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provided by Elsevier - Publisher Connector Current Biology 21, 97–105, January 25, 2011 ª2011 Elsevier Ltd All rights reserved DOI 10.1016/j.cub.2010.12.014 Article Complexin Has Opposite Effects on Two Modes of Synaptic

Jesse A. Martin,1,3 Zhitao Hu,2,3 Katherine M. Fenz,1 explored. Synaptic transmission is initiated by the fusion of Joel Fernandez,1 and Jeremy S. Dittman1,* a with the presynaptic membrane, a highly 1Department of Biochemistry, Weill Cornell Medical College, regulated process involving a host of specialized presynaptic 1300 York Avenue, New York, NY 10065, USA proteins [8–10]. SV fusion is catalyzed by the assembly of 2Department of Molecular Biology, Simches 7, Massachusetts a four-helix bundle containing three major SNARE proteins: General Hospital, 185 Cambridge Street, Boston, MA 02114, , SNAP-25, and syntaxin. The detailed molecular USA chain of events progressing from SNARE assembly to SV fusion is not fully understood, but several SNARE-binding proteins have been proposed to regulate this sequence [10, 11]. Summary In particular, the protein complexin has garnered much interest because of its ability to bind to the assembled ternary SNARE Background: Synaptic transmission can occur in a binary or complex with high affinity. Complexin is a 15–18 kDa cyto- graded fashion, depending on whether transmitter release is plasmic protein that contains a central a-helical region that triggered by action potentials or by gradual changes in binds within the groove between synaptobrevin and syntaxin membrane potential. Molecular differences of these two types in the assembled SNARE complex [12–16]. How the binding of fusion events and their differential regulation in a physiolog- of complexin to the SNARE complex regulates the probability ical context have yet to be addressed. Complexin is a of SV fusion is controversial. conserved SNARE-binding protein that has been proposed In vitro studies using SNARE-mediated fusion of proteolipo- to regulate both spontaneous and stimulus-evoked synaptic somes or cell-cell fusion have suggested that complexin either vesicle (SV) fusion. inhibits [17–19] or stimulates [20, 21] fusion. In several synaptic Results: Here we examine complexin function at a graded preparations, complexin function was explored in the context in C. elegans. Null complexin (cpx-1) mutants are of two types of SV events: action potential-trig- viable, although nervous system function is significantly gered fusion initiated by a depolarizing stimulus (evoked impaired. Loss of CPX-1 results in a 3-fold increase in the release) and stochastic fusion events monitored in the absence rate of tonic synaptic transmission at the neuromuscular junc- of activity (spontaneous release). Neuronal cultures and brain tion, whereas stimulus-evoked SV fusion is decreased 10-fold. slices derived from complexin knockout mice exhibited A truncated CPX-1 missing its C-terminal domain can rescue a reduction in evoked release, as well as stimulus-evoked synaptic vesicle exocytosis but fails to decreased spontaneous fusion events [22–25]. In contrast, suppress tonic activity, demonstrating that these two modes complexin knockdown by RNA interference led to elevated of exocytosis can be distinguished at the molecular level. A spontaneous release in conjunction with decreased evoked CPX-1 variant with impaired SNARE binding also rescues release [26]. Genetic deletion of complexin in Drosophila mela- evoked, but not tonic, neurotransmitter release. Finally, tonic, nogaster also increased tonic fusion at the neuromuscular but not evoked, release can be rescued in a syntaxin point junction (NMJ) while simultaneously reducing evoked release mutant by removing CPX-1. Rescue of either form of exocy- [27, 28]. Cross-species rescue experiments and chimeric tosis partially restores locomotory behavior, indicating that variants of complexin demonstrated that mouse and fly com- both types of synaptic transmission are relevant. plexins share domains that can both enhance and inhibit Conclusion: These observations suggest a dual role for exocytosis, indicating that complexin is a multifunctional CPX-1: suppressing SV exocytosis, driven by low levels of protein and possibly has multiple duties at the synapse [25]. endogenous neural activity, while promoting synchronous In this study, we investigated the consequences of removing fusion of SVs driven by a depolarizing stimulus. Thus, patterns the complexin ortholog CPX-1 on neurotransmission in of synaptic activity regulate complexin’s inhibitory and permis- C. elegans. We provide evidence that CPX-1 inhibits the sive roles at a graded synapse. continuous release of neurotransmitter driven by low levels of endogenous neural activity (tonic release). In contrast, Introduction CPX-1 is required for SV fusion evoked by a depolarizing stim- ulus electrode. Both of these modes of synaptic transmission Neurotransmitter release is predominantly driven by action are shown to be behaviorally relevant. Each mode of exocy- potentials in the vertebrate nervous system, whereas graded tosis could be independently rescued, suggesting that com- neurotransmission is typically observed at sensory plexin operates through distinct mechanisms during synaptic and at dendrodendritic connections [1–4]. Invertebrate transmission. Thus, CPX-1 can function to regulate synaptic nervous systems display both impulse-driven and graded transmission in an activity-dependent manner. A detailed synaptic transmission, sometimes at the same synapse [5–7]. understanding of complexin’s role may help delineate the It is widely thought that the underlying mechanism of synaptic molecular mechanisms underlying SV exocytosis. vesicle (SV) exocytosis is identical in these two forms of signaling, although the molecular differences have yet to be Results

