing 25 mM HEPES at pH 7 . 5 , 150 mM NaCl and 1 mM DTT .", "The beads were further incubated with a solution of Atg19 at 20 µM for at least 30 min .", "Beads were washed twice and pipetted directly to a 96-well-plate glass bottom well pre-filled with a solution of Atg12~Atg5-Atg16-mCherry at a final concentration of 5 µM .", "After imaging , GFP-Atg8 solution was added to the well at a final concentration of 5 µM ( ratio 1:1 with initial concentration of Atg12~Atg5-Atg16-mCherry ) .", "The reaction was allowed to reach the equilibrium for 20 min and imaged as described above .", "GFP-Atg8 solution was added to the well at a final concentration of 50 µM ( ratio 1:10 with initial concentration of Atg12~Atg5-Atg16-mCherry ) .", "The proteins were allowed to reach the equilibrium of binding and imaged immediately .", "SUVs employed in the Atg8 conjugation assay were composed of 39% POPC ( Avanti Polar Lipids ( Alabaster , AL , USA ) , Inc . , 850457C , 10 mg/ml ) , 35% POPS ( Avanti Polar Lipids , Inc . , 840034C , 10 mg/ml ) , 21% POPE ( Avanti Polar Lipids , Inc . , 850757C , 10 mg/ml ) , 5% PI3P ( Avanti Polar Lipids , Inc . , 850150P , 1 mg/ml ) .", "PI3P stock was prepared by resuspension in CHCl3 and subsequent drying under an argon stream and further drying for 1 hr in a dessicator .", "PI3P was then resuspended in CHCl3:MeOH:1M HCl ( molar ratio 2:1:0 . 1 ) and incubated for 15’ for protonation .", "The lipid was again dried under an argon stream and subsequently for one hour in a dessicator , and then resuspended in CHCl3:MeOH ( 3:1 ) and dried again under an argon stream .", "After one wash with CHCl3 , PI3P was resuspended in CHCl3 to a final concentration of 1 mg/ml .", "Corresponding amounts of the lipid stocks were transferred into a glass vial and mixed well before they were dried under an argon stream .", "The dried lipids were further dried for an additional hour in a desiccator .", "Subsequently , the dried lipids were rehydrated with liposome buffer ( 25 mM HEPES pH 7 . 5 , 137 mM NaCl , 2 . 7 mM KCl and 1 mM DTT ) for 15 min .", "The lipids were resuspended by tapping and gently sonicated for 2 min in a water bath sonicator .", "The resuspended SUVs were then extruded 21 times through 0 . 4 μm membrane followed by extrusion through a 0 . 1 μm membrane ( Whatman , Nucleopore , UK ) using the Mini Extruder from Avanti Polar Lipids Inc . .", "The final SUVs suspension has a concentration of 1 mg lipids/ml buffer .", "SUVs are stable for 2–3 days when stored at 4°C .", "Lipid mixture used for the in vitro reconstitution of Atg8 lipidation on cargo-mimetic beads and for experiment in Figure 6—figure supplement 2 , was composed of 39–35% DOPC , 35% DOPS , 20% DOPE , 5% PI3P , 1–5% of ATTO390-DOPE and buffer was composed of 25 mM HEPES pH 7 . 5 , 150 NaCl and 1 mM DTT .", "Lipid composition of SUVs used in Figure 6—figure supplement 1 consists of 39% POPC , 35% POPS , 20% POPE , 5% PI3P , 1% of Rhodamine-DOPE and buffer was composed of 25 mM HEPES pH 7 . 5 , 150 NaCl and 1 mM DTT .", "The conjugation reactions were performed at 30°C and all buffers , solutions and the SUVs with the exception of the proteins were pre-warmed to this temperature .", "Atg3 and Atg7 were used at final concentrations of 1 µM , whereas Atg8∆R117 was used at a final concentration of 5 µM and Atg12~Atg5 wild type and mutants were used at 0 . 2 µM .", "ATP was used at a final concentration of 100 µM , while MgCl2 was used at a final concentration of 1 mM .", "The reactions were stopped by the addition of loading dye ( 12% SDS , 6% beta-mercaptoethanol , 30% Glycerol , 0 . 05% Coomassie Brilliant blue G-250 , 150 mM Tris-HCl pH 7 ) .", "The reactions were run on 11% SDS/polyacrylamide gels containing 4 . 5 M urea in the separating parts .", "The gels were then stained with Coomassie staining solution ( 40% methanol , 10% acetic acid , 0 . 2% Coomassie Brilliant Blue ) .", "For Figure 5E , the gels of three independent experiments were quantified using the Analyze Gel tool of ImageJ .", "Statistical analysis was done in Prism by multiple t tests ( unpaired , two-tailed , Holm-Sidak method ) .", "A p-value < 0 . 05 was considered to be significant .", "Small unilamellar vesicles ( SUVs ) were composed of 35% DOPC , 35% DOPS , 20% DOPE , 5% PI3P , 5% of ATTO390-DOPE ( Avanti Polar Lipids , Inc . ) and prepared as described above .", "After the drying step the lipids were resuspended in 150 mM NaCl , 50 mM HEPES pH 7 . 5 , 1 mM DTT buffer .", "For the Atg12~Atg5-Atg16-mCherry and point mutants thereof binding to lipids , 25 µl of freshly prepared SUVs were mixed with 5 µg of protein at the final reaction volume of 50 µl in 150 mM NaCl , 50 mM HEPES pH 7 . 5 , 1 mM DTT buffer .", "The reaction was incubated for 30 min at room temperature .", "Next , the liposome bound protein was pelleted by ultracentrifugation for 10 min at 100 , 000xg at 22°C .", "Supernatants and pellets were separated and equal amounts were applied on 12% SDS/polyacrylamide gel and visualized by Coomassie Brilliant Blue staining ( 40% ethanol , 10% acetic acid , 0 . 2% Coomassie Brilliant Blue ) .", "For Figure 6A , B: Glutathione Sepharose 4B beads were coated with GST-prApe1 ( 1–45 ) at a final concentration of 25 µM , incubated for 30 min at 4°C and washed twice with buffer containing 25 mM HEPES at pH 7 . 5 , 150 mM NaCl and 1 mM DTT .", "Beads were further incubated with Atg19 at a final concentration of 15 µM , washed two times and further incubated with Atg12~Atg5-Atg16-mCherry at a final concentration of 12 . 5 µM .", "After 2x washings , 2 µl of the beads were pipetted into the well of a 384-wells plate , pre-filled with 22 µl buffer and subsequently 1 µl ATTO390-containing SUVs was added to the reaction .", "Conjugation reaction in Figure 6A , B was conducted in the presence of 0 . 5 mM MgCl2 , 0 . 3 µM Atg7 , 0 . 3 µM Atg3 and 0 . 1 µM meGFP-Atg8∆R117 with/without 1 mM ATP over night at 4°C with gentle mixing on an orbital shaker .", "For the experiments shown in Figure 6C and Figure 6—figure supplement 2 , beads were prepared as for Figure 6A regarding the GST-protein , Atg19 and Atg12~Atg5-Atg16-mCherry .", "Beads were incubated overnight at 4°C under gentle rolling with an excess of ATTO390-SUVs membranes .", "The day after beads were washed twice using 25 mM HEPES at pH 7 . 5 , 150 mM NaCl and 1 mM DTT buffer ( beads pelleted by sedimentation for 10 min on ice ) and overnight conjugation reaction was set up as described for Figure 6A , B .", "Imaging was performed using a Zeiss Confocal LSM700 microscope equipped with a 20x/0 . 8 Plan-Apocromat Objective .", "The FRAP experiments shown in Figure 6B and D were conducted under following conditions: 10 ms FRAP time/pixel; laser beam diameter 10 pixels .", "Acquisition was performed either every 5 or 10 s .", "For the de-conjugation reaction Atg4 was added to the well containing beads , prepared as in Figure 6A , at a final concentration of 0 . 3 µM together with EDTA at a final concentration of 1 mM .", "Beads were imaged with a Spinning Disk microscope at the indicated time points of reaction .", "For the experiments shown in Figures 1B , 2F–G 20 µl glutathione Sepharose 4B beads slurry ( GE Healthcare ) were mixed with GST-fused bait proteins ( GST-Atg19 or GST-Atg19 variants ) to the final concentration of 20 µM and incubated on a rotating wheel at 4°C for at least 30 min .", "Subsequently the beads were washed twice with 25 mM HEPES at pH 7 . 5 , 150 mM NaCl and 1 mM DTT .", "In these experiments the prey Atg12~Atg5-Atg16-mCherry was added at the final concentration of 5 µM .", "After 30 min of incubation at 4°C a 5 µl aliquot of beads was transferred into the well of a 96-well glass-bottom microplate ( Greiner Bio-One ) pre-filled with 35 µl of 25 mM HEPES at pH 7 . 5 , 150 mM NaCl and 1 mM DTT and immediately imaged with a Spinning Disk microscope .", "For the experiments shown in Figure 3B , GST-fused proteins were incubated with Sepharose beads at a final concentration of 30 µM and Atg5-mCherry-Atg16 ( 1–46 ) was used at a final concentration of 18 µM .", "For the experiments shown in Figure 5B , Sepharose beads were incubated with GST-proteins at 25 µM , washed twice with 25 mM HEPES at pH 7 . 5 , 150 mM NaCl and 1 mM DTT buffer and further incubated with Atg19 at 15 µM .", "After two washings , the beads were incubated with Atg12~Atg5-Atg16-mCherry ( wild type and mutants ) at a final concentration of 8 . 8 µM .", "For the experiments shown in Figure 3—figure supplement 2 and Figure 6—figure supplement 1 , GST-prApe1 ( 1–45 ) was incubated with Sepharose beads at 5 µM concentration .", "Beads were washed twice and further incubated with Atg19 wild type and mutant at a final concentration of 5 µM .", "Beads were washed twice and further incubated with Atg12~Atg5-Atg16-meGFP at a final concentration of 5 µM .", "The pictures shown in Figure 1B , Figure 2F , G , Figure 3B , E , Figures 5B and 6B , D and those acquired for quantifications ( including Figure 3G ) were obtained using a LD Achroplan 20x/0 . 4 Corr ObjeFigure 3ctive mounted on a confocal spinning disc microscope ( Visitron ) installed with VisiView 2 . 1 . 1 software and processed with ImageJ .", "To quantify the protein and membrane recruitment to beads the maximum brightness along a straight line drawn through a single bead was taken ( maximal fluorescence ) .", "Next , the average brightness of an empty portion of each picture was measured ( background fluorescence ) and subtracted from the maximal fluorescence for each bead .", "All intensities were normalized to the signal of the wild type protein .", "The M-Track methylation assay was conducted as previously described ( Zuzuarregui et al . , 2012 ) ( Papinski et al . , 2014 ) .", "For blots shown in Figures 1D and 3D , yeast strain BY4741-atg8∆atg19∆ with integrated Atg5-TAP was transformed with empty vector pRS315 or vector containing 6xmyc-Atg19 wild type or mutants .", "Pre-cultures of yeast were grown in selective medium to log phase and then used to inoculate YPD cultures for an overnight growth in log phase .", "Cells were harvested by centrifugation for 15 min at 3000xg and then washed once with PBS with 2% glucose and 0 . 5% ammoniumsulfate .", "Subsequently , the cells were resuspended in a volume of IP-Buffer corresponding to the volume of the pellet ( 20 mM PIPES pH 6 . 8 , 50 mM KCl , 100 mM K Acetate , 10 mM MgSO4 , 10 µM ZnSO4 , 1 mM PMSF , 1 mM NaF , 1 mM Na3VO4 , 20 mM beta-GP , 0 . 5 mM DTT , complete PI tablet ( Roche ) ( two tablets/100 ml solution ) , 0 . 1% Triton X100 , and frozen in droplets in liquid nitrogen .", "The cells were then disrupted with a freezer mill ( 6770; SPEX ) , the extract was thawed in lysis buffer and cleared by centrifugation .", "The cleared extract was incubated with 30 µL of magnetic beads ( Dynabeads M-270 Epoxy , Invitrogen , Norway ) coupled to rabbit IgG from serum ( I5006-10MG , Sigma ) for 1 hr at 4°C with rotation .", "The beads were washed five times for 10 min in lysis buffer with rotation and then incubated for 10 min at 95°C with urea loading buffer ( 116 mM Tris pH 6 . 8 , 4 . 9% Glycerol , 8 M Urea , 8% SDS ) .", "Proteins were separated by SDS-PAGE and subjected to Western blotting .", "For detection , rabbit anti-TAP ( ThermoScientific , #CAB1001 , 1:1 000 for lysates or unbound fractions and 1:10 000 for Co-IP samples respectively in 3% milk/TBST ) , mouse anti-Myc antibody ( clone 4A6 , 1:500 in 3% milk/TBST ) and mouse-anti Pgk1 ( Invitrogen , #459250 , California , USA; 1:20 000 in 3% milk/TBST ) were used to incubate the Western blots at 4°C overnight .", "Goat anti-mouse IgG HRP ( Dianova , #115–035 , Germany; 1:10 000 in 3% milk/TBST ) and goat anti-rabbit IgG-HRP ( Dianova , #111-035-003 , Germany; 1:10 000 in 3% milk/TBST ) were used to incubate the membranes for 1 hr at room temperature .", "All the washing steps were conducted using TBS-Tween 0 . 1% .", "The gels were quantified using the Analyze Gel tool of ImageJ .", "Values were normalized to the wild type , three independent experiments were quantified .", "Statistical analysis was done in Prism by Welch’s t test .", "A p-value < 0 . 05 was considered to be significant .", "For blots shown in Figure 5F , yeast strain BY4741-atg16∆atg19∆ with integrated Atg5-TAP or Atg5 K57E , N84E-TAP was transformed with a vector containing either Atg16 wild type or Atg16 E102A .", "In addition , they were also transformed with empty vector pRS313 or vector containing 6xmyc-Atg19 wild type .", "Pre-cultures of yeast were grown in selective medium in log phase and then used to inoculate YPD cultures for a 6 hr growth in log phase .", "Cells were harvested by filtration on a 90 mm glass filter with pore size 0 . 45 µm ( SterliTech ) followed by freezing in liquid nitrogen .", "Subsequently , a volume of IP-Buffer corresponding to the volume of the pellet was frozen in droplets in liquid nitrogen and added .", "The cells were then disrupted with a freezer mill ( 6770; SPEX ) , and co-immunoprecipitation was performed as described for the Atg5 - Atg19 co-immunoprecipitation with Atg19 mutants above .", "A list of constructs for the yeast experiments can be found in ( Table", "1 . ) .", "( Table", "2 . ) lists the yeast strains used in this study .", "For the prApe1 processing assay of Atg5 mutants , yeast strains BY4741 wild type and BY4741-atg5△ were transformed with empty vector pRS316 or vector containing wild type or mutant Atg5-6xmyc .", "For the prApe1 processing assay of Atg5 mutants in combination with Atg16 mutants Atg5-TAP and Atg5 K57E , N85E-TAP were integrated stably into the genome of atg5∆atg16∆ strains and transformed with empty vector pRS315 or vector containing wild type or mutant forms of Atg16 wild type or mutants .", "Pre-cultures of yeast were grown in selective medium to log phase and then used to inoculate complete medium for an overnight growth in log phase .", "The Atg5 with the Atg16 mutants were additionally subjected to nitrogen-starvation for 6 hr as described in ‘GFP-Atg8 cleavage assay’ .", "Whole cell lysates were prepared by trichloroacetic acid extractions .", "Proteins were separated by SDS-PAGE and subjected to Western blotting .", "For detection , rabbit-anti Ape1 antiserum ( Romanov et al . , 2012 ) ; 1:20000 in 3% milk/TBST ) , mouse anti-Myc antibody ( clone 4A6 , 1:1000 in 3%milk/TBST ) and mouse-anti Pgk1 ( Invitrogen , #459250; 1:20000 in 3%m/TBST ) were used to incubate the Western blots at 4°C overnight or at room temperature for 1 hr .", "Goat anti-mouse IgG HRP ( Dianova , #115–035; 1:10000 in 3%m/TBST ) and goat anti-rabbit IgG-HRP ( Dianova , #111-035-003; 1:10000 in 3%milk in TBST ) were used for a subsequent incubation of 30 min at room temperature .", "All the washing steps were conducted using TBS-Tween 0 . 1% .", "Quantification of prApe1 processing was performed using Analyze Gel tool in ImageJ software .", "Band intensities of pr- and mature Ape1 were measured and the ratio of mature Ape1 to prApe1 was calculated .", "Values calculated for wild type samples were set to 100% .", "At least three independent replicates were considered for each set of mutants tested .", "Yeast wild type and atg5∆ BY4741 strains ( Table", "2 . ) were co-transformed with empty vector ( pRS316 ) or wild type or mutant K57E , N84E Atg5 , together with the GFP-Atg8 expressing plasmid ( SMC199 ) .", "Cells were grown to log-phase in selective medium ( Formedium , UK ) and further grown for an overnight in log phase in the same selective medium .", "After two washes in SD-N , cells were transferred to SD-N ( Formedium , UK ) and subjected to nitrogen starvation for 5 hr .", "Lysates were prepared as described in ´prApe1 processing assay´ and samples were analysed by Western blotting .", "Mouse anti-GFP antibody ( 1:2000 dil . in 3 . 5% milk/PBST , Roche , Germany ) was used for detection of GFP-Atg8 .", "Mouse anti-Myc , anti-Pgk1 antibodies and anti-mouse secondary antibody were used as described in ´prApe1 processing assay´ .", "Yeast strains SMy33 and SMy62 ( Table", "2 . ) were transformed with empty vector pRS316 or pRS316 containing wild type or the mutant Atg5-6xmyc .", "Pre-cultures were grown to log-phase in selective medium and subsequently transferred to complete medium for an overnight-log-culture .", "20 OD-units were taken as Log-aliquots , washed with 0 . 85% NaCl , 1 mM PMSF and frozen in lysis-buffer ( 20 mM PIPES pH6 . 8 , 0 . 5% TritonX100 , 50 mM KCl , 100 mM KAcetate , 10 mM MgSO4 , 10 μM ZnSO4 , 1 mM PMSF , protease inhibitor mix tablet , Roche ) .", "The cultures were exposed to nitrogen-starvation for 5 hr and aliquots were washed and frozen as described above .", "The total proteins were extracted by bead-beating .", "The concentration of the lysate was determined by Bradford .", "All lysates were adjusted with lysis buffer to a final concentration of 500 µg/ml .", "The enzymatic assay was performed using 4-nitrophenol phosphate powder ( Sigma , 71768–5G ) diluted to final concentration of 1 . 25 mM in reaction buffer ( 0 . 4% TritonX100 , 10 mM MgSO4 , 10 µM ZnSO4 , 250 mM TrisHCl , pH 8 . 5 ) as a substrate .", "The formation of the product 4-nitrophenol was measured using a spectrophotometer plate-reader at 405 nm .", "An enzyme blank ( composed of lysate and reaction buffer without substrate ) was measured and subtracted for every sample .", "The molarity of the reaction product was determined with a standard curve using 4-nitrophenol 10 mM solution ( Sigma , N7660-100ML ) .", "An enzyme blank ( containing substrate in reaction buffer and lysis buffer without enzyme ) was subtracted to the standard curve .", "The reaction was stopped at a determined time point ( same for every sample ) between 5 and 25 min with 1M glycine , pH 11 adjusted with 5 M KOH .", "The activity-units ( AU ) were calculated using the following formula:activity [ AU ]=c ( pNP ) [nM]t[ min ]* protein [mg] HeLa cells ( CCL-2 ) were directly purchased from ATCC ( Manassas , Virginia , USA ) and their identity was not authenticated after purchase .", "Cells were routinely tested for mycoplasma contamination by PCR ( GATC , Konstanz , Germany ) and tested negative .", "Cells were cultured in Dulbecco’s modified Eagle medium ( DMEM ) high glucose , GlutaMAX , pyruvate ( Gibco , Waltham , MA , USA ) supplemented with 10% heat inactivated fetal bovine serum ( FBS , Sigma , MO , USA ) , 100 units/mL penicillin and 100 μg/mL streptomycin ( Gibco ) at 37°C and 5% CO2 .", "Cells were used from passage 5 to 20 .", "A list of constructs for transfections can be found in ( Table", "1 . ) .", "HeLa cells were seeded to 6-well plates on day", "1 . Transfection with siRNA against SQSTM1/p62 ( sip62 ) or non-targeting siRNA ( siControl ) was performed on day 2 , transfection with plasmids containing siRNA resistant mCherry-p62 and GFP-ATG5 or GFP was performed on day", "4 . Cells were lysed on day", "5 . For one reaction 50 pmol of sip62 ( J-010230–05 , Dharmacon , Buckinghamshire , UK ) or siControl ( D-001810–10 , Dharmacon ) were pre-incubated with 2 . 5 µl of Lipofectamine RNAimax ( Invitrogen , Waltham , MA , USA ) in 500 µl serum-free medium at RT for 20 min .", "The formed complex was added to cells supplied with 2 ml fresh DMEM containing serum and antibiotics and incubated for two days .", "Thereafter co-transfection of plasmids containing a siRNA resistant p62 variant in pmCherry-C1 ( SMC516 , Wurzer et al . , 2015 ) and Atg5 in pEGFP-C1 ( SMC255 ) or pEGFP-C1 vector only was performed .", "1 to 1 . 5 µg of plasmid-DNA were mixed with Fugene6 ( Promega , WI , USA ) in a 1 μg:3 μl ratio ( DNA:Fugene6 ) in serum-free medium and incubated at RT for 15 to 45 min .", "The mixture was added to cells supplied with 2 ml fresh DMEM containing serum and Pen/Strep and incubated for 24 hr .", "For experiments shown in Figure 1H 2 × 10^5 cells/well were seeded in 6-well plates on day 1 , and transfected with FuGene6 ( Promega ) according to manufacturer’s instructions on day", "2 . 0 . 5 µg of each pmCherry-based vector plus 0 . 5 µg of empty pEGFP-C1 or 1 µg of pEGFP-ATG5 were employed per well , two wells were transfected per condition .", "Cells were lysed as described below on day", "3 . For lysis cells were washed with cold PBS , 100 µl lysis buffer ( 20 mM Tris pH 8 . 0 , 10% glycerol , 135 mM NaCl , 0 . 5% NP-40 , complete protease inhibitor ( EDTA-free , Roche ) , 2 . 5 mM MgCl2 , DNase ) was added per well and lysis performed for 20 min at 4°C .", "Lysates from two wells were pooled , cell debris was removed by centrifugation at 16000 g for 10 min at 4°C and supernatant kept frozen at −80°C until use .", "Lysates for the microscopy based assay needed to be more concentrated and were therefore prepared by trypsinization of cells followed by a PBS wash and lysis of cell pellets from two pooled wells with 100 µl lysis buffer containing 0 . 2% NP-40 .", "The protein concentration in all HeLa cell lysates was measured with Bradford’s method and lysates were normalized to each other accordingly .", "For the pull-down shown in Figure 1F , GFP-Trap_A beads ( ChromoTek ) were mixed with empty Sepharose beads ( Sigma ) in a 1:4 ratio and equilibrated in wash buffer ( 20 mM Tris pH 8 . 0 , 10% glycerol , 135 mM NaCl ) .", "Lysates were diluted with 1 . 5 volumes of wash buffer and incubated with 40 µl equilibrated bead slurry for 1 hr at 4°C with gentle agitation .", "Beads were washed three times with wash buffer , taken up in 40 µl Laemmli loading buffer , boiled 10 min at 95°C and bound proteins were separated by SDS-PAGE and analysed by Western blotting .", "For visualizing protein interaction at equilibrium ( Figure 1G ) 50 µl lysate were incubated with 5 µl equilibrated GFP-Trap_A beads for 1 hr at 4°C .", "15 µl of this bead dispersion were added to 20 µl of the corresponding residual lysate prepared in a 96-well plate and imaged using a spinning disc microscope ( Visitron ) .", "Quantification of microscopy GFP-TRAP experiments ( Figure 1G ) was performed using ImageJ Software .", "A z-projection using the maximum intensity values was generated from every stack .", "Intensity values of 30 pixels along the rim of each bead ( oval selected in the GFP-channel but values measured in the mCherry-channel ) were measured with Oval_Profile . java plugin , averaged and the background from a representative empty area in the image was subtracted .", "For experiments shown in Figures 1H and 5 µL of GFP-TRAP beads ( Chromotek , Germany ) were mixed with 15 µL of empty sepharose 4B beads per each sample , and washed three times in IP wash buffer ( 20 mM Tris pH8 , 135 mM NaCl , 10% glycerol ) .", "15 µL of lysates were taken as input ( approx . 7 . 5% of the total volume ) .", "The remaining lysate was added to the beads and incubated for 1 hr rotating at 4°C .", "Beads were washed three times with IP wash buffer and finally resuspended in 15 µL SDS loading dye .", "Input and bead samples were subjected to Western blot analysis .", "Membranes were ultimately developed with Clarity ECL substrate ( BioRad ) or with SuperSignal West Femto substrate ( ThermoFisher Scientific , Rockford , IL , USA ) if needed .", "The signal was recorded with a ChemiDOC Touch ( Biorad ) imager .", "For quantification of band intensities , only non-saturated exposures were considered , lanes were defined in ImageJ and the lane profile plotted .", "The area under the peak of relevant bands was taken as readout .", "The beads/input enrichment factor ( EF ) was calculated according to the following equation:BEADSGFP−ATG5/BEADSGFPINPUTGFP−ATG5/INPUTGFP*GAPDHGFP−ATG5GAPDHGFP Where BEADS and INPUT indicate the cargo receptor’s band intensity in the respective fraction , GAPDH indicates the band intensity of the anti-GAPDH blot , and GFP-ATG5 and GFP indexes denote the sample .", "Interactions were considered reliable only for EF", "> 2 . The following antibodies were used for detection of proteins in GFP-TRAP experiments on HeLa lysates are .", "Mouse anti-p62 ( BD Bioscences , #610832 , Franklin Lakes , NJ , USA ) was used at 1:1000 dilution; Rabbit anti-NDP52 ( Cell Signaling , #9036 ) was used at 1:1000 dilution; Rabbit anti-OPTN ( Sigma , HPA003279 ) was used at 1:500 dilution; Mouse anti-GFP ( Roche , cat . 11 814 460 001 ) was used at 1:1000 dilution; Mouse anti-GAPDH ( Sigma , Clone GAPDH-71 . 1 ) was used at 1:25000 dilution .", "HRP-conjugated goat anti-Rabbit and anti-Mouse ( Jackson ImmunoResearch , #111-035-003 and 115-035-003 , respectively ) were used at 1:10000 dilution .", "All strains of S . cerevisiae S288C BY474x genetic background are derived from the diploid strain BY4743 and carry the following markers: his3∆1; leu2∆0; met15∆0; ura3∆0 except if stated otherwise .", "ATG5-TAP , ATG5-K57E , N84E-TAP , ATG16-TEV-2xProtA-4xH3-5xHA , ATG17-TEV-2xProtA-4xH3-5xHA strains were generated by homologous recombination of the tagged protein into the respective deletion strains .", "All other strains were generated by crossing of single strains .", "The initial coordinates of Atg5 ( with a part of Atg16 bound to it ) were obtained from the protein data bank ( PDB ) with identifier 2DYO ( Matsushita et al . , 2007 ) .", "The missing loops and atoms were modeled using the SWISS-MODEL server ( Arnold et al . , 2006; Biasini et al . , 2014; Bordoli et al . , 2009 ) .", "The final model contained residues 1–285 of Atg5 and 22–57 of Atg16 ( numbered according to 2DYO ) .", "The protonation states of the histidine residues were determined with the WHATIF server ( Vriend , 1990 ) .", "In order to capture the flexibility of the protein , molecular dynamics simulations were used to generate an ensemble of protein structures .", "These simulations , as well as the preparatory energy minimizations , were performed using GROMOS11 ( Schmid et al . , 2012 ) in combination with the Gromos force field 54a8 ( Reif et al . , 2012 ) .", "Initially , the Atg5-Atg16 complex was minimized in vacuum using steepest descent for 2000 steps .", "Subsequently , the complex was solvated in a rectangular box with 22 , 250 SPC water molecules ( Berendsen et al . , 1981 ) .", "The overall system was electrostatically neutral and therefore no ions were added .", "The solvent configurations were relaxed with another round of energy minimization where the solute atoms were position-restrained .", "Initial random velocities were drawn from a Maxwell-Boltzmann distribution at 50 K . Position restraints on the solute atoms were applied with an initial force constant of 2 . 5 × 104 kJ mol−1 nm−2 .", "With each step of equilibration performed at constant volume , the temperature was increased by 50 K , the force constant of the position restraints was reduced by a factor of 10 and the system was simulated for 20 ps .", "The final equilibration step was simulated for 40 ps at 298 K , without any position restraints .", "After these equilibration steps , the system was simulated for 1 ns at a constant temperature of 298 K using weak coupling at constant volume ( Berendsen et al . , 1984 ) .", "Two separate temperature baths were used for the solute and solvent and the relaxation time was set to 0 . 1 ps .", "All bond lengths were constrained using the SHAKE algorithm ( Ryckaert et al . , 1977 ) with a geometric accuracy of 1 × 10−4 , which enabled the use of a two fs time step .", "The center of mass translation motion was removed every 1000 steps .", "A triple range cut-off scheme was used to calculate the non-bonded interactions and a pair-list was generated every fifth time step .", "Interactions within 0 . 8 nm were calculated at every time step , whereas the interactions between 0 . 8 and 1 . 4 nm were evaluated only when the pair-list was updated and kept constant at intermediate time steps .", "The interactions beyond 1 . 4 nm were approximated by a reaction field contribution , representing a homogeneous medium with a dielectric constant of 61 , as appropriate for SPC water molecules ( Heinz et al . , 2001 ) .", "The molecular dynamics simulation , as described above , was used to obtain different configurations of the Atg5-Atg16 complex that could be used for docking .", "The initial configuration of Atg5-Atg16 , as well as ten snapshots obtained from the simulation ( sampled every 100 ps ) were used to take the flexibility of the proteins into account during the subsequent docking procedure .", "The C-terminal peptide TWEEL of Atg19 was modeled with NH and COO- termini .", "The NH terminus was chosen to represent the NH group of the peptide bond that would be present in the complete Atg19 .", "AutoDock Vina ( Trott and Olson , 2010 ) was used to dock the peptide into the configurations of the Atg5-Atg16 complex .", "The exhaustiveness was set to 50 and the search space was defined such that the whole complex was searched .", "The peptide was completely flexible during the docking process , whereas the Atg5-Atg16 complex was kept rigid .", "For each of the configurations of Atg5-Atg16 , the nine best poses of TWEEL were evaluated .", "The docking results were manually examined in order to discard any poses in which the Thr amino acid of the peptide was completely buried .", "These poses would not be possible with the complete Atg19 and are therefore not of interest .", "Three major interaction sites were found to be reoccurring in multiple snapshots of the Atg5-Atg16 complex .", "One of them involved residues of both Atg5 and Atg16 .", "Since the focus of the present study was identification of the interactions of TWEEL with Atg5 , this interaction site was no longer considered .", "For other binding sites , the hydrogen bonding patterns were examined for all the poses of the docked peptide in all of the configurations of Atg5-Atg16 .", "Generally , several configurations of the peptide were found in each of the binding sites , but here we focused on the residues that were most prone to be involved in salt bridges or hydrogen bonds to the TWEEL peptide .", "The first binding site , which was also found in the initial configuration of the Atg5-Atg16 complex , was characterized by persistent salt bridges and hydrogen bonds between the glutamic acids of the peptide with K57 and K137 .", "In the second binding site , the residues N84 and R208 were the ones that were most often involved hydrogen bonds and salt bridges with TWEEL .", "The Atg8 ( PDB: 2ZPN , ( Noda et al . , 2008 ) and Atg5 ( PDB: 2DYO , ( Matsushita et al . , 2007 ) structures were superposed by secondary-structure matching ( SSM ) ( Krissinel and Henrick , 2004 ) using the Coot software ( Emsley and Cowtan , 2004 ) .", "The Atg8 molecule in complex with Atg19 AIM motif was superposed onto the two ubiquitin-like Atg5 bundles separately .", "The resulting shift of Atg19 AIM motif was depicted omitting the original Atg8 structure for clarity , indicating putative AIM binding pockets on Atg5 ." ] ]

[ "Selective autophagy is mediated by cargo receptors that link the cargo to the isolation membrane via interactions with Atg8 proteins .", "Atg8 proteins are localized to the membrane in an ubiquitin-like conjugation reaction , but how this conjugation is coupled to the presence of the cargo is unclear .", "Here we show that the S . cerevisiae Atg19 , Atg34 and the human p62 , Optineurin and NDP52 cargo receptors interact with the E3-like enzyme Atg12~Atg5-Atg16 , which stimulates Atg8 conjugation .", "The interaction of Atg19 with the Atg12~Atg5-Atg16 complex is mediated by its Atg8-interacting motifs ( AIMs ) .", "We identify the AIM-binding sites in the Atg5 subunit and mutation of these sites impairs selective autophagy .", "In a reconstituted system the recruitment of the E3 to the prApe1 cargo is sufficient to drive accumulation of conjugated Atg8 at the cargo .", "The interaction of the Atg12~Atg5-Atg16 complex and Atg8 with Atg19 is mutually exclusive , which may confer directionality to the system ." ]

[ "A living cell must remove unhealthy or excess material from its interior in order to remain healthy and operational .", "Cells pack this waste into membrane-bound compartments named autophagosomes in a process called autophagy .", "So-called autophagy proteins make sure that only the unwanted material is eliminated .", "However , it was not completely clear how these proteins achieve this .", "What was known was that proteins called cargo receptors recognize and bind to specific waste materials .", "At the same time , so-called autophagy enzymes tag the membranes of the autophagosome with a protein known as Atg8 , so that cargo receptor molecules can bind this membrane .", "Now , Fracchiolla , Sawa-Makarska et al . report that , in yeast , an autophagy enzyme links these two events by binding to the cargo receptor and promoting the tagging of the autophagosome’s membrane at the same place .", "The enzyme in question is a complex made from three autophagy proteins ( called Atg12 , Atg5 and Atg16 ) , and its activity ensures that the membrane is tagged only next to those materials that need to be disposed of .", "Although it is now clearer how a cell’s waste ends up in the autophagosome , it is still puzzling how this process is regulated and how the other autophagy-related components contribute to this highly coordinated process .", "In particular , an important next step will be to find out what is the source of membrane that gives rise to the autophagosome ." ]

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

[ "stem cells and regenerative medicine" ]

