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Thomas C. Südhof - One of the best experts on this subject based on the ideXlab platform.
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the primed snare Complexin synaptotagmin complex for neuronal exocytosis
Nature, 2017Co-Authors: Qiangjun Zhou, Thomas C. Südhof, Minglei Zhao, Axel T. Brunger, Peng Zhou, Austin L. WangAbstract:Synaptotagmin, Complexin, and neuronal SNARE (soluble N-ethylmaleimide sensitive factor attachment protein receptor) proteins mediate evoked synchronous neurotransmitter release, but the molecular mechanisms mediating the cooperation between these molecules remain unclear. Here we determine crystal structures of the primed pre-fusion SNARE-Complexin-synaptotagmin-1 complex. These structures reveal an unexpected tripartite interface between synaptotagmin-1 and both the SNARE complex and Complexin. Simultaneously, a second synaptotagmin-1 molecule interacts with the other side of the SNARE complex via the previously identified primary interface. Mutations that disrupt either interface in solution also severely impair evoked synchronous release in neurons, suggesting that both interfaces are essential for the primed pre-fusion state. Ca2+ binding to the synaptotagmin-1 molecules unlocks the complex, allows full zippering of the SNARE complex, and triggers membrane fusion. The tripartite SNARE-Complexin-synaptotagmin-1 complex at a synaptic vesicle docking site has to be unlocked for triggered fusion to start, explaining the cooperation between Complexin and synaptotagmin-1 in synchronizing evoked release on the sub-millisecond timescale.
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The primed SNARE–Complexin–synaptotagmin complex for neuronal exocytosis
Nature, 2017Co-Authors: Qiangjun Zhou, Thomas C. Südhof, Minglei Zhao, Peng Zhou, Austin L. Wang, Axel T. BrungerAbstract:Synaptotagmin, Complexin, and neuronal SNARE (soluble N-ethylmaleimide sensitive factor attachment protein receptor) proteins mediate evoked synchronous neurotransmitter release, but the molecular mechanisms mediating the cooperation between these molecules remain unclear. Here we determine crystal structures of the primed pre-fusion SNARE-Complexin-synaptotagmin-1 complex. These structures reveal an unexpected tripartite interface between synaptotagmin-1 and both the SNARE complex and Complexin. Simultaneously, a second synaptotagmin-1 molecule interacts with the other side of the SNARE complex via the previously identified primary interface. Mutations that disrupt either interface in solution also severely impair evoked synchronous release in neurons, suggesting that both interfaces are essential for the primed pre-fusion state. Ca2+ binding to the synaptotagmin-1 molecules unlocks the complex, allows full zippering of the SNARE complex, and triggers membrane fusion. The tripartite SNARE-Complexin-synaptotagmin-1 complex at a synaptic vesicle docking site has to be unlocked for triggered fusion to start, explaining the cooperation between Complexin and synaptotagmin-1 in synchronizing evoked release on the sub-millisecond timescale.
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Evolutionary conservation of Complexins: from choanoflagellates to mice
EMBO reports, 2015Co-Authors: Xiaofei Yang, Yea Jin Kaeser-woo, Jimin Pei, Taulant Bacaj, Nick V. Grishin, Thomas C. SüdhofAbstract:Complexins are synaptic SNARE complex-binding proteins that cooperate with synaptotagmins in activating Ca(2+)-stimulated, synaptotagmin-dependent synaptic vesicle exocytosis and in clamping spontaneous, synaptotagmin-independent synaptic vesicle exocytosis. Here, we show that Complexin sequences are conserved in some non-metazoan unicellular organisms and in all metazoans, suggesting that Complexins are a universal feature of metazoans that predate metazoan evolution. We show that Complexin from Nematostella vectensis, a cnidarian sea anemone far separated from mammals in metazoan evolution, functionally replaces mouse Complexins in activating Ca(2+)-triggered exocytosis, but is unable to clamp spontaneous exocytosis. Thus, the activating function of Complexins is likely conserved throughout metazoan evolution.