Mutants Lacking CPX-1 Have Motor System Defects *Correspondence: [email protected] The C. elegans genome contains two complexin homologs: 3These authors contributed equally to this work cpx-1 and cpx-2. Of these two genes, cpx-1 shares a higher Current Biology Vol 21 No 2 98

Figure 1. cpx-1 Mutants Display Motor System Defects (A) Protein sequence alignment for C. elegans CPX-1 (worm), D. melanogaster dmCplx Gen- Bank AAF69518.1 (fly), and mouse Cplx1 (mouse) using a ClustalW algorithm. The N-terminal domain (NTD), accessory domain (AD), central helix (CH), and C-terminal domain (CTD) are indi- cated in gray. Identically conserved residues across all three species are shaded in red, whereas similar residues are shaded in blue. (B) Percent sequence identity for each domain is indicated for each pairwise comparison: worm (w), fly (f), and mouse (m). (C) Representative trajectories for 20 animals for wild-type, cpx-1(ok1552), cpx-1 expressing a rescuing cDNA under a GABA promoter, and cpx-1 expressing a rescuing cDNA in all neurons. (D) Summary of average speed for the four strains. The number of animals measured for each average is indicated within the bar. (E) Paralysis time course on 1.0 mM aldicarb for wild-type (black circles), cpx-1 (red triangles), rescue in GABA neurons (open green squares), and rescue in all neurons (open blue circles). Data are mean 6 standard error of the mean (SEM). # denotes significantly different from wild-type (p < 0.01) but not significantly different from cpx-1. ** denotes significantly different from cpx-1 (p < 0.01) but not significantly different from wild-type. Significance determined by Tukey- Kramer method.