[ [ "Cnidarians are renowned for their remarkable ability to regenerate any missing body part .", "Classical work on the freshwater polyp Hydra has shown that both head and foot regeneration can occur without a significant contribution from cell proliferation ( i . e . , through morphallaxis ) ( Park et al . , 1970; Marcum and Campbell , 1978a , 1978b; Cummings and Bode , 1984; Dübel and Schaller , 1990; Holstein et al . , 1991 ) .", "In planarians , by contrast , proliferation of pluripotent stem cells ( called neoblasts ) and formation of a mass of undifferentiated cells ( called blastema ) are required for head , tail , and pharynx regeneration ( Reddien and Sanchez Alvarado , 2004; Baguñà , 2012; Reddien , 2013; Adler et al . , 2014 ) .", "The establishment of a blastema in regeneration has been observed in other taxa including annelid worms ( Bely , 2014 ) and echinoderms ( Candia Carnevali , 2006; Kondo and Akasaka , 2010 ) , but the nature of the cells involved is unclear .", "Urodele amphibians are the only vertebrate tetrapods that can regenerate amputated limbs as adults .", "They are similar to planarians in their requirement for cell proliferation and blastema formation to complete regeneration , but the cellular source for urodele regeneration is different .", "In newts , dedifferentiation of cells in the stump provides progenitor cells , but in the axolotl , resident stem cells fulfill the same task ( Sandoval-Guzman et al . , 2014 ) .", "Furthermore , amphibian blastema cells are lineage restricted rather than being pluripotent ( Kragl et al . , 2009 ) .", "The ability to regenerate varies greatly among animals ( Sánchez Alvarado , 2000; Sánchez Alvarado and Tsonis , 2006; Galliot and Ghila , 2010; Tanaka and Reddien , 2011 ) , with substantial differences sometimes found between closely related taxa: Amphibians , urochordates , planarians , and cnidarians all include both groups or species with excellent regenerative capabilities and their poorly regenerating close relatives ( Sánchez Alvarado , 2000; Galliot and Ghila , 2010 ) .", "One possible explanation for these observations is that the basic genetic toolkit for regeneration is primitive and present in all animals , but that modulation or loss of some components can modify the ability of a given taxon to regenerate .", "This has recently been shown to be the case in planarians where changes in canonical Wnt signaling underlie differences in regenerative ability between closely related species ( Liu et al . , 2013; Sikes and Newmark , 2013; Umesono et al . , 2013 ) .", "Hence , studying regeneration in a broad variety of animal models might reveal both regeneration mechanisms that are primitive and widely shared among animals as well as evolutionarily derived ones and could assist in addressing a major question in regenerative medicine , namely why humans are not capable of regenerating many tissues .", "A specific difficulty in the study of tissue and organ renewal in higher animals is the fact that , like embryonic development , regeneration is a dynamic process .", "Therefore , understanding regeneration requires the analysis of individual cells over long time periods covering the duration of the regenerative process .", "The large size and opaque nature of many animals impede in vivo regeneration research at such resolution in most model organisms .", "In this study , we show that Hydractinia echinata , which is a common colony-forming cnidarian in the European North Atlantic ( Figure 1 ) , provides a powerful model system to study the cellular and molecular basis of animal regeneration .", "Indeed , Hydractinia is easy to culture in the lab and allows whole mount gene expression analysis , cellular analyses , transgenesis , and gene knockdown ( Plickert et al . , 2012 ) .", "The animal reproduces sexually on a daily basis , but also grows clonally by elongation of gastrovascular tubes , called stolons , and asexual budding of new polyps ( Figure 1 ) .", "Finally , they are small and optically translucent , and post-metamorphic animals are sessile and can grow on microscope slides enabling in vivo imaging of cellular processes .", "Like many cnidarians , Hydractinia displays a remarkable regenerative ability and growth plasticity , but the molecular and cellular mechanisms underlying these capabilities are not well understood .", "We have studied both oral ( i . e . , head ) and aboral ( i . e . , stolon ) regeneration in Hydractinia and show that stem cell migration and proliferation underlie head regeneration in this animal .", "Surprisingly , stolon regeneration is achieved through a fundamentally different cellular and morphogenetic process , demonstrating that a single species can apply different mechanisms to regenerate different tissues or body parts . 10 . 7554/eLife . 05506 . 003Figure 1 . Hydractinia life cycle and colony structure . Scale bar 200 μm . DOI: http://dx . doi . org/10 . 7554/eLife . 05506 . 003" ], [ "Our first aim was to characterize Hydractinia head regeneration .", "For this , polyps were isolated from their colony by a transverse cut close to the polyp-stolon boundary and decapitated ( n > 300; Figure 2A ) .", "The remaining cylinder-like body column was then followed until a new head developed ( Figure 2B ) and the animals regained the ability to feed .", "Lesion closure by stretching out of epidermal epithelial cells occurred at both cut ends within 6 hr following decapitation in all cases .", "No further development occurred at the aboral end where the stolons were removed ( see section below ) .", "About 24 hr later , a dome-like tissue appeared at the oral pole .", "This was followed by the development of a new mouth and tentacles 48–96 hr post decapitation ( in over 95% of cases; Figure 2B ) .", "Anti-acetylated tubulin antibody staining confirmed that the nervous system had completely regenerated with both neurons and nematocytes ( cnidarian-specific mechanosensory/effector cells ) appearing normal as in control animals ( Figure 2C–F ) .", "The polyps regained the ability to catch prey about two to 3 days post decapitation when a new head fully formed , but tentacle elongation sometimes continued for an additional few days .", "The time course of head regeneration ( n > 300 ) was variable among polyps , lasting between 1 and 4 days , depending on age ( young post metamorphosis animals may regenerate faster ) , genetic background ( we use a polymorphic wild type laboratory population ) , and general state of health ( malnourished or otherwise unhealthy animals may display delayed regeneration ) .", "No indications for stolon regeneration were observed within the time course of head regeneration .", "Regeneration of decapitated polyps that remained attached to their colonies was indistinguishable from isolated polyps within the natural variability stated above . 10 . 7554/eLife . 05506 . 004Figure 2 . Head regeneration in Hydractinia .", "( A ) Experimental setup .", "( B ) Live images of regenerating polyp .", "Scale bar 100 μm .", "( C–F )", "Anti acetylated tubulin ( green ) —phalloidin ( red ) —DAPI ( blue ) staining of regenerating polyp .", "Asterisks are depicted at approximately the same position in each panel .", "( C ) Intact polyp .", "( D ) 4 hpd .", "( E ) 24 hpd .", "( F ) 72 hpd .", "Nem = nematocyte; Neur = neuron .", "Scale bars 100 μm . DOI: http://dx . doi . org/10 . 7554/eLife . 05506 . 004 Head regeneration after decapitation in the freshwater cnidarian , Hydra , can occur in the absence of cell proliferation ( Park et al . , 1970; Marcum and Campbell , 1978a , 1978b; Dübel and Schaller , 1990; Holstein et al . , 1991 ) .", "We therefore decided to analyze cell proliferation in Hydractinia head regeneration .", "In intact Hydractinia polyps , cell proliferation was almost exclusively restricted to a band at the lower part of the polyp body column , with little or no proliferating cells outside of this band .", "This was shown with both EdU incorporation as well as anti-phospho-Histone 3 ( pH3 ) antibody labeling to visualize mitotic cells ( Figure 3A ) .", "We then decapitated polyps and allowed them to regenerate .", "During the course of regeneration , we incubated the polyps in EdU for 40 min at different times following decapitation .", "Animals were immediately fixed and processed for EdU visualization or anti pH3 staining .", "These experiments showed , first , that wound closure was not associated with enhanced cell proliferation ( Figure 3—figure supplement 1 ) .", "However , a striking shift in the spatial distribution of cycling cells was evident 24–48 hpd ( hours post-decapitation ) .", "In contrast to the lower body column band-fashion distribution of cycling cells in intact polyps , the post decapitation distribution of cycling cells concentrated at the oral pole where the new head was about to form ( n = 100; Figure 3A ) .", "Intact polyps that were labeled by EdU while still connected to their stolonal network showed the same pattern of S-phase cell distribution as polyps with intact heads isolated from their colonies ( n = 20; Figure 3—figure supplement 1 ) .", "Hence , head but not stolon amputation in Hydractinia is followed by the formation of a blastema with highly proliferative cells . 10 . 7554/eLife . 05506 . 005Figure 3 . Cell proliferation during head regeneration .", "( A ) EdU ( upper panes ) and phospho-Histone 3 ( pH3 ) ( lower panes ) labeling of cells .", "Scale bar 200 μm .", "( B ) Maceration of animals and labeling of cycling cells .", "Scale bar 10 μm .", "( C ) Percentages of epithelial and i-cells out of the total mitotic cells .", "( D ) Effect of gamma irradiation on cell proliferation and regeneration .", "( E ) Effect of irradiation on nervous system regeneration .", "Green , anti-acetylated tubulin; red , phalloidin; blue , DAPI . DOI: http://dx . doi . org/10 . 7554/eLife . 05506 . 00510 . 7554/eLife . 05506 . 006Figure 3—figure supplement 1 . EdU labeling of polyps .", "( A ) S-phase cells during wound closure .", "( B ) S-phase cells in a polyps connected to a colony .", "( C ) S-phase cells in a polyp 24 hr post isolation .", "Scale bars 200 μm . DOI: http://dx . doi . org/10 . 7554/eLife . 05506 . 00610 . 7554/eLife . 05506 . 007Figure 3—figure supplement 2 . A selection of dissociated Hydractinia cells . DOI: http://dx . doi . org/10 . 7554/eLife . 05506 . 00710 . 7554/eLife . 05506 . 008Figure 3—figure supplement 3 . Effect of different absorbed doses of gamma irradiation on head regeneration and cell proliferation . DOI: http://dx . doi . org/10 . 7554/eLife . 05506 . 00810 . 7554/eLife . 05506 . 009Figure 3—figure supplement 4 . TUNEL staining of control and irradiated polyps post decapitation . DOI: http://dx . doi . org/10 . 7554/eLife . 05506 . 00910 . 7554/eLife . 05506 . 010Figure 3—figure supplement 5 . Effect of 30 μM mitomycin C on cell proliferation and head regeneration . DOI: http://dx . doi . org/10 . 7554/eLife . 05506 . 010 We then set to address the question of which cell types proliferated during regeneration .", "For this , we decapitated animals and 24 hr later macerated them as previously described ( David , 1973 ) .", "We also macerated intact animals as control .", "Cells were spread on glass slides and stained with anti pH3 antibodies .", "Cnidarians have relatively few cell types including epidermal and gastrodermal myoepithelial cells , several types of neurons , stinging cells ( nematocytes ) , gland cells , and stem cells ( Figure 3—figure supplement 2 ) .", "Hydrozoan stem cells are called interstitial cells , or i-cells for short .", "In Hydractinia , i-cells reside in interstitial spaces between epithelial cells ( mostly epidermal ) , and at a population level are thought to be pluripotent life long , giving rise to all somatic lineages as well as germ cells ( Müller et al . , 2004 ) .", "This is very different from Hydra where i-cells do not contribute to the two self-renewing epithelial lineages ( Bode , 1996 ) .", "Most cell types are easily distinguishable morphologically ( Figure 3—figure supplement 2 , 3B ) .", "We have counted mitotic cells in intact vs regenerating animals 24 hpd in three separate experiments and found that on average epithelial cells , as identified by morphology , form less than 4% of the total mitotic cell complement , whereas the vast majority of S-phase cells are i-cells ( Figure 3B , C ) .", "There was no significant difference in the relative proportion of mitotic epithelial cells in intact vs regenerating animals .", "Hence , i-cells form the major proliferative cellular component in Hydractinia head regeneration , but the contribution of epithelial cells to this process through proliferation and/or transdifferentiation cannot be ruled out based on these data .", "To address the requirement for cell proliferation for head regeneration we exposed Hydractinia polyps to gamma irradiation .", "We first established that absorbed doses of up to 300 Gy did not completely abolish cell proliferation in decapitated polyps ( n = 30; Figure 3—figure supplement 3 ) .", "At 500 Gy , S-phase cells were no longer detectable ( n = 30; Figure 3—figure supplement 3 ) but the animals remained responsive to mechanical stimulation .", "To analyze the direct effect of gamma irradiation and assess cell death by apoptosis we performed TUNEL staining on intact and decapitated animals that had or had not been irradiated .", "We found small numbers of apoptotic cells in non-irradiated animals between 6 and 24 hpd ( n = 30 ) .", "This is consistent with previous work on other animals , where apoptotic cells were shown to play a role in the regenerative process ( Hwang et al . , 2004; Tseng et al . , 2007; Chera et al . , 2009; DuBuc et al . , 2014 ) .", "In irradiated animals , the distribution of apoptotic cells was similar to the distribution of proliferating i-cells in non-irradiated animals ( n = 30; Figure 3—figure supplement 4; compare with Figure 3A ) .", "We concluded that cycling i-cells are sensitive to gamma irradiation .", "We then irradiated animals at 500 Gy followed by decapitation , and returned them to their culture tanks .", "None of the animals regenerated at 48 hr following this treatment ( n = 30; Figure 3D , E; Figure 3—figure supplement 3 ) .", "Animals irradiated at 300 Gy or lower did regenerate , but at a markedly slower pace ( n = 30; Figure 3—figure supplement 3 ) .", "Importantly , the lack of EdU incorporation showed that no proliferative blastema developed at the oral pole of animals irradiated at 500 Gy ( Figure 3D ) .", "Treatment with mitomycin C , a cytostatic drug that was shown to kill i-cells in Hydractinia ( Müller et al . , 2004 ) , had similar effects , with animals failing to regenerate following treatment ( Figure 3—figure supplement 5 ) .", "Therefore , cell proliferation and blastema formation are essential for Hydractinia head regeneration .", "This is markedly different from Hydra head regeneration , which can occur through morphallaxis in the complete absence of cycling cells .", "Our next step was to study gene expression during regeneration .", "We focused on Piwi1 , Vasa , Myc2 and Pl10 , all standard stem cell markers in cnidarians and other metazoans ( Reddien et al . , 2005; Rebscher et al . , 2008; Voskoboynik et al . , 2008; Alie et al . , 2011; Collins et al . , 2013; Juliano et al . , 2014 ) .", "In addition , we studied Ncol1 , an early nematocyte differentiation marker in cnidarians ( David et al . , 2008; Millane et al . , 2011 ) .", "We first established the normal expression pattern in intact polyps using in situ hybridization .", "As shown for Vasa previously ( Rebscher et al . , 2008 ) , all i-cell marker genes were expressed in a band fashion at the lower part of the polyp body column , co-localizing with S-phase and mitotic cells ( Figure 4A , B; compare with Figure 3A; n = 40 ) . 10 . 7554/eLife . 05506 . 011Figure 4 . Gene expression during head regeneration .", "( A ) Expression of i-cell marker genes in intact polyps .", "Scale bar 200 μm .", "( B ) Higher magnification of positive cells in the band .", "Scale bar 10 μm .", "( C ) Expression of marker genes 24 hr post decapitation .", "( D ) Higher magnification of blastema cells expressing i-cell marker genes .", "( E ) Effect of gamma irradiation on i-cell marker gene expression .", "( F and G )", "Downregulation of marker genes during regeneration by RNAi .", "( F ) Effect on regeneration and quantitative analysis of knockdown .", "( G ) Effect on cell proliferation . DOI: http://dx . doi . org/10 . 7554/eLife . 05506 . 01110 . 7554/eLife . 05506 . 012Figure 4—figure supplement 1 . Effect of histones H2A and H4 knockdown and quantification of Piwi1 mRNA following RNAi knockdown .", "( A ) Control RNAi .", "( B ) Histone H2A RNAi .", "( C ) Histone H4 RNAi .", "( D ) Relative quantity of Piwi1 expression in control vs Piwi1 RNAi .", "Expression levels are normalized to 18S rRNA .", "Error bars = SD . DOI: http://dx . doi . org/10 . 7554/eLife . 05506 . 012 We then decapitated polyps and fixed them at different time points during head regeneration and performed in situ hybridization with cRNA probes for i-cell genes .", "We found that decapitation had a major effect on the distribution of i-cells .", "Instead of being restricted to the band area , we now saw i-cells at more oral positions .", "Most strikingly , about 24 hpd , coinciding with the major proliferative peak in the blastema , there was strong expression of i-cell markers and Ncol1 in the blastema ( n = 40; Figure 4C , D ) .", "Hence , the blastema that forms during head regeneration at the oral pole contains large numbers of i-cells and early nematoblasts in contrast to the absence of these cells in oral areas in intact animals .", "Irradiation of animals at 500 Gy resulted in a marked reduction of i-cells and nematoblast marker expression at 24 hr post decapitation ( n = 10; Figure 4E ) .", "This further shows that i-cells are sensitive to irradiation and are required for head regeneration .", "To study the role of stem cell genes in head regeneration we used RNAi to knockdown Piwi1 , Vasa , Pl10 and Ncol1 .", "RNAi was performed as previously described ( Duffy et al . , 2010 , 2011; Millane et al . , 2011 ) .", "Briefly , polyps were removed from their colony and were then decapitated .", "Following decapitation the cylindrical body columns were incubated in seawater containing 30–50 ng/μl double stranded RNA ( dsRNA ) corresponding to 200 bp coding sequence of Piwi1 , Vasa , Pl10 and Ncol1 .", "Control experiments were performed with dsRNA corresponding to the backbone sequence of the pBlueScript cloning vector , which is not encoded by the Hydractinia genome .", "qPCR analysis of Piwi1 RNAi treated regenerating animals revealed a significant reduction in Piwi1 mRNA levels comparing to control RNAi ( Figure 4—figure supplement 1 ) .", "Regeneration in animals in which Piwi1 , Vasa , Pl10 or Ncol1 were knocked down was compromised , with the frequency of regenerating RNAi animals significantly different from the control RNAi ( chi-square test , p = 0 . 00001; Figure 4F ) .", "Animals treated with control ( i . e . , non-coding ) dsRNA regenerated normally .", "The experiments were repeated four times with 10 animals for each treatment .", "To address the role of these genes in i-cell proliferation , we treated regenerating animals with dsRNA as described above .", "Twenty-four hours after decapitation we incubated them in EdU for 20 min followed by fixation and EdU visualization .", "We found that knockdown of Piwi1 , Vasa , Pl10 , or Ncol1 had no visible effect on cell proliferation ( Figure 4G ) .", "In these RNAi animals the head blastema formed normally yet regeneration was significantly affected , suggesting that these genes are required for differentiation .", "The specificity of the RNAi treatment was further confirmed by knockdown of histones H2A or H4 which strongly reduced EdU incorporation in regenerating animals ( Figure 4—figure supplement 1 ) .", "Hence , Piwi1 , Pl10 , and Vasa expression is not required for i-cell proliferation and for the formation of the head blastema .", "Ncol1 , which is a nematogenesis marker , is not expressed in cycling i-cells and its downregulation is therefore not expected to affect S-phase cells .", "The experiments described above show that decapitation results in head blastema formation that includes numerous proliferating i-cells .", "In fully grown , intact animals , i-cells are more or less restricted to the band area in the lower polyp body column , and are nearly absent from the head , which also does not include significant numbers of proliferating cells ( n = 75/83 ) .", "We therefore reasoned that in the near absence of resident stem cells in the intact head ( Figure 4A ) , the source of stem cells in the head blastema could either be migration , that is , i-cells moving from the band in the lower polyp body column , or dedifferentiation of local differentiated cells in the stump .", "To discriminate between these two options , we performed two types of experiments .", "First , we pulse-labeled intact polyps with EdU for 60 min , followed by decapitation .", "The animals were intensively washed to remove EdU and left to initiate head regeneration .", "They were fixed and processed for EdU visualization at 6 and 24 hpd .", "We found that animals fixed 6 hpd had a strong signal in the band at the lower body column and only a few EdU+ cells were scattered at more oral positions ( Figure 5A ) .", "However , those animals fixed 24 hpd had also a strong EdU signal in the developed blastema ( Figure 5B; Figure 5—figure supplement 1 ) .", "Because no EdU could be incorporated after washing the animals , the EdU+ cells in the blastema must have been the very same cells that were in S-phase in the lower band 24 hr earlier and had migrated to the stump following decapitation .", "Analysis of pH3 immunoreactivity in EdU pulse chased animals revealed double positive cells in the blastema , indicating that S-phase cells in the band continued to proliferate even after reaching the stump and contributed new cells to the blastema through mitosis ( Figure 5—figure supplement 1 ) . 10 . 7554/eLife . 05506 . 013Figure 5 . The cellular source for head regeneration .", "( A and B )", "EdU pulse-chase .", "( A ) 6 hpd—most EdU+ cells restricted to the band .", "Scale bar 50 μm .", "( B ) 24 hpd—many EdU+ cells migrated to the blastema .", "( C–G )", "Live images of Piwi1-GFP+ transgenic cells .", "( C ) Transgenic Piwi1-GFP+ polyp .", "Scale bar 200 μm .", "( D ) Higher magnification of live GFP+ i-cells in vivo .", "Scale bar 10 μm .", "( E ) Live transgenic polyp pictured at 10 ( left ) and 24 ( right ) hpd .", "( F ) Live images of a single , Piwi1-GFP+ i-cell migrating to the forming blastema ( arrow ) .", "( G ) Live image of a single , Piwi1-GFP+ i-cell dividing during migration ( encircled ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 05506 . 01310 . 7554/eLife . 05506 . 014Figure 5—figure supplement 1 . Proliferation of migrating cells . Animals were EdU pulse labeled for 60 min and fixed either intact or at different time points post decapitation .", "They were then stained with anti-pH3 antibody .", "( A–D )", "Intact animals .", "( E–H ) 24 hpd .", "( I–P ) 48 hpd .", "( A–L ) represent projections of multiple confocal stacks; ( M–P ) are single confocal sections .", "Scale bars 100 μm ( A–L ) and 10 μm ( M–P ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 05506 . 01410 . 7554/eLife . 05506 . 015Figure 5—figure supplement 2 . The structure of the construct used to generate transgenic , Piwi1-GFP+ animals . DOI: http://dx . doi . org/10 . 7554/eLife . 05506 . 01510 . 7554/eLife . 05506 . 016Figure 5—figure supplement 3 . Piwi1 immunohistochemistry and co-localization of gene expression and S-phase cells .", "( A ) Co-staining of anti-Piwi1 antibody staining ( green ) and DAPI ( blue ) .", "Scale bar 200 μm .", "( B ) Co-staining of anti-Piwi1 antibody staining ( green ) and EdU ( red ) .", "Scale bar 20 μm .", "( C ) Co labeling of Ncol1 ( blue ) and EdU ( green ) .", "Scale bar 20 μm . DOI: http://dx . doi . org/10 . 7554/eLife . 05506 . 016 Second , to gain a more dynamic view on i-cell migration during regeneration we established transgenic animals expressing GFP under the Hydractinia endogenous Piwi1 genomic control elements .", "For this , we cloned 2 . 5 kb upstream and 1 . 1 kb downstream of the Piwi1 genomic coding sequence locus and inserted the GFP coding sequence instead of Piwi1 ( Figure 5—figure supplement 2 ) .", "This construct was microinjected to one-cell stage embryos as described previously ( Künzel et al . , 2010 ) .", "Genomic integration and stable GFP expression occurs in Hydractinia within 24 hr ( Künzel et al . , 2010 ) .", "GFP expression in transgenic polyps ( Figure 5C , D ) was consistent with the endogenous expression of Piwi1 as assessed by in situ hybridization ( Figure 4A ) and immunohistochemistry ( Figure 5—figure supplement 3 ) .", "Because genomic integration does not occur in all cells , the animals were mosaics and not all Piwi1+ i-cells expressed GFP .", "This feature was useful because the density of GFP+ cells was not as high as would be expected in a fully transgenic animal , facilitating tracking of single cells in vivo ( Figure 5C , D ) .", "Transgenic polyps were isolated from their colonies by a transverse cut close to the polyp-stolon boundary .", "Polyps were decapitated and then viewed while the head blastema was developing over several hours using a fluorescence stereomicroscope ( Figure 5C ) , time-lapse DeltaVision deconvolution microscope ( Figure 5F; Video 1 ) , or Andor spinning disk confocal microscope ( Figure 5G; Video 2 ) .", "Piwi1+ cells were observed migrating into the prospective head area ( Figure 5E–G; Videos 1 , 2 ) and no evidence for dedifferentiation was evident as all viewed recruited GFP+ cells at the blastema were migratory .", "Some migrating cells underwent mitosis before reaching the blastema; they stopped migrating , completed mitosis , and the two daughter cells resumed migration ( Figure 5G; Video 2 ) .", "Based on these experiments and the EdU pulse-chasing , we conclude that the primary cellular source for establishing the head blastema and , subsequently , head regeneration is migration of i-cells from the band in the polyp lower body column to the prospective head area .", "A possible contribution of existing epithelial cells to the regeneration process through mitosis and/or transdifferentiation ( body column epithelial cells to tentacle epithelial cells ) cannot be ruled out . 10 . 7554/eLife . 05506 . 017Video 1 . Follow up of individual cells migrating to forming blastema . DOI: http://dx . doi . org/10 . 7554/eLife . 05506 . 01710 . 7554/eLife . 05506 . 018Video 2 . Follow up on proliferating cell migrating to blastema . DOI: http://dx . doi . org/10 . 7554/eLife . 05506 . 018 Hydractinia polyps are not able to regenerate stolons directly from their aboral ends ( Müller et al . , 1986 ) .", "Polyps , or even fragments of them , can , instead , transform into stolons but this phenomenon is not well understood ( Putnam Hazen , 1902; Müller et al . , 1986; Lange and Müller , 1991 ) .", "To study aboral , that is , stolon , regeneration we removed polyps from their colony by a transverse cut at the lower third of the polyp body column to exclude any stolonal tissue .", "Isolated polyps healed the cut surface within hours ( Figure 6A ) .", "We then followed the polyps and photographed them every 24 hr for up to 25 weeks .", "The polyps appeared normal for days and sometimes weeks , responded to mechanical stimuli by contraction , and were able to catch , kill , and ingest brine shrimp nauplii , resembling a solitary Hydra polyp .", "No blastema developed at the aboral pole after stolon removal , and the general distribution of EdU+ cells was largely similar to polyps that were labeled on their colony .", "( Figure 3—figure supplement 1 ) .", "Over the next weeks , however , the polyps started resorbing their tentacles and thereby lost the ability to feed ( Figure 6A ) .", "Next , the entire head structure disappeared and each polyp's cylindrical body column started to elongate , thereby decreasing its diameter ( Figure 6A ) .", "New branches appeared at irregular intervals and some developed into polyps with fully functional heads and tentacles , thereby regaining the ability to feed ( n = 200; Figure 6B ) .", "Chitin secretion ( which is stolon specific in Hydractinia ) ( Lange and Müller , 1991 ) commenced ( Figure 6C ) and eventually , sexual polyps developed and produced fertile gametes ( Figure 6D ) .", "Based on chitin secretion and ability to generate polyps , it appeared that the polyp body column had transformed into stolons rather than regenerated new stolons from the aboral stump . 10 . 7554/eLife . 05506 . 019Figure 6 . Stolon regeneration .", "( A ) Time course of a single polyp transforming into a stolon and budding new polyps .", "Scale bar 200 μm .", "( B ) Sexually mature colonies derived from an isolated polyp .", "( C ) Chitin secretion ( arrow ) by a polyp that has transformed into a stolon .", "( D ) Early embryos spawned by colony derived from a single polyp .", "( E ) Loss of oral Wnt3 in polyp transforming into a stolon .", "( F ) Oral Wnt3 expression in newly bud polyps ( arrows ) .", "( G ) Stolon-like expression of i-cell markers in transformed polyps . DOI: http://dx . doi . org/10 . 7554/eLife . 05506 . 01910 . 7554/eLife . 05506 . 020Figure 6—figure supplement 1 . Expression of i-cell markers in stolons and Wnt3 in a primary polyp . Colonies are growing on glass cover slips and images are taken from below , except of Wnt3 that was taken from above .", "Scale bar 100 μm . DOI: http://dx . doi . org/10 . 7554/eLife . 05506 . 02010 . 7554/eLife . 05506 . 021Figure 6—figure supplement 2 . Expression of i-cell and nematogenesis markers in polyps that had transformed into stolons . DOI: http://dx . doi . org/10 . 7554/eLife . 05506 . 021 To characterize the molecular events associated with the transformation of polyps to stolons we first studied the expression of Wnt3 during this process .", "Wnt3 is an established oral marker in Hydractinia ( Plickert et al . , 2006; Müller et al . , 2007; Duffy et al . , 2010 ) , but is also expressed weakly in the i-cell band of polyps ( Plickert et al . , 2006 ) .", "We found that polyp heads that were losing their tentacles had also lost Wnt3 expression ( Figure 6E ) .", "By contrast , Wnt3 mRNA reappeared in the oral tip of new polyps budding from this newly transformed tissue ( Figure 6F ) .", "The transformed polyp expressed the gene in a more ubiquitous fashion ( see below ) .", "Hence , loss of oral Wnt3 expression preceded , or accompanied , the loss of head characteristics such as tentacles and mouth .", "To further analyze the polarity of polyps that transformed to stolons we performed in situ hybridization using i-cell marker cRNA probes .", "In normal , non-regenerating polyps , i-cells were largely restricted to the band area at the polyp lower body column ( Figure 4A ) .", "In stolons , by contrast , i-cells were equally distributed in the epidermal interstices along the stolon flanks ( Figure 6—figure supplement 1 ) .", "Hence , we hypothesized that a polyp to stolon transformation should be reflected by a spatial change in i-cell marker expression from restricted band-like to ubiquitous .", "Indeed , in situ hybridization of Piwi1 , Vasa , Pl10 , Myc2 and Ncol1 on isolated polyps that lost head structures showed a stolon-like expression pattern of these genes ( Figure 6G; Figure 6—figure supplement 2 ) .", "To summarize this point , Hydractinia aboral regeneration is not direct and proceeds through three stages: First , loss of anterior-posterior polarity; second , full transformation of the polyp into a stolon; third , budding new polyps and regaining oral-aboral polarity .", "Hence , Hydractinia polyps can regenerate a head through i-cell migration and blastema formation , but they cannot directly regenerate stolons; they can transform into stolonal tissue instead ." ], [ "We have studied both head and stolon regeneration in the cnidarian Hydractinia .", "Decapitation was followed by rapid wound healing that primarily involved stretching of epithelial cells without the requirement for cell proliferation ( Figure 3—figure supplement 1 ) .", "Thereafter , we monitored the migration of stem cells ( i-cells ) from their normal position in the band area at the lower polyp body column to the prospective head , and their proliferation to form a head blastema .", "Of note , our data show that not all i-cells migrate to the stump , consistent with migratory vs non-migratory i-cell sub-populations .", "New head structures developed within 2–3 days , after which most i-cells disappeared from the head area and resumed their normal position in the band .", "Gamma irradiation abolished blastema formation and regeneration altogether .", "By contrast , individual knockdown of each one of the i-cell genes Piwi1 , Vasa and Pl10 , and the early nematogenesis marker Ncol1 did not prevent blastema formation , but did inhibit regeneration .", "Hence , the role of these genes might be related to the ability of i-cells to differentiate rather than to keeping them undifferentiated .", "Similar results were obtained with Smedwi2 ( a Piwi homologue ) knockdown in planarians ( Reddien et al . , 2005 ) , but knocking out a different stem cell gene , Sox2 , in axolotl inhibits proliferation of neural progenitors ( Fei et al . , 2014 ) .", "A common view in the literature has been that cnidarians can regenerate through morphallaxis , that is , without contribution from cell proliferation ( Park et al . , 1970; Marcum and Campbell , 1978a , 1978b; Holstein et al . , 1991 ) .", "More recent studies conducted on Hydra and on the sea anemone Nematostella vectensis , however , have shown that cell proliferation accompanies the regeneration of cnidarian heads under normal circumstances ( Govindasamy et al . , 2014 ) , but the necessity of proliferation was only demonstrated in Nematostella decapitation and Hydra bisection ( Miljkovic-Licina et al . , 2007; Chera et al . , 2009; Passamaneck and Martindale , 2012; DuBuc et al . , 2014 ) .", "Our results support the new emerging view on head regeneration in the Cnidaria and are consistent with Passamaneck and Martindale's hypothesis ( Passamaneck and Martindale , 2012 ) that Hydra's ability to regenerate a head in the absence of cell proliferation is evolutionarily derived within this phylum .", "This scenario suggests that blastema formation is an evolutionarily primitive hallmark of distal regeneration in animals .", "Isolated polyps were unable to directly regenerate stolons from their aboral end like they do following decapitation from the oral end , consistent with previous studies ( Putnam Hazen , 1902; Müller et al . , 1986; Duffy et al . , 2010 ) , and no blastema formed at the aboral stump following removal of the stolons ( Figure 3—figure supplement 1 ) .", "Instead of regenerating stolons , isolated polyps lost oral-aboral polarity , and polyp identity altogether , and transformed into stolons .", "This process lasted for many weeks , thereby demonstrating their remarkable growth plasticity .", "Polyp to stolon transformation was preceded by loss of oral Wnt3 expression and oral-aboral polarity , and acquisition of a ubiquitous , stolon-like distribution of i-cells , as opposed to the band like distribution typical of polyps .", "The newly transformed stolons budded new polyps and became fully functional , sexually mature colonies .", "These data , therefore , show that Hydractinia polyps possess tissue pluripotency .", "For now , however , we cannot discriminate between the scenarios of pluripotent i-cells vs several self-renewing , but lineage restricted , progenitors .", "So why do polyps not directly regenerate stolons ?", "We suggest that tissue polarity along the oral-aboral axis prevents direct stolon regeneration .", "In a previous study , it has been shown that Wnt signaling promotes oral structures in Hydractinia , but represses stolons ( Duffy et al . , 2010 ) .", "Downregulation of Wnt3 or Tcf in decapitated polyps induces phenotypes reminiscent of polyp to stolon transformation in the present study , but requires shorter time to develop ( Duffy et al . , 2010 ) .", "We show that in the absence of experimental manipulation , Wnt3 expression and oral-aboral polarity are lost spontaneously in isolated polyps , enabling the transformation of the polyp into stolonal tissue that can bud new polyps .", "Possibly , Wnt3 not only maintains oral- but also polyp- identity , and stolons develop by default in its absence .", "A summary of the two distinct regenerative processes in Hydractinia is schematically illustrated on Figure 7 . 10 . 7554/eLife . 05506 . 022Figure 7 . A summary of Hydractinia regeneration . Red dots represent proliferating i-cells .", "( A ) A schematic of a normal colony including stolons , feeding and sexual polyps .", "( B ) Head regeneration .", "Isolation of a polyp from the colony and its decapitation result in migration of i-cells to the head stump but not to the stolon stump .", "A head blastema , but not stolon blastema , is formed and provides the progenitors for the new head .", "( C ) Transformation of polyps to stolons involves loss of polarity and ubiquitous spread of i-cells . DOI: http://dx . doi . org/10 . 7554/eLife . 05506 . 022 In conclusion , our results , and results published over the past few years by others ( Chera et al . , 2009; Kragl et al . , 2009; DuBuc et al . , 2014; Sandoval-Guzman et al . , 2014 ) , show that the mechanisms governing animal regeneration can be not only species-specific , but also tissue-specific within a single species .", "Some regeneration mechanisms , like blastema formation , are conserved in animals , and their modulation over evolutionary times may have affected the regenerative ability of different species .", "An exciting development in the study of regeneration is provided by the ability to track individual , transgenic cells in vivo using Hydractinia as an animal model .", "In vivo cell migration assays have been performed in Hydra ( Khalturin et al . , 2007 ) , but the sessile nature of adult Hydractinia facilitates long-term studies at single cell resolution ." ], [ "Colonies of H . echinata were cultured in artificial seawater at 18°C under 14/10 light/dark regime .", "They were fed brine shrimp nauplii four times a week and ground fish once a week .", "Animals were anesthetized for 30 min in 4% MgCl in seawater .", "They were then placed in a Glycerin/Acetic acid/Seawater ( 1:1:13 ) solution for 10 min , followed by incubation in Glycerin/Acetic acid/Distilled Water ( 1:1:13 ) for 2 hr .", "They were then pipetted up and down to complete maceration and fixed in 8% formaldehyde for 30 min .", "Cells were spread on a glass slide and dried overnight .", "Cell counting of macerated cells was performed by eye using a FV1000 Olympus confocal scanning laser microscope .", "EdU incorporation was performed for 20–60 min at a concentration of 150 μM .", "For visualization , animals were fixed and processed using the Click-iT EdU Alexa Fluor 488 Imaging Kit ( Life Technologies , Dun Laoghaire , Co Dublin , Ireland ) according to the manufacturer's protocol .", "Gamma-irradiation was carried out using a 137Cs source at a dose-rate of 12 Gy/min .", "TUNEL staining was complete as per the manufacture's protocol ( Life Technologies ) .", "Click-iT TUNEL Alexa Fluor 488 Imaging Assay , Cat: 10245 .", "Animals were fixed in 4% paraformaldehyde in PBS for 60 min and then washed three times in phosphate buffered saline - 0 . 3% Triton ( PBST ) and blocked for 30 min in 2% BSA/PBST .", "Primary antibodies ( anti-Hiwi [a kind gift from Dr Celina Juliano , Yale University] , anti acetylated tubulin [T7451 , Sigma] , anti-phospho H3 [ab5176 Abcam] ) were diluted 1:500–1:2000 in BSA/PBST and incubated overnight at 4°C , followed by three washes with PBST then blocked for 30 min in 5% serum in BSA/PBST .", "Secondary antibodies ( Alexa Fluor 488 goat anti-rabbit IgG [A-11008 , Invitrogen] , Alexa Fluor 546 Phalloidin LifeTec [A22283] ) were diluted 1:500 in BSA/PBST/serum and incubated for 1 hr at room temperature .", "Animals were washed three times with PBST , incubated in 1:2000 Hoescht ( 20 mg/ml ) , DAPI ( 1:5000 ) or phalloidin ( 1:2000 ) in PBST and washed a further three times in PBST .", "Animals were mounted in mounting medium ( F4680 , Sigma ) .", "In situ hybridization was performed as previously described ( Gajewski et al . , 1996 ) .", "Templates for DIG-labeled RNA probes ( Roche ) were generated by PCR .", "RNA synthesis was performed by SP6 and T7 RNA polymerases according to the manufacturer's protocol ( Fermentas ) .", "The sequence of the oligonucleotides is given in Table 1 .", "In situ hybridization was performed at 55°C . 10 . 7554/eLife . 05506 . 023Table 1 . Oligonucleotides used in this studyDOI: http://dx . doi . org/10 . 7554/eLife . 05506 . 023TargetAccessionPrimer namePrimer sequencePiwi1JG772275 . 1CniwiFort75′-gatcataatacgactcactatagggagagttgatttcacaatcggttagac-3′CniwiRevSp65′-agtgcatttaggtgacactatagaagtgtactactactactactggttattt-3′PiwiIRNAiT7Fw5′-gcgtaatacgactcactatagggagagctgtgtggaaagaccagtc-3′PiwiIRNAiSP6Rv5′-tgcatttaggtgacactatagaagtgcgtcaaatccaatcaccatc-3′Piwi1qPCRfw5′-aagtatggcctggcatctca-3′Piwi1qPCRrv5′-cactgtctgctgtcgtaaaacc-3′Piwi1qPCRprobe5′-tgcagtatgaacaagatgtgatgttgtgtgctgatgttcag-3′Pl10AB048849 . 1Pl10ForT75′-gatcataatacgactcactatagggagattctggcaaaacagctgcattt-3′Pl10RevSP65′-agtgcatttaggtgacactatagaagtgagcttcatccagacaaagaaac-3′Pl10rnaiFWT75′-gcgtaatacgactcactatagggagagcgtaacacccattttg-3′PL10rnaiRVsp65′-tgcatttaggtgacactatagaagtgtaaatcacgcgca-3′Ncol1JX486117 . 1Ncol1-T7fwd5′-gatcataatacgactcactatagggacgtccaggaccaccaggagta-3′Ncol1-Sp6rev5′-tagcaatttaggtgacactatagaactgggcaacagtattgtggacaaga-3′VasaEF467228 . 1HeVASAforT75′-taatacgactcactatagggagaaggttcaaagtggttgccattt-3′HeVASArevSP65′-atttaggtgacactatagaagagtactgccaactttaccaat-3′VasaRNAiFWT75′-gcgtaatacgactcactatagggagagttgaaatgctgggacaagaagg-3′VasaRNAiRVsp65′-tgcatttaggtgacactatagaagtggcggtagcgataagaacagtc-3′Wnt3AM279678 . 1Wnt3ForwardPrimerT75′-gatcataatacgactcactataggggagtccgccttcattagtgg-3′Wnt3ReversePrimerSP65′-tagcaatttaggtgacactatagaatgggcggagtcgtatctatc-3′cMycJF820068 . 1cMycInSituFWt75′-gatcataatacgactcactatagggcctttaacgcctcccagttct-3′H2AH2aRNAiFwd1T75′-gatcataatacgactcactatagggatgtctggacgtggaaaagg-3′H2aRNAiRv1SP65′-tagcaatttaggtgacactatagaaccaatatctcagcagataaatattccaag-3′H2aRNAiFwd2T75′-gatcataatacgactcactataggggagttggctggtaacgcag-3′H2aRNAiRv2SP65′-tagcaatttaggtgacactatagaaacttcttctgtcctttgtcgttct-3′H4H4RNAiFwd1T75′-gatcataatacgactcactatagggatgtctggacgcggaaaag-3′H4RNAiRv1SP65′-tagcaatttaggtgacactatagaactttagtacacctctggtttcctc-3′H4RNAiFwd2T75′-gatcataatacgactcactataggggtcaaacgtatctctggccttat-3′H4RNAiRv2SP65′-tagcaatttaggtgacactatagaaaacctccgaatccgtaaagag-3′cMycInsituRVsp65′-agtgcatttaggtgacactatagaattgttaacggaaaagggaaaactg-3′PlasmidgfpRv-SAC15′-aaaaagagctcctatttgtatagttcatccatgccatg-3′TerminatorFw-PacI5′-aaaaattaattaacgtacgggccctttcgtct-3′RaceNewsplicedleaderFwd5′-tactcacactatttctaagtccctgagtttaag-3′PiwiRV2SP65′-tagcaatttaggtgacactatagaaccttagcgccacctgtgc-3′CloningLigDVectorGFP-Fusion5′-gcggccgctgcagccccggt-3′BackbonelactRV15′-actggccgtcgttttacaac-3′Piwi1ProFw1new5′-cagatgatccgcagacaatagac-3′Piwipromrevin5′-gttttcttcttataatttttctaaaaactt-3′PiwiRv2SP65′-tagcaatttaggtgacactatagaaccttagcgccacctgtgc-3′Piwi1TerRV1-PacI5′-aaaaattaattaagaaggcttacgctagtgtgaattag-3′Piwi1TerFw1-SAC15′-aaaaagagctcgtagctgcgcgttgtttacg-3′GFPSeqFusRev5′-ttgcatcaccttcaccctctcc-3′PBIGFor5′-taaaaataggcgtatcacgaggccc-3′ Animals were embedded in Ibidi ( Martinsried , Germany ) μ-dish ( #81156 ) using 1% low-melt agarose in seawater .", "They were observed using either a fluorescence stereomicroscope , DeltaVision deconvolution microscope , or Andor spinning disc confocal microscope .", "For time-lapse videos , images or stacks were taken every 5 min .", "RNAi was performed as previously described ( Duffy et al . , 2010; Millane et al . , 2011; Duffy , 2012 ) .", "Templates for RNA synthesis were generated by PCR ( see oligonucleotide list on Table 1 ) .", "Sense and antisense RNA strands were generated as for in situ hybridization but were annealed by heating them together to 70°C and allowing them to cool down at room temperature .", "Animals were incubated in seawater to which dsRNA at 20–40 μg/ml was added directly after decapitation .", "The experiments run until the control animals had regenerated ( usually between 3–5 days ) .", "dsRNA solution was replaced every 24 hr . mRNA was extracted using standard Trizol/chloroform extraction technique and cleaned over RNeasy minikit ( 74104; Qiagen ) according to the manufacturer's protocol .", "RNA was reverse transcribed using Omniscript RT kit ( 205110; Qiagen ) .", "qPCR was run on a StepOne Plus ( Life Technologies ) using TaqMan chemistry .", "Experiments were performed on three colonies using three technical replicates for each .", "We cloned the genomic regions 2 . 5 kb upstream and 1 . 1 kb downstream of the Hydractinia Piwi1 coding sequence into a modified pBluescript backbone ( Künzel et al . , 2010 ) and replaced the coding sequence by GFP .", "( Figure 5—figure supplement 2 ) .", "One-cell stage embryos were microinjected with 200 pl volume of the plasmid at a concentration of 4–5 μg/μl as previously described ( Künzel et al . , 2010; Millane et al . , 2011; Duffy , 2012; Kanska and Frank , 2013 ) ." ] ]