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deconstructing Complexin function in activating and clamping ca2 triggered exocytosis by comparing knockout and knockdown phenotypes
Proceedings of the National Academy of Sciences of the United States of America, 2013Co-Authors: Xiaofei Yang, Peng Cao, Thomas C. SüdhofAbstract:Complexin, a presynaptic protein that avidly binds to assembled SNARE complexes, is widely acknowledged to activate Ca2+-triggered exocytosis. In addition, studies of invertebrate Complexin mutants and of mouse neurons with a double knockdown (DKD) of Complexin-1 and -2 suggested that Complexin maintains the readily releasable pool (RRP) of vesicles and clamps spontaneous exocytosis. In contrast, studies of mouse neurons with a double knockout (DKO) of Complexin-1 and -2, largely carried out in hippocampal autapses, did not detect changes in the RRP size or in spontaneous exocytosis. To clarify Complexin function, we here directly compared in two different preparations, cultured cortical and olfactory bulb neurons, the phenotypes of Complexin DKD and DKO neurons. We find that Complexin-deficient DKD and DKO neurons invariably exhibit a ∼50% decrease in vesicle priming. Moreover, the DKD consistently increased spontaneous exocytosis, but the DKO did so in cortical but not olfactory bulb neurons. Furthermore, the Complexin DKD but not the Complexin DKO caused a compensatory increase in Complexin-3 and -4 mRNA levels; overexpression of Complexin-3 but not Complexin-1 increased spontaneous exocytosis. Complexin-3 but not Complexin-1 contains a C-terminal lipid anchor attaching it to the plasma membrane; addition of a similar lipid anchor to Complexin-1 converted Complexin-1 from a clamp into an activator of spontaneous exocytosis. Viewed together, our data suggest that Complexin generally functions in priming and Ca2+ triggering of exocytosis, and additionally contributes to the control of spontaneous exocytosis dependent on the developmental history of a neuron and on the subcellular localization of the Complexin.
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Complexin Activates Exocytosis of Distinct Secretory Vesicles Controlled by Different Synaptotagmins
The Journal of neuroscience : the official journal of the Society for Neuroscience, 2013Co-Authors: Peng Cao, Xiaofei Yang, Thomas C. SüdhofAbstract:Complexins are SNARE-complex binding proteins essential for the Ca(2+)-triggered exocytosis mediated by synaptotagmin-1, -2, -7, or -9, but the possible role of Complexins in other types of exocytosis controlled by other synaptotagmin isoforms remains unclear. Here we show that, in mouse olfactory bulb neurons, synaptotagmin-1 localizes to synaptic vesicles and to large dense-core secretory vesicles as reported previously, whereas synaptotagmin-10 localizes to a distinct class of peptidergic secretory vesicles containing IGF-1. Both synaptotagmin-1-dependent synaptic vesicle exocytosis and synaptotagmin-10-dependent IGF-1 exocytosis were severely impaired by knockdown of Complexins, demonstrating that Complexin acts as a cofactor for both synaptotagmin-1 and synaptotagmin-10 despite the functional differences between these synaptotagmins. Rescue experiments revealed that only the activating but not the clamping function of Complexins was required for IGF-1 exocytosis controlled by synaptotagmin-10. Thus, our data indicate that Complexins are essential for activation of multiple types of Ca(2+)-induced exocytosis that are regulated by different synaptotagmin isoforms. These results suggest that different types of regulated exocytosis are mediated by similar synaptotagmin-dependent fusion mechanisms, that particular synaptotagmin isoforms confer specificity onto different types of regulated exocytosis, and that Complexins serve as universal synaptotagmin adaptors for all of these types of exocytosis independent of which synaptotagmin isoform is involved.
Josep Rizo - One of the best experts on this subject based on the ideXlab platform.
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Reconciling isothermal titration calorimetry analyses of interactions between Complexin and truncated SNARE complexes.
eLife, 2017Co-Authors: Eric A Prinslow, Chad A. Brautigam, Josep RizoAbstract:Neurotransmitter release depends on the SNARE complex formed by syntaxin-1, synaptobrevin and SNAP-25, as well as on Complexins, which bind to the SNARE complex and play active and inhibitory roles. A crystal structure of a Complexin-I fragment bearing a so-called 'superclamp' mutation bound to a truncated SNARE complex lacking the C-terminus of the synaptobrevin SNARE motif (SNAREΔ60) suggested that an 'accessory' α-helix of Complexin-I inhibits release by inserting into the C-terminus of the SNARE complex. Previously, isothermal titration calorimetry (ITC) experiments performed in different laboratories yielded apparently discrepant results in support or against the existence of such binding mode in solution (Trimbuch et al., 2014; Krishnakumar et al., 2015). Here, ITC experiments performed to solve these discrepancies now show that the region containing the Complexin-I accessory helix and preceding N-terminal sequences does interact with SNAREΔ60, but the interaction requires the polybasic juxtamembrane region of syntaxin-1 and is not affected by the superclamp mutation within the experimental error of these experiments.