defect (Figure 1D). We cloned the full- length cDNA encoding CPX-1 and expressed a CPX-1::GFP fusion protein under a neuronal promoter (snb-1 syn- aptobrevin) in cpx-1 mutants. CPX- 1::GFP restored locomotion (104% 6 5.7% of wild-type), confirming that the locomotion defect was specific to loss of complexin in the nervous system (Figure 1D). degree of homology with the mouse (32% versus 19% identity) We also examined the sensitivity of cpx-1 mutants to aldi- and fly (44% versus 28% identity) complexins (Figure 1A). The carb, a cholinesterase inhibitor. Acetylcholine (ACh) levels in protein structure and function of mouse complexin suggests the NMJ are rapidly reduced through the enzymatic action that it possesses four domains, with the central helix (CH) of acetylcholinesterases, and inhibition of these enzymes domain displaying the highest degree of conservation. The results in a buildup of ACh, followed by paralysis of the animal worm complexin protein CPX-1 contains a CH domain [29]. Animals with defects in ACh secretion are resistant to with 78% identity to the mouse Cplx1 and 91% identity to aldicarb, whereas animals secreting higher than normal levels the Drosophila complexin dmCplx (Figure 1B). In order to of ACh are hypersensitive and quickly paralyze upon expo- study the role of complexin in the worm nervous system, we sure to aldicarb [30–32]. We examined the time course of utilized the mutant cpx-1(ok1552), which carries a 1.3 kb dele- paralysis on 1.0 mM aldicarb for wild-type and cpx-1 mutant tion of the cpx-1 gene, removing one of its two exons. cpx-1 animals (Figure 1E). cpx-1 mutants were paralyzed within (ok1552) animals are viable but have significant defects in 20 min of aldicarb exposure, whereas wild-type animals locomotion, whereas heterozygous animals are indistinguish- required 2 hr to paralyze. Neuronal expression of CPX- able from wild-type. We also examined cpx-2 mutants, and, 1::GFP completely restored normal aldicarb sensitivity, sug- based on expression data and behavioral studies, we found gesting that CPX-1 normally inhibits the secretion of ACh. that CPX-2 does not function redundantly with CPX-1 (see Fig- Alternatively, if CPX-1 specifically acts in GABA motor ure S1 available online). We thus focused on CPX-1 as the neurons to promote release, decreased GABA release in the major complexin in C. elegans. cpx-1 mutant could also account for its hypersensitivity to We quantified the decrease in locomotion of cpx-1 mutants aldicarb [33]. To test this hypothesis, we attempted to rescue by recording the center-of-mass speed of individual animals cpx-1 mutants with CPX-1::GFP under a GABA-specific moving on an agar plate (Figure 1C). Compared to wild-type promoter (unc-25 GAD). Neither locomotion nor aldicarb controls, cpx-1 mutants moved at significantly slower speeds sensitivity were altered (Figures 1D and 1E), suggesting that (40% 6 2.2% of wild-type), consistent with a nervous system defects in GABA neuron function in cpx-1 mutants cannot Complexin and Synaptic Vesicle Fusion 99

Figure 2. CPX-1 Is Expressed throughout the Nervous System and Localizes to Synapses (A1) Fluorescent image of the head region expressing GFP under the cpx-1 promoter. (A2) Expression of GFP under the cpx-1 promoter in the body region. Note the cell bodies in the ventral nerve cord at the top with commissural fibers extending to the dorsal nerve cord. (B1) Expression of mCherry under a cholinergic promoter (unc-17 VAChT) and a nuclear-localized GFP under the cpx-1 promoter in the ventral nerve cord. (B2) Expanded view of the inset in (B1) showing indi- vidual cholinergic (white arrows) and GABAergic (blue arrowheads) motor neurons expressing cpx-1. (C1 and C2) Fluorescent images of the dorsal nerve cord expressing a CPX-1::GFP fusion protein (C1) and mCher- ry::Rab3 (C2). (C3) The two fluorescent images merged and color coded. Scale bars are 25 mm in (A1) and (A2), 30 mm in (B1), 10 mm in (B2), and 5 mm in (C1)–(C3).