[ "Cnidarians possess remarkable powers of regeneration , but the cellular and molecular mechanisms underlying this capability are unclear .", "Studying the hydrozoan Hydractinia echinata we show that a burst of stem cell proliferation occurs following decapitation , forming a blastema at the oral pole within 24 hr .", "This process is necessary for head regeneration .", "Knocking down Piwi1 , Vasa , Pl10 or Ncol1 expressed by blastema cells inhibited regeneration but not blastema formation .", "EdU pulse-chase experiments and in vivo tracking of individual transgenic Piwi1+ stem cells showed that the cellular source for blastema formation is migration of stem cells from a remote area .", "Surprisingly , no blastema developed at the aboral pole after stolon removal .", "Instead , polyps transformed into stolons and then budded polyps .", "Hence , distinct mechanisms act to regenerate different body parts in Hydractinia .", "This model , where stem cell behavior can be monitored in vivo at single cell resolution , offers new insights for regenerative biology ." ]

[ "Although all animals are capable of regenerating damaged tissue to some extent , a few—including jellyfish , coral , and their relatives—are able to regenerate entire lost body parts .", "Closely related species may have very different regeneration capabilities .", "This has led some researchers to propose that higher animals , such as mammals , still possess the ancient genes that allow entire body parts to regenerate , but that somehow the genes have been disabled during their evolution .", "Studying animals that can regenerate large parts of their bodies may therefore help scientists understand what prevents others , including humans , from doing so .", "An animal that is particularly useful for studies into regeneration is called Hydractinia echinata .", "These tiny marine animals make their homes on the shells of hermit crabs .", "They are small , transparent and stay fixed to one spot , making it easy for scientists to grow them in the laboratory and closely observe what is going on when they regenerate .", "Bradshaw et al . genetically engineered Hydractinia individuals to produce a fluorescent protein in their stem cells; these cells have the ability to become one of several kinds of mature cell , and often help to repair and grow tissues .", "This allowed the stem cells to be tracked using a microscope .", "When the head of Hydractinia was cut off , stem cells in the animals' mid body section migrated to the end where the head used to be and multiplied .", "These stem cells then created a bud ( known as a blastema ) that developed into a new , fully functional head within two days , allowing the animals to capture prey .", "Reducing the activity of certain stem cell genes prevented the new head from growing , but the bud still formed .", "Next , Bradshaw et al . removed a structure from the opposite end of the animal , called the stolon , which normally helps Hydractinia attach to hermit crabs shells .", "Stolons regenerated in a completely different way to heads .", "No bud formed .", "Instead , the remainder of the animal's body , which included the head and the body column , gradually transformed into a stolon rather than regenerating this structure , and only then grew a new body column and head .", "Therefore , different tissues in the same animal can regenerate in different ways .", "Understanding the ‘tricks’ used by animals like Hydractinia to regenerate may help translate these abilities to regenerative medicine ." ]

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

[ "cell biology", "genetics and genomics" ]

[ [ "Phosphorylation of translation initiation factor 2 on serine 51 of its alpha subunit ( eIF2α ) is a potent mechanism for translational regulation in eukaryotes .", "Phosphorylated eIF2 impedes the guanine nucleotide exchange activity of eIF2B thereby limiting the pool of active GTP-bound eIF2 .", "The consequences to rates of translation initiation are mRNA-specific .", "By way of this direct effect on protein synthesis and its indirect consequences to the abundance of downstream effector proteins , levels of eIF2α phosphorylation modulate gene expression translationally and transcriptionally ( Hinnebusch , 2014 ) .", "In animals four different kinases couple unrelated stress signals to eIF2α phosphorylation ( eIF2αP ) .", "eIF2αP effectively integrates these into a stereotypical downstream response referred to as the Integrated Stress Response ( ISR ) ( Harding et al . , 2003 ) .", "The ISR modulates biological processes ranging from the cell autonomous endoplasmic reticulum unfolded protein response to organismal immunity , memory and cognition ( Pakos-Zebrucka et al . , 2016; Wek , 2018 ) .", "The four eIF2a kinases , GCN2 , PERK , PKR and HRI , share a similar kinase effector domain , but diverge in the molecular mechanisms and nature of the upstream signals that regulate their kinase activities .", "General Control Non-depressible 2 ( GCN2 ) is the oldest eIF2α kinase , conserved in all known eukaryotes .", "It was discovered as the product of a gene required for yeast adaptation to starvation for any amino acid , as in its absence yeast were unable to mount a rectifying transcriptional General Control response ( the yeast counterpart to animal cell ISR ) ( Hinnebusch and Fink , 1983 ) .", "This amino acid starvation-induced , GCN2-dependent unicellular gene expression program has as its targets genes encoding transporters and biosynthetic enzymes that function to restore amino acid sufficiency , as well as tRNA synthetases that promote amino acid utilization as building blocks of proteins ( Hinnebusch , 2005 ) .", "These physiological features , in conjunction with the domain organization of the GCN2 protein ( including an eIF2α kinase module and a module highly related to histidyl-tRNA synthetases , HisRS-like ) , suggested that uncharged tRNAs may serve as activating ligands of GCN2 as part of a feed-back mechanism that defends cellular pools of charged tRNAs ( Wek et al . , 1989; Wek et al . , 1990 ) .", "This model was supported by the observations that mutations in the HisRS-like module that interfere with uncharged tRNA-binding in vitro abolished GCN2 activity in yeast ( Wek et al . , 1995; Zhu et al . , 1996 ) and by evidence that tRNA binding to the HisRS-like portion of GCN2 relieves an intramolecular repressive signal arising from its interaction with the kinase domain ( Dong et al . , 2000 ) .", "Parallel lines of enquiry indicate that GCN2 activity also relies on direct ( Ramirez et al . , 1991; Zhu and Wek , 1998 ) and indirect ( Marton et al . , 1997 ) interactions with ribosomes or ribosome-associated proteins ( Jiménez-Díaz et al . , 2013; Inglis et al . , 2019 ) .", "These latter observations suggest that GCN2 activation may not arise solely by binary interaction between GCN2 and uncharged tRNAs as activating ligands .", "Additionally , a recent genetic observation made in mice brought into question the singular role of excess uncharged tRNAs as GCN2 activating ligands and instead suggested that in some circumstances the interaction between GCN2 and ribosomes might take center stage .", "A strain of mice ( C57BL/6J ) that lacks an abundant neuron-specific isoacceptor arginyl-tRNA was noted to have higher levels of GCN2-dependent ISR activity in brain tissues compared with a reference strain that expressed normal levels of the neuron-specific arginyl-tRNA .", "ISR activity in arginyl-tRNA depleted C57BL/6J brain was not associated with globally elevated uncharged tRNAs , but was increased by a second mutation that compromises the cells ability to re-cycle stalled ribosomes ( Ishimura et al . , 2016 ) .", "Together , these observations suggest a mechanism for activating mammalian GCN2 that emanates from a ( stalled ) ribosome-generated signal that arises when pools of charged tRNAs are limiting .", "Whilst a role for localized pools of uncharged tRNAs remains possible , the findings of Ishimura ( et al . 2016 ) suggest that GCN2 activation may proceed independently of globally elevated levels of uncharged tRNAs .", "Following up on these hints for the existence of additional aspects of mammalian GCN2 activation , we took advantage of recent developments in CRISPR-Cas9 technology to search for mammalian genes whose compromise also enfeebles GCN2 activation .", "Our findings , pointing to a role for the ribosomal P-stalk in coupling an amino acid starvation-induced change in the ribosome to GCN2 activation , are described below ." ], [ "As a first step towards identifying genes implicated in GCN2-mediated ISR induction we confirmed the suitability of two reporter cell lines .", "In both HeLa and CHO cells inactivation of the GCN2-encoding Eif2ak4 gene selectively abolished responsiveness of the ISR regulated CHOP::GFP reporter to the histidinyl-tRNA synthetase inhibitor , histidinol .", "In both cell lines , the CHOP::GFP reporter remained responsive to the glycosylation inhibitor tunicamycin , a toxin that activates the ISR orthogonally , through an ER stress inducible eIF2α kinase , PERK ( Figure 1A and B ) .", "( Harding et al . , 1999; Harding et al . , 2000 ) .", "Furthermore , GCN2 ablation did not affect the tunicamycin-responsive XBP1::mCherry reporter present in the CHO cells .", "The reporter cell lines were thus deemed suitable tools to search for additional components that may contribute to GCN2-dependent ISR activation .", "The HeLa reporter cells were targeted with pooled lentivirus expressing guide RNAs targeting the entire human genome ( Sanjana et al . , 2014 ) , treated with medium lacking lysine and arginine ( -KR ) , and enriched for CHOP::GFP dull cells ( that mimic the GCN2-ablation phenotype ) using fluorescence-activated cell sorting ( FACS ) ( Figure 1C ) .", "After two rounds of sorting , sequencing of the guides confirmed that those targeting the GCN2 encoding Eif2ak4 were amongst the most highly enriched in the dull cells .", "Ontology cluster analysis also revealed enrichment for guides targeting the mRNA cap binding complex , other translation initiation factors , and ribosomal proteins , consistent with similar studies previously carried out in yeast ( Hinnebusch , 2005 ) .", "Despite the enrichment for guides targeting genes plausibly implicated in amino acid starvation-mediated ISR activation , many of the HeLa cells selected for CHOP::GFP dullness had also lost the ability to respond to tunicamycin ( not shown ) .", "Furthermore , it became evident that the clonogenic potential of stressed HeLa cells was poor , compared with CHO cells ( Figure 1—figure supplement 1A ) .", "These features were deemed to compromise the prospect of enrichment for guides targeting genes of interest by further cycles of selection in HeLa cells .", "To circumvent this problem , we drew on the sequence information derived from the targeted HeLa cells to create a CHO-based CRISPR library focused on those genes enriched in the dull HeLa cells and expanded to other members of their gene families .", "The resulting 19 , 305-guide library targeting 3 , 222 CHO genes ( ~6 guides per gene ) was used to mutagenize CHO cells .", "Flow cytometry-based sorting enriched for cells that were selectively compromised in CHOP::GFP expression in response to histidinol but retained significant responsiveness to tunicamycin ( Figure 1D ) .", "Sequencing of the integrated guides from genomic DNA isolated from the CHOP::GFP dull CHO cells confirmed enrichment of guides targeting Eif2ak4 and a subset of genes encoding translation initiation factors and ribosomal proteins .", "Among the latter was Rps10 ( encoding eS10 ) , previously implicated in GCN2 responsiveness to amino acid starvation in yeast ( Lee et al . , 2015 ) ( Figure 1—figure supplement 1B ) .", "Guides targeting two other ribosomal genes were also conspicuously enriched in the dull CHO cells: Rplp0 and Rplp1 , encoding uL10 and P1 , both components of the acidic ribosomal P-stalk , a heteropentameric structure that also includes P2 ( guides directed to the P2-encoding Rplp2 gene were also modestly enriched in the dull population ) ( Figure 1E ) .", "The enriched Rplp0 guides caught our interest , because they mapped to the region encoding the C-terminus of uL10 , which constitutes the helical spine of the P-stalk protrusion from the ribosome surface and the linker connecting it to the ribosome core .", "Guides targeting the ribosome-embedded N-terminal portion of uL10 were strongly depleted from all pools of CHO cells ( regardless of their ISR status ) consistent with a role for this portion of the protein in cell fitness ( Figure 1E and F ) .", "These findings hinted at a possible role for the P-stalk in ISR activation in response to histidinol .", "Given the proximity of the P-stalk to the ribosomal A site , we considered a role for the P-stalk in signaling event ( s ) triggered by lack of charged tRNAs or by ribosome stalling .", "To follow up on the genotype-phenotype relationship suggested by the screen , we targeted CHO cells with the Rplp0 and Rplp1 guides found to be enriched in CHOP::GFP dull cells and characterised genotypically-defined clones phenotypically .", "Histidinol induction of CHOP::GFP expression was conspicuously compromised in the targeted clones , whereas their responsiveness to ER stress-inducing agents was unaffected .", "A selective defect was also observed in Rplp0 and Rplp1 mutant cells in response to lysine and arginine depletion , albeit not to the level of GCN2 ablated cells ( Figure 2A and B ) .", "The defect in the ISR brought about by targeting cells with the Rplp0-directed guide was rescued by restoring gene function , attained by re-targeting the mutant Rplp0 locus with a homologous repair template to reestablish the coding region of the gene ( whilst adding an epitope tag ) ( Figure 2C and D ) .", "These findings formally establish a correlation between CRISPR-Cas9-induced lesions in the P-stalk and a selective defect in the inducibility of the CHOP::GFP ISR reporter in response to inhibition of tRNA charging or to starvation for amino acids .", "Attenuated translation initiation is a common feature of the ISR .", "It is readily detected by tracking the distribution of ribosomes in density gradients of cell lysates ( Nilsen et al . , 1982; Harding et al . , 2000 ) .", "Amino acid starvation of wildtype cells resulted in the expected redistribution of ribosomes from denser polysome fractions to lighter fractions and to free 40S and 60S subunits .", "This starvation-induced shift in the ribosome profile was lost in GCN2-ablated cells , but only partially attenuated even in the strongest single mutant lines ( Figure 3A and B ) .", "This partial loss of function phenotype is consistent with the partial defect in CHOP::GFP induction and with the predicted structure of the mutant uL10 , which retains the N-terminal portion of the P-stalk helical spine ( Figure 2A and B ) .", "In an effort to acquire a more penetrant lesion in the P-stalk , we targeted several Rplp0 mutant clones with guides to Rplp1 .", "Very few double mutant cells survived the procedure , but one such clone Rplp0/Rplp1m29-132 ( encoding a uL10 truncated at Y231 and a P1 protein lacking H17-D18 in helix 1 ) phenocopied GCN2 ablation both in terms of its polysome profile and activation of the CHOP::GFP reporter ( Figure 3A–3C ) .", "In wildtype cells amino-acid starvation led to a time dependent accumulation of activated hyper-phosphorylated GCN2 , which was conspicuously lacking in the Rplp0/Rplp1m29-132 cells .", "Note the retarded mobility of GCN2 from the starved wild type when resolved on a PhosTag gel , compared with GCN2 from mutant cells ( Figure 3D and Figure 3—figure supplement 1A ) .", "Phosphatase treatment of the lysate , prior to gel loading , confirmed that the heterogenous mobility shift indeed reflected multiple phosphorylation events ( Figure 3E and Figure 3—figure supplement 1B ) .", "Like the GCN2 ablated cells , the compound P-stalk Rplp0/Rplp1m29-132 double mutant cells were also selectively defective in induction of the endogenous ISR markers CHOP and ATF4 in response to amino acid starvation but retained inducibility of the markers to ER stress ( Figure 3F ) .", "Immunoblotting of extracts from the wildtype and P-stalk mutant cells confirmed the truncation of uL10 ( detected with an antisera to the N-terminal portion of the protein ) and the loss of its C-terminus ( revealed with a monoclonal antibody , 3BH5 , reactive with a C-terminal peptide conserved in all three P-stalk proteins ) in both the single Rplp0m14 and the compound Rplp0/Rplpm29-132 double mutant ( Figure 4A ) .", "Coomassie-stained SDS-PAGE gels of ribosomes isolated from the mutant cells was conspicuous for the presence of a 34 . 2 kD protein ( the predicted size of uL10 ) in the wildtype that was missing from both mutants ( Figure 4B ) .", "Furthermore , ribosomes isolated from the mutant cells had diminished P1/P2 content ( conspicuous in the Rplp0/Rplp1m29-132 double mutant but also apparent in the Rplp0m14 single mutant ( Figure 4B , middle panel ) , consistent with a destabilizing effect of the mutations on the association of P1 and P2 with the ribosome .", "While these studies were ongoing , we learned of findings , now published , indicating a physical interaction between GCN2 and the mammalian ribosomal P-stalk and providing evidence that intact ribosomes , or their isolated P-stalk , can stimulate GCN2-mediated phosphorylation of eIF2α in vitro ( Inglis et al . , 2019 ) .", "At the concentrations used , isolated ribosomes had negligible associated eIF2α kinase activity .", "However , in our hands too , nanomolar concentration of ribosomes isolated from wildtype CHO cells markedly stimulated eIF2α phosphorylation by GCN2 ( Figure 5A and Figure 5—figure supplement 1A ) .", "Interestingly , ribosomes isolated from the Rplp0 and compound Rplp0/Rplp1 mutant cells were impaired in GCN2-dependent eIF2α phosphorylation .", "This was evident in multiple preparations of wildtype and mutant ribosomes ( Figure 5A–5C and Figure 5—figure supplement 1A-C ) .", "Furthermore , the hierarchy of the defect in GCN2 activation by the different ribosome preparations in vitro correlated with the ISR impairment of their source mutant cells in vivo , in that the in vitro defect was more severe in preparations of ribosomes from compound Rplp0/Rplp1m29-132 mutant cells than its Rplp0m9 single mutant parent or the unrelated Rplp0m14 single mutant ( Figure 5A–5C and Figure 5—figure supplement 1C ) .", "The stimulatory effect of ribosomes appeared to be GCN2 specific , as the related PERK kinase was only minimally activated in vitro ( Figure 6A ) .", "This finding is consistent with the lack of effect of the P-stalk lesions on the PERK-dependent ISR induction by ER stress in cells ( Figures 2A and 3B–3F ) .", "To further characterize ribosome P stalk-dependent GCN2 activation , we compared experimentally accessible enzymatic features of GCN2 alone with those of a compound enzyme constituted of GCN2 and ribosomes .", "At physiological substrate concentrations of 2 µM eIF2α ( Chen et al . , 2015 ) , the approximately 10-fold increase in the rate of GCN2-dependent eIF2α phosphorylation brought about by the presence of wildtype ribosomes ( Figure 5C ) could be mimicked by a 10-fold increase in the concentration of GCN2 in in vitro reactions without ribosomes .", "However , the relationship between reaction velocity and substrate concentration proved very different when comparing GCN2 ( 2 . 5 nM ) with ribosomes ( 50 nM ) to GCN2 alone ( at 25 nM ) .", "While the former reaction saturates at less than 50 μM ( substrate Km of ~7 . 8 µM ( 95% confidence 5 . 2–10 . 4 µM ) and a Vmax of 130 min−1 ( 95% confidence 116–140 min−1 ) ) the latter reaction was not saturated with substrate concentrations attainable experimentally ( Figure 6B and C ) .", "The kinetic properties of GCN2 , reacted with ribosomes from the mutant Rplp0/Rplp1m29-132 cells , resembled GCN2 alone , establishing a role for the P-stalk in this shift in the enzymatic properties of the kinase ( Figure 6—figure supplement 1A and B ) .", "In keeping with recent observation ( Inglis , 2018 and Inglis et al . , 2019 ) , we also noted that ribosome-mediated potentiation of GCN2’s eIF2α-directed kinase activity was not associated with a consistent difference in the known GCN2 auto-activation mark , phosphorylation of activation-loop residue Thr899 ( Romano et al . , 1998 ) ( Figure 6D ) .", "It is noteworthy that in vitro , ribosomes promote GCN2 phosphorylation on residues other than threonine 899 ( Inglis , 2018 ) .", "Unfortunately the antiserum directed towards human GCN2 pThr899 used here fails to recognise the hamster protein , thus we are unable to ascertain if the defect in GCN2 phosphorylation observed in amino acid deprived , P-stalk mutant cells ( Figure 3D and E and Figure 3—figure supplement 1 ) encompasses that residue .", "However , given that the phos-tag gels report on multiple phosphorylation events in the amino acid deprived wildtype cells that are absent from P-stalk mutant cells , it seems reasonable to conclude that the ribosome-mediated signal also results in phosphorylating events other than pThr899 in vivo .", "Their relationship to GCN2’s activity as an eIF2α kinase remains to be determined .", "Together with the altered kinetics of the ribosome-associated enzyme , these features suggest a ribosome-dependent process extending beyond activation-loop autophosphorylation ." ], [ "The correlation , established here , between a selective defect in GCN2-mediated ISR activation in cells with genetic lesions to their P-stalk and a defect in ability of their ribosomes to activate GCN2 in vitro implicates the P-stalk in propagating a signal from ribosomes perturbed by amino acid starvation to GCN2 .", "This finding bridges recent genetic and cell biological observations implicating stalled ribosomes in GCN2 activation ( Ishimura et al . , 2016; Darnell et al . , 2018 ) with biochemical evidence that the ribosome P-stalk can activate GCN2 in vitro ( Jiménez-Díaz et al . , 2013; Inglis et al . , 2019 ) , establishing that the in vitro observation , suggestive of a role for the ribosome in GCN2 activation , relate to a process required for GCN2 activation by amino acid starvation in cells .", "The implication of the P-stalk as an agent in GCN2 activation brings up interesting questions relating to the coupling between a process common to amino acid starvation and inhibition of tRNA charging and alteration in the state of the P-stalk .", "The location of the P-stalk on the surface of the ribosome adjacent to the A site and its functional role in recruiting elongation factors to the ribosome ( Helgstrand et al . , 2007; Nomura et al . , 2012 ) and in stimulating their GTPase activity ( Mohr et al . , 2002 ) , implicate the P-stalk in translation elongation .", "In elongating ribosomes , charged cognate tRNAs ( in complex with eEF1A ) followed by eEF2 , cycle through the A site ( reviewed in Brown and Shao , 2018; Dever et al . , 2018 ) .", "It is tempting to speculate on a scenario whereby the cycling of these factors in proximity to the P-stalk restrains the latter’s GCN2 stimulatory activity .", "This may occur through elongation factor mediated steric blocking of the interaction between GCN2 and domain II of uL10 ( shown to be essential for GCN2-P-Stalk binding , Inglis et al . , 2019 ) , by competition for the attention of the acidic C-terminal tails of the P-stalk proteins ( which are important for activation of GCN2 , Inglis et al . , 2019 ) , or by eliciting conformational changes in the P-stalk .", "Disruption of this elongation cycle by lack of cognate charged tRNA presumably stalls ribosomes in a conformation that relieves such restraint ( s ) , exposing the dormant capacity of the P-stalk to activate GCN2 ( Figure 7 ) .", "A ribosome-centered activation event , mediated by the P-stalk , fits the genetic data whereby in neurons lacking an abundant isoacceptor arginine tRNA , GCN2 activation is favored by lesions in GTPBP2-a mammalian ribosome rescue factor whose absence stabilizes stalled ribosomes ( Ishimura et al . , 2014; Ishimura et al . , 2016 ) .", "The notion that the activating signal arises from stalled ribosomes is also in keeping with the positive correlation noted between GCN2’s response to single amino acid starvation in different mammalian cell lines and the extent of ribosome pausing ( Darnell et al . , 2018 ) .", "Indeed , the relationship between stalling and GCN2 activity is likely homeostatic as GCN2∆ cells exhibit more stalling in response to amino acid starvation ( Darnell et al . , 2018 ) .", "Ribosomes isolated from actively-translating fed cells ( with repressed GCN2 ) , were nonetheless potent activators of GCN2 in vitro ( our observation and Inglis et al . , 2019 ) .", "Presumably ribosome isolation disrupts the aforementioned constraints on the P-stalk and exposes its latent ability to activate the kinase in vitro .", "In this vein , it is interesting to compare the P-stalk’s role in yeast and mammalian GCN2 activation .", "Activation by the mammalian P-stalk requires both the ribosome-associated N-terminal domain II of uL10 and the acidic C-termini of the three P-stalk components that extend from the ribosomes surface ( Inglis et al . , 2019 ) .", "By contrast , the yeast P1/P2 proteins are sufficient for GCN2 activation in vitro ( Jiménez-Díaz et al . , 2013 ) .", "These observations fit the genetic evidence of a role for the free ( not ribosome-associated ) pool of yeast P2 in GCN2 activation in vivo .", "Interestingly , the free pool of yeast P2 contributes to GCN2 activation in response to osmotic stress and glucose deprivation , but is dispensable for GCN2 activation by histidine depletion .", "The latter observation is consistent with the notion that GCN2’s response to amino acid starvation might depend on a ribosome-coupled P-stalk mediated signal , which may be provided adequately by yeast uL10 even in the absence of associated P1/P2 ( Jiménez-Díaz et al . , 2013 ) .", "The importance of the physical coupling between the P-stalk and the ribosome to mammalian GCN2 activation is suggested by the strong phenotype of the compound Rplp0/Rplp1m29-132 mutant in which the truncation of uL10 and the internal deletion of P1 conspire to deplete ribosomes of associated P-stalk acidic C-termini , whilst retaining a substantial pool of cytosolic P proteins with intact C-termini .", "The notion that efficient delivery of the P-stalk’s message to GCN2 relies on proximity to the ribosome is consistent with the importance of other contacts made between GCN2 and the mammalian ribosome , such as those dependent on eS10 ( Lee et al . , 2015 ) ( a product of a gene also ‘hit’ in our screen for CHOP::GFP dull cells ) .", "Whilst the ribosome takes central stage in GCN2 activation by amino acid starvation , it is interesting to consider that in mammals too there may be a role for the free pool of P1/P2 proteins in GCN2 activation in response to other non-ribosomal stress signals .", "The nature of the P-stalk dependent activating event raises other questions .", "Yeast P1/P2 proteins markedly stimulated GCN2 autophosphorylation in vitro ( Jiménez-Díaz et al . , 2013 ) .", "However , mammalian ribosomes had no consistent effect on the phosphorylation status of GCN2 Thr899 ( here , Inglis , 2018 and Inglis et al . , 2019 ) .", "Nonetheless , mammalian ribosomes imparted on GCN2 new enzymatic properties , reflected in the lowering of the enzymes Km .", "The nature of this activating event ( s ) is an open question , as is the linked question whether stimulation of the eIF2α-directed kinase activity of GCN2 requires the continued presence of ribosomes , or if the activated state survives dissociation of GCN2 from ribosomes .", "The importance of GCN2’s relationship with ribosomes to activation of the ISR ( known in yeast as the GCN4-dependent General Control gene expression program ) was first recognized before identification of eIF2α as GCN2’s substrate ( Ramirez et al . , 1991; Zhu and Wek , 1998 ) and subsequently buttressed by identification of ribosome-associated factors such as GCN1 and GCN20 as important GCN2 co-factors ( Marton et al . , 1997 ) .", "These seminal early findings , together with more recent work ( of which this paper is part ) , point to a role for a stalled ribosome-initiated P-stalk-mediated activation signal .", "Moreover , these observations suggest the possibility that GCN2 , the first eIF2α kinase , evolved initially as part of a simple feed-back loop attenuating translation initiation in response to ribosome stalling .", "The coupling of GCN2’s activity to a gene expression program that acts physiologically to relieve some of the more common causes of stalling , may have emerged later and eventually evolved into the ISR we recognize today in multicellular organism ." ], [ "Standard cloning techniques were used to create the recombinant DNA vectors listed in the Key Resources Table ( supplementary Table 1 ) .", "The table also lists the antibodies , cell lines , reagents , oligonucleotides and software used in this study .", "HeLa cells were maintained in DMEM supplemented with 10% Fetalclone II serum ( Hyclone ) , 0 . 5% β-mercaptoethanol , 1x MEM-non-essential-amino-acids , and 1X pen-strep .", "CHO cells were maintained in Hams’ F12 supplemented with 10% Fetalclone II serum ( Hyclone ) and 1x pen-strep .", "For lysine and arginine starvation , cells were washed 3x in PBS+ Mg2+ Ca2+ incubated in Advanced DMEM/F-12 ( 12634010 , Thermo-Fisher ) with 10% dialyzed FCS for 2–4 hr before the start of the experiment .", "Treated samples were then washed 3x with PBS + Mg2+ Ca2+ and incubated in SILAC Advanced DMEM/F-12 Flex Media ( A2494301 , Thermo-Fisher ) supplemented with 17 . 5 mM glucose , and 10% dialyzed FCS for the indicated period .", "HeLa cells were stably transfected with a linearized CHOP::GFP vector ( lab m31 ) and pRc/RSV ( Invitrogen ) 10:1 and selected for G418 resistance and ISR induction .", "The selected line was then stably transfected with a constitutive mCherry expression vector to yield ( Hela CGC55 ) .", "A previously-described CHOP::GFP-C30 cell line ( Novoa et al . , 2001 ) was transfected with plasmid UK1313 pCAX-F-XBP1∆DBD-mCherry ( MP5 ) 10:1 with pbabe-puro ( Addgene 1764 ) and a puromycin-resistant clone with tunicamycin induced XBP1::mCherry expression identified and the puromycin resistance was removed by transient transfection with sgCRISPR-RNA-expression vectors targeting the puromycin N-acetyltransferase gene ( UK1901+UK1902 ) to yield the DP19 clone used throughout this study .", "Cas9 expressing derivatives of the HeLa CGC55 and CHO DP19 reporter lines were made by infecting the cells with lentivirus prepared from packaging Lenti-Cas9-Blast ( UK1674 , Addgene 1000000049 ) by co-transfection with helpers pMD2 . g ( UK1700 , Addgene 12259 ) , psPAX2 ( UK 1701 , Addgene 12260 ) , in HEK 293 T cells and selecting for blasticidin resistance ( 10 and 30 μg/ml for HeLa and CHO respectively ) .", "Using the pooled human GECKO 2 . 0 CRISPR library and following an established protocol ( Addgene 1000000049 , Sanjana et al . , 2014 ) we infected HeLa Cas9 expressing reporter cells library lentivirus at a MOI <0 . 3 and selected for Puro resistance resulting in a final representation of >60 surviving cells/guide for each of library segments A and B . After a week of expansion , cells were treated in arginine and lysine free medium for 20 hr followed by collection in PBS-EDTA , washing in PBS + 0 . 2% BSA and 8 . 47*107 cells sorted with the dullest ~3% ( 2 . 6 × 106 ) collected on an Influx or Melody cell sorter ( BD biosciences ) .", "An equal number of treated cells were passed without sorting as a control group .", "The collected cells were replated , expanded and aliquots frozen or passed for another round of treatment and sorting .", "Genomic DNA was prepared from 3 . 6 × 107 cells from each round using the DNAzol method ( Chomczynski et al . , 1997 ) from the separately maintained A and B library segments .", "PCR inserts were prepared for NGS sequencing with two rounds of PCR with primers UK 1433 and UK 1434 for the first round and primers UK 1432 and one of the barcoded reverse primers ( UK1418-UK1432 ) for the second round .", "After quantification the products were subjected to NGS sequencing using custom primer UK1435 and the illumina indexing primer with single-end reads of 50 bp on a hiseq 4000 .", "The sequences were processed and guide counts , gene rankings and statistics were generated using MAGECK software ( Li et al . , 2014 ) .", "The top 1% ( 200 hits ) were submitted to metascape ( Zhou et al . , 2019 ) for gene annotation analysis and genes in the top enriched ontology clusters were added to the list along with the next ~9% of genes selected in the screen .", "Guides to 1500 unrelated CHO genes and 200 non-targeting control guides were also included in the library ( NCBI Geo database; accession numbers awaiting assignment ) .", "CHO homologues were identified from a gene list from the CHO-K1 reference genome ( GCF_000223135 . 1 ) .", "Guides based on improved efficiency rules ( Doench et al . , 2014 ) were designed against the selected genes and pooled single stranded oligonucleotides ( see oligo UK2580 ) were synthesized as part of a full genome library made with two sets of arms for independent PCR amplification and cloning ( Twist Biosciences , California USA ) .", "The 19305 guide library ( CHO-mini-library ) targeting 3222 CHO genes ( six guides per gene ) was PCR amplified from the pool in 8 rounds of PCR with primers UK1855 and UK1856 ) , digested with Bbs1 , and the 27 bp fragments purified by PAGE and cloned into a BbsI cut UK1789 pKLV-U6gRNA ( BbsI ) -PGKpuro2ABFP vector ( Addgene 50946 ) as described ( Koike-Yusa et al . , 2014 ) .", "The resulting focused library was used to mutagenize the CHO-CHOP::GFP , XBP1::mCherry double reporter cells at a MOI of 0 . 1 and a representation of ~350 cells for each library guide in duplicate pools .", "In the first round , 4–5% of the dullest cells following 20 hr induction with histidinol were selected and corresponding unselected pools maintained .", "In the second round three levels of dull cells corresponding to the lowest ~4 . 6 , 11 , and 24% were collected from each duplicate along with pools of treated unsorted cells .", "The selected guides were PCR amplified from genomic DNA as above but using primers UK1758 and UK1434 for the first round and primers UK1432 and one of the barcoded reverse primers ( UK1759-UK1776 ) for the second round of PCR .", "The PCR products were subjected to NGS sequencing and analysis by MAGECK software as above for the HeLa screen .", "The figures show an average of the duplicates for both the dullest and medium dull selected cells ( n = 4 ) compared to the average for the duplicate unselected controls ( n = 2 ) .", "The CHO CRISPR screening data has been submitted to NCBI Geo database ( study accession number GSE134917 ) .", "Individual CRISPR-Cas9 mutant CHO cell lines were made by co-transfecting the indicated sgRNA expression vector with the Cas9-Blast plasmid ( UK1674 ) into parental DP19 ( CHOP::GFP; XBP1::mCherry reporter cells ) , selecting for the puromycin resistance encoded by the sgRNA expression vector ( 6 μg/ml ) for two days .", "Reporter induction was measured by flow cytometry of at least 10000 gated live singlets on a Becton Dickinson LSR Fortessa on 2–4 independent experiments for each condition and mutant line .", "The cell lines used in this study were all made in our lab from HeLa ( ATCC Cat# CRL-7924 , RRID:CVCL_0058 ) or CHO . K1 ( ATCC Cat# CRL-9618 , RRID:CVCL_0214 ) cell lines obtained from and authenticated by the ATCC .", "The identity of both the human ( HeLa ) the non-human ( CHO . K1 ) cell lines has been authenticated using the criteria of A . successful targeting of essential genes using species specific CRISPR whole genome library , and B . sequencing of the wildtype or mutant alleles of the genes studied that confirmed the sequence reported for the corresponding genome .", "The cell lines have tested negative for mycoplasma contamination using a commercial kit ( MycoAlert ( TM ) Mycoplasma Detection Kit , Lonza ) .", "None of the cell lines is on the list of commonly misidentified cell lines maintained by the International Cell Line Authentication Committee .", "Cells were treated as indicated and lysates prepared and ATF4 and CHOP detected using rabbit antiserum as previously reported except that proteins were transferred to PVDF and IR-800 ( LI-COR ) conjugated secondary antisera were used ( Harding et al . , 1999; Harding et al . , 2000 ) .", "Anti P-protein immunoblots were probed with a mouse monoclonal 3BH5 ( 1:5 ) that recognizes the conserved acidic C-terminus of all three P-proteins ( Vilella et al . , 1991 ) followed by anti-mouse IR800 and detection by LI-COR scanning and then the blot was incubated with rabbit monoclonal anti uL10 1–200 ( AbCam , Cambridge , UK ab192866 , 1:1000 ) , followed by anti-rabbit HRP and detection by chemiluminescence .", "Samples containing CHO cell extracts ( mock or λ phosphatase treated where indicated ) or human GCN2 were denatured and separated on 5% Tris-acetate phos-tag or 7% Tris-acetate gels respectively and transferred in bicine transfer buffer ( Cubillos-Rojas et al . , 2012 ) and probed with anti-GCN2 ( phospho T899 ) antibody ( 1/1000 , AbCam , Cambridge , UK ab75836 ) or an antibody raised against the bacterially-expressed kinase domain of mouse GCN2 antibody ( lab name NY168 ) mixed 5:1 with non-affinity purified P-GCN2 890–904 ( phospho T899 ) ( human numbering , but the peptide is identical in mouse and human ) ( lab name 6779 ) in the case of CHO cell extracts ( Harding et al . , 2000 ) used at 1:3000 .", "For polysome analysis cell lysis and 10–50% sucrose gradients were carried out as described in Johannes and Sarnow ( 1998 ) .", "Ribosomes were purified as described ( Khatter et al . , 2014 ) using modified buffers as follows: Cells were lysed in ( 15 mM Tris , pH 7 . 5 , 0 . 5% IGEPAL , 6 mM MgCl2 , 150 mM NaCl , 1 mM DTT , and 10 μl/ml RNAsin Plus ( Promega ) , and protease inhibitors ( Sigma S8830 ) ) , cleared 4 times at 18000g × 5 min and layered on a 3 ml sucrose cushion buffer D-30% ( 50 mM HEPES pH 7 . 5 , 2 mM Mg ( OAc ) 2 , 150 mM KOAc , 30% sucrose ) , and centrifuged for 660 min at 40000 rpm in a TLA110 rotor .", "The pellet was resuspended in wash buffer ( 50 mM HEPES pH 7 . 5 , 4 . 4 mM Mg ( OAc ) 2 , 35 mM KOAc , 314 mM KCL , 6% sucrose 0 . 5 mg/ml puromycin , 1 . 2 mM GTP and 5 μl/ml RNAsin Plus ) and rotated for 30 min at 4 °C to release nascent peptides , cleared for 10 min at 18000 g and then the supernatant re-pelleted through a sucrose cushion as described above .", "The final pellet was resuspended in the buffer D made with 6 . 8% sucrose and 2 mM TCEP .", "GCN2 , expressed as a StrepII-tagged protein was purified from insect Sf9 cells as described ( Inglis et al . , 2019 ) and stored in small aliquots at −80 ºC .", "Phosphorylation reactions were carried out in PCR tubes at a total volume of 20 µL in an assay buffer consisting of 20 mM HEPES ( pH 7 . 4 ) , 50 mM potassium acetate , 5 mM magnesium acetate , 2 mM ATP , 0 . 5 mM tris ( 2-carboxyethyl ) phosphine ( TCEP ) , 0 . 05 mg/mL bovine serum albumin .", "Ribosomes isolated from CHO cells ( or an equal volume of ribosome resuspension buffer D , see above ) were combined with GCN2 ( to a concentration of 2 . 5 nM in the final assay ) and allowed to equilibrate at 17 ˚C for 10 min .", "The reaction was initiated by adding the N-terminal domain of human eIF2α ( residues 1–185 ) purified from bacteria ( Ito et al . , 2004 ) to a final concentration of 2 µM , and allowed to progress at 17 ˚C for the indicated time until terminated by adding 6 . 6 µL of 4% SDS , 200 mM dithiothreitol in a tris-glycine buffer .", "Samples were resolved on a 50 µM phos-tag/100 µM manganese chloride ( Apex Biotechnology , Hsinchu City , Taiwan cat # F4002 ) 15% SDS-PAGE .", "The gel was soaked for 20 min in 50 mM EDTA , 0 . 1% SDS in tris-glycine buffer to chelate the phos-tag reagent and transferred onto a PVDF membrane and immunoblotted with a primary rabbit serum directed to the N-terminus of eIF2a ( residues 1–185 ) ( lab name NY1308 ) and an IRDye fluorescently labeled secondary anti-rabbit IgG ( LI-COR ) ( Chen et al . , 2015 ) .", "The fluorescence signals were detected with an Odyssey near-infrared imager ( LI-COR ) and quantified by ImageJ ( NIH ) ." ] ]