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Re-examining how Complexin inhibits neurotransmitter release
eLife, 2014Co-Authors: Thorsten Trimbuch, Josep Rizo, David K. Flaherty, Diana R. Tomchick, Christian RosenmundAbstract:Complexins play activating and inhibitory functions in neurotransmitter release. The Complexin accessory helix inhibits release and was proposed to insert into SNARE complexes to prevent their full assembly. This model was supported by ‘superclamp’ and ‘poor-clamp’ mutations that enhanced or decreased the Complexin-I inhibitory activity in cell–cell fusion assays, and by the crystal structure of a superclamp mutant bound to a synaptobrevin-truncated SNARE complex. NMR studies now show that the Complexin-I accessory helix does not insert into synaptobrevin-truncated SNARE complexes in solution, and electrophysiological data reveal that superclamp mutants have slightly stimulatory or no effects on neurotransmitter release, whereas a poor-clamp mutant inhibits release. Importantly, increasing or decreasing the negative charge of the Complexin-I accessory helix inhibits or stimulates release, respectively. These results suggest a new model whereby the Complexin accessory helix inhibits release through electrostatic (and perhaps steric) repulsion enabled by its location between the vesicle and plasma membranes. DOI: http://dx.doi.org/10.7554/eLife.02391.001
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Subtle Interplay between synaptotagmin and Complexin binding to the SNARE complex.
Journal of molecular biology, 2013Co-Authors: Kyle D. Brewer, Raquel Perez-castillejos, Josep RizoAbstract:Ca²⁺-triggered neurotransmitter release depends on the formation of SNARE complexes that bring the synaptic vesicle and plasma membranes together, on the Ca²⁺ sensor synaptotagmin-1 and on Complexins, which play active and inhibitory roles. Release of the Complexin inhibitory activity by binding of synaptotagmin-1 to the SNARE complex, causing Complexin displacement, was proposed to trigger exocytosis. However, the validity of this model was questioned based on the observation of simultaneous binding of Complexin-I and a fragment containing the synaptotagmin-1 C2 domains (C2AB) to membrane-anchored SNARE complex. Using diverse biophysical techniques, here we show that C2AB and Complexin-I do not bind to each other but can indeed bind simultaneously to the SNARE complex in solution. Hence, the SNARE complex contains separate binding sites for both proteins. However, total internal reflection fluorescence microscopy experiments show that C2AB can displace a Complexin-I fragment containing its central SNARE-binding helix and an inhibitory helix (Cpx26-83) from membrane-anchored SNARE complex under equilibrium conditions. Interestingly, full-length Complexin-I binds more tightly to membrane-anchored SNARE complex than Cpx26-83, and it is not displaced by C2AB. These results show that interactions of N- and/or C-terminal sequences of Complexin-I with the SNARE complex and/or phospholipids increase the affinity of Complexin-I for the SNARE complex, hindering dissociation induced by C2AB. We propose a model whereby binding of synaptotagmin-1 to the SNARE complex directly or indirectly causes a rearrangement of the Complexin-I inhibitory helix without inducing Complexin-I dissociation, thus relieving the inhibitory activity and enabling cooperation between synaptotagmin-1 and Complexin-I in triggering release.
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Binding of the Complexin N terminus to the SNARE complex potentiates synaptic-vesicle fusogenicity
Nature structural & molecular biology, 2010Co-Authors: Mingshan Xue, Hsiaotuan Chao, Josep Rizo, Timothy K. Craig, Christian RosenmundAbstract:Complexins facilitate and inhibit neurotransmitter release through distinct domains, and their function was proposed to be coupled to the Ca(2+) sensor synaptotagmin-1 (Syt1). However, the mechanisms underlying Complexin function remain unclear. We now uncover an interaction between the Complexin N terminus and the SNARE complex C terminus, and we show that disrupting this interaction abolishes the facilitatory function of Complexins in mouse neurons. Analyses of hypertonically induced exocytosis show that Complexins enhance synaptic-vesicle fusogenicity. Genetic experiments crossing Complexin- and Syt1-null mice indicate a functional interaction between these proteins but also show that Complexins can promote Ca(2+)-triggered release in the absence of Syt1. We propose that the Complexin N terminus stabilizes the SNARE complex C terminus and/or helps release the inhibitory function of Complexins, thereby activating the fusion machinery in a manner that may cooperate with Syt1 but does not require Syt1.