rate (Figures 3A and 3C), with no change in the tEPSC amplitude (Figure 3D). Even in low calcium, spontaneous fusion was significantly increased in cpx-1 mutants (Figure S3). account for the observed behavioral phenotypes. Consistent Surprisingly, EPSCs evoked by a depolarizing stimulus elec- with a role for CPX-1 in ACh release, we found that rescue trode (eEPSCs) were decreased by about 90% in cpx-1 of CPX-1::GFP under a cholinergic promoter (unc-17 VAChT) mutants (Figures 3B and 3E), suggesting a requirement for nearly completely rescued wild-type aldicarb sensitivity CPX-1 during SV fusion evoked by high levels of activity. Total (Figure S2A). synaptic charge was decreased to a similar degree, indicating that the eEPSC peak was not decreased simply because of CPX-1 Is a Synaptic Protein Broadly Expressed asynchrony of the evoked fusion events in the cpx-1 mutant in the Nervous System (data not shown). Consistent with the behavioral assays, To examine the expression of cpx-1 in C. elegans, we drove CPX-1 expression in GABA neurons failed to rescue either expression of GFP under the control of the cpx-1 promoter the tEPSC rate or eEPSC, whereas expression in all neurons (3 kb upstream region). Expression was observed in a large rescued both modes of exocytosis. Thus, SV exocytosis is number of neurons in the head, ventral cord, and tail, including suppressed by CPX-1 during low levels of activity, whereas all cholinergic and GABAergic motor neurons in the ventral high levels of activity require CPX-1 to promote synchronous cord (Figures 2A and 2B). In contrast, cpx-2 is expressed in exocytosis. only a small number of neurons having little or no overlap with cpx-1 expression (Figure S1B). To examine the subcel- Synaptic Alterations of cpx-1 Mutants Are Not the Result lular localization of CPX-1, we imaged CPX-1::GFP in the of Changes in Nervous System Development dorsal nerve cord under the control of a modified unc-129 Complexin has been proposed to play a role in synapse promoter, which expresses in a subset of DA and DB cholin- development in mouse and fly [27, 35], so it is important to ergic motor neurons [34]. CPX-1::GFP was diffusely expressed examine whether the nervous system has been altered in throughout the axon with some enrichment at presynaptic cpx-1 mutants. In particular, developmental changes in the terminals based on colocalization with an SV marker mCherry:: motor neurons and NMJs may explain the behavioral and elec- Rab-3 (Figure 2C). trophysiological effects described above. The number of cholinergic motor neurons in the ventral cord was identical Tonic Activity Is Elevated, and Evoked Release between wild-type and cpx-1 mutant animals (Figure 4A). Is Decreased, in the Absence of Complexin Furthermore, we counted the number of active zones per The behavioral assays and expression pattern indicate that unit length of axon by imaging the fluorescently tagged active CPX-1 plays a major role in the nervous system. To examine zone (AZ) protein ELKS-1::CFP in wild-type and cpx-1 mutant the effects of removing CPX-1 on synaptic transmission, we animals and found nearly identical AZ densities in the two recorded synaptic activity at the NMJ in dissected animals. strains (Figure 4B). Thus, neither the number of motor neurons The worm NMJ is a graded synapse, and basal activity in the nor the synapse number is altered appreciably in the absence nervous system of the dissected animal continuously drives of cpx-1. neurotransmitter release, as measured by whole-cell patch- clamp recordings from body wall muscle [7]. This tonic excit- Overexpression of CPX-1 Does Not Affect the NMJ atory postsynaptic current (tEPSC) rate depended on external The experiments described above establish that loss of CPX-1 calcium, because over 90% of these fusion events were elim- at the synapse directly leads to dysregulation of tonic and inated in zero calcium external solution (Figure S3). cpx-1 stimulus-evoked neurotransmission. If complexin functions mutants display a large increase (290% of wild-type) in tEPSC as a fusion clamp by binding the trans-SNARE complex, then Current Biology Vol 21 No 2 100

Figure 3. Tonic Release Increases while Evoked Release Decreases at the NMJ in the Absence of Complexin (A and B) Examples of tonic (A) and evoked (B) EPSCs for wild-type, cpx-1(ok1552), rescue in GABA neurons, and rescue in all neurons. (C–E) Average tonic EPSC rate (C), tEPSC ampli- tude (D), and evoked EPSC peak (E) for each of the four strains shown above. See Experimental Procedures for details of recordings. The tEPSC rate increased in cpx-1 mutants and remained elevated in the GABA rescue transgenics. Rescue in all neurons decreased the tonic release rate slightly below wild-type. The evoked EPSC decreased in cpx-1 mutants, and rescue in GABA neurons failed to restore the EPSC, whereas rescue in all neurons restored the EPSC. Data are mean 6 SEM. # denotes significantly different from wild-type (p < 0.01) but not significantly different from cpx-1. ** denotes significantly different from cpx-1 (p < 0.01) but not significantly different from wild-type. Significance determined by Tukey-Kramer method.

it is possible that overexpressing CPX-1 will inhibit synaptic indistinguishable over a 5-fold range of protein expression, transmission and alter behavior by driving the formation as measured by axonal fluorescence levels (Figures S5A and of excess CPX-1/trans-SNARE complexes [22, 25, 36]. We S5D). There was also no effect on aldicarb sensitivity when examined the aldicarb sensitivity of cpx-1 mutant animals CPX-1 was overexpressed in subsets of motor neurons or in expressing different amounts of a fluorescently tagged synaptic mutants with elevated rates of transmitter release rescuing complexin (CPX-1::pG). We found that rescue was (Figures S5B and S5C). Finally, we observed no change in locomotory behavior in these overexpression strains (data not shown). Thus, CPX-1 does not appear to be a limiting factor in the regulation of synaptic transmission.