[ "The eukaryotic translation initiation factor 2α ( eIF2α ) kinase GCN2 is activated by amino acid starvation to elicit a rectifying physiological program known as the Integrated Stress Response ( ISR ) .", "A role for uncharged tRNAs as activating ligands of yeast GCN2 is supported experimentally .", "However , mouse GCN2 activation has recently been observed in circumstances associated with ribosome stalling with no global increase in uncharged tRNAs .", "We report on a mammalian CHO cell-based CRISPR-Cas9 mutagenesis screen for genes that contribute to ISR activation by amino acid starvation .", "Disruption of genes encoding components of the ribosome P-stalk , uL10 and P1 , selectively attenuated GCN2-mediated ISR activation by amino acid starvation or interference with tRNA charging without affecting the endoplasmic reticulum unfolded protein stress-induced ISR , mediated by the related eIF2α kinase PERK .", "Wildtype ribosomes isolated from CHO cells , but not those with P-stalk lesions , stimulated GCN2-dependent eIF2α phosphorylation in vitro .", "These observations support a model whereby lack of a cognate charged tRNA exposes a latent capacity of the ribosome P-stalk to activate GCN2 in cells and help explain the emerging link between ribosome stalling and ISR activation ." ]

[ "Often thought of as “workhorse” molecules , proteins take part in almost every structure and activity in a living cell .", "They are constructed from smaller building blocks called amino acids by molecular machines called ribosomes .", "Each cell needs a constant supply of amino acids to make new proteins .", "If cells are running low on amino acids , they can change their internal biochemistry to use amino acids more economically .", "GCN2 is one protein that helps activate these biochemical changes , but it was unclear how a shortage of amino acids could activate GCN2 .", "Earlier in 2019 , researchers reported that , in a test tube at least , isolated ribosomes could themselves activate GCN2 .", "They also identified a part of the ribosome called the P-stalk as playing an important role in the interaction .", "Now , Harding et al . – who include some of the researchers involved in the earlier study – explore the activation of GCN2 further , but this time based on experiments with mammalian cells .", "First , a genetic screen was conducted to identify genes that if mutated specifically prevented the activation of GCN2 in cells that were starved of amino acids .", "This screen identified a few genes , several of which are involved in creating the P-stalk of the ribosome .", "By isolating the mutant ribosomes from these cells and studying them in the laboratory , Harding et al . then showed that these ribosomes are unable to activate GCN2 .", "These findings confirm that the P-stalk of the ribosome plays an essential role in activating GCN2 in response to a shortage of amino acids .", "They shed light on a fundamental biological system , and further work will undoubtedly seek to uncover the details of the process by which GCN2 is activated ." ]

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

[ "neuroscience" ]