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distinct domains of Complexin i differentially regulate neurotransmitter release
Nature Structural & Molecular Biology, 2007Co-Authors: Kerstin Reim, Nils Brose, Xiaocheng Chen, Hsiaotuan Chao, Hui Deng, Josep Rizo, Christian RosenmundAbstract:Complexins constitute a family of four synaptic high-affinity SNARE complex–binding proteins. They positively regulate a late, post-priming step in Ca2+-triggered synchronous neurotransmitter release, but the underlying molecular mechanisms are unclear. We show here that SNARE complex binding of Complexin I (CplxI) via its central α-helix is necessary but, unexpectedly, not sufficient for its key function in promoting neurotransmitter release. An accessory α-helix on the N-terminal side of the SNARE complex–binding region has an inhibitory effect on fast synaptic exocytosis, whereas sequences N-terminally adjacent to this helix facilitate Ca2+-triggered release even in the absence of the Ca2+ sensor synaptotagmin-1. Our results indicate that distinct functional domains of CplxI differentially regulate synaptic exocytosis and that, through the interplay between these domains, CplxI carries out a crucial role in fine-tuning Ca2+-triggered fast neurotransmitter release.
Axel T. Brunger - One of the best experts on this subject based on the ideXlab platform.
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The primed SNARE–Complexin–synaptotagmin complex for neuronal exocytosis
Nature, 2017Co-Authors: Qiangjun Zhou, Thomas C. Südhof, Minglei Zhao, Peng Zhou, Austin L. Wang, Axel T. BrungerAbstract:Synaptotagmin, Complexin, and neuronal SNARE (soluble N-ethylmaleimide sensitive factor attachment protein receptor) proteins mediate evoked synchronous neurotransmitter release, but the molecular mechanisms mediating the cooperation between these molecules remain unclear. Here we determine crystal structures of the primed pre-fusion SNARE-Complexin-synaptotagmin-1 complex. These structures reveal an unexpected tripartite interface between synaptotagmin-1 and both the SNARE complex and Complexin. Simultaneously, a second synaptotagmin-1 molecule interacts with the other side of the SNARE complex via the previously identified primary interface. Mutations that disrupt either interface in solution also severely impair evoked synchronous release in neurons, suggesting that both interfaces are essential for the primed pre-fusion state. Ca2+ binding to the synaptotagmin-1 molecules unlocks the complex, allows full zippering of the SNARE complex, and triggers membrane fusion. The tripartite SNARE-Complexin-synaptotagmin-1 complex at a synaptic vesicle docking site has to be unlocked for triggered fusion to start, explaining the cooperation between Complexin and synaptotagmin-1 in synchronizing evoked release on the sub-millisecond timescale.
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the primed snare Complexin synaptotagmin complex for neuronal exocytosis
Nature, 2017Co-Authors: Qiangjun Zhou, Thomas C. Südhof, Minglei Zhao, Axel T. Brunger, Peng Zhou, Austin L. WangAbstract:Synaptotagmin, Complexin, and neuronal SNARE (soluble N-ethylmaleimide sensitive factor attachment protein receptor) proteins mediate evoked synchronous neurotransmitter release, but the molecular mechanisms mediating the cooperation between these molecules remain unclear. Here we determine crystal structures of the primed pre-fusion SNARE-Complexin-synaptotagmin-1 complex. These structures reveal an unexpected tripartite interface between synaptotagmin-1 and both the SNARE complex and Complexin. Simultaneously, a second synaptotagmin-1 molecule interacts with the other side of the SNARE complex via the previously identified primary interface. Mutations that disrupt either interface in solution also severely impair evoked synchronous release in neurons, suggesting that both interfaces are essential for the primed pre-fusion state. Ca2+ binding to the synaptotagmin-1 molecules unlocks the complex, allows full zippering of the SNARE complex, and triggers membrane fusion. The tripartite SNARE-Complexin-synaptotagmin-1 complex at a synaptic vesicle docking site has to be unlocked for triggered fusion to start, explaining the cooperation between Complexin and synaptotagmin-1 in synchronizing evoked release on the sub-millisecond timescale.