Effects of CPX-1 Domain Deletions on Behavior Having established that overexpressing CPX-1 does not alter aldicarb sensitivity or locomotion, we analyzed the ability of four domain-deletion mutants and a double point mutant to rescue the aldicarb hypersensitivity and locomotion defects of cpx-1 mutants. The rescue constructs were fused to GFP in order to assess relative protein expression levels in the axon. For the strains presented here, all mutant CPX-1 variants were expressed at the synapse with a comparable abundance to the full-length rescue construct (Figure S5D). In agreement with mammalian complexin studies, the central helix was required for CPX-1 function in that its deletion (DCH) completely eliminated rescue of both aldicarb sensitivity and locomotion (Figure 5). A double point mutation near the end of the central helix (K71A, Y72A) significantly decreases SNARE binding in this highly conserved region of complexin in both mouse and fly [25, 36]. As expected from the DCH Figure 4. No Gross Changes in Motor Neuron Number or Synapse Density Were Observed in cpx-1 Mutants rescue, the KY/AA mutation eliminates rescue of aldicarb (A1–A3) The ventral nerve cord was imaged in animals expressing mCherry sensitivity (Figure 5B). However, we observed a significant under a cholinergic promoter in wild-type (A1) and cpx-1(ok1552) mutant recovery of locomotory function in the KY/AA mutant, sug- (A2) backgrounds. There was no significant difference in the number of gesting that some aspect of CPX-1 function persists in this ventral cord motor neurons in both genotypes, as summarized in (A3). SNARE-binding mutant (Figure 5C). (B1–B3) The dorsal nerve cord was imaged in animals expressing the active Interestingly, loss of the N-terminal domain (DNTD) had no zone protein ELKS-1::CFP in a subset of DA/DB motor neurons in wild-type effect on rescue, whereas loss of the C-terminal domain (B1) and cpx-1 mutant (B2) backgrounds. Loss of CPX-1 did not significantly D alter the NMJ density, as summarized in (B3). ( CTD) impaired rescue in both behavioral assays (Figure 5). Data are mean 6 SEM. Number of animals analyzed is indicated within each Deletion of the accessory domain (DAD) impaired rescue of bar. Scale bars are 5 mm. aldicarb sensitivity to some extent, whereas locomotion was Complexin and Synaptic Vesicle Fusion 101

Effects of CPX-1 Domain Deletions on Synaptic Transmission We directly tested the contributions of the CH and CTD domains to the regulation of synaptic transmission by moni- toring SV fusion at the NMJ in these domain-deletion rescue animals. Rescue of cpx-1 with the SNARE-binding mutant KY/AA failed to rescue the tonic rate of fusion (Figures 6A and 6C). Likewise, a truncated CPX-1 variant lacking its CTD also failed to inhibit tonic neurotransmission, suggesting that both the SNARE-binding region and the C terminus are impor- tant for complexin’s inhibitory function (Figures 6A and 6C). In contrast, the eEPSC was partially restored in both DCTD and KY/AA rescue animals, indicating that these regions contribute, but are not essential, to CPX-1 function during stimulus-evoked SV fusion (Figures 6B and 6E). In both cases, restoration of evoked neurotransmitter release correlated with rescue of locomotion, demonstrating the behavioral relevance of evoked release. In all cases, no effect on the tEPSC ampli- tude was observed (Figure 6D). Thus, the eEPSC can be rescued independently of the tEPSC, further supporting the notion that complexin separately regulates these two modes of exocytosis.