[ [ "The statistics of sensory input vary over time , due to moving objects , background motion as would arise from optic flow , and due to active sensation such as sniffing ( Shusterman et al . , 2011 ) , whisking ( Hill et al . , 2011 ) or eye movements ( Tatler et al . , 2006 ) .", "To achieve better performance in the current condition , many sensory systems measure the recent sensory statistics and adjust their responses to the expected sensory input .", "For example , adaptation in the visual system adjusts a cell’s dynamic range based on the expected stimulus distribution , including the stimulus mean ( Barlow and Levick , 1969 ) and variance ( Victor and Shapley , 1979; Smirnakis et al . , 1997; Nagel and Doupe , 2006 ) .", "In addition , the retina adapts to spatiotemporal correlations so as to remove predictable signals and enhance the response to novel input ( Hosoya et al . , 2005 ) .", "These expectations derive from correlations in visual input , which can extend over a wide range of scales due to extended textures , motion of large objects , and from eye and body movements .", "For example , object motion sensitive ganglion cells receive peripheral inhibition to suppress the predictable excitatory input due to small , fixational eye movements and transmit unpredictable , novel signals from moving objects ( Olveczky et al . , 2003 ) .", "In addition , inhibition from fast , large global shifts may reflect the instantaneous expectation that an eye movement is occurring , and play a role in saccadic suppression ( Roska and Werblin , 2003; Geffen et al . , 2007 ) .", "In addition to peripheral inhibition , it has long been known that changes in the retinal image far away from the receptive field center produce excitation ( known as the ‘periphery’ or ‘shift’ effect ) ( Krüger and Fischer , 1973; Mcilwain , 1964 ) in various species , including cat , rabbit , and primate ( Watanabe and Tasaki , 1980; Krüger and Fischer , 1973 ) .", "The functional importance of long-range excitation , however , is unclear despite numerous studies on the spatiotemporal properties of this input .", "Many studies have focused on the stimulus parameters that generate excitation ( Barlow et al . , 1977; Ikeda and Wright , 1972; Li et al . , 1992; Passaglia et al . , 2009 ) .", "However , few studies have measured how long-range excitation changes the neural code for local stimuli ( Passaglia et al . , 2009 ) , and none has considered how image statistics might relate to such long-range excitation .", "We examined how peripheral stimulation changes how a ganglion cell encodes the central part of its receptive field .", "Numerous studies in the salamander retina have characterized the properties of the ganglion cell receptive field center ( Smirnakis et al . , 1997; Hosoya et al . , 2005; Olveczky et al . , 2003; Geffen et al . , 2007; Kastner and Baccus , 2011; Werblin , 1972 ) , and have studied the effects of peripheral stimuli on the neural code as related to eye movements ( Olveczky et al . , 2003; Geffen et al . , 2007; Baccus et al . , 2008 ) .", "Our experiments were performed in the isolated intact retina , and ganglion cell spiking activity was recorded using a multielectrode array .", "We find that peripheral stimulation amplifies information transmission about the local stimulus in ganglion cells .", "Underlying this increase in information in neural responses is a more complete adaptation to the local stimulus , allowing for both low and high local contrast environments to be encoded with a similar response range .", "This rapid change in the neural code causes the cell to encode the intensity sequence of the stimulus and the contrast at different times relative to the global shift , thus causing peripheral motion to act as a timing signal to coordinate the encoding across a population of cells .", "We further show that these effects can be produced by a simple model combining local and peripheral inputs prior to a threshold and an adaptive stage .", "Finally , using the same model we show that the pulse of increased information that we observed when stimulating the periphery matches in timing the expected arrival of novel information generated by a global image shift as would occur during motion of a large object or an eye/head movement .", "Our results show that global motion switches the neural code from one that conserves energy , encoding only strong stimuli , to one that transmits greater information and encodes both weak and strong stimuli .", "These findings reveal a principle of adaptation that acts to allocate energy resources in the form of neural activity to times that are expected to contain novel information ." ], [ "To measure how peripheral motion changes the response of ganglion cells , we presented a stimulus to simulate image movement in the retina due to two different conditions: a moving object in a static scene or global motion as would be caused by movement of the eye or a large object .", "We chose a set of brief stimuli that could produce a wide variation in excitation – from extremely weak to very strong – depending on a cell’s location , to determine the effect of peripheral excitation , and how that effect varies with the cell’s central excitation .", "The stimulus consisted of a central square object with a constant luminance in front of a checkerboard peripheral pattern .", "The object covered the classical linear receptive field center plus part of the surround of most cells ( Figure 1A , top panel ) .", "To present the same central stimulus in the presence and absence of strong peripheral stimulation , either the object alone ( object shift ) or the whole stimulus ( global shift ) was shifted abruptly by one peripheral square , 50 µm in length , ~1 degree of visual field in the salamander .", "We then varied the central luminance level to measure the effect of the strong peripheral stimulus in encoding the luminance of the central stimulus .", "When the object moved alone , many cells showed transient activity , which depended on the location of the cell relative to the object border ( Figure 1B , left column ) .", "As expected , those cells near the center of the object showed the weakest activity because they experienced little change in light intensity , and overall , the response was insensitive to the central luminance value .", "In the global shift condition , however , brief strong firing events occurred during both right and left shifts for most cells including those in the center of the object ( Figure 1B , right column ) .", "To assess the effect of the peripheral shift on the ability of a cell to distinguish different central stimuli , we computed the slope of a line fit to the firing rate as a function of the log of central intensity , and found this slope to be much greater during the global shift ( Figure 1C ) .", "We examined which cells were most strongly affected by the periphery by comparing responses to all constant luminance objects under both peripheral conditions .", "In doing so , we found that the cells with the weakest response to the object condition showed the greatest enhancement of sensitivity from peripheral motion , with 39 out of 76 cells at least doubling the slope of their firing rate vs . the log of central luminance ( Figure 1D ) .", "This increased activity during the global shift condition could not be attributed to the linear receptive field of the cell overlapping the object border , as steps to the right and left would have linear contributions of opposite signs , even if such stimuli might be within the spatial region occupied by the classical receptive field surround ( Demb et al . , 2001; Borghuis et al . , 2013 ) .", "Accordingly , the effect of the global shift was mostly insensitive to the phase of the checkers in the periphery ( see Materials and methods ) .", "Thus , peripheral stimulation enhanced the sensitivity to weak central stimuli during abrupt global image motion . 10 . 7554/eLife . 09266 . 003Figure 1 . Global image shifts increase sensitivity to weak local input .", "( A ) ( Top ) A diagram of the stimulus is shown .", "The central square representing an object shifted left or right either in the presence of a static periphery ( still periphery , moving object , left in bottom panel ) or in conjunction with the periphery ( global shift , right in bottom panel ) .", "In both conditions , the central stimulation was the same .", "Shifts occurred every 0 . 5 s , and the luminance level in the object changed every 110 s to one of four values spaced logarithmically .", "Lower panel shows the central stimulus region under both peripheral conditions .", "One checker is colored red ( not used in actual stimulus ) to help the reader identify the relationship between this particular checker and the central stimulus .", "( B ) Average firing rate response of four different cells from different preparations to four different luminance values under both peripheral conditions: object shift ( left ) , global shift ( right ) .", "Stimulus shifts to the right ( 0 s ) and left ( 0 . 5 s ) are marked with dotted lines .", "The classical ( linear ) receptive field center , computed from a white noise checkerboard stimulus is shown as a colored oval .", "( C ) Average firing rate computed between 50 and 150 ms after the stimulus shifted to the left and right for the cell shown in ( B , top panel ) , colors of dots show different luminance levels corresponding to the curves in ( B ) .", "A linear fit ( lines ) to the data was used to compute the sensitivity m to the luminance of the central region , computed as the slope of the firing rate vs . the log of the central luminance for left and right object shifts with periphery still ( open circles , mL , Still , mR , Still ) and for global shifts ( filled circles , mL , Shift ) .", "( D ) The ratio of the luminance sensitivity m during global and object shifts compared for each cell to the firing rate in the object shift condition , indicating the strength of the object shift stimulus .", "Axes are logarithmic .", "Results for mStill and mShift were averaged over shifts to the left and right .", "Cells above the dotted line increased the slope of firing vs . central luminance by more than a factor of two during a global shift compared with an object shift .", "Colored dots correspond to the cells shown in ( B ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 09266 . 003 To analyze the dynamics of the effects of peripheral stimuli on the processing of central input , we decoupled the central and peripheral inputs by presenting a stimulus with a continuously flickering light intensity in the center , combined with brief peripheral motion .", "Although this stimulus differs from a global image shift , which simultaneously changes central and peripheral regions as in ( Figure 1 ) , the independent control of central and peripheral stimuli allowed us to assess the dynamics of how the periphery changes the encoding of a central stimulus .", "To create a local naturalistic input , the central object intensity flickered with a temporal power spectrum resembling natural scenes , which was inversely proportional to the frequency – called ‘pink noise’ ( Simoncelli and Olshausen , 2001 ) ( Figure 2A , right panel inset ) .", "The periphery was a checkered pattern that was either still , producing no temporal input , or reversed every 0 . 5 s , producing strong synchronized peripheral stimulation .", "For most recorded cells , shortly after peripheral stimulation there was an excitatory effect – indicated by an increase in firing ( Krüger and Fischer , 1973 ) – followed by strong inhibition as might underlie a previously reported component of saccadic suppression ( Roska and Werblin , 2003 ) , and then a slower recovery to the baseline state ( Figure 2B and Figure 2—figure supplement 1A ) .", "This effect also occurred with both left and right shifts of the checkers ( Figure 2B , inset ) , indicating that this effect was primarily not caused by the classical ( linear ) receptive field surround , which would have produced opposite effects for the two background phases . 10 . 7554/eLife . 09266 . 004Figure 2 . Peripheral gating of information transmission .", "( A-i )", "Spatial stimulus design showing central and peripheral regions .", "( A-ii )", "The temporal sequence in each region .", "The center stimulus flickered randomly with a naturalistic amplitude spectrum proportional to 1/f ( inset ) .", "In the periphery , the stimulus either shifted ( reversed in sign ) every 0 . 5 s or was still .", "Most cells had linear receptive field centers fully contained in the central region ( yellow oval indicates receptive field center ) .", "( B ) Peristimulus time histogram aligned to the time of peripheral stimulation .", "Inset shows the two different peripheral shifts averaged separately , indicating that both excitation and inhibition occur for both peripheral phases .", "( C ) Filters and nonlinearities of a linear-nonlinear model computed from the spike times and the center signal .", "In the Shift case , only spikes from the high firing rate window were used ( gray box in B ) .", "The dashed nonlinearity is the curve that would have resulted from a vertical shift of the Still case to account for the observed increase in activity in the high firing rate window .", "( D ) Mutual information between the spike count in a 50 ms time window and the central region as a function of time after the peripheral shift .", "Inset shows information computed separately for left and right shifts of the grating .", "( E ) Average for different cell types of the normalized information in the Shift condition for three different cell types; biphasic Off ( n = 95 cells ) , slow Off ( n = 10 ) and slow On ( n = 7 ) .", "Information was normalized by the value in the last bin .", "( F ) Average across cells of the information that the spike count carries about the peripheral signal I ( R; P ) or about the central region once information about peripheral input has been removed ( see Materials and methods ) .", "By the chain rule of mutual information , the two quantities add to the total amount of information the spike count conveys about the stimulus , I ( R; P , C ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 09266 . 00410 . 7554/eLife . 09266 . 005Figure 2—figure supplement 1 . Peripheral shift increases the response to central stimuli with a natural temporal spectrum .", "( A ) Scatter plot of the firing rate for each cell during gating ( 50–100 ms ) and baseline ( 450–500 ms ) time windows .", "( B ) Stability of information calculations over the amount of data size .", "The increase in the information ratio in the gating window ( Figure 2 ) was computed for all of the data , and various inverse fractions shown on the horizontal axis .", "A second-order polynomial fit was used to extrapolate the curve to the limit of infinite data ( Strong and Koberle , 1998 ) .", "( C ) Ratio between the mutual information in the gating window and the information in the last time window right before peripheral excitation as a function of the bin size used for counting spikes .", "Shown are values for On cells , monophasic Off cells and Biphasic Off cells .", "Dotted line represents bin size used in Figure 2 . DOI: http://dx . doi . org/10 . 7554/eLife . 09266 . 005 The presence of excitatory input , however , does not reveal how peripheral input changes the neural code for central input .", "For example , peripheral excitation could potentially saturate the cell , and thus mask central input .", "We therefore measured the sensitivity to the central region using a linear-nonlinear ( LN ) model ( see Materials and methods ) , consisting of a linear temporal filter representing the average feature preferred by the cell followed by a static nonlinearity capturing the cell’s average threshold and sensitivity , defined as the average slope of the nonlinearity ( Chichilnisky , 2001; Baccus and Meister , 2002 ) .", "In this case , although the LN model does not capture all of the nonlinear dynamics of the receptive field center ( Gollisch and Meister , 2010 ) ( some of which is captured in a model below ) , the nonlinearity can be used as a statistical measure of sensitivity to the central stimulus ( Kastner and Baccus , 2011; Baccus and Meister , 2002; Rieke , 2001; Zaghloul et al . , 2005 ) at different times relative to the peripheral shift .", "To observe changes in the neural code associated with peripheral excitation , we computed two LN models: one when the periphery was still and the second one under the shift condition using only spikes from a 50 ms gating window centered on the peak of excitation ( Figure 2C ) .", "Even though the firing rate greatly increased during 50–100 ms after the peripheral shift , the temporal filter changed little when compared to the still condition , indicating that the cell continued to convey the same average feature about the visual stimulus during the 50 ms high firing rate interval ( Figure 2C ) .", "We defined the sensitivity to the central stimulus as the average slope of the nonlinearity ( Kastner and Baccus , 2011; Baccus and Meister , 2002 ) and found that the sensitivity was the greatest 50–100 ms after a peripheral shift , during the high firing rate window .", "This indicates that the increase in firing rate after the peripheral shift is not due to a response independent of the center stimulus , as such an effect would have shifted the nonlinearity vertically , leaving the sensitivity to the center unchanged ( Figure 2C , dashed line ) .", "Instead , we find that an abrupt peripheral shift dynamically gates the response of a cell , enhancing the cell’s sensitivity to its preferred visual feature near the receptive field center .", "However , the amount of information conveyed is influenced not only by sensitivity but also by noise , and thus an increase in sensitivity does not necessarily imply an increase in information transmission .", "Therefore , to confirm that the increased activity and sensitivity were accompanied by an increase in transmitted information , we estimated the mutual information between the stimulus sequence in the object region and the cell’s response as measured by the spike count at different times relative to the peripheral shift .", "This quantity is I ( R;C|p ) , the mutual information between the response , R , and the central intensity , C , given a particular peripheral stimulus p∈P , where p is the time relative to the peripheral shift ( see Materials and methods ) .", "We found that just after a peripheral shift , information about the central stimulus sharply increased as compared to when the periphery was still , indicating that a signal from the periphery increases information transmission from the central region ( Figure 2D , Figure 2—figure supplement 1B and C ) .", "After this increase , information transmission then decreased ( 100–300 ms after the global shift ) and then recovered to the baseline state .", "All cell types showed a sharp increase of information during the gating window , with the peak time depending on the cell type ( Figure 2E ) .", "The leftward shift of the nonlinearity ( Figure 2C ) increased the slope of the nonlinearity and information transmission because the threshold of the nonlinearity in the baseline condition was positioned to the right of the mean stimulus , which is the case for ganglion cells of diverse species including mammalian and primate retina ( Chichilnisky , 2001; Keat et al . , 2001 ) .", "In this analysis , by considering I ( R;C|p ) we are taking the more traditional point of view that cells encode signals in the center of their receptive fields and are modified by other ( peripheral ) signals .", "However , one could argue that ganglion cells are actually encoding the periphery and being modified by the center .", "The total information between the response and both central and peripheral stimuli , I ( R;P , C ) can be separated into two components by the chain rule for mutual information ( Cover and Thomas , 1991 ) ( see Materials and methods ) .", "( 1 ) I ( R;P , C ) =I ( R;P ) +I ( R;C|P ) =IR;P+IR;C|ppεP However , on average , for all cell types studied , the information that the cell carried about the peripheral stimulus I ( R;P ) was substantially smaller than the information the cell carried about the center given the peripheral stimulus ( I ( R;C|P ) ) , averaging 27% of I ( R;C|P ) ( Figure 2F ) .", "These results support the view that peripheral input gates information transmission from the central region .", "Although it may seem puzzling that little information is conveyed about the periphery even though there is a large timed increase in the average firing rate , this is because the large peak at ~100 ms represents only the average response to the stimulus .", "On any given trial , the decision to fire is primarily controlled by the central input and in many cases , no spikes occurred when the periphery moved .", "Nonetheless , the brain could use a population of cells to identify the gating window as a time when activity increases synchronously throughout the retina .", "Our analysis using the number of spikes in a time bin neglects more complex encoding due to latency ( Gollisch and Meister , 2008 ) and firing patterns in the population ( Schnitzer and Meister , 2003 ) .", "Yet , this analysis suggests that the brain could extract this increased information simply by counting spikes , without the need for a more complex decoding scheme across time bins or using the population .", "We then compared the amount of information transmission with the theoretical maximum given the cell’s stochastic firing properties .", "We assessed the theoretical maximum by computing the sigmoidal nonlinearity that maximized information about the stimulus , given a cell’s maximum firing rate and measured spiking noise as defined by its spike-count distribution for a given average response , P ( r|⟨r⟩ ) .", "The average firing rate however was not constrained at a particular value , in contrast to previous studies , meaning that a leftward shift of the nonlinearity with the same maximum would generate a higher average firing rate ( Pitkow and Meister , 2012 ) ( see Materials and methods ) .", "After a shift , the information conveyed was 0 . 47 ± 0 . 04 , ( n = 18 cells ) of the maximal value , compared with 0 . 24 ± 0 . 05 before the shift .", "Thus , after the shift , the neural code used more of the capacity of the cell given its noise properties and the spike count code .", "However , this came at the increased cost of energy in terms of a higher firing rate ( 6 . 9 ± 1 . 7 Hz after the shift , and 1 . 9 ± 0 . 3 before the shift ) .", "Previous results indicate that the high threshold of ganglion cells allows them to conserve spikes at the expense of maximal information transmission ( Pitkow and Meister , 2012 ) .", "Our analysis indicates that after a peripheral shift , the neural code shifts away from energy conservation and towards high-throughput information transmission .", "To examine which properties of the neural code changed between the shift and still conditions , we presented a Gaussian white noise sequence with a fixed mean and different contrasts , defined as the standard deviation divided by the mean .", "This allowed us to compute and compare separate LN models for each contrast .", "We observed that both excitation and inhibition from a peripheral shift depended on the central contrast , with a much stronger increase in firing observed at low contrast ( Figure 3A ) .", "This result is consistent with the observation that cells with the weakest response to a moving square had the strongest effect from peripheral stimuli ( Figure 1 ) .", "We then fitted LN models as stated above at different contrasts during 50 ms time windows corresponding to gating , suppression , and recovery ( Figure 3B–C ) .", "As the contrast in the central region decreased , the filter in some cells became slower and more monophasic as previously reported ( Baccus and Meister , 2002 ) .", "However , although at low contrast , the gating window’s firing rate exceeded the recovery window’s firing rate by more than 20-fold , the filters were markedly similar .", "Thus , peripheral stimulation affected the sensitivity , but had a minimal effect on the average local features preferred by the ganglion cells . 10 . 7554/eLife . 09266 . 006Figure 3 . A more adapted representation underlies an increase in information transmission .", "( A ) The spatial stimulus was the same as in Figure", "2 . The time course of the central stimulus was a Gaussian white noise stimulus with one of four different contrasts or 100% binary contrast , consisting of black and white intensity values .", "PSTHs are shown for the different conditions .", "( B ) Filters computed using only spikes from 50 ms time windows , corresponding to color boxes in ( A ) .", "Purple , gating window; Olive green , suppression window; Orange , recovery window .", "( C ) Input distributions ( left ) and nonlinearities in the same three 50 ms time windows as in ( B ) .", "Upper curves are all in units of the linear prediction; lower curves show the same data but in units of standard deviation of the linear prediction .", "The abscissa is displayed on a logarithmic scale , such that normalization by the standard deviation produces a lateral shift .", "( D ) Average adaptation index across cells that exhibited peripheral excitation ( see Materials and methods , n = 400 ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 09266 . 00610 . 7554/eLife . 09266 . 007Figure 3—figure supplement 1 . Periphery induced changes in adaptation .", "( A ) Left , schematic nonlinearities for a hypothetical perfectly adapted cell , where the slope of the nonlinear is inversely proportional to the contrast .", "Right , normalized slope of the nonlinearities vs . normalized inverse contrast , showing the two quantities should be equal for an ideally adapting cell .", "( B ) Same as ( A ) for a non-adapting cell , showing that for a non-adapting cell the slope does not change .", "( C ) Average nonlinearity slope during the 50 ms time window corresponding to just after the peripheral shift ( purple in Figure 3B–D ) vs . the average nonlinearity slope during the 50 ms corresponding to the recovery time window ( orange ) for both high ( 24% ) and low ( 3% ) contrast in the center region . DOI: http://dx . doi . org/10 . 7554/eLife . 09266 . 007 Changes in sensitivity are also known to be caused by adaptation to the local contrast .", "We therefore tested how peripheral stimulation influenced adaptation to the central contrast by measuring changes in adaptation as a function of time since the peripheral shift .", "For an ideally adapting cell , the sensitivity would scale in inverse proportion to the contrast ( Figure 3—figure supplement 1A–B ) .", "Previous results , however , have shown that ganglion cells adapt less than this ideal amount , in particular at low contrasts ( Ozuysal , 2012 ) .", "We found that in the gating window , responses to different contrasts were much more similar to each other , indicating a greater level of adaptation to the central contrast ( Figure 3C ) .", "This effect arose because at low contrast , the slope of the nonlinearities changed more than at high contrast , similar to the stronger effects of gating seen with cells that responded more weakly to a shifting object ( Figure 1D ) .", "At 3% contrast , the slope changed by 5 . 5 ± 1 . 1 Hz per contrast unit ( one s . d . of the nonlinearity input was 0 . 03 contrast units , or 3% ) and at 24% contrast the slope changed by 0 . 20 ± 0 . 06 Hz per contrast unit ( one s . d . was 0 . 24 contrast units , or 24% ) ( Figure 3—figure supplement 1C ) .", "As a result of these effects at different contrasts , during the gating window nonlinearities reached a more similar height across contrasts .", "Accordingly , when normalized by the stimulus standard deviation at each contrast , nonlinearities also had a more similar shape across contrasts in the gating window .", "We computed an index of adaptation that takes the value of 1 for an ideally adapting cell and 0 for a non-adapting cell ( see Materials and methods and Figure 3—figure supplement 1A–B ) , and found that most cells increased their adaptation to contrast during the gating window , such that all contrasts were represented with more similar responses than during the recovery window ( Figure 3D ) .", "This indicates that an increase in adaptation underlies the increase in information during the gating window , such that near the receptive field center , both weak and strong signals are conveyed .", "Cells exhibiting contrast adaptation – by changing their sensitivity when the contrast changes – will increase information about fluctuations in intensity , but potentially lose information about the contrast itself ( Fairhall et al . , 2001 ) .", "Because many cells showed increased adaptation after a peripheral shift , we tested whether the cells encoded different properties of the stimulus – the sequence of light intensities and the contrast – at different times relative to a peripheral shift .", "We designed a flickering stimulus that had a relatively small number of conditions to facilitate the estimation of information about the stimulus sequence and/or the contrast .", "The center followed a binary white noise M-sequence , at four possible contrasts σ , where the instantaneous intensity value was μ+σ⋅m and μ is the mean intensity , fixed throughout the experiment and m=±1 are the instantaneous values of the M - sequence ( see Materials and methods ) .", "All combinations of binary sequences ( up to four frames , lasting 400 ms , m ( 4 ) ∈M ( 4 ) , contrasts and times relative to peripheral excitation were presented an equal number of times ( see Materials and methods and Figure 4A ) .", "Figure 4B shows raster plots for one-cell ordered according to contrast and mixing the responses to all M - sequences .", "We estimated the mutual information between the response and two stimulus parameters at different times relative to the peripheral shift – the light intensity sequence in the previous four frames ( M ( 4 ) ) , I ( R;M ( 4 ) |p ) , and the center’s contrast ( ∑ ) , I ( R;∑|p ) , where σ∈∑ ( Figure 4C ) .", "We found that the responses coming from the same cell at different times carry different types of information .", "When computing I ( R;M ( 4 ) |p ) , the analysis was conducted as if the brain was decoding the stimulus sequence without knowing the contrast .", "The results were similar , however , if we considered that the brain might decode the contrast , and use this knowledge to better decode the stimulus sequence ( eq ( 10 ) and Figure 4—figure supplement 1 ) .", "Whereas a static neural code would typically face the choice between adaptation and preserving the adapting statistic , a dynamic neural code avoids this tradeoff by rapidly switching between complementary representations of the same stimulus . 10 . 7554/eLife . 09266 . 008Figure 4 . Different stimulus properties are conveyed with different dynamics .", "( A ) Experimental design for the measurement of sequence and contrast information .", "The center object follows a binary M-sequence at four different contrasts .", "Each position in the sequence and contrast combination occurs at all possible times relative to a peripheral shift .", "( B ) Raster plots for an example cell aligned to the time of the peripheral shift and ordered according to contrast .", "Many different sequences are shown for each contrast value .", "Luminance values in the center change every 100 ms , generating temporally discrete responses .", "Vertical lines show the times used to extract responses for the information calculation .", "( C ) Average across cells ( n = 94 ) of the normalized information conveyed about the contrast ( four different levels , solid line ) or the four frame stimulus sequence M ( 4 ) ( dashed line ) as a function of time since the shift . DOI: http://dx . doi . org/10 . 7554/eLife . 09266 . 00810 . 7554/eLife . 09266 . 009Figure 4—figure supplement 1 . Different components of the stimulus are conveyed with different dynamics . Information that the response carries about the contrast at a particular time relative to peripheral stimulation ( solid black trace , left vertical axis ) and information that the response carries about the center sequence M ( 4 ) given that the response occurs at a given contrast and time relative to peripheral stimulation ( red trace , right vertical axis ) .", "These two terms sum to the total information given by the response about the stimulus at a given time relative to peripheral stimulation , I ( R;∑ , M ( 4 ) |p ) .", "Information that the response carries about the time , p relative to peripheral stimulation , I ( R;P ) ( dotted black trace , left axis ) .", "Although this quantity is a number and not a function of time , it is shown here for comparison . DOI: http://dx . doi . org/10 . 7554/eLife . 09266 . 009 To identify the minimum components required to produce the dynamic neural code we observed , we began with the known structure of excitatory input to a ganglion cell consisting of a central pathway comprising a linear filter , a static nonlinearity and an adaptive stage .", "This central pathway model was analogous to an accurate model of contrast adaptation ( Ozuysal , 2012 ) , where the rectified nonlinearity and adaptation likely occur at the bipolar cell synaptic terminal , although here we used a simplified adaptive stage .", "Nonlinear peripheral input is delivered only in the inner retina , as horizontal cells do not respond to fine gratings ( Baccus et al . , 2008 ) .", "Therefore , the only remaining choice in the model structure was the level at which to combine the peripheral input .", "Because a peripheral shift caused the overall nonlinearity of the LN model to shift laterally , rather than vertically with respect to the central pathway’s linear input ( Figures 2 and 3 ) , the peripheral pathway delivers input prior to the nonlinearity , corresponding to input to the bipolar cell terminal ( Figure 5A ) .", "The peripheral pathway can be represented by small rectified subunits that cause the response to be insensitive to the peripheral pattern ( Victor , 1979; Olveczky et al . , 2003 ) .", "Rather than explicitly simulate spatiotemporal dynamics of the periphery , we modeled the net effect of the abrupt peripheral stimulus via a timed biphasic signal .", "The model effectively replicated the data for Gaussian stimuli at different contrasts ( Figure 5C , D ) , as well as for constant luminance stimuli ( Figure 5—figure supplement 1 ) . 10 . 7554/eLife . 09266 . 010Figure 5 . Gating of information through a shift in an internal threshold .", "( A ) Model of a cell where two pathways are combined prior to a threshold and an adaptive block , implemented here as a feed forward divisive effect with a memory .", "Peripheral pathway is composed of many nonlinear subunits making the pathway insensitive to the stimulus spatial pattern and delivers biphasic input to the central pathway ( first positive , then negative ) .", "The stimulus is Gaussian white noise at 3–24% contrast matching the experiment ( and colors ) in Figure", "3 . The central pathway is composed of a linear temporal filter , because stimulus is only a function of time .", "( B ) Signals arising at points", "( a ) ,", "( b ) and", "( c ) whose locations in the model are marked in panel ( A ) .", "When the peripheral input is positive ( gating window , purple bar ) or negative ( suppression window , olive green bar ) the central input is shifted to higher or lower values with respect to the baseline state ( recovery window , orange bar ) and fixed threshold .", "Right , the Gaussian distribution of the filtered stimulus occurring at point", "( c ) in ( A ) compared to the threshold nonlinearity occurring after point", "( c ) in ( A ) during the gating ( purple ) , suppression ( olive green ) and recovery ( orange ) windows .", "The peripheral input effectively shifts the Gaussian mean with respect to the fixed threshold .", "( C ) Model responses to the same Gaussian stimulus used in Figure 3 at the times of the corresponding color bars in ( B ) .", "( D ) Adaptation index for the model’s output as a function of time after the shift . DOI: http://dx . doi . org/10 . 7554/eLife . 09266 . 01010 . 7554/eLife . 09266 . 011Figure 5—figure supplement 1 . Model responses to peripheral gating for steady central stimuli .", "( A ) Traces show the signals at points ( a–c ) indicated in the model in Figure 5A .", "Different steady intensity levels from the center", "( a ) are summed with dynamic peripheral input", "( b ) to generate distinct transient responses in the model", "( c ) prior to the threshold .", "Dotted line represents the cell’s threshold , which is only crossed with the aid of peripheral excitation .", "( B ) PSTHs generated by the model when constant luminance levels are delivered to the model .", "( C ) Average PSTH ( n = 21 cells ) of the firing rate produced by a central 1 mm square at one of four constant luminance levels logarithmically spaced , with the periphery shifting every 0 . 5 s .", "In contrast to Figure 1 , the central square is fixed in space and does not move with the periphery .", "Error bars are SEM . DOI: http://dx . doi . org/10 . 7554/eLife . 09266 . 011 A key aspect of the model is the order in which the signals are combined .", "Because the peripheral input is delivered prior to the threshold and adaptive stage , it is summed with the unadapted measure of the central input .", "This causes the peripheral input to have a larger effect on weak central stimuli than on strong ones as experimentally observed ( Figures 1 and 3 ) .", "Furthermore , weak stimuli only cross threshold when peripheral input is applied ( Figure 5B ) , allowing the adaptive stage to encode and adapt to signals of all strengths , including those from a central stimulus with a constant intensity ( Figure 5—figure supplement 1B ) .", "Thus , when peripheral excitation is present ( Figure 5B , Figure 5—figure supplement 1A ) , the model applies a lower threshold relative to the central input , conveying more information about the full range of contrast environments , but less information about the contrast level .", "When peripheral input is absent or inhibitory , the model applies a high threshold with respect to the central input ( Figure 5B and Figure 5—figure supplement 1A ) , conveying primarily higher contrasts and encoding information about the contrast level .", "An important conclusion we derived from the model is that a single amplitude of peripheral input ( equivalent to a fixed threshold shift with respect to the central input ) replicates the results at all central contrasts .", "Thus even though the effects of peripheral input differ with the central contrast , this is because of the differing downstream effects of adaptation; the peripheral signal itself delivered prior to the threshold does not depend on the central contrast .", "We then measured the scale of excitation and compared it to the scale of transient peripheral inhibition , which is thought to play a role in suppressing the effects of eye movements ( Olveczky et al . , 2003; Roska and Werblin , 2003 ) .", "To measure the distance over which lateral excitation acts , we designed a stimulus to measure peripheral gating of sensitivity to a central object as a function of distance from the peripheral stimulus .", "The stimulus had three different components .", "First , the periphery was composed of 50 μm checkers that covered the whole screen .", "Second , on top of the periphery , a mean intensity gray mask covered the peripheral checkers over the central region; the size of the mask , L , was varied .", "Third , the central object consisting of a pink noise flickering sequence was then added on top of the central gray region and was presented as a fenestrated checkerboard pattern of fixed size ( Figure 6A–B ) .", "By decreasing the mask size , more peripheral checkers were present , and the distance between peripheral checkers to any measured cell was decreased .", "At the smallest mask sizes , peripheral checkers were intercalated with the central region object , and occupied all space not covered by the central object ( Figure 6B , bottom ) .", "The excitatory influence of gating acted at distances of up to 1 mm ( Figure 6C ) .", "This further confirms that the effect was distinct from the linear receptive field , which would not be activated by a fine checkerboard at this distance .", "The observed changes in sensitivity indicate that excitatory and inhibitory influences acted over a similar scale , equivalent to a radius of ~20 deg .", "of visual angle . 10 . 7554/eLife . 09266 . 012Figure 6 . Similar spatial scale for peripheral excitation and inhibition .", "( A ) Experimental design for measuring the spatial scale of peripheral excitation and inhibition ( see Materials and methods ) .", "( Top )", "The periphery was a checkerboard pattern with squares of 50 μm covering all the screen that reversed in intensity at 1 Hz .", "( Middle )", "A gray mask with no temporal component and variable size , L , was drawn on top of the checkers .", "( Bottom )", "The object was a checkered pattern with a square size of 100 μm ( shown in green for illustration ) .", "Object squares in the center flickered with a pink noise distribution , with an equivalent contrast of 10% , changing every 30 ms . ( B ) Top , schematic of how the different components of the stimulus were layered .", "Middle and Bottom , a sample cell’s spatial receptive field for the object stimulus is shown in red with the color representing sensitivity to that particular square of the object for two different sizes of the intermediate mask .", "With this design , the object does not change across the different conditions and any change in the sensitivity to the object is due to the distance of the peripheral checkers .", "( C ) Average over cells ( n = 66 ) of the normalized sensitivity to the object stimulus , which was computed as the average slope S ( d , t ) of the nonlinearity of a linear-nonlinear model normalized by the average slope of the nonlinearity when the background was at infinity , S ( ∞ , t ) as a function of time bin t , relative to the background shift for two different mask sizes .", "( D ) Average of the normalized sensitivity as in panel ( C ) as a function of distance between the cell and the mask during the gating window ( 50–100 ms after the shift ) and an inhibitory window ( 150–200 ms after the shift ) .", "Each point in a line corresponds to the minimum distance between the cell’s linear receptive field and the background checkers for a particular background condition mask L . For the gating window , the baseline of sensitivity at 0–50 ms , which is too soon after the shift to be affected by it , was subtracted for each distance d .", "This subtraction was not done for the recovery window because at distances less than 500 µm , residual inhibition creates a saturating decrease in sensitivity , causing many cells to have zero slope at this time .", "See Figure 6—Figure supplement 1 for sensitivity before the subtraction of this baseline . DOI: http://dx . doi . org/10 . 7554/eLife . 09266 . 01210 . 7554/eLife . 09266 . 013Figure 6—figure supplement 1 . Similar spatial scale for peripheral excitation and inhibition . Average normalized sensitivity as in Figure 6D as a function of distance between the cell and the mask during three different time windows .", "At 0–50 ms , the sensitivity had not yet recovered from the previous shift .", "Therefore , the sensitivity at 0–50 ms was taken as a baseline , which was subtracted from the sensitivity at 50–100 ms , as shown in Figure 6D . DOI: http://dx . doi . org/10 . 7554/eLife . 09266 . 013" ], [ "Our results indicate that transient peripheral input increases information transmission about the visual feature in the receptive field center .", "However , for natural images and an abrupt global shift in the image as from motion of a large object , the eye or head , the transient effect of gating would be combined with transient changes in image statistics created by the image shift .", "We therefore compared the timing of our gating results ( Figure 2 ) to the dynamics of the expected information transmission for natural scenes during an abrupt shift in the image .", "The statistics over the receptive field center depend both on the image and the sequence of eye movements , and have strong correlations over time .", "Consequently , attaining representative statistics of the distributions of both light intensity and responses over multiple time points requires many trials .", "Owing to the difficulty in sampling the high dimensional distribution of the stimulus and responses over multiple time points ( see Materials and methods ) , such an experiment would be prohibitively long .", "We therefore addressed this question using a spatiotemporal version of our model of gating with simulated eye movements and a large number of natural images .", "To estimate the timing of information transmission under natural images , we combined natural images with simulated global shifts of the image ( Figure 7A-i ) .", "The spatiotemporal model used for fast Off-type ganglion cells had the same structure as the reduced model ( Figure 5 ) , but we replaced the initial linear filter with the spatiotemporal receptive field measured from a fast Off-type bipolar cell ( Baccus et al . , 2008 ) .", "Because peripheral gating is delivered prior to the threshold in the model , it is likely that it represents an input presynaptic to the bipolar cell terminal .", "Furthermore , because strong adaptation to contrast is thought to arise in the presynaptic terminal , the model is effectively of the synaptic release from bipolar cells , although the actual density of bipolar cells was not modeled .", "Thus , although further nonlinearities exist in the inner retina that would make the responses of ganglion cells to natural scenes more complex , we expect this model will be informative as to the relative timing of bipolar cell release and peripheral gating .", "Fast Off-type bipolar cells are roughly linear at a constant mean intensity for a stimulus that flickers ( Figure 7—figure supplement 1 ) or jitters as in fixational eye movements , and previous models indicate that these cells may convey the primary input to fast Off-type ganglion cells ( Baccus et al . , 2008 ) .", "Because the model was used to estimate the information transmitted after a global image shift , we measured the noise in bipolar cells at different contrasts .", "The signal-to-noise ratio ( SNR ) increased with contrast , which was incorporated into the model ( Figure 7A-iii , see Materials and methods ) .", "This model does not capture all nonlinearities of the bipolar cell response , including luminance adaptation , a slightly saturating nonlinearity for high contrast stimuli , and weak contrast adaptation .", "Furthermore , because we did not include luminance adaptation , the model effectively assumes that during the fixation period , adaptation has reached a steady state , and that the global shift is too brief to cause substantial luminance adaptation .", "The main goal of the model , however , was to gain insight into the dynamics of information transmission under sudden image shifts .", "The input to the model consisted of a set of natural images combined with fixational drift eye movements interrupted by a sudden image shift of 6 degrees ( Figure 7A ) , sufficient to exceed most local image correlations ( Figure 7—figure supplement 1A ) .", "Because natural images have strong correlations , and because bipolar cell receptive fields are biphasic both in space and time , fixational drift created a relatively small change in the filtered stimulus ( Figure 7A-ii ) .", "However , for a brief window of time after the shift , the light intensity seen by the bipolar cell was less correlated with its previous values ( Figure 7B , D and F ) .", "During this window , a wider range of possible filtered stimuli occurred , but still with most values remaining close to zero and under the fixed threshold ( Figure 7A-ii , B ) .", "Based on the noise measurements in bipolar cells , because of the higher variance after the shift , the SNR of the bipolar cell membrane potential would be expected to increase .", "Note that these simulations do not include the effects of moving objects in the environment , which would cause the stimulus to more closely resemble experiments in Figures 2–4 , where central and peripheral inputs are uncorrelated . 10 . 7554/eLife . 09266 . 014Figure 7 . Model of central information for natural scenes with eye movements .", "( A ) Spatiotemporal model used in the simulation .", "( A-i )", "An eye movement trajectory overlaid on a natural image , consisting of fixational drift , and a sudden eye movement ( green line ) that takes the center of each cell from one image location ( insets ) to another .", "The image series is convolved ( ⊗ ) with a separable spatiotemporal filter previously measured from a fast Off-type bipolar cell ( Baccus et al . , 2008 ) , yielding a linear prediction for a bipolar cell at each spatial location .", "( A-ii ) ( Top ) The linear prediction for 100 model bipolar cells over different images as a function of time .", "A sudden eye movement occurs at 0 s ( dotted line ) .", "Vertical scale is in arbitrary units .", "( Middle and Bottom )", "The linear prediction is shown for a population of bipolar cells ( one at each spatial location ) at 100 ms before and after the sudden image shift responding to the image and eye movement trajectory shown in ( A-i ) .", "Color scale is the same in both images .", "( A-iii )", "Noise model .", "( Top ) signal to noise ratio measured experimentally in a bipolar cell from repeats of a spatially uniform Gaussian white-noise stimulus under different stimulus contrasts ( Figure 7—figure supplement 1 ) ( Ozuysal , 2012 ) .", "( Bottom )", "Noise generated with this model , shown in the same arbitrary units as in ( A-ii ) Top .", "Black line is the standard deviation of the noise at each point in time .", "( A-iv ) ( Top ) After the linear central input is summed with the noise , the result is passed through a rectifying nonlinearity and then through a feedforward divisive operation representing a simplified version of adaptation , as in the model in Figure 5 .", "( B ) Distributions of linear prediction values over many images at different times , compared with the rectifying nonlinearity ( black line ) from ( A-iv ) .", "Distributions before t = 0 s and after 350 ms are identical .", "( C ) Dynamics of information transmission after a sudden eye movement .", "The Shannon mutual information I ( Gt;Rt|p ) between the linearly filtered stimulus , g ( t ) , and the model output , r ( t ) ( black dashed line ) at a given delay from the shift , p , and the conditional mutual information I ( Gt;Rt|Rt−Δ , p ) between the same quantities when conditioning on the response at a previous time r ( t−Δ ) ( black solid line ) .", "Both stimuli and responses were averages over bins of 50 ms . Also shown is the standard deviation of the linear prediction from ( A-iii ) ( green line ) .", "( D ) Comparison of the expected conditional mutual information from the model at each time after an image shift ( black line ) with the time course of information transmission measured during experiments for several cell types ( reproduced from Figure 2E ) .", "DOI: http://dx . doi . org/10 . 7554/eLife . 09266 . 01410 . 7554/eLife . 09266 . 015Figure 7—figure supplement 1 . Noise measurements in bipolar cells .", "( A ) Top .", "Three membrane potential responses of a fast Off bipolar cell responding to a repeated Gaussian white noise of 5% contrast .", "Middle .", "Mean of the three repeats , the standard deviation of which was an estimate of the signal carried by the bipolar cell , along with a linear model of the response , consisting of the stimulus convolved with a linear temporal filter .", "Bottom , the residual membrane potential , which is the difference between each individual response and the mean value .", "The standard deviation of these residual responses is estimated to be the noise at each contrast .", "( B ) Same as A . for 21% contrast . DOI: http://dx . doi . org/10 . 7554/eLife . 09266 . 01510 . 7554/eLife . 09266 . 016Figure 7—figure supplement 2 . Correlations and information in a spatiotemporal model of gating .", "( A ) Correlation coefficient between image patches of 1° ( the size of the simulated bipolar cell’s center ) as a function of their distance .", "Vertical dotted line shows the sudden eye movement used in the simulation , exceeding most positive correlations between images patches .", "( B ) Conditional mutual information I ( Gt;Rt|Rt−Δ , p ) between the filtered stimulus and the output of the spatiotemporal model in Figure 7 at a given delay from the shift , p , at three different noise levels .", "Doubling or halving the standard deviation of the noise did not change the results qualitatively .", "Gray trace indicates the near-zero level of information when randomly permuting the response with respect to the filtered stimulus , indicating that the joint distribution of filtered stimulus and response was sampled sufficiently .", "( C ) Same as ( B ) but changing the bins over which linear predictions and responses are computed .", "( D ) Correlation coefficient between linear prediction samples , g ( t ) and g ( t−50 ms ) , shown at different times , t .", "Significant decorrelation is seen even as early as 50 ms after the shift .", "( E , F )", "Joint probability distribution of g ( t ) and g ( t−50 ms ) , at two times separated by 50 ms , showing that prior to a sudden shift in the image linear inputs are highly correlated in time and more decorrelated just after the shift . DOI: http://dx . doi . org/10 . 7554/eLife . 09266 . 016 We first estimated the mutual information between the set of linearly filtered stimuli , G={g ( t ) } , and the model’s response , R={r ( t ) } , as a function of time from the shift ( Figure 7C , dashed line ) .", "Because of strong correlations in the stimulus ( discussed further below ) , sequential measurements of the intensity in a new environment are largely redundant , and thus may not convey added information .", "To account for this effect , we estimated the novel information learned about the stimulus from a response given that the system has access to the previous response .", "This is the conditional mutual information I ( Gt;Rt|Rt−Δ , p ) between the set of linear predictions Gt and the responsesRt at the same time t given the response at the previous time point Rt−Δ , computed for a given delay p from the shift ( see Materials and methods ) .", "As expected , because during fixational eye movements responses are highly correlated in time , each additional sample conveyed little novel information .", "However , after the sudden shift moved the receptive field to a new location , the conditional mutual information abruptly increased ( Figure 7C , solid line ) .", "Unlike the mutual information at different times relative to the shift , I ( G;R|p ) , the conditional mutual information captures the intuition that most of the information about the new environment should occur in a short amount of time ( Figure 7—figure supplement 2 ) .", "Most importantly , the conditional mutual information showed faster dynamics ( Figure 7C , continuous black line ) , with new information arriving faster than the peak of the linear prediction ( Figure 7C , green line ) .", "We then compared the timing of the increase in information from gating ( Figures 2–3 ) with the timing of the expected increase in the conditional information following the shift ( Figure 7D ) .", "We found that these greatly overlapped , meaning that the active increase in information produced by gating is timed to match the expected increase in novel information generated by the shift .", "Given the statistics of natural scenes and the measured noise in bipolar cells , our model indicates that gating represents a mechanism that increases information transmission at the expected peak of novel information after a global shift in the image .", "Adaptation is typically considered to be a process that optimizes information transmission given the current environment , and previous studies have focused on which threshold response curve would maximize information in the current environment ( Laughlin , 1981; Brenner et al . , 2000 ) .", "However , it is clear that information transmission is not the only objective , as the threshold of retinal ganglion cells is much higher than predicted by this ideal .", "Consequently , it has been proposed as an alternative factor that ganglion cells conserve spikes at the expense of maximal information transmission ( Pitkow and Meister , 2012 ) .", "We propose that neither view of a neural code optimized for a single current environment – either for maximal information transmission or for energy efficiency – is fully representative of natural vision .", "Our findings indicate that a peripheral shift causes a switch from a code that conserves energy to one of increased information transmission , with higher information transmission occurring at the expected time of higher signal-to-noise ratio and higher information content .", "These results suggest a general principle of neural coding – the dynamic allocation of neural activity to times most likely to contain novel information .", "This principle of adaptation acts to allocate resources across environments , and in fact is analogous to known principles of communication theory that act to enhance dynamic information transmission under an energy constraint .", "An energy-efficient communications channel that carries signals over a range of frequencies should allocate power such that signal power plus noise power is a constant , except for frequencies where noise exceeds a value set by the available power ( Shannon , 1998; Warland and Rieke , 1999 ) .", "This concept of efficient power allocation is known as ‘water-filling’ , as suggested by the notion of pouring power allocated to signal transmission into a basin whose depth varies according to noise but whose surface level is constant .", "It is less well appreciated in neuroscience that the same water-filling principle applies to efficiently allocate power over time when the noise level dynamically varies as can occur during wireless transmission ( Goldsmith and Varaiya , 1997 ) .", "In this case , greater power should be allocated to times of higher SNR .", "Because a higher variance signal has a higher SNR in the bipolar cell ( Figure 7A-iii [Ozuysal , 2012] ) , this principle agrees with our observation that additional spiking is allocated to times of expected high variance of the filtered stimulus ( Figure 7C–D ) .", "However , because both signal and noise are changing dynamically , further studies are needed to compare dynamic changes in information transmission to an estimated optimal allocation of power over time given the changing stimuli and noise .", "Our results indicated that gating occurs at the time when the expected statistics of the central input will change .", "This expectation may arise from the sudden movement of large objects , and we expect that peripheral gating will be important during natural saccadic eye movements .", "Previous results have strongly implicated transient nonlinear peripheral inhibition in suppressing the effects of fixational eye movements on object motion sensitive ganglion cells ( Olveczky et al . , 2003 ) , and the similar spatial scale of peripheral nonlinear gating and nonlinear inhibition ( Figure 6 ) is consistent with a role of gating in eye movements .", "Although salamanders and other amphibians have differences in their eye movements in that they make head saccades to target prey ( Manteuffel and Roth , 1993 ) , they have similar fixational drift ( Manteuffel et al . , 1977 ) and optokinetic head movements ( Manteuffel , 1984 ) to mammals .", "Accordingly , the property of object motion sensitivity related to fixational eye movements is common to both salamanders and mammals .", "Similarly , the basic phenomenon of peripheral retinal excitation has been observed in mammals , and we expect that effects on neural coding we observe here will be similar .", "The duration of the ~1 degree global image shifts we have used ( Figure 1 ) is one stimulus frame ( ~30 ms ) , similar to the duration of a ~1 degree saccade in a number of species , for rabbit , cat and monkey , 20–50 ms ( Collewijn and Zuidam , 1977; Evinger et al . , 1981; Fuchs and Johns , 1967 ) ; humans , ~20 ms ( Baloh et al . , 1975 ) ; and fish , ~70 ms ( Easter , 1975 ) .", "Although larger saccadic eye movements are longer in duration , the key property we find is that the timing of the linear filter in the receptive field center is coincident with temporal filtering from the peripheral input .", "Thus , even if the global shift is more smooth as in the case of a larger saccade or an amphibian head saccade , we expect that the excitation from both center and periphery will still coincide .", "Timing signals that indicate a changing stimulus have been observed in other systems that use active sensation , including sniffing in olfaction and whisking in the vibrissae system ( Shusterman et al . , 2011; Hill et al . , 2011 ) .", "In these cases , an efferent copy of a motor command can provide the timing signal .", "But because the retina lacks such an efferent copy , a signal that the stimulus is changing must be computed from the sensory input .", "Inhibitory amacrine cells are known to deliver signals laterally across long distances , and could increase the firing rate through synaptic disinhibition ( Barlow et al . , 1977 ) .", "We note that a similar organization is found in the hippocampus , where a common signal is generated by oscillations in inhibitory neurons ( Buzsáki , 2002 ) .", "On the principle that the threshold should be lowered when greater information is expected , synchronous oscillations between brain regions may perform a similar function of allocating sensitivity to time intervals of greater information content .", "The neural code embodies a choice between tradeoffs .", "A high threshold may be efficient in terms of energy , at the expense of the amount of information ( Pitkow and Meister , 2012 ) .", "A biphasic filter and a threshold may emphasize novelty in natural scenes ( Srinivasan et al . , 1982 ) , but certain stimuli such as a constant luminance will be rejected .", "An adaptive system may improve information transmission across an entire set of stimuli , but the particular statistic that triggers adaptation may be lost .", "It is commonly assumed that a cell makes a single choice among these alternatives , whatever the benefits and consequences .", "Our results , however , show that cells can sequentially switch between complementary representations to capture the benefits of both ." ], [ "Larval tiger salamander retinal ganglion cells were recorded using an array of 60 electrodes ( Multichannel Systems ) as described ( Kastner and Baccus , 2011 ) .", "Intracellular recordings from bipolar cells were performed using sharp microelectrodes as previously described ( Ozuysal , 2012 ) A video monitor projected stimuli at 60 Hz , and values of intensity changed at 30 Hz .", "The monitor was calibrated using a photodiode to ensure the linearity of the display .", "Stimuli had a constant mean intensity of ~10 mW/m2 .", "Contrast was defined as the standard deviation divided by the mean of the intensity values , unless otherwise noted .", "To measure the difference between object and global shifts ( Figure 1 ) , the stimulus consisted of a square object 1200 µm on a side and a constant luminance of one of four logarithmically spaced values , and was presented in front of a black and white background checkerboard ( 50-µm squares ) .", "Either the object alone or the entire image was suddenly displaced 50 µm left and right at 1 Hz .", "The experiment was repeated with both phases of the background checkerboard , for a total of 16 combinations of shifts .", "Each combination was presented for 110 s twice in interleaved format with movements happening every 0 . 5 s .", "The first 10 s of each presentation were discarded leaving 200 trials per condition , with an equal number of left and right shifts that were analyzed independently ( see Figure 1C ) .", "LN models for Gaussian stimuli ( Figure 3 ) consisted of the light intensity passed through a linear temporal filter , which describes the average response to a brief flash of light in a linear system , followed by a static nonlinearity , which describes the threshold and sensitivity of the cell ( Baccus and Meister , 2002 ) .", "To compute LN models for white noise stimuli , we first computed linear filters , F ( t ) , which were the time-reverse of the spike-triggered average .", "Then , we calculated linear prediction , g ( t ) , as the convolution of the temporal filter and the central stimulus , s ( t ) , ( 2 ) g ( t ) =∫F ( τ ) s ( t−τ ) dτ A static nonlinearity , N ( g ) , was computed by averaging the value of the firing rate , r ( t ) , over bins of g ( t ) .", "The filter , F ( t ) , was normalized in amplitude such that it did not amplify the stimulus , i . e . the variance of s and g were equal ( Baccus and Meister , 2002 ) .", "Thus , the linear filter contained only relative temporal sensitivity , and the nonlinearity represented the overall sensitivity of the transformation .", "For pink noise stimuli ( Figure 2 ) , a sequence was generated with an amplitude spectrum that was inversely proportional to the frequency ( 1/f ) .", "Because the stimulus contained temporal correlations , the linear filter was computed by reverse correlation while normalizing by the autocorrelation of the stimulus ( Baccus and Meister , 2002 ) .", "In our experimental designs , the full stimulus S consisted of two components , the periphery , P , and a center stimulus C . In all experiments , the periphery was either still ( with zero entropy and thus the set of responses R contained no information about it ) , or reversed at 1 Hz .", "By discretizing time in 50 ms bins we create 20 different peripheral conditions p ∈ P , each of which represents a time relative to the period of the peripheral stimulus .", "By the chain rule of information ( Cover and Thomas , 1991 ) ( 3 ) I ( R;P , C ) =I ( R;P ) +I ( R;C|P ) The last term can be understood as the average information that the response carries about the central stimulus if the peripheral stimulus was known .", "This equation can also be written as ( Cover and Thomas , 1991 ) ( 4 ) I ( R;P , C ) =I ( R;P ) +⟨I ( R;C|p ) ⟩p∈P where the last term is an average over all instances of the peripheral stimulus p ∈ P . We computed I ( R;C|p ) ( the quantity inside the .", "p∈P in eq ( 8 ) ) for each time bin p relative to the peripheral period ( Figure 2D , inset ) so that for each p there is no information between the cell’s response and the peripheral stimulus ( because under the set of stimuli analyzed there is only one peripheral stimulus , which has zero entropy ) .", "To analyze how information varies as a function of time relative to the peripheral shift , we show I ( R;C|p ) averaged over both phases of the periphery , each of which had a similar time course ( Figure 2D , inset ) .", "A 200 s sequence of a Gaussian pink noise ( 1/f amplitude spectrum ) stimulus with an equivalent contrast of 10% was repeated 10–20 times .", "For the stability of information calculations with this number of repeats , see ( Figure 2—figure supplement 1B ) .", "In the Still condition , the periphery was static and in the Shift condition the peripheral checkerboard shifted every 0 . 5 s .", "As stated above , each time relative to the peripheral period was analyzed separately , so that under each condition there was no information between the response of a cell and the periphery’s position .", "Although the central sequences were not the same for each time bin relative to the peripheral shift , by having 200 central sequences per periphery we limit the chance of biasing any particular periphery by associating it with more ( or less ) discriminable central stimuli .", "Center sequences were identical between the Still and the Shift conditions , and therefore the differences between I ( R;C|p ) under still and shift for any peripheral stimulus p is only attributable to the one difference in the experimental conditions , the presence or absence of peripheral stimulation .", "The response , ri , during trial i was defined as the number of spikes in a 50 ms time bin; other intervals from 12 to 160 ms yielded similar results ( Figure 2—figure supplement 1B ) .", "The mutual information , I ( R;C|p ) , was computed by taking the difference between the total response entropy , H ( R|p ) , and the conditional ( noise ) entropy , HR;C|p ( Cover and Thomas , 1991 ) , ( 5 ) I ( R;C|p ) =H ( R|p ) −H ( R|C , p ) where ( 6 ) H ( X1|X2 ) =−∑x1 , x2p ( x1 , x2 ) log2 ( p ( x1|x2 ) ) Entropy values were calculated from a histogram estimate of probability distributions .", "The central stimulus followed a 4-bit M-sequence , with each stimulus frame having one of two values , μ+ΔI and μ−ΔI , and a Michelson contrast ( ΔI/μ ) of one of four possible values , 3 , 6 , 12 and 24% .", "Each four frame sequence occurred once in a repetition of the M-sequence , where M ( 4 ) is the set of all 16 possible combinations of four binary digits .", "The luminance in the center was updated at 10 Hz , and therefore one presentation of the M-sequence lasted 0 . 1 s⋅24=1 . 6 s .", "The sequence was repeated for 11 trials at a given contrast and the responses for the first trial after a transition to a new contrast were discarded from the analysis .", "Contrasts were picked randomly without replacement and then a different order chosen once all four contrasts were tested .", "A stimulus was defined as the combination of the center ( both sequence and contrast ) and the periphery .", "By binning time in 100 ms there are 10 possible peripheral stimuli ( time p relative to peripheral period ) , 16 possible sequences , m ( 4 ) and 4 possible contrasts ( σ ) for a total of 640 different stimuli .", "Each stimulus was measured at least 10 times .", "When the central stimulus is divided into the stimulus sequence , m ( 4 ) ∈M ( 4 ) and the contrast , σ ∈ ∑ , the total information from eq ( 1 ) can be further expanded into: ( 7 ) I ( R;P , ∑ , M ( 4 ) ) = I ( R;P ) +I ( R;∑|P ) +I ( R;M ( 4 ) |∑ , P ) = I ( R;P ) + I ( R;∑|p ) +I ( R;M ( 4 ) |∑ , p ) p∈P Where I ( R;∑|p ) and I ( R;M ( 4 ) |∑ , p ) are functions of time p .", "The quantity I ( R;M ( 4 ) |∑ , p ) represents the information the brain could extract about the stimulus-sequence at a given time relative to the peripheral stimulus if it knew the contrast .", "Whereas I ( R;M ( 4 ) |p ) represents the information that the brain could extract about the stimulus sequence at a given time if it did not know the contrast .", "Comparisons of the time course of I ( R;M ( 4 ) |p ) and I ( R;∑|p ) ( Figure 4C ) , as well as between I ( R;M ( 4 ) |∑ , p ) and I ( R;∑|p ) ( Figure 4—figure supplement 1 ) , indicate that the dynamics of information about sequence and contrast are different whether the brain can use contrast information to decode the sequence or not .", "Spatial extent of peripheral changes in sensitivity .", "To measure the spatial extent of increases and decreases in sensitivity , the central stimulus consisted of a mask in the pattern of a checkerboard , with squares 100 µm in size , that flickered with a pink noise stimulus intensity that was the same in all squares ( Figure 5A ) .", "The overall size of the mask was 1 . 2 mm .", "The central stimulus was identical in all conditions .", "The background was a more finely scaled checkerboard , with squares 50 µm in size , and a central blank region that was varied in size from the full size of the monitor ( no checkers in the periphery ) to 0 µm ( checkers everywhere except in central stimulus ) .", "At smaller values of the central blank region , the background was intercalated with the central region ( Figure 5B , bottom ) .", "For each location of the peripheral stimulus , we computed an LN model between the center stimulus and the cell’s response and calculated the average slope of the nonlinearity for different time windows .", "The model of long-range excitation consisted of a central stimulus s ( t ) that was passed through a linear filter F ( τ ) , yielding the filtered central input ( 8 ) b ( t ) =∫01F ( τ ) s ( t−τ ) dτ The filtered central input was combined with a signal , a ( t ) , that depended on background motion .", "Because the goal of this model was to investigate the integration of central and peripheral signals , and not the origin of the peripheral signal as has been studied elsewhere in greater detail ( Passaglia et al . , 2009 ) , we defined a ( t ) to be a biphasic function of time that began at the time t = 0 , representing the time of the saccade .", "As to a plausible origin of this input , the peripheral effect occurred for a high spatial frequency checker and did not depend on the direction of the shift .", "Such a response could be generated by a group of rectified subunits as found in the receptive fields of polyaxonal amacrine cells ( Baccus et al . , 2008 ) , but this was not explicitly implemented here .", "Because a ( t ) had two phases , positive and negative , whereas polyaxonal amacrine cells are thought only to deliver inhibition , it is expected that the positive phase would arise through disinhibition delivered through a second intervening amacrine cell that provides tonic inhibition .", "The central and peripheral inputs were then passed through a threshold nonlinearity N ( . ) .", "( 9 ) c ( t ) = N ( b ( t ) +a ( t ) ) The nonlinearity was chosen with a slope of one and the threshold equal to 0 . 9 times the maximum amplitude of the peripheral signal a ( t ) · .", "The output of the threshold function was then scaled by feedforward divisive adaptation , yielding the model output ( 10 ) y ( t ) = c ( t ) α+∫Fα ( τ ) c ( t-τ ) dτ Fα ( t ) was an exponentially decaying filter with an integral of one , and a time constant set to 10 s , although this parameter could be varied from 1 to 100 s with little effect .", "Smaller values of α yield more complete adaptation .", "However , for constant luminance experiments , equation ( 10 ) reduces to a constant independent of the luminance value when α = 0 , therefore non-zero values of α are needed .", "Thus , the value of alpha was optimized to yield changes in adaptation as observed in the data , as well as responses to different levels of constant luminance .", "We used α = 0 . 30 , but values from 0 . 05 to 2 . 00 could be used with similar results .", "From previous studies , the first sharp threshold encountered in the retina is at the bipolar cell synaptic terminal ( Mennerick and Matthews , 1996 ) .", "Thus , the filter in the model should correspond to the spatiotemporal receptive field of a bipolar cell , which we took from our previous measurements of fast-Off bipolar cells ( Baccus et al . , 2008 ) ( Figure 7A ) .", "In order to use the model to assess information transmission , we measured the noise in the membrane potential of bipolar cells as a function of contrast and found that the level of noise increases roughly linearly with contrast ( Figure 7A-iii ) .", "This allowed us to choose a noise level at each time point that depended on the filtered stimulus , approximating measured bipolar cell noise .", "A set of 342 images were taken from a database of natural scenes ( Tkačik et al . , 2011 ) .", "The bipolar cell membrane potential was simulated by combining a linear receptive field pathway ( Figure 7A-i ) and noise ( Figure 7A-iii ) .", "Because the filtering and noise of bipolar cells was measured at a fixed mean intensity , to avoid the need to incorporate luminance adaptation into the model , we normalized the mean intensity of all images .", "The linear receptive field center and surround were modeled independently , with each being a filter separable in space and time .", "Spatial linear predictions were made by convolving each image with a spatial disk of 1 . 0 and 2 . 5 degrees of visual space corresponding to center and surround .", "To compute the complete linear prediction of cells , images were jittered around according to a random walk with mean velocity of 0 . 33° per second simulating fixational eye movements and an instantaneous saccade simulated by a step of 6° in a random orientation in the image location .", "The temporal receptive fields for the center and surround were convolved with this image series and summed , generating the complete linear prediction for each location as a function of time; gx , t where x denotes the location and t the time .", "From the 342 images , a total of 82 , 863 identical bipolar cells with non-overlapping centers were simulated .", "Because bipolar cell noise depends on the stimulus contrast ( Figure 7A-iii ) , we used a model of the noise whereby the instantaneous standard deviation of the noise at each point in time relative to the shift depended on the standard deviation of the linear prediction across the bipolar cell population ( Figure 7A-iii , lower panel , black trace ) .", "This created the greatest noise during the gating window , and thus potentially underestimates the actual information conveyed .", "From the signal standard deviation at a particular time , an equivalent Gaussian contrast was found that would generate a linear prediction with the same standard deviation .", "With the equivalent Gaussian contrast , a level of noise was chosen such that the signal to noise ratio was the same in the simulation and in the bipolar cell’s measured membrane potential noise under repetitions of Gaussian stimulation at different contrasts ( Figure 7A-iii , upper panel ) .", "Each cell in the model received independent noise which was generated from a Gaussian distribution .", "The linear prediction g ( t ) and model output r ( t ) were binned to compute mutual information and conditional mutual information .", "The linear prediction g ( t ) and the response r ( t ) were divided into 16 unequal bins , positioned to maximize information about the total range of g ( t ) and r ( t ) .", "The same bins were kept for all delays , p relative to the shift .", "The information that the response carries about the linear prediction at a particular delay p relative to the shift , was computed as I ( Gt;Rt|p ) by taking the difference between the total response entropy at a given delay , H ( Rt|p ) , and the conditional ( noise ) entropy , H ( Rt|Gt , p ) .", "The conditional mutual information between the linear prediction g ( t ) and the response r ( t ) given the previous response r ( t−Δ ) at a given delay , p , was computed as ( 11 ) I ( Gt;Rt|Rt−Δ , p ) =H ( Gt|Rt−Δ , p ) −H ( Gt|Rt−Δ , Rt , p ) To compute a change in adaptation at different times relative to a shift , an index was computed that compared the measured change in the slope of the nonlinearity to the change expected from complete adaptation .", "After presenting a series of contrasts σi , we computed the nonlinearities N ( . ) of an LN model , and from each nonlinearity extracted the slope mi .", "Picking one contrast as reference , we normalized the slopes and the contrasts by those of the reference m~= m/m0 and σ~=σ/σ0 and fitted a line to m~ vs . 1σ .", "The adaptive index is the slope of the fitted line , which will be one for an ideally adapting cell and zero for a nonadapting cell ( Figure 3—figure supplement 1A–B ) .", "The goal was to find the nonlinearity that maximized the mutual information I ( G;R ) , between the set of linear predictions , G and the set of spike counts , R , given the noise properties of the cell .", "We began with the linear prediction as a function of time g ( t ) , the spike count distribution at that time , P ( r ( t ) ) , computed over trials , and the average rate at that time , ⟨r ( t ) ⟩ , computed by averaging over trials .", "The nonlinearity N0 ( g ) maps g ( t ) at a time t onto a model average firing rate ⟨r′ ( t ) ⟩ at that time , but to include noise that was most consistent with the observed noise we computed from the data the distribution of spike counts for a given average rate , P ( r ( t ) |⟨r⟩ ) .", "This function mapped each average rate ⟨r ( t ) ⟩ at each time onto a distribution P ( r ( t ) ) .", "The optimized nonlinearity , N0 ( g ) , was a sigmoid parameterized by a slope , x1−1 and a midpoint , x0 , and was constrained to have the same minimum ( zero ) and maximum rate as the measured data N0 ( x ) =rmax1+e- ( x-x0 ) /x1 .", "To find for each candidate N0 ( . ) the best estimated joint distribution P ( g ( t ) , r′ ( t ) ) between g ( t ) and the model distribution of spike counts r′ ( t ) , we used N0 ( . ) to mapg ( t ) onto ⟨r′ ( t ) ⟩ , and then used the function P ( r ( t ) |⟨r⟩ ) to map ⟨r′⟩ onto P ( r′ ( t ) |g ( t ) ) for a particular value of g ( t ) , i . e . P ( r′ ( t ) |g ( t ) ) =P ( r ( t ) |N0 ( g ) ) .", "Then , we weighted this conditional distribution by the marginal probability of the linear prediction g , P ( g ) , which has a Gaussian distribution , to compute the full joint distribution of g ( t ) and r′ ( t ) , ( 12 ) P ( g ( t ) , r′ ( t ) ) =P ( g ( t ) ) P ( r′ ( t ) |g ( t ) ) from which the mutual information was computed .", "Then we performed a grid search of the parameters of N0 ( ) and found from P ( g ( t ) , r′ ( t ) ) the nonlinearity that maximized I ( Gt , Rt′ ) .", "The maximum value of I ( Gt , Rt′ ) was taken as the maximum amount of information given the measured noise of the cell , and its minimum and maximum firing rate ." ] ]