Loss of Complexin Rescues a Syntaxin Mutant The elevated rate of tonic neurotransmission observed in cpx-1 mutants is distinct among known synaptic mutants in C. elegans. Two hypersecretion mutants have previously been characterized at the worm neuromuscular junction: slo-1 BK channel and tom-1 Tomosyn. Interestingly, evoked SV fusion is significantly increased in both of these mutants, whereas tonic SV fusion is relatively unaltered [37–39], further supporting the notion that these two modes of exocytosis can be distinguished at the molecular level. Because some SNARE mutations that disrupt assembly of the four-helix bundle are thought to slow the forward rate of fusion [26, 40], we wondered whether removing complexin’s inhibitory function would counteract a SNARE mutation and restore fusion. To test this hypothesis, we utilized a mutant syntaxin 1 in which a membrane-proximal conserved SNARE residue (alanine 248) is substituted with valine: unc-64 Syx(A248V) [41]. This mutation is not in the region of syntaxin 1, which interacts with complexin so that initial trans-SNARE assembly and com- Figure 5. Effects of Removing Complexin Protein Domains on Behavior plexin binding would not be predicted to be altered. However, (A) Cartoon depicting the four protein domains of CPX-1: N-terminal domain the unc-64 Syx(A248V) nearly eliminates both tonic and stim- (NTD), accessory domain (AD), central helix (CH), and C-terminal domain ulus-evoked SV fusion, suggesting that a late stage in SNARE (CTD). assembly is impaired [39]. (B) Average percentage of animals paralyzed on 1.0 mM aldicarb after Intriguingly, whereas both unc-64 Syx(A248V) and cpx-1 40 min for wild-type (WT), cpx-1, full-length rescue CPX-1 (FL), rescue moved at much lower speeds than wild-type animals with each of the domains individually deleted as indicated, and a double (17% 6 1.9% and 37% 6 2.1% of wild-type, respectively), point mutant K71A Y72A (KY/AA), all in cpx-1(ok1552). Number of aldicarb assays for each strain is indicated either within or above the bar. the cpx-1;unc-64 Syx(A248V) double mutant displayed near- (C) Average speed for the same strains. Number of animals analyzed for wild-type locomotion (78% 6 6.9%; Figures 7A and 7B). each strain is indicated within or above the bar. Thus, loss of CPX-1 appears to restore motor system function Data are mean 6 SEM. # denotes significantly different from wild-type in the syntaxin mutant. In addition to slower locomotion, (p < 0.01) but not significantly different from cpx-1. ** denotes significantly unc-64 Syx(A248V) mutants sometimes fail to initiate move- different from cpx-1 (p < 0.01) but not significantly different from wild- ment and remain quiescent throughout the locomotion assay type. * denotes significantly different from both cpx-1 and wild-type (p < 6 6 0.01). Significance determined by Tukey-Kramer method. Details of the (17.1% 4% quiescent versus 2.7% 1.6% for wild-type; domain deletions are described in the Experimental Procedures. see Figure S6A). The cpx-1;unc-64 Syx(A248V) double mutant failed to reduce the elevated quiescence, demonstrating that some locomotor phenotypes are not restored in the absence almost fully restored (Figure 5). Note that we refer to this region of complexin. of complexin as the accessory domain rather than the helix Although it is possible that mutants with enhanced synaptic because the C. elegans ortholog contains a proline residue transmission may generally rescue the syntaxin mutant, loss of within this region (see Figure 1); thus, a purely a-helical confor- complexin had a significantly larger impact on the unc-64 Syx mation would not be expected in this region of CPX-1. (A248V) mutant than the hypersecretion mutants slo-1 BK Current Biology Vol 21 No 2 102

Figure 6. Central Helix-SNARE Interactions and the C-Terminal Domain of CPX-1 Are Required for Its Inhibition of Tonic, but Not for Evoked SV Fusion (A and B) Representative traces for tonic (A) and evoked (B) EPSCs are shown for wild-type, cpx-1(ok1552), rescue with a central helix dele- tion (DCH), rescue with a C-terminal domain dele- tion (DCTD), and rescue with a double point mutant in the central helix predicted to decrease SNARE binding (KY/AA), all in cpx-1(ok1552). (C–E) Average values for the frequency (C) and amplitude (D) of tEPSCs and peak eEPSC amplitude (E) are shown for each strain. Data are mean 6 SEM. # denotes significantly different from wild-type (p < 0.01) but not significantly different from cpx-1. * denotes significantly different from both cpx-1 and wild-type (p < 0.01). Significance determined by Tukey-Kramer method.

Discussion

The results of this study lead us to several conclusions about the role of complexin in C. elegans. First, animals develop normally in the absence of CPX-1. Second, CPX-1 has both negative and positive functions in channel and tom-1 Tomosyn. Although evoked SV exocytosis neurotransmission: it inhibits fusion of SVs driven by endog- is increased in each mutant [37–39], neither could restore loco- enous activity while simultaneously promoting synchronous motion to the same extent as loss of CPX-1 (Figure 7C). Finally, neurotransmitter release evoked by a strong depolarizing rescue of locomotion in cpx-1 might arise simply from an stimulus. Third, the positive and negative functions can be impairment of SV fusion that protects a pool of vesicles from separated within the structure of complexin. The N-terminal depletion. We examined three mutants that are defective domain does not appear to play a major role in CPX-1 in distinct aspects of SV exocytosis (snb-1 synaptobrevin, function, whereas the SNARE-interacting central helix is unc-2 Ca2+ voltage-dependent channel, and snt-1 synaptotag- essential for both regulatory functions of CPX-1. The C-ter- min) and found that locomotion was not significantly rescued minal domain and tight SNARE binding are required only (Figure S6B). for complexin’s inhibitory role. Fourth, complexin’s function We next examined the effects of removing CPX-1 in the in the final stages of fusion is underscored by its genetic A248V syntaxin mutant on SV fusion. Whereas unc-64 Syx interactions with syntaxin. Specifically, a syntaxin ‘‘zipper- (A248V) almost eliminated tEPSC fusion events, the cpx-1; ing’’ mutant can be rescued by removing complexin. These unc-64 Syx(A248V) double mutant displayed wild-type rates data suggest that complexin bound to the trans-SNARE (Figures 7D and 7F). In contrast, the eEPSC amplitude was complex raises the energy barrier for SV fusion. The not restored in the cpx-1;unc-64 Syx(A248V) double mutant, opposing effects of complexin removal on tonic and evoked highlighting the distinct role of CPX-1 in supporting this release suggest that these are distinct exocytic events that mode of exocytosis (Figures 7E and 7H). The average tEPSC can be separated at the molecular level (see model in amplitude was decreased by about 43% in the unc-64 Syx Figure S7A). (A248) mutant and restored in the cpx-1;unc-64 Syx(A248V) double mutant (Figure 7G). Although smaller tonic fusion Separating Functions of CPX-1 Domains events might cause an apparent decrease in tEPSC frequency N-Terminal Domain if a significant number falls below detection threshold, a 43% There is evidence from murine culture studies that the NTD decrease in tEPSC amplitude is expected to cause only facilitates evoked SV fusion and that a few residues within a 0.2% decrease in tEPSC frequency under our recording the NTD are identical across several phyla (68% identity conditions (Figures S6C and S6D). Thus, the decrease in between worm and fly), suggesting a conserved function of tEPSC rate (and its restoration in the double mutant) repre- this domain [24, 25]. The fly complexin NTD, however, does sents a change in the frequency of fusion rather than a change not appear to affect synaptic transmission when expressed in tEPSC amplitude. These observations suggest that in mouse neurons [25]. Similar to dmCplx, we observed removing CPX-1 in the unc-64 Syx(A248V) mutant can alle- complete rescue of function with a version of CPX-1 missing viate the defect in tonic fusion, whereas evoked EPSCs are the entire NTD. Thus, this domain does not play a major role not restored. Furthermore, restoration of tonic neurotransmis- in complexin function in C. elegans. sion was accompanied by an increase in locomotion, demon- Accessory Domain strating that this mode of exocytosis is relevant to locomotory Prior studies on the AD have suggested an inhibitory role in behavior. fusion [22, 25, 42, 43]. The nematode CPX-1 AD contains Complexin and Synaptic Vesicle Fusion 103