[ "Sensory stimuli have varying statistics influenced by both the environment and by active sensing behaviors that rapidly and globally change the sensory input .", "Consequently , sensory systems often adjust their neural code to the expected statistics of their sensory input to transmit novel sensory information .", "Here , we show that sudden peripheral motion amplifies and accelerates information transmission in salamander ganglion cells in a 50 ms time window .", "Underlying this gating of information is a transient increase in adaptation to contrast , enhancing sensitivity to a broader range of stimuli .", "Using a model and natural images , we show that this effect coincides with an expected increase in information in bipolar cells after a global image shift .", "Our findings reveal the dynamic allocation of energy resources to increase neural activity at times of expected high information content , a principle of adaptation that balances the competing requirements of conserving spikes and transmitting information ." ]

[ "To see an object , light must travel from it and be focused onto the retina at the back of the eye .", "The image projected onto each retina is then processed by neurons known as ganglion cells , which transmit a processed version of the image to the brain .", "Each ganglion cell responds to a specific section of the retinal image , in particular to intensity changes or movements that occur within that region , known as the cell’s receptive field .", "However , ganglion cells in the retina of many species can also become active if rapid movements occur in parts of the retinal image that are far away from the receptive field of that ganglion cell .", "Jadzinsky and Baccus have now investigated how this peripheral motion affects the response of salamanders’ retinal cells .", "The images consisted of a central object surrounded by a checkerboard pattern , the brightness of which could be varied to change the contrast of the image ( higher contrast images stimulate the ganglion cells more strongly ) .", "Then , either the entire image or only the central object moved .", "Moving the whole image represents the pattern that would be seen if a salamander moved its eye or head to look at a new part of a scene .", "Jadzinsky and Baccus found that when only the central object moved , the ganglion cells only responded to high-contrast images that strongly stimulated the cells , effectively conserving energy by only responding to strong signals .", "However , when the whole image moved , the cells also responded to lower-contrast images , showing that they had switched to processing the local region of the scene in more detail .", "These effects could be reproduced in a simple mathematical model .", "The model suggests that the ganglion cells increase their information transmission at times when a large amount of new information is expected to be received: for example , immediately after the salamander has moved its eyes .", "The next challenges in this research are to identify the specific retinal neurons that generate this change in processing in the ganglion cells , and to further understand how sensory input influences how the nervous system allocates energy resources ." ]

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

[ "plant biology" ]

[ [ "Species around the globe face rapidly changing environments , often in combination with habitat loss and fragmentation .", "These factors are expected to have a negative impact on biodiversity ( Lindenmayer et al . , 2010 ) .", "Three processes enable species to survive altered conditions: migration , adaptation , and phenotypic plasticity ( Aitken and Whitlock , 2013; Aitken et al . , 2008; Hoffmann et al . , 2015; Nicotra et al . , 2010 ) .", "An important conservation strategy is to assist these natural processes to help build more resilient communities .", "We can help populations to become better adapted to future environmental conditions by assisting migration of gene pools across the landscape ( Aitken and Whitlock , 2013; Aitken et al . , 2008 ) .", "We can aid populations to survive in situ by ensuring that sufficient genomic variation exists for adaptation to changing environments ( Hoffmann et al . , 2015 ) .", "We can enable individuals to respond to a greater range of environments by conserving existing phenotypic plasticity ( Nicotra et al . , 2010 ) .", "Seed sourcing during landscape restoration provides an ideal opportunity to apply scientific knowledge to enable these key processes and improve conservation outcomes ( Broadhurst et al . , 2008; Prober et al . , 2015 ) .", "For example , seed sources can be selected to restore historical patterns of gene flow across fragmented landscapes and to incorporate high levels of available genomic diversity .", "If plasticity varies among populations , seed can be selected to augment the phenotypic plasticity of individuals at restoration sites .", "Seed sources can also be matched with current or predicted future climates , enabling assisted migration to favorable environments ( Aitken and Whitlock , 2013; Williams et al . , 2014 ) .", "Historically , restoration has often focused on geographically restricted local sources of seed under the premise that this would improve restoration outcomes by reducing the risk of maladaptation to local conditions and by preventing outbreeding depression ( Broadhurst et al . , 2008 ) .", "However , there are several potential drawbacks to this narrow local focus .", "In a fragmented system , narrow local seed sourcing reduces the number of potential source populations , thereby reducing the pool of available genetic material .", "This reduced gene pool may result in inbreeding depression in future generations , especially if combined with small population size ( Broadhurst et al . , 2008 ) .", "Obtaining seed from a wider geographical area can increase genomic and phenotypic diversity , thereby increasing the ability of the species to survive in situ ( Broadhurst et al . , 2008 ) .", "Additionally , the focus on maintaining local adaptation assumes a static environment , not the rapidly changing environments that occur today .", "When local conditions change , traits and genes that have conferred an advantage in the past may not be suitable in future environments .", "In recent years , climate adjusted provenancing has been proposed , providing a seed sourcing strategy that focuses on both genetic diversity and adaptability under predicted future conditions ( Byrne et al . , 2013; Prober et al . , 2015 ) .", "This strategic assisted migration of variation across the landscape can aid in the establishment of populations that are more adaptable to future environments ( Prober et al . , 2015 ) .", "To identify an appropriate seed sourcing strategy for a reforestation project , it is useful to characterize genomic variation in the target species with empirical data .", "These data can be used to infer patterns of Isolation By Distance ( IBD ) and Isolation By Environment ( IBE ) .", "IBD describes the correlation between genomic distance and geographic distance , which arises when gene flow occurs more often between populations that are in close geographic proximity .", "IBE describes the correlation between genomic distance and environmental distance , while controlling for geographic distance ( Wang and Bradburd , 2014 ) .", "IBE arises because environmental drivers can influence gene flow , so that migration rate is effectively modulated by the environment .", "This means IBE is detectable in genome-wide variation , and not just at loci mediating adaptation .", "Landscape genomic models can be generated that describe the relationship between genetic differentiation and both spatial and environmental distances ( representing IBD and IBE ) .", "These predictive models can be used to optimize the genetic material selected for restoration and should improve long term outcomes ( Hoffmann et al . , 2015; Williams et al . , 2014 ) .", "The extent of phenotypic plasticity in potential seed sources can be measured in growth assays of seedling traits across contrasting environmental conditions .", "The magnitude of the environmental response can be compared among maternal lines or populations and may identify populations that differ in their response to the environment .", "Such differing responses have been seen in some species of Eucalyptus ( Andrew et al . , 2010; Byrne et al . , 2013; McLean et al . , 2014 ) , which typically have high levels of within-population genetic variation and moderate-high rates of outcrossing ( Byrne , 2008 ) .", "Eucalyptus melliodora ( A . Cunn . ex Schauer ) , commonly called yellow box , is an iconic Australian tree that is the subject of extensive restoration efforts across its distribution .", "It is a foundation species of a critically endangered ecological community: the White Box–Yellow Box–Blakely’s Red Gum Grassy Woodland and Derived Native Grassland ( Department of Environment and Climate Change and Water , 2011; Department of the Environment and Heritage , 2006; Threatened Species Scientific Committee , 2006 ) .", "This woodland community exists in a fragmented landscape , with less than 5% of its original distribution remaining , mostly in small remnant patches ( Department of Environment and Climate Change and Water , 2011; Department of the Environment and Heritage , 2006; Threatened Species Scientific Committee , 2006 ) .", "Efforts to restore this endangered woodland community are ongoing and restoration practitioners are seeking scientific recommendations to improve seed sourcing .", "Climate change is an important consideration in seed sourcing decisions because species distribution modelling predicts that most eucalypts will need to shift their distributions considerably in response ( González-Orozco et al . , 2016 ) .", "In particular , ecological niche modelling for E . melliodora predicts that by 2090 the species distribution will shift toward the southeast and suitable areas will decrease by 77% as a result of environmental changes ( Broadhurst et al . , 2018 ) .", "Here we survey genomic variation in 275 individuals from 36 sites across the present range of E . melliodora .", "To help determine an appropriate seed sourcing strategy , we fit the genotypic data to geographic distance and key environmental variables at the sites of origin .", "This enables characterization of isolation by distance across a broad area , providing an empirical estimate of ‘local’ for comparison with current practice for local provenancing .", "We also identify features of the abiotic environment that can further explain genomic differentiation after accounting for geographic distance .", "Additionally , we examine seedling growth under different simulated climate conditions to test for variation in growth traits and phenotypic plasticity both within and among sites .", "Our landscape genomic model , which can empirically define local provenances and identify variation suitable for predicted future climates , can help build resilient populations through scientifically based restoration ." ], [ "We selected leaf material from 39 sites , sampling 3–10 trees per site ( Supplementary file 1 ) .", "For each sample we Illumina sequenced a Genotyping by Sequencing ( GBS ) library ( Elshire et al . , 2011 ) and used a reference alignment approach to call genotypes .", "We conducted a preliminary analysis based on 123 , 227 SNPs and removed 69 samples due to greater than 60% missing data .", "Visual examination of a cluster dendrogram of genomic distance between samples showed that technical replicates cluster closely together ( Figure 1—figure supplement 1 ) .", "A preliminary principal coordinate analysis ( PCoA ) identified 19 samples that were strong genomic outliers ( Figure 1—figure supplement 2 ) , likely misidentified samples or recent hybrids .", "This result is consistent with minor morphological differences noted in these samples , as well as previous microsatellite work ( Broadhurst et al . , 2018 ) .", "After removal of poor quality and geographic and genomic outlier samples , we re-ran the genotyping with the remaining 280 samples , resulting in 9 , 781 SNPs after filtering .", "A second preliminary PCoA identified an additional five outlier samples that we considered sufficiently differentiated from the main E . melliodora cluster to merit removal from downstream analyses ( Figure 1—figure supplement 3 ) .", "We removed these samples and reran the missing data filter .", "The final data set included 275 samples from 36 sites ( Figure 1A ) , genotyped at 9 , 378 physically distinct SNPs ( >300 bp apart ) .", "To help determine an appropriate seed sourcing strategy , we examined the effects that geography and environment have on the distribution of genomic variation across the landscape .", "The genomic analyses focused on the effects on the genome as a whole , rather than individual genes .", "The study of individual genes is beyond the scope of the current study .", "The PCoA of genomic distance among samples showed continuous variation with little suggestion of discrete population structure ( Figure 1B ) .", "This analysis , which was based on genomic data with no geographic information included , showed that the samples largely formed a single cluster , with the first PCoA axis correlating with latitude ( Figure 1B ) .", "Outside of the main cluster , samples from the northernmost site separated out along the first PCoA axis ( vertical axis ) and a few samples from two other sites separated out along the second PCoA axis ( horizontal axis ) .", "Together , the first two PCoA axes explained 3 . 0% of the genomic variation among individuals .", "The Mantel test , examining the correlation between geographic and genetic distance matrices , estimated that geographic distance between samples explained 2 . 3% of the variation in individual genomic distance , indicating weak , but statistically significant , isolation by distance ( p=0 . 0001 ) .", "We summarized genomic diversity between sampling sites using pairwise Fst .", "For all comparisons Fst was low ( mean Fst = 0 . 04 , sd = 0 . 02 ) ( Supplementary file 2 ) .", "The maximum Fst of 0 . 10 occurs between sites 3 and 13 , which are separated by over 1200 km .", "Similar to the individual-level PCoA of genomic distance among samples ( Figure 1B ) , the site-level PCoA of Fst between sampling sites also corresponded roughly to latitude ( Figure 1—figure supplement 4 ) .", "In contrast , the first two axes of the PCoA of Fst between sampling sites explained a higher percentage of variation ( 37 . 1% ) .", "All sites with more than four individuals genotyped had similar levels of allelic diversity and expected heterozygosity ( Supplementary file 1 ) .", "Overall , these results highlight the low level of genetic structure over a large spatial scale in E . melliodora .", "The site-by-site Fst matrix was used to test for geographic and environmental correlations using generalized dissimilarity modelling ( GDM ) ( Ferrier et al . , 2007; Fitzpatrick and Keller , 2015; Thomassen et al . , 2011 ) .", "Of the 28 environmental variables considered for the model , we removed 12 variables because the single variable model explained less than 5% of the deviance ( bioclimatic variables 2 , 5 , 6 , 9 , 10 , 14 , 17 , 19; elevation; water at depth; Prescott Index; and MrVBF ) .", "We removed an additional nine variables due to high correlation and lower explanatory power than another remaining variable ( bioclimatic variables 1 , 4 , 7 , 12 , 13 , 15 , 18; surface nitrogen; and surface phosphorus ) ( Supplementary file 3 ) .", "We ran permutation testing on a model with the remaining seven variables , along with geographic distance .", "This highlighted an additional two variables with low statistical significance and low explanatory power .", "We removed these two variables ( surface water and bioclimatic variable 8 ) from the final model .", "We also removed phosphorus at depth because , although it explained a substantial amount of genomic variation , the sampled sites were not well distributed across the range of phosphorus values .", "As a result , we included four environmental variables in the final model: isothermality ( bioclimatic variable 3 ) , mean temperature of the coldest quarter ( bioclimatic variable 11 ) , precipitation of the wettest quarter ( bioclimatic variable 16 ) , and total soil nitrogen at 100–200 cm ( nitrogen at depth ) ( Figure 2 ) .", "The correlation coefficients between these variables were all less than 0 . 13 , with the exception of the precipitation variable , which showed a moderate correlation with isothermality ( r = 0 . 53 ) and nitrogen ( r = 0 . 45 ) ( Supplementary file 3 ) .", "The GDM model with these four variables plus geographic distance explained 40% of the genetic differentiation ( Fst ) between sampling sites .", "The GDM model showed a positive non-linear relationship between environmental distance and genomic distance ( Figure 2A ) .", "Visual examination of the genomic distances predicted from the model versus the observed values indicated the model had reasonable predictive power ( Figure 2B ) .", "To quantify the predictive power of the GDM model , we used a cross validation approach by generating 1000 models with a random 30% of sampling sites removed .", "GDM proved satisfactory at predicting genomic differences between removed sites ( cross validation correlation mean = 0 . 73 , standard deviation = 0 . 12 ) .", "Geographic distance showed a non-linear relationship with genomic distance .", "The geographic spline predicted no genomic differentiation until close to 500 km , at which point an increase in geographic distance predicted an increase in genomic distance ( Figure 2C ) .", "Randomly subsampling sites showed that the predicted genomic distance for large geographic distances was quite variable , but for sites less than 500 km apart , all iterations consistently predicted little genomic differentiation between sites ( Figure 2D ) .", "Of the four environmental variables , nitrogen at depth showed the strongest relationship with genomic distance , with changes in genomic distance predicted across the range of nitrogen values ( Figure 2E ) .", "Mean temperature of the coldest quarter was the second strongest predictor , showing changes in genomic distance predicted across the range of temperature values ( Figure 2F ) .", "Precipitation of the wettest quarter was the third strongest environmental predictor , predicting the largest change in genomic distance between 250 and 400 mm of precipitation ( Figure 2G ) .", "Isothermality ( mean diurnal range divided by annual temperature range ) was the final predictor , predicting the most change in genomic distance at higher values ( Figure 2H ) .", "To project the final GDM model onto the current environmental landscape , we first delineated the geographic extent of the analysis by defining an E . melliodora distribution polygon .", "We then projected the GDM model onto this region based on the current values of the environmental variables across the landscape .", "For visualization , the dimensionality was reduced using principal component analysis ( PCA ) and the first three axes were assigned to RGB colors to represent genomic composition , with similar color for similar predicted genomic composition .", "The resulting map partitioned the landscape into a number of regions with different predicted genomic compositions , including northern coastal , northern inland , and southern regions ( Figure 3A ) .", "While the biggest differences occurred in regions with few sampling sites , the northern and southern sites have distinct genomic compositions ( Figure 3A ) .", "These projections highlight where environmental filtering of genotypes may have occurred due to differences in selective pressures .", "We compared the GDM model projected onto current conditions to the GDM model projected onto 2070 climate predictions as an indication of the amount of genomic change required to keep pace with changes in selective pressures resulting from environmental change ( ‘genomic vulnerability’ , ( Bay et al . , 2018 ) ) ( Figure 3B ) .", "For the middle north region and the southern areas towards the coast ( red in Figure 3B ) , the model predicted more intense natural selection in response to climate change , thus indicating that these areas should be prioritized for assisted migration .", "We also used the GDM model to compare the genomic composition under future environmental conditions at a single location to the genomic composition under current climate conditions across the landscape .", "This comparison is useful for identifying optimal seed sources for restoration sites given climate change scenarios .", "We demonstrated this utility by selecting two hypothetical reforestation sites and identifying distinct regions that would provide favorable seed sources for each site ( Figure 4 ) .", "The analysis for the southern reforestation site identified a large portion of the southern distribution , centered at the reforestation site .", "For this site it appears that the selected areas are largely a result of the pattern of isolation by distance , in particular the lack of genetic differentiation for long geographical distances .", "The analysis for the northern reforestation site identified a more limited range of areas across the landscape , although this could be driven in part by a decreased power due to lower sampling intensity in the north .", "Within 500 km of the site , the analysis identified a core region centered on the reforestation site and small regions along the northern coast .", "There were a number of areas within 500 km of the site that were not good matches .", "In addition , a number of more distant areas along the southern coast were also identified , indicating these selected areas are driven more by patterns of isolation by environment than isolation by distance .", "Overall , the map suggests that there is a lower availability of seed sources to match the northern reforestation site .", "These analyses suggest that for seed sourcing in woodland restoration , a model-based approach incorporating genomic variation , geographic distance , and environmental variables would allow for more genetic diversity and enable better matching of the selected genotypes to current and predicted future environmental conditions at the reforestation site .", "We conducted a climate controlled growth experiment to examine phenotypic variation among sampling sites and assay phenotypic plasticity .", "We grew seedlings from six sites , with six maternal lines per site , at two different climate regimes ( average summer conditions and 5°C hotter than summer conditions ) .", "We measured variation in three seedling growth traits: seedling height , total leaf length , and relative height increment .", "For analysis of seedling height and total leaf length , we analyzed a total of 291 seedlings ( from 32 maternal lines representing six sampling sites ) that were determined to be well established at the five week measurement .", "For analysis of the relative height increment , we analyzed a total of 560 seedlings ( from all 36 maternal lines ) for which we were able to calculate this metric .", "There were four seedlings that were outliers for the relative height increment .", "These outliers had little effect on the results of the linear models , so we included them in the final analysis .", "The models for all three response variables showed that all fixed effects ( sampling site , maternal line nested within sampling site , and experimental condition ) were statistically significant at the p=0 . 05 level ( Figure 5 and Supplementary file 4 ) .", "Experimental condition explained a small percentage of the variation ( 1 . 2–8 . 1% ) , as did sampling site ( 1 . 8–17 . 7% ) .", "Maternal line tended to explain a larger amount of variation ( 10 . 6–27 . 6% ) .", "However , most of the variation remained unexplained ( 56 . 6–71 . 5% ) ( Figure 5 and Supplementary file 4 ) .", "None of the three response variables showed significant variation in phenotypic plasticity across sites ( p>0 . 50 for all maternal line/sampling site by experimental condition interactions ) ( Figure 5 and Supplementary file 5 ) .", "We then conducted an outdoor drought experiment using a subset of seedlings from the chamber experiment .", "We analyzed 146 seedlings representing 20 maternal lines from five sampling sites .", "These seedlings were grouped into 73 pairs , with one of each pair assigned to each treatment—well-watered versus drought .", "We analyzed variation in four response variables: stomatal conductance , leaf length to width ratio , relative chlorophyll content ( SPAD index ) , and specific leaf area ( SLA , leaf area divided by dry mass ) .", "The drought-treated seedlings had significantly lower stomatal conductance rates than the well-watered ones , indicating that the seedlings were affected by the watering treatment ( p<0 . 00001 ) ( Figure 6 and Supplementary file 6 ) .", "Treatment explained most of the variation in stomatal conductance ( 62 . 3% ) , while maternal line and sampling site explained only a small amount of variation ( 5 . 8% and 0 . 9% respectively ) .", "For the remaining three response variables ( leaf length to width ratio , SPAD , and SLA ) , much of the variation was unexplained ( 40 . 5%–70% ) .", "Treatment was not statistically significant and explained little to no variation ( 0 . 0–4 . 4% ) .", "Sampling site and maternal line were statistically significant in the linear models at the p=0 . 05 level and explained some variation ( 6 . 7–21 . 2% ) ( Figure 6 and Supplementary file 6 ) .", "Smaller , thicker leaves , and thus lower SLA values , were expected for drought-treated seedlings and for seedlings grown from seed collected from drier areas .", "Consistent with this expectation , the seedlings subjected to drought conditions showed lower SLA values .", "However , seedlings from drier sampling sites ( D and T3 ) showed higher SLA values than more mesic sites ( B , G , and 11 ) , contrary to expectation ( Figure 6 ) .", "None of the four response variables showed significant variation in phenotypic plasticity across sites ( p>0 . 13 for all maternal line/sampling site by experimental condition interactions ) ( Figure 6 and Supplementary file 7 ) .", "Both our climate controlled growth experiment and our outdoor drought experiment found high levels of phenotypic variation in all measured traits .", "While most of the variation remained unexplained , sampling site explained a small , but statistically significant , amount of the variation .", "We determined whether phenotypic divergence between sites could be due to local selection using a Qst-Fst analysis ( Gilbert and Whitlock , 2015; Leinonen et al . , 2013 ) .", "We estimated Qst for each trait under each experimental condition and compared these values to the genome-wide distribution of Fst values ( Supplementary file 8 ) .", "Qst and Fst were not significantly different , indicating that phenotypic differences between sites could be a result of genetic drift alone .", "While not statistically significant , seedling height did show differences between Qst and Fst in both hot ( Qst-Fst = 0 . 33 , p=0 . 11 ) and warm ( Qst-Fst = 0 . 24 , p=0 . 14 ) chambers .", "This indicates that local selection could be driving the divergence in height between sites , but our analysis lacked statistical power due to small sample sizes .", "In addition to measuring growth traits , we also examined the shape of the leaves of seedlings from the drought experiment .", "We noted substantial variation in leaf shape , both among sites and within sites ( Figure 7 ) .", "The remarkable amount of phenotypic variation in the seedlings is consistent with the high levels of genomic variation measured ." ], [ "Eucalyptus melliodora is a foundation species in a critically endangered woodland community that now occupies a fraction of its former distribution and is the subject of restoration projects across its native range .", "Our examination of the distribution of genomic and phenotypic variation across the range of this species provides valuable information for sourcing seed for restoration , including empirically defining local provenances and matching genotypes to predicted future environmental conditions .", "We found little genomic divergence between sampling sites ( mean Fst = 0 . 04 ) , which is consistent with microsatellite analysis of this species ( Fst = 0 . 03 , ( Broadhurst et al . , 2018 ) ) and population genetic analyses of other tree species ( E .", "camaldulensis , Fst = 0 . 05 , 0 . 08 , ( Butcher et al . , 2009 ) ; E . globulus , Fst = 0 . 08 , ( Jones et al . , 2002 ) ; Corymbia calophylla , Fst = 0 . 03 , ( Sampson et al . , 2018 ) ; Pinus taeda , Fst = 0 . 04 , ( Eckert et al . , 2010 ) ; Quercus robur , Fst = 0 . 07 , ( Vakkari et al . , 2006 ) ; Quercus engelmannii , Fst = 0 . 04 , ( Ortego et al . , 2012 ) ; Populus tremuloides , Fst = 0 . 03 , ( Wyman et al . , 2003 ) ) .", "Examining the relationship between genomic and geographic distance , we are able to empirically define ‘local’ in this species to be on the order of 500 km , which is substantially farther than the current practice .", "These results mean restoration projects can and should source seed more broadly across the landscape , with limited risk of mixing highly evolutionarily diverged material .", "In a highly fragmented landscape this will increase the number of favorable source sites , enabling the collection of higher quality seed with increased genetic diversity ( Broadhurst et al . , 2008 ) .", "Incorporating more naturally occurring genomic variation can increase the adaptive potential of the restored populations by providing the substrate for adaptation to rapidly changing environmental conditions .", "In addition to isolation by distance , our model identified soil nitrogen , temperature of the coldest quarter , precipitation of the wettest quarter , and isothermality as significant environmental drivers of genome-wide patterns of variation across the landscape .", "Of these variables , the climate variables are predicted to change rapidly over time .", "Change in soil nitrogen content might occur over longer time scales , but it is difficult to forecast due to complex biotic feedbacks ( Brevik , 2013 ) .", "This suggests that optimal seed sourcing will need to balance the tracking of rapidly changing climate variables with the need to account for variables that are more stable due to their dependence on stable features of geology , topography , or hydrology .", "The different time scales also highlight the important concern that key environmental variables may become uncoupled , resulting in less than ideal conditions for this species across the landscape .", "Previous niche modelling of E . melliodora examined environmental drivers of the distribution of the species ( Broadhurst et al . , 2018 ) .", "Similar to our analysis , that analysis also found temperature and precipitation variables to be important , but the exact bioclimatic variables identified did not overlap .", "This is not unexpected given that niche modelling identifies drivers that define the environmental tolerance of the species , while the analysis presented here identifies drivers for genomic variation within the species .", "Many studies of within-species genetic variation in trees find temperature and precipitation variables to be the most important drivers ( Aitken et al . , 2008 ) ; however , the exact variables vary and other variables are often found to play important roles .", "A quantitative genetics study of Eucalyptus delegatensis in Australia found that the variables that contributed most to the adaptive variability of the species were related to solar radiation ( Garnier-Géré and Ades , 2001 ) , which was not assessed in our study .", "Additionally , they found that the variability of temperature and rainfall played an important role ( Garnier-Géré and Ades , 2001 ) .", "One of our top predictors was isothermality , which is a composite variable of temperature ranges .", "A genetic study of ecologically relevant loci in 13 alpine plant species in the European Alps found that , after accounting for broad spatial patterns , temperature and/or precipitation variables were the primary drivers of genetic variation in all but one species ( Manel et al . , 2012 ) .", "In contrast , a genetic study of putatively neutral loci in three tree species in Central America found different drivers in different species ( Poelchau and Hamrick , 2012 ) .", "In one species , an integrated environmental measure , incorporating temperature and precipitation , was the primary driver; in the second species the primary driver was geographic distance; in the third species the results were ambiguous ( Poelchau and Hamrick , 2012 ) .", "This indicates that environmental drivers of within-species genetic diversity are likely to be somewhat species specific .", "The focus of this study was the whole-genome population structure that reflects historical adaptation , gene flow , and demography .", "Analyses of individual genes was beyond the scope of this study due to the low resolution GBS genotyping and the limited extent of linkage disequilibrium in Eucalyptus ( Silva-Junior and Grattapaglia , 2015; Thumma et al . , 2005 ) .", "However , our results demonstrate a lack of strong population structure , indicating that using whole genome sequencing to identify adaptive alleles is feasible in this species .", "For instance , the Qst-Fst analysis indicates the possibility of local adaptation for seedling height and a future study could identify the adaptive loci underlying plant height by targeting sampling sites segregating for this trait .", "Specific alleles that potentially confer increased fitness in the face of a rapidly changing climate would be useful targets for restoration projects .", "Our analyses of phenotypic variation found no site-specific variation in phenotypic plasticity that would enable us to identify provenances better able to cope with rapid environmental change .", "However , plasticity is trait-specific , so traits that are hypothesized to be important for establishment and survival should continue to be investigated because they may provide valuable information for restoration projects .", "Importantly , our growth experiments support the results of the genomic analyses , showing the remarkable extent of variation both among sites and within sites , further supporting our recommendation that seed sources incorporate the high level of variation that occurs naturally in E . melliodora .", "The results of this study are promising for the future of E . melliodora across its native distribution .", "We found high genomic and phenotypic diversity within sites , as well as shared across the range .", "This naturally occurring variation can provide a basis for adaptation to rapidly changing environments and it should be incorporated into restoration projects through strategic seed sourcing .", "It is important to note that our genomic analyses were based on mature trees that predate extensive land clearing for agriculture .", "It remains to be determined whether human modifications of the landscape have disrupted the historical patterns of gene flow , resulting in more fragmented and inbred populations .", "Genomic analyses of seedlings or saplings at these sites may show different results , although our phenotypic studies using seedlings produced results concordant with our genomic analyses .", "Our landscape genomic model can guide seed selection by empirically defining local provenances and identifying variation suitable for predicted future climates .", "This understanding of the relationship between environmental and genomic variation can be combined with other types of information , such as basic biological knowledge of the ecological community and best agronomic practices in restoration , to establish foundation species and ecosystems with the highest probability of success in rapidly changing environments ." ], [ "We obtained E . melliodora leaf samples from mature trees at 39 sampling sites—38 sites across the species' native range and a single site in Western Australia , well outside the species' natural distribution .", "We collected samples through a community science project described in Broadhurst et al . , 2018 ( Supplementary file 1 ) .", "From each site , a citizen scientist collected leaf samples from up to 30 trees , put the samples in silica gel for drying , and shipped them to CSIRO for processing .", "In addition to leaf material , they also collected seeds from the sampled trees when available .", "We selected 3 to 10 trees per sampling site for sequencing and we processed each of the seven trees from Western Australia twice , using different leaves from the same tree to serve as technical replicates .", "No power analysis was used to determine sample size during the design of the study .", "Sample size was determined based on our experience and judgment , with consideration of the availability of samples .", "We sequenced these 379 samples using a modified Genotyping-By-Sequencing ( GBS ) protocol ( Elshire et al . , 2011 ) .", "Briefly , we extracted genomic DNA from approximately 50 mg of leaf tissue using the Qiagen DNeasy Plant 96 Kit , digested with PstI for genome complexity reduction , and ligated with a uniquely barcoded sequencing adapter pair .", "We then individually PCR amplified each sample to avoid sample bias .", "We pooled samples in equimolar concentrations and extracted library amplicons between 350 and 600 bp from an agarose gel .", "We sequenced the library pool on an Illumina HiSeq2500 using a 101 bp paired-end protocol at the Biomolecular Resource Facility at the Australian National University , generating almost 260 million read pairs .", "We checked the quality of the raw sequencing reads with FastQC ( v0 . 10 . 1 , [Andrews , 2012] ) .", "We used AXE ( v0 . 2 . 6 , [Murray and Borevitz , 2017a ) ) to demultiplex the sequencing reads according to each sample's unique combinatorial barcode and were unable to assign 11% of read pairs to a sample . We used trimit from libqcpp ( v0 . 2 . 5 , [Murray and Borevitz , 2017b] ) to clean the reads for each sample , using default parameters , except q = 20 .", "This involved removing adapter contamination due to read-through of small fragments ( 20% of read pairs ) and merging overlapping pairs ( 49% of read pairs ) , with both steps using algorithms based on a global alignment of the read pair .", "We also used trimit for sliding window quality trimming ( 11% of reads ) .", "We then used a custom script to remove sequencing reads that did not begin with the expected restriction site sequence ( 16% of reads ) .", "We aligned sequencing reads to the E . grandis reference genome ( v2 . 0 , [Bartholomé et al . , 2015; JGI , 2015; Myburg et al . , 2014] ) , including all nuclear , chloroplast , mitochondrial , and ribosomal scaffolds , but used only nuclear scaffolds for downstream analyses .", "We aligned reads using bwa-mem ( v0 . 7 . 5a-r405 , [Li , 2013] ) , as paired reads ( -p ) and treating shorter split hits as secondary alignments ( -M ) , with 88% of reads successfully mapped .", "We used GATK's HaplotypeCaller in GVCF mode ( v3 . 6–0-g89b7209 , [McKenna et al . , 2010] ) to call variants for each sample with heterozygosity ( -hets ) increased to 0 . 005 , indel heterozygosity ( -indelHeterozygosity ) increased to 0 . 0005 , and the minimum number of reads sharing the same alignment start ( -minReadsPerAlignStart ) decreased to 4 .", "We used GATK's GenotypeGVCFs ( v3 . 6–0-g89b7209 , [McKenna et al . , 2010] ) for a preliminary round of joint genotyping across all samples based on the individual variant calls and again increasing the heterozygosity ( -hets ) to 0 . 005 and the indel heterozygosity ( -indelHeterozygosity ) to 0 . 0005 .", "For basic filtering , we used GATK to remove variants that were indels , had no variation relative to the reference , were non-biallelic SNPs , had QD < 2 . 0 ( ‘variant call confidence normalized by depth of sample reads supporting a variant’ ) , MQ > 40 . 0 ( ‘Root Mean Square of the mapping quality of reads across all samples’ ) , or MQRankSum < -12 . 5 ( ‘Rank Sum Test for mapping qualities of REF versus ALT reads’ ) .", "We removed samples with more than 60% missing data and SNPs with more than 80% missing data .", "We examined the genomic distance between samples to verify that technical replicates clustered closely together .", "We used a preliminary PCoA , based on genomic distance between samples , to identify outlier samples .", "We removed outlier samples and poorly sequenced samples from the dataset for final genotyping and all downstream analyses .", "We reran GATK's joint genotyping on the final sample set .", "We again used GATK to remove variants that were indels , SNPs with no variation relative to the reference , and non-biallelic SNPs .", "We determined final filtering thresholds by examining parameter distributions .", "A locus was retained for subsequent analysis if ExcessHet < 13 . 0 ( ‘phred-scaled p-value for exact test of excess heterozygosity’ ) , -0 . 3 < InbreedingCoeff < 0 . 3 ( ‘likelihood-based test for the inbreeding among samples’ ) , MQ > 15 . 0 ( ‘Root Mean Square of the mapping quality of reads across all samples’ ) , -10 . 0 < MQRankSum < 10 . 0 ( ‘Rank Sum Test for mapping qualities of REF versus ALT reads’ ) , and QD > 8 . 0 ( ‘variant call confidence normalized by depth of sample reads supporting a variant’ ) .", "We ran a second preliminary PCoA analysis to identify additional outlier samples .", "Finally , we used VCFtools ( v0 . 1 . 12b , [Danecek et al . , 2011] ) to remove SNPs with greater than 60% missing data and thin the SNPs so that none were closer than 300 bp .", "To examine the genomic structure of E . melliodora and how it is influenced by geography , we conducted individual-based analyses .", "For these analyses , we converted the final genotypic data ( a vcf file ) to a sample-by-SNP matrix and imported it into a genind object ( R adegenet v2 . 0 . 1 , [Jombart , 2008] ) .", "We calculated the pairwise genomic distances between individuals using a euclidean distance in dist ( R stats v3 . 1 . 2 , [R Core Team , 2015] ) .", "To visualize the genomic distance among samples , we ran a PCoA using dudi . pco ( R ade4 v1 . 7–4 , [Dray and Dufour , 2007] ) .", "We plotted the first two PCoA axes , with samples colored in a rainbow gradient based on sample latitude .", "We calculated the linear regression and correlation between latitude and the first PCoA axis using lm ( R stats 3 . 1 . 2 , [R Core Team , 2015] ) .", "We calculated the geographic distance between samples based on their GPS coordinates using earth . dist ( R fossil v0 . 3 . 7 , [Vavrek , 2011] ) .", "We used a mantel test ( R vegan v2 . 4–0 , [Oksanen et al . , 2016] ) , which examines the correlation between two distance matrices , to quantify the linear relationship between the genomic distance between individuals and the natural logarithm of the geographic distance .", "We then conducted site-based analyses .", "To estimate within-site genomic diversity , for each sampling site we calculated the number of alleles and the expected heterozygosity using summary and Hs ( R adegenet v2 . 0 . 1 , [Jombart , 2008] ) .", "We used the sample-by-SNP matrix to calculate pairwise Fst ( Weir and Cockerham , 1984 ) using pairwise . WCfst ( R hierfstat v0 . 04–22 , [Goudet and Jombart , 2015] ) .", "We ran a sampling-site level PCoA on the pairwise Fst matrix using dudi . pco ( R ade4 v1 . 7–4 , [Dray and Dufour , 2007] ) and calculated the percent of variation explained for each PCoA axis .", "To examine the role that environmental factors played in driving the genomic structure across the landscape , we used Generalized Dissimilarity Modelling ( GDM ) , which uses matrix regression to estimate the non-linear relationship between genomic distance and environmental distance ( Ferrier et al . , 2007; Fitzpatrick and Keller , 2015; Thomassen et al . , 2011 ) .", "We then used this model to predict the distribution of genomic variation across the landscape under current environmental conditions , as well as predicted future conditions .", "We obtained environmental data for the GDM from climate , elevation , soil , and landscape raster layers .", "Climate variables included 19 bioclimatic variables for the current time period ( 1960–1990 ) , at 30 arc second resolution ( WorldClim , 2016b ) .", "Elevation was from a digital elevation model aggregated from 90 m resolution ( CGIAR-CSI , 2016 ) .", "Soil data included available water capacity , total nitrogen , and total phosphorus sampled at the surface ( 0–5 cm ) and at depth ( 100–200 cm ) ( CSIRO , 2016 ) .", "Landscape data included the Prescott Index ( a measure of water balance ) and MrVBF ( a topographic index ) ( CSIRO , 2016 ) .", "For future predictions , we used the 19 bioclimatic variables predicted for 2070 at 30 arc second resolution based on GCM MIROC5 for representative concentration pathway 8 . 5 ( WorldClim , 2016a ) , which is a greenhouse gas concentration trajectory showing continual increase in emissions over time .", "We determined the values for each variable at each sampling site based on GPS coordinates and used those values to calculate the environmental distances between sites .", "To determine the genomic distances between sampling sites for the GDM , we scaled the Fst matrix to between 0 and 1 by subtracting the minimum value and then dividing by the maximum value .", "We generated the GDM model using gdm ( R gdm v1 . 2 . 3 , [Manion et al . , 2016] ) with the scaled Fst matrix , geographic distances between sites , and environmental distances for the 28 variables for the current time period .", "Initially , we generated a GDM model for each environmental variable separately and excluded variables from further analysis if the deviance explained by the model was less than 5% .", "For the remaining variables , we calculated Pearson's correlation for site values between pairwise sets of variables .", "If a pair of variables had a correlation greater than 60% , we excluded the variable with the lowest explanatory power from subsequent analysis .", "We conducted permutation testing using gdm . varImp ( R gdm v1 . 2 . 3 , [Manion et al . , 2016] ) with 1000 permutations to determine the explanatory power and statistical significance of the remaining variables and to excluded additional inconsequential variables .", "We generated a final GDM model with the remaining environmental variables .", "We cross validated the GDM model using a random 70% of the spatial sampling sites as training data and the remaining 30% of sites as test data and ran 1000 resampled iterations .", "We used the GDM models from the training sites to predict the genomic dissimilarity between the test sites and used Pearson's correlation to compare the predicted values to the observed values .", "To test the robustness of the geographic prediction from the GDM model , we visualized the geographic splines from 100 of these GDM models .", "To project the final GDM model onto the current environmental landscape , we first delineated the geographic extent of the analysis by defining an E . melliodora distribution polygon .", "We downloaded 14 , 977 E . melliodora occurrence records from the Atlas of Living Australia ( ALA , 2016 ) , of which we removed 189 because they were well outside the expected distribution or were sparse records on the distribution margin .", "We generated the polygon using ahull ( R alphahull v2 . 1 , [Pateiro-López and Rodríguez-Casal , 2010] ) , with alpha = 15 and gBuffer ( R rgeos v0 . 3–21 , [Bivand and Rundel , 2016] ) , with a 20 km buffer .", "We then transformed the environmental rasters based on the model splines ( gdm . transform ) , performed a PCA of the transformed layers ( prcomp R stats v3 . 1 . 2 , [R Core Team , 2015] ) , and predicted across space ( predict ) .", "We visualized the result by graphing the first three components of a PCA using a red-green-blue plot ( Fitzpatrick and Keller , 2015 ) .", "We also projected the model onto a predicted future environmental landscape with the same procedure , except we replaced the current bioclimatic rasters with the future ones for 2070 that were predicted under a high CO2 emission scenario .", "We calculated ‘genomic vulnerability’ ( Bay et al . , 2018 ) , which is the amount genomic change required to track environmental change over time , using the predict function with time = TRUE .", "We examined the implications of the GDM model for seed sourcing decisions by selecting two hypothetical reforestation sites .", "We compared predicted future GDM values at these two hypothetical reforestation sites to current climate GDM values across the landscape of potential seed sources .", "This enabled us to generate a map of the predicted genomic similarity of potential seed sources to the hypothetical reforestation sites under climate change .", "To examine the effect of provenance and environment on phenotype , we conducted experiments in climate controlled growth chambers under two different climate regimes .", "No power analysis was used to determine sample size during the design of the experiment .", "Sample size was determined based on our experience and judgment , with consideration of the availability of seed and space in the growth chambers .", "We selected six sites ( 11 , B , D , G , T1 , T3; asterisks in Figure 1A ) and six maternal lines per site that had sufficient seed .", "For each of the 36 maternal lines , we grew a minimum of 64 replicate seedlings , with four seeds planted per pot ( 6 . 5 cm x 6 . 5 cm x 20 cm pots with soil that was 80% Martin's mix and 20% sand ) .", "We germinated seeds in climate controlled chambers with 12 hr of light at 25°C and 12 hr of dark at 15°C .", "We set lights to mimic summer morning light ( photosynthetic photon flux 370 nm = 82 , 400 nm = 83 , 420 nm = 78 , 450 nm = 37 , 530 nm = 31 , 620 nm = 72 , 660 nm = 28 , 735 nm = 34 , 850 nm = 89 , 6500 K = 94 µmol/m2/s ) .", "We watered all seeds twice daily to keep the soil moist .", "We culled to one seedling per pot 12–14 days after planting .", "Three weeks after germination , we sorted seedlings into treatment chambers using a randomized block design based on maternal line .", "In each of the two climate chambers , we grew eight or nine replicate seedlings from each maternal line .", "Climate conditions were determined with SolarCalc ( Spokas and Forcella , 2006 ) to mimic average summer conditions ( sampling site 11 ) and hotter conditions ( 5°C temperature increase; sampling site T3 ) .", "We ran the experimental conditions for 12–14 weeks and took phenotypic measurements at five time points:1 , 2 , 3 , 5 , and 11 weeks after the experimental treatment began .", "Measurements included seedling height , number of leaves , and total leaf length .", "For the analysis of seedling height and total leaf length , we used the measurements at five weeks after the experimental treatment began and used only seedlings that were determined to be well established at that time .", "We also calculated a relative height increment for each seedling by determining the last measurement when the seedling had two or fewer leaves and the first measurement with eight or more leaves .", "The relative height increment is the difference between the natural logarithm of the two height measurements , divided by the difference in time .", "We investigated phenotypic plasticity by examining interaction plots between maternal line and experimental condition for three response variables: seedling height , total leaf length , and relative height increment .", "We statistically tested for an interaction between sampling site/maternal line and experimental condition with linear mixed-effect models using lmer ( R lme4 v1 . 1–10 , [Bates et al . , 2015] ) for each of the three response variables .", "Due to a lack of power to consider maternal line nested within sampling site , we ran two models for each response variable—one with maternal line and one with sampling site .", "These models included the experimental condition , sampling site or maternal line , and their interactions as fixed effects .", "We included germination chamber and block as random effects .", "We visually identified four outliers with a relative height increment over 0 . 035 .", "We ran the models with and without outliers to determine if they affected the results .", "We visualized the distribution of values for the three response variables across the six sampling sites using box plots .", "We quantified the distribution of phenotypic variation with linear mixed-effect models using lmer ( R lme4 v1 . 1–10 , [Bates et al . , 2015] ) .", "For each of the three response variables , the model included maternal line nested within sampling site and experimental condition as main effects , with no interaction term , and germination chamber and block as random effects .", "After completion of the chamber experiment , we conducted an outdoor covered drought experiment on the 16 week old seedlings .", "No power analysis was used to determine sample size during the design of the experiment .", "Sample size was determined based on our experience and judgment , with consideration of the availability of space in the covered growth facility .", "We selected 160 seedlings from five sampling sites , with four maternal lines per site .", "We paired each seedling with a seedling of similar size from the same maternal line and treatment chamber .", "We randomly assigned each seedling of the pair to a different treatment group .", "We transplanted the seedlings to PVC tubes ( 9 cm diameter x 50 cm height with sand , perlite , and slow release osmocote ) and kept them well watered for seven weeks , allowing them to acclimate to the outdoor conditions .", "Then we imposed two treatments: well-watered and drought .", "For the well-watered treatment , we watered the seedlings to saturation as needed ( between three times per week and twice per day , depending on the weather ) .", "For the drought treatment , we watered as necessary to reach ( but not exceed ) 50% saturation .", "We measured leaf traits on each seedling three weeks after the initiation of treatment .", "We measured stomatal conductance with a porometer ( SC-1 Leaf Porometer by Decagon Devices ) and determined that water stress was induced in the drought-treated seedlings .", "We determined the leaf length to width ratio from a scan of the most recent fully expanded leaf from each seedling using image analysis software ( WD3 WinDIAS Leaf Image Analysis System by Delta-T Devices ) .", "This leaf was initiated prior to the start of treatment , but expanded while under treatment conditions .", "We took additional measurements two months after the initiation of treatment .", "We used a chlorophyll meter ( SPAD – 502 by Konica Minolta ) to determine the SPAD index , which measures relative chlorophyll content; reduction in SPAD index would indicate detrimental effects of water limitation .", "We calculated specific leaf area ( SLA , leaf area divided by dry mass ) by scanning a single leaf from each seedling to determine the leaf area ( WD3 WinDIAS Leaf Image Analysis System by Delta-T Devices ) and weighing oven dried leaves .", "For analysis , we excluded data for seedlings that died during the experiment .", "We also excluded the experimental treatment partner of any dead seedlings .", "We visualized phenotypic plasticity by examining interaction plots between maternal line and experimental condition for four response variables: stomatal conductance , leaf length to width ratio , SPAD index , and SLA .", "We statistically tested for an interaction between sampling site/maternal line and experimental condition with linear mixed-effect models using lmer ( R lme4 v1 . 1–10 , [Bates et al . , 2015] ) for each of the four response variables .", "Due to a lack of power to consider maternal line nested within sampling site , we ran two models for each response variable—one with maternal line and one with sampling site .", "These models included the experimental condition , sampling site or maternal line , and their interactions as fixed effects .", "We included block and sample pairings as random effects .", "We visualized the distribution of values for the four response variables across the five sampling sites using box plots .", "We quantified the distribution of phenotypic variation with linear mixed-effect models using lmer ( R lme4 v1 . 1–10 , [Bates et al . , 2015] ) .", "For each of the four response variables , the model included maternal line nested within sampling site and experimental condition as main effects , with no interaction term , and block and sample pairings as random effects .", "Due to a lack of power , the p-value for the sampling site term was determined from a model without maternal line .", "We examined local adaptation using a Qst-Fst analysis ( R QstFstComp v0 . 2 , [Gilbert and Whitlock , 2015] ) for each phenotypic trait measured under each experimental condition .", "For each comparison , we estimated Qst under the model for offspring related as half-siblings through shared mothers and compared that value to the distribution of Fst values for the sampling sites included in the experiment .", "Statistical significance was determined based on the predicted null distribution of Qst-Fst using 10 , 000 simulation replicates .", "GBS sequencing reads are available at the NCBI Sequence Read Archive ( SRA ) ( http://www . ncbi . nlm . nih . gov/sra ) under BioProject PRJNA413429 .", "Growth experiment data and scripts for genomic and phenotypic analyses are available at https://github . com/LaMariposa/emelliodora ( Supple , 2018; copy archived at https://github . com/elifesciences-publications/emelliodora ) ." ] ]