Figure 7. Loss of CPX-1 Restores Tonic Fusion in a Syntaxin 1 Mutant (A) Representative locomotion trajectories for a hypomorphic allele of syntaxin 1 unc-64(e246) corresponding to A248V, as well as cpx-1 and the cpx-1 syntaxin(A248V) double mutant (cpx-1; unc-64). (B) Summary of average speed for wild-type (WT) animals, in addition to the three strains described above. (C) Summary of average speed for syntaxin 1(A238V) unc-64 mutants and three double mutants in which unc-64 is paired with cpx-1 (cpx-1;unc-64), slo-1 BK channel (slo-1;unc-64), and tom-1 Tomosyn (tom-1;unc-64). Number of animals analyzed for each strain is indicated within the bar. ## denotes significantly different from unc-64 (p < 0.01). (D–H) Representative traces for tonic (D) and evoked (E) EPSCs are shown for all four strains. Average values for the frequency (F) and amplitude (G) of tEPSCs and peak eEPSC amplitude (H) are shown for each strain. Data are mean 6 SEM. # denotes significantly different from wild-type (p < 0.01) but not signifi- cantly different from cpx-1. ** denotes signifi- cantly different from cpx-1 (p < 0.01) but not significantly different from wild-type. * denotes significantly different from both cpx-1 and wild- type (p < 0.01). Significance determined by Tukey-Kramer method.

CTD was able to rescue synaptic trans- mission in the Cplx1/2 double knockout [22]. In contrast, the fly dmCplx CTD is required for suppression of sponta- neous and evoked SV fusion at the fly NMJ [28] and in mouse autaptic cultures [25]. We find that without its CTD, CPX-1 a proline and therefore will probably not adopt a purely helical can no longer suppress tonic release, establishing a require- structure. Regardless, aldicarb sensitivity cannot be fully ment for the CTD in the fusion clamp activity of complexin. In rescued in the absence of this domain, so this region contrib- contrast to tonic release, the CTD deletion mutant retains utes to the suppression of ACh release, in agreement with fly 50% normal evoked responses, demonstrating that this and mouse complexin. domain is not essential for the facilitation of evoked SV fusion Central Helix by complexin. The physical interaction of complexin with the SNARE complex is mediated by residues of the CH and residues lining A Distinct Role of Complexin during Stimulus-Evoked the groove between syntaxin and synaptobrevin. The CH- Release deleted CPX-1 failed to rescue in all of our behavioral and elec- Decreased evoked release has been observed in mouse trophysiological assays, suggesting an essential requirement [14, 22, 26], fly [27], and worm (this study) synapses lacking for complexin function in the worm, a conclusion that holds complexin. This deficit could have simply reflected the deple- for all studies of complexin function. SNARE binding is tion of a readily releasable pool of SVs in the wake of an reduced by mutating two key residues (K71A Y72A) [22, 36]. elevated spontaneous fusion rate. Indeed, Hobson et al. (this This mutation eliminated the facilitatory and inhibitory func- issue of Current Biology) report a substantial decrease in SV tions of mouse Cplx1, as well as the inhibitory function of fly number in cpx-1 mutant worms, suggesting that vesicle pool dmCplx in mouse cultured neurons [22, 25, 26]. In contrast, depletion contributes to this defect in exocytosis [45]. we found that the defective CPX-1(KY/AA) can still support However, our data reveal a separate role for complexin in sup- evoked fusion to a significant degree at the C. elegans NMJ, porting evoked fusion. We show here that evoked release can suggesting that tight SNARE binding is not essential for be restored to a significant degree even when tonic release promoting neurotransmission. remains elevated (KY/AA and CTD deletion rescue). The C-Terminal Domain central helix is required for rescue of evoked release, indi- Little is known about the structure or function of the C-terminal cating that the central helix has additional functions during domain of complexin. An in vitro study suggests that this evoked fusion. Perhaps complexin interacts with another region of complexin contributes to its function by binding to target at the synapse to promote fusion when calcium levels membranes and stimulating liposome fusion [44]. However, are elevated. Further studies will be needed to address this in mouse autaptic cultures, a truncated Cplx1 missing its entire possibility. Current Biology Vol 21 No 2 104

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