[ "As species face rapid environmental change , we can build resilient populations through restoration projects that incorporate predicted future climates into seed sourcing decisions .", "Eucalyptus melliodora is a foundation species of a critically endangered community in Australia that is a target for restoration .", "We examined genomic and phenotypic variation to make empirical based recommendations for seed sourcing .", "We examined isolation by distance and isolation by environment , determining high levels of gene flow extending for 500 km and correlations with climate and soil variables .", "Growth experiments revealed extensive phenotypic variation both within and among sampling sites , but no site-specific differentiation in phenotypic plasticity .", "Model predictions suggest that seed can be sourced broadly across the landscape , providing ample diversity for adaptation to environmental change .", "Application of our landscape genomic model to E . melliodora restoration projects can identify genomic variation suitable for predicted future climates , thereby increasing the long term probability of successful restoration ." ]

[ "Yellow box , or Eucalyptus melliodora , is an emblematic Australian tree that is essential to many native ecosystems .", "Some of these environments are now critically endangered , and replanting yellow box trees is one of the first steps to try to restore them .", "However , it can be difficult for reforestation practitioners to decide where to collect the seeds they will use to replant an area .", "They have to select seeds that carry the genetic information that gives the trees the best chances of surviving now and in the future .", "This is a complex task because climate change creates fast-changing environments .", "Here , Supple et al . collect genetic material from 275 E . melliodora trees at 36 different sites .", "Genetic analyses show that the yellow box trees from these sites exchange their genetic material and do not form isolated populations .", "This means that the seeds do not need to be sourced from near the reforestation site , but can be collected from areas much further away .", "This results in higher quality seeds for reforestation because seeds from more sites will include more genetic diversity .", "Supple et al . then use information about the local climate , such as temperature and rain levels , at the sites where the samples were gathered to create a model that describes the relationship between genetic , geographical , and environmental factors .", "This helps identify which sites produce the seeds that will grow best under particular conditions .", "In addition , the seedlings from these sites are grown in the laboratory to see how well they fare in different types of environments .", "It therefore becomes possible to match a reforestation site with the seeds that will thrive in the future climate of the area .", "Sharing this knowledge with conservationists will help to guide their replanting strategies for E . melliodora .", "The method can also be applied to other plant species and restoration projects , so it could ultimately shape resilient ecosystems that can cope with climate change ." ]

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

[ "structural biology and molecular biophysics", "physics of living systems" ]

[ [ "The ring-shaped oligomeric ATPases control key biological processes including protein folding , transcription , DNA replication , cellular cargo transport , and protein turnover ( Erzberger and Berger , 2006; Puchades et al . , 2020; White and Lauring , 2007 ) .", "The 26S proteasome is an ATP-dependent protein degradation machine in the AAA+ ( ATPases associated with diverse cellular activities ) family of ATPases ( Collins and Goldberg , 2017 ) .", "The proteasome holoenzyme consists of a barrel-shaped 20S core particle ( CP ) capped by 19S regulatory particles ( RPs ) on one or both ends ( Bard et al . , 2018 ) .", "Each RP features a nine-member Lid subcomplex and a heterohexameric ring of AAA+ ATPases assembled from six distinct gene products ( RPT1–RPT6 ) that share 85% sequence identity .", "These ATPases use the energy from ATP hydrolysis to mechanically unfold substrates and translocate them into the CP for proteolysis ( Figure 1A ) .", "Processive translocation and degradation of protein substrates is critical for the biological functions of the ubiquitin-proteasome system ( Collins and Goldberg , 2017; Hershko and Ciechanover , 1998 ) .", "In previous investigations of the structural mechanism of proteasomal degradation , cryo-electron microscopy ( cryo-EM ) of the substrate-engaged proteasome captured seven states , EA1–ED2 .", "EA1 and EA2 likely represent the resting states of proteasome; ED1 and ED2 are hypothesized to be involved in substrate translocation ( Figure 1—figure supplement 1; Dong et al . , 2019; de la Peña et al . , 2018 ) .", "In these structures , an unfolded substrate primarily interacts with aromatic residues on the pore-1 loop ( PL1 ) of each ATPase .", "These short structured loops form a right-handed helical ‘staircase’ delineating the interior of the translocation channel .", "Changes in bound nucleotides at the ATPase interfaces are associated with rearrangements of the architecture of the PL1 staircase and the proteasome-substrate interaction , as a result of both translational and pivot movements of these ATPases ( Figure 1B and Figure 1—figure supplement 1; Dong et al . , 2019; de la Peña et al . , 2018 ) .", "One or two PL1s are disengaged from substrate interaction in each state .", "These disengaged PL1s usually occupy distal , or top , positions in the staircase away from the CP ( Figure 1A ) .", "To account for substrate translocation , we and others have proposed that when certain PL1s disengage and move to the top , substrate-engaged PL1s may move in the opposite direction , toward the CP .", "This conformational rearrangement may provide the power stroke to promote axial translocation of substrate ( Figure 1B; Dong et al . , 2019; de la Peña et al . , 2018 ) .", "In spite of these structural insights , important questions regarding the dynamics of proteasomal structures remain , in particular how the six proteasomal ATPases coordinate during conformational transitions to achieve substrate translocation .", "Based on these cryo-EM structures , we previously proposed that the conformation and nucleotide-binding status of the ATPase hexamer may cycle consecutively through six different states with rotational equivalence , thus driving processive substrate translocation ( Dong et al . , 2019; Hua et al . , 2002 ) .", "A similar model of sequential transitions was proposed in an independent study of yeast proteasome structures ( de la Peña et al . , 2018 ) .", "Only two out of the six conformations proposed in the sequential-transition model were identified in these structural studies , and direct experimental study of the transition sequence has not been reported ( Dong et al . , 2019; de la Peña et al . , 2018 ) .", "Transiting into the next state involves multiple changes in nucleotide status and a complex conformational rearrangement of the ATPase hexamer .", "We still do not know the order of these events and how the chemical energy from ATP hydrolysis may be harvested to drive these transitions .", "In addition , there are experimental findings that appear inconsistent with the predictions of a strict sequential-transition model .", "For example , mutations of the Walker-A ( WA ) or Walker-B ( WB ) motifs on an ATPase impede its nucleotide-binding or hydrolysis activity , and are therefore predicted to inactivate the proteasome by blocking the transition sequence .", "However , Walker mutations on some ATPases of the proteasome are in fact well-tolerated in yeast ( Rubin et al . , 1998; Eisele et al . , 2018; Kim et al . , 2013 ) .", "Similarly , mutations of other functional motifs on different proteasomal ATPases have varying effects on protein degradation , leading to the hypothesis that the six ATPases may have nonequivalent roles in the proteasome activities despite their high levels of similarity in sequence and structure ( Rubin et al . , 1998; Beckwith et al . , 2013; Erales et al . , 2012 ) .", "These functional properties of the proteasome are not interpreted by the previous models and so far no alternative model that is consistent with both structural and functional observations has been suggested .", "Computational approaches , such as molecular dynamics simulations , are frequently employed to decipher the structural dynamics of proteins ( Brini et al . , 2020 ) .", "However , despite recent advances , it is still impractical to perform a full-atom simulation of a large system such as the proteasome at a biologically relevant time scale ( ~100 ms ) ( Gecht et al . , 2020 ) .", "We therefore developed a novel approach to simulate the conformational dynamics of the proteasomal ATPase hexamer by constructing a physical model based on the nucleotide-dependent free-energy landscape ( FEL ) of the ATPase complex .", "To obtain the FEL , we first performed comparative analysis on known proteasome structures to identify the primary degrees of freedom ( DOFs ) of proteasome’s conformational changes , and applied these DOFs as the conformational coordinates of the FEL .", "We then parameterized the free energy surface based on the mode of nucleotide-ATPase interactions , and experimentally determined the nine parameters of the FEL .", "Conformational changes of the ATPase complex are mapped to simple stochastic transitions on the FEL which evolves as the nucleotide status changes , driven by independent chemical processes at each nucleotide-binding pocket .", "To address whether the simulated dynamics based on the FEL model recapitulates the actual dynamics of the ATPase complex , we experimentally tested the model predictions in a wide range of conditions that are distinct from the results used for model construction , as well as comparing the predictions with published results .", "We found that the FEL predictions were widely congruent with the experimental measurements in this and previous studies .", "Our work introduces a new method for studying the dynamics of a complex protein machine such as the 26S proteasome , and provides a coherent explanation for a variety of structural and kinetic observations .", "It also provides a satisfying picture of the underlying mechanism by which the AAA+ hexamer on the proteasome operates in driving substrate translocation ." ], [ "In this work , we elect a combination of the ATPase complex’s conformation and the nucleotide distribution in the six ATPase pockets to specify a ‘state’ of the proteasome , for consistency with nomenclature in structural studies .", "A description of the FEL of proteasome is represented by the potential of mean force of a specific proteasome population measured as a bivariate function of its conformational coordinates and the nucleotide distribution ( Kirkwood , 1935; Rodnina et al . , 2011 ) .", "The conformational coordinates of the FEL are defined by the primary DOFs of proteasome’s conformational changes , identified by comparing proteasome structures .", "We designate each conformation in the FEL by whether the interfaces of the six ATPase domains on proteasome are open or closed , based on the observation that the solvent-excluded surface area ( SESA ) of these interfaces mostly adopts binary values in these cryo-EM structures ( Figure 1C and Figure 1—figure supplement 2 ) .", "A large SESA is associated with closed interfaces that appear to arrange the PL1s on neighboring ATPases into a staircase that interacts with substrate peptide .", "In contrast , a smaller SESA suggests an open interface and the relative geometry of the neighboring ATPases tends to vary ( Figure 1—figure supplement 1 ) .", "An ATPase subunit that is flanked by two open interfaces is disengaged from substrate interaction , as observed in the cryo-EM structures ( Figure 1—figure supplement 1 ) .", "In the FEL , we constrain the total number of open interfaces in each hexamer conformation to be either 2 or 3 , as observed in the cryo-EM structures except for the EA states which involve only one open interface ( Figure 1—figure supplement 1 ) ( see Discussion ) .", "This defines 30 conformations , after excluding 5 with ambiguity in assigning ATPase-substrate interaction due to symmetry ( Supplementary file 1; Materials and methods 'Defining a discrete conformational space of the proteasome ATPase complex by extrapolating the cryo-EM observations' ) .", "States in the FEL are also differentiated by the nucleotide distribution in these ATPases .", "Each nucleotide pocket may be occupied by ATP , ADP , ATP-γS ( a slowly-hydrolyzing ATP analog ) , or no nucleotide .", "We ignore the transient ADP•Pi state due to the very weak affinity between free phosphate and the proteasome ( Figure 2—figure supplement 1 ) ; also , no such state has been identified so far in proteasome structures .", "The total number of distinct states in the FEL model is therefore 30×46=122 , 880 .", "The molecular details of the nucleotide-ATPase interactions suggest a strategy to parameterize the free energy of each conformation as a function of its nucleotide status .", "Each nucleotide-ATPase interaction on the proteasome involves several conserved elements: the WA , WB , sensor I , and sensor II motifs from the cis ATPase and the arginine fingers from the trans ATPase ( Figure 2A; Ogura and Wilkinson , 2001 ) .", "The arginine fingers are the major components interacting with the γ- and β-phosphate groups on ATP at a closed interface .", "We found that the cis elements exhibited rather minor rearrangements among different states of either substrate-free or substrate-engaged proteasomes ( root-mean-square deviation [RMSD] ~0 . 58 Ȧ ) ( Figure 2—figure supplement 2 ) .", "We therefore parameterized the total free energy of the ATPase hexamer as a sum of the contributions from each individual ATPase interface .", "Each interface’s contribution is subdivided into three components: the basal energy Eb , which derives from direct interactions between adjacent ATPases at a closed interface; the pocket energy Ep from the nucleotide-cis-element interactions , which are similar for ADP and ATP; and the bridge energy EBr , which differentiates ADP from ATP and originates from the engagement of arginine fingers and other trans elements with the γ- and β-phosphate on the nucleotide at a closed interface ( Figure 2B ) .", "The cis pockets of disengaged ATPases exhibit either low or no nucleotide density in cryo-EM maps , and are associated with slightly rearranged cis motifs ( p=0 . 014 ) ( Figure 1—figure supplement 1 and Figure 2—figure supplement 2; Dong et al . , 2019 ) .", "We therefore assign a separate pocket energy EpAPO for disengaged ATPases to reflect their low affinity for nucleotides .", "The chemical energy in ATP is excluded from the free energy calculation , since it does not explicitly contribute to the simulation of ATPase dynamics ( see the next section ) .", "These energy parameters can in principle vary for the different ATPase subunits Rpt1–Rpt6 .", "The magnitude of this variation is unclear .", "To reduce the model’s complexity , we made an approximation that all six ATPases share an identical set of parameters .", "As described below , this approximation provides excellent agreement with kinetic measurements and is compatible with the asymmetric cryo-EM occupancies and effects of WB mutations .", "In a following section , we analyze the contribution of the proteasome Lid-ATPase interaction to the symmetry breaking among these ATPases .", "We determined all nine parameters in the FEL model from the measurements in this study and previous work ( see Materials and methods 'Determining the FEL and kinetic parameters' and 'Determining the parameters related with substrate translocation' ) .", "A critical parameter is the difference in the bridge energies ( EBr ) between ATP and ADP interfaces , which we find is key for determining the directionality of translocation .", "This parameter was measured using a single-molecule binding assay , based on the relationship between the dissociation constant ( Kd ) of the nucleotides and these energy terms .", "In the FEL framework , the nucleotide pockets are classified into three groups , according to how the Kd depends on the energy parameters ( Figure 3A ) .", "This designation varies with the status of the cis ATPase and the interface .", "We used a very low concentration ( 200 nM ) of Alexa647-conjugated ATP to limit the interaction to the strongest binding pockets ( group 1 in Figure 3A ) on the human 26S proteasome which was immobilized on a passivated coverslip , and observed the interaction between ATP and the proteasome by TIRF ( Total Internal Reflection Fluorescence ) microscopy .", "The proteasome was fluorescently labeled on the 19S particle using a SNAP tag to allow accurate detection of colocalization with Alexa647-ATP .", "We varied the concentrations of competing unlabeled nucleotides ( ATP , ADP , and ATP-γS ) in a steady-state measurement , to circumvent the possibility that conjugation with fluorophore may alter the nucleotide-ATPase affinity ( Figure 3B ) .", "ATP-γS was employed to minimize nucleotide hydrolysis .", "This analysis yielded the following ratio of the inhibitor constant ( Ki ) for ATP:ADP:ATP-γS=1:7 . 9 ( ±2 . 0 ) :0 . 5 ( ±0 . 15 ) ( at 90% confidence interval ) , giving eBr , ATP−eBr , ADP=−1 . 6 ( ±0 . 4 ) kcal/mol .", "After determining the FEL parameters , we explored the basic features of the landscape .", "For the 12 ATPase-hexamer conformations that are characterized by either a single or two adjacent disengaged ATPase units , as in the observed ED or EC states , uniform ATP binding generates a flat FEL .", "The presence of ADP in a single pocket breaks the symmetry and lowers the relative free energy of a subset of conformations by 1 . 6 kcal/mol .", "The further addition of an empty pocket next to the ADP pocket produces a well-separated energy-minimum conformation in which the empty and ADP cis pockets are found in the disengaged ATPase and its counterclockwise neighbor ( Figure 2C ) .", "Although we did not impose any coupling between nucleotide status and ATPase conformation , this energy-minimum arrangement of nucleotide and conformation is identical to those observed in the ED1 and ED2 states of proteasome , possibly explaining the prevalence of this arrangement in proteasome structures as well as in related AAA+ ATPases ( Figure 1—figure supplement 1; Dong et al . , 2019; Majumder et al . , 2019; Cooney et al . , 2019 ) .", "We simulated the dynamics of the ATPase complex as stochastic transitions in a discrete 12 ( 6 conformational+6 nucleotide ) dimensional space ( Figure 3—figure supplement 1 ) .", "The transitions between conformations were described as a simple bi-state process with a rate constant determined by the Arrhenius equation .", "The ATP cycle at each pocket proceeds independently , as described by the basic chemical rate equations .", "The off-rate of a nucleotide is calculated from its Kd as a function of the energy parameters , with the on-rate set at a constant ( Figure 3A; Materials and methods 'Determining the FEL and kinetic parameters' and 'Determining the parameters related with substrate translocation' ) .", "The detailed process of a conformational change is not considered explicitly ( i . e . , as ‘adiabatic’ ) in that the change of nucleotide distribution alters the FEL and causes repopulation of the conformational space of proteasome .", "We do not include any assumption on the coordination of the nucleotide cycles , or the coupling of the nucleotide and conformational changes , or any predetermined sequence of transitions .", "To generate testable predictions from simulated ATPase dynamics , we introduced a substrate peptide that is mechanically coupled with the continuous segment of the PL1 staircase .", "In these simulations , the disengaged PL1s occupy the top staircase position , as observed in cryo-EM structures ( Figure 1B; Dong et al . , 2019; de la Peña et al . , 2018 ) .", "The unit step of translocation is defined by the axial separation between PL1s , corresponding to approximately two amino acids ( AAs ) in the substrate polypeptide ( Figure 1B ) .", "The force coupling with a translocating substrate may affect the rates of ATPase conformational changes .", "We represent this effect as a titling of the FEL and simplify by ignoring the stochastic and sequence-dependent variations of these forces and introduce two constant values to capture the average effect on the forward and backward processes ( Materials and methods 'Determining the parameters related with substrate translocation' ) .", "To experimentally determine these two force parameters and another parameter for defining the activation energy barrier , we employed a quantitative degradation assay based on the decay of a fluorescent reporter of the ubiquitylated cyclinB N-terminus fused with an infrared fluorescent protein iRFP ( Figure 3D; Lu et al . , 2017 ) .", "The N-terminal cyclinB is an unstructured protein that is efficiently degraded by the proteasome starting from its N-terminus ( King et al . , 1996; Yamano et al . , 2004 ) .", "We find that degradation of cyclinB-iRFP follows Michaelis-Menten-like kinetics with KM=5 . 6 nM ( Figure 3E ) .", "We performed each measurement at substrate concentrations much higher than the KM to maximize the signal-to-noise ratio , and tested five substrate concentrations , each with three replicas , to verify the saturation condition .", "It took an average of 55 s ( i . e . , the turnover time ) for the proteasome to degrade a 46 kDa cyclinB-iRFP molecule in a process that could be rate-limited by several steps including substrate commitment , unfolding , and translocation ( Figure 3D; Collins and Goldberg , 2017 ) .", "To determine the actual rate-limiting step , we engineered a mutant of cyclinB containing only three lysine residues , and conjugated fluorescent ubiquitin chains onto these lysines ( Lu et al . , 2015 ) .", "We examined this substrate using a single-molecule translocation assay developed previously ( Figure 3C; Lu et al . , 2015; Hon and Lu , 2019 ) .", "The processive translocation of a substrate coincides with the stepwise removal of entire ubiquitin chains by the deubiquitinase Rpn11 on the proteasome , which was detected by TIRF to measure the ubiquitin copies on a substrate molecule ( Figure 3—figure supplement 2 ) .", "The decay rate of fluorescent ubiquitins on three-lysine cyclinB suggests a translocation rate of 10 . 5 ( ±0 . 8 ) AA/s ( Materials and methods 'Single-molecule proteasome assay' ) .", "A similar translocation rate was found in a measurement of Vmax of proteasomal degradation ( Peth et al . , 2013b ) .", "With this translocation rate , a cyclinB-iRFP peptide containing 445 AAs would take at least 44 s to progress into the CP , that is , 80% of the 55 s total turnover time .", "This suggests that translocation is the limiting step of the entire degradation process for this substrate .", "Degradation of these substrates is unlikely to be limited by deubiquitylation because substrates with multiple Ub chains are degraded faster than the same substrate with fewer chains ( Lu et al . , 2015 ) .", "Therefore , in the experiments described below , we used the measured degradation rate of cyclinB-iRFP as an approximation for the translocation rate .", "This reporter assay also gives consistent results with direct single-molecule measurements under perturbed conditions , such as in the presence of ADP or ATP-γS ( Figure 3C ) .", "We next determined the translocation rate of cyclinB-iRFP in the presence of 500 μM ATP and different concentrations of ADP , and estimated the three translocation-related parameters using these data ( Figure 3F and Materials and methods 'Determining the parameters related with substrate translocation' ) .", "Directly probing the dynamical behavior of the proteasomal ATPases is challenging .", "If the FEL model can recapitulate the actual ATPase dynamics , it may contribute valuable insights into the functional mechanism of the proteasome .", "To evaluate the consistency of the model with reality , we seek to examine the predictions of the simulated dynamics in experiments that are independent of those for model construction , as discussed below .", "The workflow and the main results are summarized in Figure 4—figure supplement 1 .", "We found that the simulated ATPase dynamics at a steady state promotes a directional translocation of substrate into the CP , with variations due to the stochasticity of the dynamics ( Figure 4A ) .", "We first compared the simulated translocation rates of cyclinB-iRFP at different ATP concentrations with experimental measurements ( Figure 4B ) .", "Interestingly , the translocation-rate curve is non-monotonic and peaks around the physiological concentration of ATP .", "The FEL model accurately captures the quantitative features in both the up and down phases of the rate curve , each yielding a different insight .", "The EC50 value for ATP in the up phase is 45 μM , far from the Kd value of ATP or ADP at any binding pocket ( Figure 3A: ~100 nM for group 1 , ~3 μM for group 2 , ~2 mM for group 3 ) .", "Guided by the FEL model , we found that the expression of this EC50 value is given by the ratio between the total rate of ATP hydrolysis and the on-rate of ATP ( Materials and methods 'Deriving a formula for the EC50 value in the ATP titration experiment' ) .", "The observed inhibition of translocation at high ATP concentrations ( Figure 4B ) is likely due to the loss of ATPase cooperativity .", "This inhibition is not due to extra Mg2+ , proteasome disassembly , degradation-independent iRFP inactivation , or a general slowdown of ATP hydrolysis ( Figure 4—figure supplement 2 to Figure 4—figure supplements 3–5 ) .", "As the FEL model suggests , coordinated movements of the ATPase subunits require the ATPase complex to have a unique energy-minimum conformation for each nucleotide status typically occurring during physiological operation , so that , when the nucleotide status changes , these ATPase’s conformations undergo a well-defined collective change , leading to translocation .", "Very high ATP concentrations bias the proteasome toward all-ATP status and a flat FEL , resulting in a loss of ATPase cooperativity and abrogating the directionality of translocation ( Figures 2C and 4A ) .", "This result is in contrast to the predictions of a sequential-transition model , which would predict a monotonic increase of translocation rate at increasing ATP concentrations , regardless of the parameters and reaction details , inconsistent with the observation ( Materials and methods 'The translocation rate at different ATP concentrations as predicted by a strict sequential-transition model' ) .", "The FEL-predicted translocation kinetics are also consistent with the results of competition experiments involving ATP-γS .", "In the simulation ( Figure 4A ) , ATP-γS introduces pauses between processive phases of substrate translocation , longer at higher concentrations , which closely resembles the ATP-γS-induced translocation pauses in single-molecule force measurements of ClpXP , a proteasome-like ATPase in prokaryotes ( Sen et al . , 2013 ) .", "In the reporter experiment , 36 μM ATP-γS inhibited the translocation rate by 50% in the presence of 500 μM ATP ( Figure 4C ) , despite the fact that the difference in the apparent Ki values between ATP and ATP-γS is only twofold ( Figure 3B ) .", "ATP-γS alters the ATPase dynamics which in turn affects the kinetics of nucleotide turnover .", "This process is required to interpret the low IC50 for ATP-γS , as suggested by the FEL model .", "We also performed an ATP-γS competition experiment at a level where the high-ATP-inhibition effect is apparent ( 15 mM ATP ) .", "Despite an overall reduction in translocation and a dramatic shift in IC50 , the FEL-predicted rates still closely match the experimental results ( Figure 4D ) .", "The FEL model also predicts that the translocation rate should linearly depend on the rate of ATP hydrolysis at varying ATP-γS concentrations ( Figure 4C ) , consistent with a previous observation ( Peth et al . , 2013b ) .", "For structurally stable substrates , unfolding may be a limiting step in proteasomal degradation .", "To test the FEL model in the context of such substrates , we created a fluorescent reporter by inserting a dihydrofolate reductase ( DHFR ) domain from Escherichia coli between cyclinB and iRFP .", "We found that adding a DHFR ligand , folic acid , led to a dose-dependent stabilization of the ubiquitylated cyclinB-DHFR-iRFP in the presence of the proteasome , while the degradation of the original cyclinB-iRFP was unaffected ( Figure 5—figure supplement 1 ) .", "In the presence of folic acid , cyclinB-DHFR-iRFP degradation is still complete , or processive ( Figure 5—figure supplement 2 ) .", "The IC50 value for folic acid in inhibiting the degradation is 800 μM , much higher than the 1 μM dissociation constant between DHFR and folic acid ( Posner et al . , 1996 ) .", "This is likely because unfolding of the DHFR domain by the ATPase’s actions primarily occurs when DHFR is transiently unliganded , consistent with the linear relationship between folic acid concentration and the inverse of the degradation rate ( Figure 5—figure supplement 3 and Materials and methods 'Interpreting the degradation kinetics of unfolding-limited substrates' ) .", "The FEL model suggests that folic acid lowers the overall efficiency of ATP utilization in degrading DHFR-containing substrates as reported previously ( Figure 5A; Peth et al . , 2013b ) .", "We tested the degradation rates of ubiquitylated cyclinB-DHFR-iRFP with 800 μM folic acid in an ATP-titration experiment , and found that folic acid did not affect the EC50 value of ATP in the up-phase of the rate curve , although it lowered the peak degradation rate .", "However , folic acid significantly reduced the degree of degradation inhibition at high ATP concentrations ( Figure 5A and B ) .", "This is likely because the ATPase activity unfolding the substrate is less affected by high ATP concentrations than is translocation ( Figure 4—figure supplement 5 and Figure 5—figure supplement 4 ) .", "To predict the degradation rate of cyclinB-DHFR-iRFP , we introduced one additional parameter to describe the unfolding rate of unliganded DHFR by proteasome in the FEL model .", "These qualitative features of the predicted degradation-rate curve in the ATP-titration experiment are insensitive to the choice of this parameter ( Materials and methods 'Interpreting the degradation kinetics of unfolding-limited substrates' ) .", "Molecular machines may occasionally run backward due to the stochasticity of single-molecular dynamics .", "Simulation by the FEL model suggests that substrates with an unfolding-resistant domain would not stably engage with the proteasome but would instead escape at a rate determined by the backward kinetics ( Figure 5C ) .", "A stable ligand , such as methotrexate , can inhibit the degradation of cyclinB-DHFR-iRFP at a low concentration by preventing the unfolding of DHFR , resulting in partial cleavage of the substrate ( Figure 5—figure supplement 1; Johnston et al . , 1995 ) .", "In a competition assay , degradation of the reporter cyclinB-iRFP was reduced by a nonfluorescent competitor cyclinB-DHFR-iRFPdark ( Figure 5D ) .", "This inhibition was exacerbated in the presence of methotrexate ( Figure 5E ) .", "We measured the turnover time of the stable DHFR substrate in this assay and found the value comparable with the model prediction which was calculated as a first-passage time on a 20–30 AA peptide track measured from the PL1s to the proteolysis sites in the CP ( Figure 5F ) ( Materials and methods 'Monte Carlo simulation of the FEL model of proteasome' and 'Measuring the residence time of an unfolding-resistant substrate on the proteasome' ) .", "Adding ATP-γS or ADP in the simulation reduces the first-passage time and facilitates the escape of stable substrates ( Figure 5C ) .", "These results are consistent with the experimental values for the turnover time in the presence of ATP-γS or ADP ( Figure 5F ) .", "In summary , we found that predictions from the FEL model closely match new experimental observations at nucleotide concentrations ranging across three orders of magnitude , in both the forward and the backward processes .", "The overall consistency with experiments is not sensitive to parameter uncertainty at a typical value of ~30% ( Figure 5—figure supplement 5 ) .", "We further explore the features of the simulated ATPase dynamics and compare the predictions with additional results to examine this FEL approach .", "This analysis also provides insights into the mechanism of the proteasome and rationalizes the effects of ATPase mutations in previous studies .", "Simulating the ATPase dynamics gives the steady-state distribution of the proteasome at each conformation and the frequency of every conformational transition .", "The FEL model is built on fundamental physical and chemical processes and does not a priori specify the occupancy of any conformation , the transition pathway between conformations , or how the ATPases cooperate .", "Interestingly , in the simulation , we found that the ATPase conformations self-organize into a transition network , with the ED-like and EC-like conformations predominating , as they do in the cryo-EM analysis ( Figure 6A; Dong et al . , 2019 ) .", "Although the main transition pathway among EDs resembles the previous sequential model , there are several side transitions , or branches , that are key to understanding some experimental observations .", "To simulate the effects of an ATPase mutation , we abolished the ATP-hydrolyzing activity of one ATPase , for example Rpt3 , in the simulation , and then analyzed the global dynamics of the mutated proteasome ( Miller and Enemark , 2016 ) .", "ATP hydrolysis at the Rpt3–Rpt4 pocket is important for the transition from the ED1 to ED2 cryo-EM state; its inactivation mimics a WB mutation and strongly reduces the transition frequency from the ED1- to the ED2-equivalent conformation in an FEL simulation .", "This loss of ‘flow’ was compensated by an increase in the transition to an EC-like conformation , so that the overall translocation rate was only reduced by 11% ( Figure 6B and Figure 6—figure supplement 1 ) .", "Transition networks with an inactivated ATPase also show an expansion of the ED-like conformations in which the ATPases at the –2 or –3 positions are flanked by open interfaces ( 0=mutant , negative=counterclockwise ) ; this trend is consistent with and may explain a previous cryo-EM analysis on WB mutant proteasomes ( Eisele et al . , 2018 ) .", "The order of the elementary steps of reactions and transitions is an important part of the ATPase mechanism and is challenging to observe directly .", "Different sequences of these steps have been hypothesized to drive the orderly conformational transitions of the proteasome and other AAA+ ATPases ( Dong et al . , 2019; Majumder et al . , 2019; Monroe et al . , 2017; Lyubimov et al . , 2011 ) .", "Our simulation identifies the most-likely reaction sequence by enumerating all the possible sequences that can promote transitions between two states and calculating their probabilities .", "For the ED1-to-ED2 transition , the most-likely reaction sequence begins with ATP hydrolysis at the Rpt3–Rpt4 pocket , followed by ATP binding to Rpt5 and a conformational rearrangement of the ATPase complex .", "This conformational change exchanges the ADP-bound Rpt3–Rpt4 interface for an ATP-bound Rpt5–Rpt1 interface and drives a unit-step translocation of two AAs by rearranging the PL1 staircase ( Figure 6—figure supplement 2 ) .", "In this process , 1 . 6 kcal/mol energy is dissipated .", "This energy-dissipating step is important for establishing the directionality of translocation in the simulation .", "This conformational change also drives Rpt4 into a disengaged APO state with a weak nucleotide affinity , allowing rapid release of bound ADP .", "Otherwise , the rate of ADP release would be incompatible with the fast kinetics of substrate translocation .", "Phosphate release is unlikely to be limiting since phosphate has a Ki value of 70 mM measured in a competition assay , a much weaker affinity than ADP for the ATPases ( Figure 2—figure supplement 1 ) .", "Identifying this ‘most-likely’ reaction sequence is critical for obtaining the formula for the EC50 value of ATP ( Materials and methods 'Deriving a formula for the EC50 value in the ATP titration experiment' ) .", "In a proteasome with non-hydrolyzing Rpt3 , the ED1-to-EC transition that partially rescues the ‘flow’ is initiated by ATP hydrolysis at the Rpt6–Rpt3 pocket .", "The associated conformational change drives a translocation of two unit-steps , or four AAs ( Figure 6—figure supplement 3 ) .", "These larger steps occur stochastically even in the wild-type ( WT ) proteasome , though they are less energetically favorable .", "Nonetheless , these occasional larger steps likely explain the experimentally observed translocation efficiency of 2 . 6–3 . 0 AAs per ATP consumption ( Figure 5A; Peth et al . , 2013b ) .", "We next address the question of whether our model can help to interpret the observed functional disparity among the proteasomal ATPases .", "The model described above is symmetric , with all ATPases having identical parameter .", "One potential source of symmetry-breaking is the proteasome Lid-ATPase interaction .", "The Lid subcomplex interacts extensively with the ATPase domains , primarily on Rpt3/6 , in the EA-like or EA-like states in which the PL1s on Rpt3 and Rpt2 respectively occupy the top and bottom niches in the staircase and Rpt6 is in a disengaged position ( Dong et al . , 2019; Chen et al . , 2016; Zhu et al . , 2018; Beck et al . , 2012 ) .", "This arrangement closely resembles one ED-like conformation ( Figure 6—figure supplement 4 , gray node ) .", "Weakening the Lid-ATPase interaction by mutations reduces the fraction of proteasome in the EA-like states ( Greene et al . , 2019 ) .", "We therefore hypothesize that the Lid-ATPase interaction may effectively stabilize this specific ATPase conformation by lowering its standard free energy .", "We implemented such a stabilizing effect in the FEL simulation by lowering the free energy of this ED-like conformation by an arbitrary value , and found that this resulted in significant expansion of the occupancy of the ED1- and ED2-conformations and a simultaneous shrinkage in other ED-like conformations ( Figure 6—figure supplement 4 ) , consistent with the cryo-EM observations ( Dong et al . , 2019; de la Peña et al . , 2018 ) .", "To study whether the Lid-ATPase interaction may contribute to the different growth phenotypes in yeast WB mutants , we examined how the translocation rate changed after simulated inactivation of the ATP-hydrolysis activity of individual ATPases in the presence of the Lid .", "We found that the predicted translocation rate is generally correlated with the corresponding growth phenotype in a range of the Lid-ATPase interaction strength , which suggests that this interaction may contribute to the growth phenotypes of yeast WB mutants , potentially in conjunction with other compensatory mechanisms ( Figure 6—figure supplement 5 ) ." ], [ "Protein machines accomplish complex tasks , driven by their exquisite structural dynamics at the molecular level .", "Studying these dynamics is challenging for both experimental investigation and molecular simulation .", "In this work , we exercised the principle of parsimony to reconstruct the FEL of the proteasomal ATPase complex and experimentally determined its parameters in an attempt to uncover the mechanism in polypeptide translocation .", "The FEL is an intrinsic property of a protein or a complex and is a key component of physical models that have provided important insights into the mechanisms of molecular motors ( Wang and Oster , 1998; Tu and Cao , 2018; Bustamante et al . , 2001 ) .", "FELs can be derived from conformational occupancies in cryo-EM studies through the Boltzmann equation Dashti et al . , 2014; Fischer et al . , 2010 .", "Identification of the very large number of proteasomal states is challenging and the nonequilibrium transitions further complicate the analysis .", "The FEL-based simulation introduced in our study generates various predictions which we used to examine the validity of the simulated ATPase dynamics and this overall approach .", "We found that the simulation recapitulated a number of important experimental observations including degradation kinetics , state distributions in cryo-EM datasets and the growth phenotypes of WB mutants ( Figure 4—figure supplement 1 ) .", "Moreover , this study reveals that these varieties of phenomena are in fact driven by a coherent and simple principle embodied in our model , thus offering mechanistic insights into the ATP-driven cooperative actions of the ATPases in promoting the translocation of substrate polypeptides .", "The observation that the nucleotide status of three consecutive ATPase subunits on the proteasome represents a continuous sequence in an ATP cycle has led to a sequential rotary model and ‘hand-over-hand’ translocation for proteasomal ATPase activity ( Puchades et al . , 2020; Dong et al . , 2019; de la Peña et al . , 2018 ) .", "A strict sequential mechanism requires that the ATP-bound subunit adjacent to the ADP subunit must hydrolyze ATP first .", "We compared the nucleotide-interacting motifs in all high-resolution structures of the proteasome but failed to identify a consistent trend that could suggest an allosteric effect between the ATP-pocket and other parts of the complex .", "As an alternative , we considered the possibility that the apparent cooperativity between different subunits might emerge from the modulation of their collective FEL by ligand binding , as proposed in the celebrated Monod-Wyman-Changeux concerted model for allosteric regulation ( Changeux , 2012 ) .", "Loss of ATPase cooperativity due to flattening of the FEL at above-physiological ATP concentrations reduces the rate of substrate degradation ( Figure 4B ) .", "A similar inhibitory effect has been reported for a bacterial pilus assembly motor , though the exact mechanism is still unclear ( Sukmana and Yang , 2018 ) .", "Although sacrificing some translocation efficiency , the flat FEL of the all-ATP state facilitates interchange among proteasomal conformations , even under physiological ATP concentrations .", "This property may be important for bypassing an occasional stuck or defective ATPase subunit that would completely inhibit proteasomal activity in a strict sequential-transition model .", "The resulting network of conformational transitions is analogous to the ‘ring-resetting’ model for the ClpXP ATPase complex ( Stinson et al . , 2013 ) .", "It is interesting to note that the peptidase activity of the proteasome is also inhibited by ATP concentrations above a threshold; however , this is likely to be due to a different mechanism since the critical concentration for peptidase inhibition is ~50× lower than that for translocation ( Smith et al . , 2011 ) .", "One or two ATPase units are disengaged from substrate peptide or from the translocation channel in all identified states of the proteasome .", "These disengaged ATPases are flanked by two open interfaces except in EA states , where the ‘disengaged’ subunit is associated with one open interface .", "We speculate that this is due to the extensive Lid-ATPase interaction in these states ( Chen et al . , 2016 ) .", "The presence of disengaged ATPases ensures that there is a unique energy-minimum conformation , and thus ensures a high level of cooperativity among the ATPase subunits .", "The smaller pocket energy Ep of these disengaged units also allows a fast nucleotide exchange , which is required for efficient translocation .", "Disengaged ATPase units are frequently observed in the structures of ring-shaped ATPases and may have a similar role in their activities ( Majumder et al . , 2019; Fei et al . , 2020; Puchades , 2017 ) .", "The nonequilibrium transitions of the ATPase complex identified by the FEL approach reveal important features of proteasome functions .", "Our model shows that substrate translocation steps are directly coupled to an energy-dissipating conformational transition which swaps an ADP-bound closed interface with another ATP closed interface ( Figure 6—figure supplement 2 ) .", "We propose that this coupling may dictate the directionality of translocation .", "The simulated translocation process deviates from a strict ‘hand-over-hand’ mechanism in that the step size in our model can adopt multiples of 2× AA , depending on which ATP molecule is first hydrolyzed ( Figure 6—figure supplement 2 and Figure 6—figure supplement 3 ) .", "Variations in step size have been observed in substrate translocation by ClpXP , though its connection with our simulation is unclear ( Sen et al . , 2013; Shi et al . , 2016 ) .", "We used the cryo-EM structures of proteasome as the basis for model construction .", "These structures are mainly for identifying the DOFs of the ATPase’s conformational change ( Figure 4—figure supplement 1 ) .", "Other aspects of the structural information , such as the conformational occupancy and nucleotide distribution , are not required for model construction but are still consistent with its predictions .", "The inter-subunit signaling motif has been suggested to mediate ATPase communication and coupling with nucleotides and may contribute to the bridge energy in the FEL model ( Chang et al . , 2017 ) .", "We did not include explicit allosteric coupling between ATPases in our model , as we find that it is not essential for explaining the current experimental observations .", "Adding allosteric effects to the model would be straightforward and may accommodate the ‘coordinated bursts’ mechanism to extend this model to other ATPase systems ( Fei et al . , 2020 ) .", "Partial degradation , or processing , by the proteasome is a natural process in the maturation of certain protein factors ( Nassif et al . , 2014 ) .", "The backward process on proteasome is much less understood and may contribute to efficient release of partially degraded substrates .", "In our study , modeling the substrate-escape kinetics as a first-passage time problem yields predictions consistent with the measurements .", "Other mechanisms , such as loss of grip on certain substrates , may also contribute to the escape kinetics ( Nassif et al . , 2014 ) .", "In the current model , we simplified the mechanical force on a substrate during translocation as a constant .", "Incorporating the precise unfolding landscape of a substrate and the interaction energy with the ATPase into the FEL model would be straightforward ( Saha and Warshel , 2021 ) , and may shed light on the mechanism of partial or nonprocessive degradation of certain substrates .", "The same mutation in the six highly-similar ATPases often have different effects on proteasome activities ( Rubin et al . , 1998; Eisele et al . , 2018; Beckwith et al . , 2013 ) .", "Previous studies lead to no consensus on whether certain subunits play a more important role in substrate degradation and the extent to which the sequence divergence among the six ATPases contributes to these functional heterogeneities is unclear .", "Interestingly , we found that it is possible to rationalize at least some of these functional heterogeneities without invoking disparity at the level of individual ATPases .", "The introduction of Lid-ATPase interactions as a simple conformation-stabilizing parameter in the FEL simulation , recapitulates the asymmetric cryo-EM state distributions and may explain the different phenotypes of WB mutants .", "Direct interpretation of WA mutants remains challenging , as these mutations often lead to proteasome assembly defects ( Beckwith et al . , 2013 ) .", "In the simulation , large heterogeneities of energy parameters among the six ATPases can alter the ATPase dynamics ( Figure 6—figure supplement 6 ) .", "Interestingly , this effect is significantly blunted by incorporating the Lid-ATPase interaction ( Figure 6—figure supplement 6 ) .", "This may contribute to the inessentiality of parameter variations in recapitulating the experimental results .", "Including the Lid-ATPase interaction in the model yields predictions that are compatible with all the kinetic measurements in our study and indicates that the proteasome may occasionally visit an EA-like state during translocation .", "However , the peak translocation rate is underestimated by 20%–25% in this case , suggesting that our understanding of the symmetry-breaking mechanism is still incomplete .", "The FEL presented here is undoubtedly an approximation of the actual FEL of the proteasome .", "Results in future studies may identify additional mechanisms , such as allosteric effects , to increase the accuracy of the simulated ATPase dynamics .", "As one example of the results that are not well recapitulated by the current model , protein substrates and ubiquitin chains activate the ATPase activity of the proteasome by ~2-fold , larger than the predicted 9% increase by the FEL model ( Peth et al . , 2013a ) .", "This activation step may be independent of the processive translocation phase described by the FEL model , and may involve a transition from the resting to translocating states of the proteasome ( Bard et al . , 2019 ) .", "In the EA states of the proteasome , all ATPases bind nucleotides , suggesting that the disengaged subunit in the resting states may have higher nucleotide affinity than the disengaged ones in the translocating states ( Dong et al . , 2019 ) .", "This high affinity may limit nucleotide turnover in the resting states .", "Similarly , our model may not accurately describe the behaviors of substrates whose degradation is limited by steps other than translocation , such as the DHFR-containing substrates in this study .", "Extending the FEL model to cover such substrates may reveal other important aspects of the proteasome’s activities .", "We expect this FEL approach to have many applications in guiding the experimental design and data analysis , and to provide valuable insights into the mechanism of proteasomal ATPases and other AAA machines ." ], [ "The nucleotide-binding pockets were defined based on the structure of EA1 state .", "Briefly , for each Rpt subunit of EA1 state , the amino acids within 6–9 Å of the bound nucleotide were selected and defined as the nucleotide-binding pocket .", "Structures of each nucleotide-binding pocket in different states of the proteasome were aligned in Pymol and the RMSD of the aligned atoms in the nucleotide pocket was reported .", "To gauge the interface between two ATPase subunits , the SESA was used to report the buried area formed by the interacting residues .", "Using Pymol , the solvent-accessible surface area ( SASA ) for individual ATPase domains in isolation and the SASA value for the complex were calculated .", "Then the SESA between two ATPase domains was calculated using this formula ( using Rpt1 and Rpt2 as an example ) : SESA of Rpt1-Rpt2=[ ( SASA of Rpt1 ) + ( SASA of Rpt2 ) – ( SASA of Rpt1-Rpt2 complex ) ]/2 .", "Recombinant human N-terminal ( 1–88 ) cyclinB1 ( cycB_NT ) , 3-lysine cycB_NT ( K18 , K36 , and K64 ) , cycB_NT-iRFP , cycB_NT-DHFR-iRFP were purified from E . coli cells using a polyHis tag and Ni2+-affinity column .", "PKA ( protein kinase A ) sites ( RRASV ) was placed at both the N- and C-terminus of cycB_NT-iRFP and cycB_NT-DHFR-iRFP for detection by autoradiography in degradation assays .", "Human ubiquitin with a cysteine residue and a polyHis-tag at the N-terminus was purified from E . coli cells using cation exchange chromatography ( GE , 17-1152-01 ) and was labeled with Dylight-550-maleimide ( Pierce , 62290 ) .", "After removing unreacted dyes , labeled ubiquitin was subjected to anion exchange chromatography ( GE , 17-1153-01 ) to separate labeled and unlabeled ubiquitin .", "Finally , the N-terminal polyHis tag was cleaved off using thrombin .", "Anti-20S antibody ( MCP21 ) was biotinylated using biotin-NHS ( Pierce , 20217 ) , and was purified using a desalting column .", "Radioactive ( Lyubimov et al . , 2011 ) p-ATP was used to label substrates with a PKA site at the N-terminus for in vitro ubiquitylation and degradation assays .", "Human E1 , E2 UbcH10 , WT-ubiquitin were purchased from BostonBiochem .", "Purified streptavidin was from Invitrogen .", "Protein concentrations were determined using Bio-Rad protein assay; ubiquitin concentration was determined by UV A280 absorption .", "Purification of recombinant APC-Cdh1 from insect cells has been described elsewhere ( Brown et al . , 2016; Weissmann et al . , 2016 ) .", "Briefly , viruses expressing 14 APC components were generated by transfecting Sf9 insect cells with the recombinant baculoviral genome based on a biGBac system using Fugene 6 reagent .", "Amplified viruses were added to HighFive insect cell culture for protein expression .", "APC was expressed with a Twin-Step ( II ) -tag on the C-terminus of APC4 , and was isolated from cell lysate using Strep-Tactin sepharose , and then was polished by ion-exchange chromatography and gel filtration .", "Myc-6xHis-Cdh1 was purified from HighFive cells using Ni2+ agarose .", "Human proteasomes were affinity-purified on a large scale from a stable HEK293T cell line harboring HTBH tagged hRPN11 ( a gift from L . Huang ) .", "The cells were Dounce-homogenized in a lysis buffer ( 50 mM NaH2PO4 [pH7 . 5] , 100 mM NaCl , 10% glycerol , 5 mM MgCl2 , 0 . 5% NP-40 , 5 mM ATP , and 1 mM DTT ) containing protease inhibitors .", "The lysate was cleared and incubated with NeutrAvidin agarose beads ( Thermo Fisher Scientific ) overnight at 4°C .", "The beads were then washed with excess lysis buffer followed by a wash buffer ( 50 mM Tris-HCl[pH7 . 5] , 1 mM MgCl2 , and 1 mM ATP-Mg2+ ) .", "Usp14 on proteasome was washed off using the wash buffer plus 100 mM NaCl for 30 min . 26S proteasomes were eluted from the beads by cleavage using TEV protease ( Invitrogen ) .", "For purifying SNAP-tagged proteasome , the HTBH-Rpn11 cell line was stably transfected with a lentivirus carrying FLAG-SNAP-Rpn3 under a CMV promoter , and the SNAP-proteasome was purified from the transfected cell line using the above procedure .", "Identity of the cell lines is authenticated using STR profiling and mycoplasma contamination was regularly tested by PCR .", "The APC ubiquitylation reactions were carried out in the UBAB buffer ( 25 mM Tris-HCL[pH 7 . 5] , 50 mM NaCl , and 10 mM MgCl2 ) containing 30 nM APC-Cdh1 , 100 nM E1 , 2 µM UbcH10 , 2 mg/ml BSA , the energy regenerating systems , 1 µM substrate ( cycB_NT , cycB_NT-iRFP , or cycB_NT-DHFR-iRFP ) , and 100 uM WT-ubiquitin or 15 µM Dylight550-ubiquitin , incubated for 4 hr at 25° .", "In case , the substrates were ( Lyubimov et al . , 2011 ) p-labeled , calyculin A ( EMD , 19–139 ) was added at 10 µg/ml to prevent dephosphorylation .", "Ubiquitylated iRFP substrates were diluted in a buffer containing 1× UBAB , 10% glycerol , 1 mM DTT , 0 . 5 ml/ml γ-globulin ( Sigma-Aldrich , G5009 ) , 0 . 05% NP-40 , and nucleotide-Mg2+ at the experimental concentrations , and were aliquoted to a 384-well plate ( Corning 3544 ) .", "Purified human 26S proteasome was added at 1 . 0–3 . 0 nM final concentration to start the reaction .", "Degradation kinetics was monitored using a microplate reader ( BioTek Synergy H1 ) once every 90 s at 35° .", "For each buffer condition , the degradation measurement was performed at five substrate concentrations ( 40 nM , 60 nM , 80 nM , 120 nM , and 200 nM ) , with three replicas for each concentration .", "A standard reaction with 500 µM ATP-Mg2+ was included in every batch of reactions to control for sample batch-to-batch variations .", "The initial degradation rate ( v0 ) was obtained using a linear fitting of the degradation curve in the first 15 min after temperature stabilization .", "The turnover time is the inverse of the degradation rate divided by the proteasome concentration .", "The detailed procedure of single-molecule proteasome assay has been described before ( Lu et al . , 2015; Hon and Lu , 2019 ) .", "Briefly , 15 nM 26S proteasome and 15 nM biotinylated MCP21 antibody were mixed and incubated at room temperature for 15 min .", "The proteasome-antibody mix was loaded onto PEG-passivated slides coated with streptavidin .", "After a brief incubation , unbound protein was washed off , and was exchanged into an imaging buffer ( 1× UBAB , 20 mM Imidazole , 2 mg/ml BSA , and nucleotides ) containing diluted ubiquitylated substrate at ~1 nM .", "Images were acquired at 100 ms per frame on a custom TIRF microscope equipped with three laser lines of 488 nM ( 150 mW ) , 561 nM ( 150 mM ) , 638 mM ( 100 mW ) , and a Pco SCMOS camera .", "The single-molecule experiment was performed at room temperature of ~27° .", "The basic image processing and single-particle identification were performed as described previously ( Lu et al . , 2015; Hon and Lu , 2019 ) .", "The ‘stepped’ traces as in Figure 3—figure supplement 2 which signaled the co-translocation deubiquitylation mediated by Rpn11 were identified and aligned by the time of substrate-proteasome interaction .", "The average intensity of fluorescent ubiquitins among these ‘stepped’ traces was calculated for each time point , and the rate of translocation was calculated using a linear fitting of the averaged trace between 1 and 3 s after proteasome-substrate interaction .", "Data analysis was carried out in Matlab 2018 .", "The coverslip surface was passivated with Tween20 and functionalized with biotinylated BSA as described previously ( Hua et al . , 2014 ) .", "50 nM SNAP-Rpn3 proteasome was incubated with 15 nM biotinylated MCP21 antibody and 1 uM SNAP-surface 549 dye for 30 min at room temperature .", "The proteasome-antibody mix was buffer-exchanged into buffer W ( 1× UBAB , 0 . 05% Tween20 , and 0 . 5 mM DTT ) + 0 . 4 mg/ml BSA using a 30-kD concentrator , and then was buffer-exchanged into buffer W + 1 µM Alexa647-ATP at 4° .", "The sample was diluted by five times in an imaging buffer ( buffer W + competitive nucleotides + 200 nM Alexa647-ATP ( Thermo Fisher Scientific ) + PCA/PCD as the oxygen scavenging system ) and was incubated for 20 min on ice before loading onto the passivated surface via streptavidin .", "Images acquisition started after a 3-min incubation .", "We did not observe obvious 19S–20S dissociation as suggested by the fluorescent SNAP-Rpn3 signal in a 30-min incubation .", "The basic image processing and single-particle identification were performed as described previously ( Lu et al . , 2015; Hon and Lu , 2019 ) .", "We performed colocalization ( <1 pixel ) analysis of the SNAP-tag signal with Alexa647-ATP .", "The fraction of SNAP-tagged proteasome particles that interacted with Alexa647-ATP under a steady-state condition was calculated as the colocalization ratio ( CR ) .", "The inhibitor constant Ki value for each type of nucleotide was obtained by a linear regression using the following formula: ( 1 ) C . R=[ATP∗]KATP∗ ( 1+[NT]K ) +[ATP∗] CR: colocalization ratio; [ATP*]: concentration of Alexa647-ATP; KATP*: dissociation constant of Alexa647-ATP; [NT]: concentration of competing nucleotide .", "Data analysis was performed in Matlab 2018 .", "The sequences of fundamental steps underlying a productive translocation were shown in Figure 5—figure supplement 4 .", "The total time for one translocation step is ( 4 ) ttr=1/khall+<1/rA−>B>+1/ ( kon[ATP] ) +1/koffAPO where khall is the overall rate of ATP hydrolysis of the hexamer; ‘ < 1/rA→B >’ is the average conformational transition time .", "Under limiting ATP concentration , 1/ ( kon[ATP] ) becomes significant .", "Therefore ( 5 ) EC50 ( ATP ) =1kon ( 1/koffAPO+1/khall+<1/rA−>B> ) ∼khallkon The previously proposed sequential model does not contain quantitative details ( Dong et al . , 2019; de la Peña et al . , 2018 ) .", "As a comparison with the FEL model , we adopt a simple form of sequential model without losing generality .", "This sequential model involves six equivalent kinetic segments in a cycle; each contains two processes:", "1 . binding/unbinding of ATP;", "2 . ATP hydrolysis and ADP release .", "If assuming functional symmetry of the six ATPases , the steady-state occupancy of An=An+1…= A and Bn=Bn+1…= B , therefore under the steady-state condition: ( 6 ) 0=A*[ATP]*kon−B*koff−B*kh Considering A+B=C* is a constant due to the symmetry .", "The rate of translocation B*kh=[ATP]*C**kon*khkoff+kh+kon*[ATP] is a monotonic function of the ATP concentration .", "The total turnover time of a cyclinB-DHFR-iRFP molecule under a substrate-saturating condition involves the time for translocation and the time for unfolding .", "( 7 ) ttotal=ttr ( [ATP] ) +tuf ( [ATP] ) Unfolding of the DHFR domain mostly happens when DHFR is transiently unliganded .", "Because if that is the case , the inverse of the degradation rate of DHFR should linearly depend on the concentration of folic acid [FA] at any ATP concentration , according to Equation 6 .", "This is indeed supported by the observation described in Figure 5—figure supplement 1 . ( 8 ) ttotal=1/rdeg=1/rtr ( [ATP] ) +1/ruf ( 1+konFA*[FA]koffFA ) where rdeg is the degradation rate , ruf is the unfolding rate of unliganded DHFR on the ATPase complex .", "konFA , koffFA are the rate constants of folic acid on DHFR .", "Substrate unfolding is likely achieved by the ATPase power strokes ( Iosefson et al . , 2015 ) .", "Although the exact mechanism is still poorly understood , the unfolding rate should be generally in proportion to the ATPase activity in the absence of unfolding intermediates ( Martin et al . , 2008 ) .", "The overall degradation rate is predicted to be ( 9 ) rdeg=rtr ( [ATP] ) 1+rtr ( [ATP] ) Cuf*rh ( [ATP] ) ( 1+konFA*[FA]koffFA ) One extra parameter Cuf was introduced as the unfolding rate coefficient in front of the ATPase activity rh , which should generally depend on the folic acid concentration .", "Translocation rate rtr and ATPase activity rh as functions of ATP concentrations were simulated using the FEL model .", "We adjusted Cuf to match the measured degradation rate of cyclinB-DHFR-iRFP in the presence of folic acid ( Figure 4E ) .", "The qualitative features of the degradation-rate curve in Figure 4F , i , that is , nearly-identical with that of the FA-free curve in the up-phase and weaker effect of high-ATP inhibition , are independent of the choice of Cuf .", "100 nM purified human 26S proteasome was incubated in a buffer ( 1× UBAB , 0 . 5 mg/ml BSA , 0 . 05% NP-40 , and 0 . 5 mM DTT ) with varying concentrations of ATP-Mg2+ for 30 min at 30° .", "No-proteasome controls were incubated under an identical condition .", "20 µM denatured ovalbumin was added as a generic substrate of proteasome ( Cascio et al . , 2001 ) .", "30 nM and 50 nM proteasome was used for 100 µM and 300 µM ATP , respectively .", "After incubation , the concentration of free phosphate was quantified using Malachite green assay on a plate reader ( BioTek Synergy H1 ) .", "An iRFP substrate degradation assay is set up as described ( Materials and methods 'iRFP substrate degradation assay' ) , involving 100 nM polyubiquitylated cyclinB-iRFP reporter substrate and 50 nM polyubiquitylated cyclinB-DHFR-iRFPdark as the competitor , either in the presence or absence of 300 nM methotrexate ( MTX ) .", "cyclinB-DHFR-iRFPdark is nonfluorescent due to the lack of biliverdin in iRFP .", "The residence time TB , that is , the mean time between when translocation reaches L0 and escaping from the proteasome , of cyclinB-DHFR ( MTX ) -iRFP is calculated based on the formula: ( 10 ) TB=rRI0rRI-1RCTR+rRI0rRI×TI0-Lvt" ] ]

[ "The ring-like ATPase complexes in the AAA+ family perform diverse cellular functions that require coordination between the conformational transitions of their individual ATPase subunits ( Erzberger and Berger , 2006; Puchades et al . , 2020 ) .", "How the energy from ATP hydrolysis is captured to perform mechanical work by these coordinated movements is unknown .", "In this study , we developed a novel approach for delineating the nucleotide-dependent free-energy landscape ( FEL ) of the proteasome’s heterohexameric ATPase complex based on complementary structural and kinetic measurements .", "We used the FEL to simulate the dynamics of the proteasome and quantitatively evaluated the predicted structural and kinetic properties .", "The FEL model predictions are consistent with a wide range of experimental observations in this and previous studies and suggested novel mechanistic features of the proteasomal ATPases .", "We find that the cooperative movements of the ATPase subunits result from the design of the ATPase hexamer entailing a unique free-energy minimum for each nucleotide-binding status .", "ATP hydrolysis dictates the direction of substrate translocation by triggering an energy-dissipating conformational transition of the ATPase complex ." ]

[ "In cells , many biological processes are carried out by large complexes made up of different proteins .", "These macromolecules act like miniature machines , flexing and moving their various parts to perform their cellular roles .", "One such complex is the 26S proteasome , which is responsible for recycling other proteins in the cell .", "The proteasome consists of approximately 31 subunits , including a ring of six ATPase enzymes that provide the complex with the energy it needs to mechanically unfold proteins .", "To understand how the proteasome and other large complexes work , researchers need to be able to monitor how their structure changes over time .", "These dynamics are challenging to probe directly with experiments , but can be assessed using computer simulations which track the movement of individual molecules and atoms .", "However , currently available computer systems do not have enough power to simulate the dynamics of large protein assemblies , like the 26S proteasome: for example , it would take longer than a thousand years to model how each atom in the complex moves over a timescale in which a biological change would happen ( roughly 100ms ) .", "Here , Fang , Hon et al . have developed a new approach to simulate the structural dynamics of the proteasome’s ring of ATPase enzymes .", "Different known structures of the proteasome were used to identify the range of possible movements and shapes the complex can make .", "Fang , Hon et al . then used this data to calculate the energy level of each structure – also known as the ‘free energy landscape’ – and the rate of transition between them .", "This made it possible to simulate how the different ATPase enzymes move within the ring under a wide range of conditions .", "The simulated ATPase movements predicted how the proteasome machine would behave during various tasks , including degrading other proteins .", "Fan , Hon et al . carefully examined these predictions and found that they were consistent with experimental observations , validating their new simulation method .", "This work demonstrates the feasibility of simulating the actions of a large protein complex based on its free energy landscape .", "The results offer important insights into the functional mechanics of the 26S proteasome and related protein machines .", "Further work may help to simplify this process so the approach can be used to investigate the dynamics of other protein assemblies ." ]