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Complexin induces a conformational change at the membrane-proximal C-terminal end of the SNARE complex.
eLife, 2016Co-Authors: Ucheor B. Choi, Minglei Zhao, Yunxiang Zhang, Ying Lai, Axel T. BrungerAbstract:Complexin regulates spontaneous and activates Ca(2+)-triggered neurotransmitter release, yet the molecular mechanisms are still unclear. Here we performed single molecule fluorescence resonance energy transfer experiments and uncovered two conformations of Complexin-1 bound to the ternary SNARE complex. In the cis conformation, Complexin-1 induces a conformational change at the membrane-proximal C-terminal end of the ternary SNARE complex that specifically depends on the N-terminal, accessory, and central domains of Complexin-1. The Complexin-1 induced conformation of the ternary SNARE complex may be related to a conformation that is juxtaposing the synaptic vesicle and plasma membranes. In the trans conformation, Complexin-1 can simultaneously interact with a ternary SNARE complex via the central domain and a binary SNARE complex consisting of syntaxin-1A and SNAP-25A via the accessory domain. The cis conformation may be involved in activation of synchronous neurotransmitter release, whereas both conformations may be involved in regulating spontaneous release.
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Complexin inhibits spontaneous release and synchronizes Ca2+-triggered synaptic vesicle fusion by distinct mechanisms
eLife, 2014Co-Authors: Ying Lai, Jiajie Diao, Yunxiang Zhang, Daniel J. Cipriano, Mark S. Padolina, Richard A. Pfuetzner, Axel T. BrungerAbstract:Previously we showed that fast Ca2+-triggered vesicle fusion with reconstituted neuronal SNAREs and synaptotagmin-1 begins from an initial hemifusion-free membrane point contact, rather than a hemifusion diaphragm, using a single vesicle–vesicle lipid/content mixing assay (Diao et al., 2012). When Complexin-1 was included, a more pronounced Ca2+-triggered fusion burst was observed, effectively synchronizing the process. Here we show that Complexin-1 also reduces spontaneous fusion in the same assay. Moreover, distinct effects of several Complexin-1 truncation mutants on spontaneous and Ca2+-triggered fusion closely mimic those observed in neuronal cultures. The very N-terminal domain is essential for synchronization of Ca2+-triggered fusion, but not for suppression of spontaneous fusion, whereas the opposite is true for the C-terminal domain. By systematically varying the Complexin-1 concentration, we observed differences in titration behavior for spontaneous and Ca2+-triggered fusion. Taken together, Complexin-1 utilizes distinct mechanisms for synchronization of Ca2+-triggered fusion and inhibition of spontaneous fusion. DOI: http://dx.doi.org/10.7554/eLife.03756.001
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Complexin 1 enhances the on rate of vesicle docking via simultaneous snare and membrane interactions
Biophysical Journal, 2014Co-Authors: Jiajie Diao, Daniel J. Cipriano, Axel T. BrungerAbstract:In synaptic terminals, Complexin is thought to have inhibitory and activating roles for spontaneous mini-release and evoked synchronized neurotransmitter release, respectively. We used single vesicle-vesicle microscopy imaging to study the effect of Complexin-1 on the docking on-rate between vesicles that mimic synaptic vesicles and vesicles that mimic the plasma membrane. We found that Complexin-1 enhances the on-rate of docking between synaptic vesicle mimics containing full-length synaptobrevin-2 and full-length synaptotagmin-1 and plasma membrane mimicking vesicles containing full-length syntaxin-1A and SNAP-25A. This effect requires the C-terminal domain of Complexin-1 which binds to the membrane, the presence of PS in the membrane, and the core region of Complexin-1 which binds to the SNARE complex.References1. Diao, J., P. Grob, ⋯, A. T. Brunger. 2012. Synaptic proteins promote calcium-triggered fast transition from point contact to full fusion. Elife 1:e00109.2. Diao, J., D. J. Cipriano, ⋯, A. T. Brunger. 2013. Complexin-1 enhances the on-rate of vesicle docking via simultaneous SNARE and membrane interactions. J. Am. Chem. Soc. 135: 10.1021/ja407392n.
Kerstin Reim - One of the best experts on this subject based on the ideXlab platform.
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Acute Complexin Knockout Abates Spontaneous and Evoked Transmitter Release
Cell reports, 2019Co-Authors: Francisco José López-murcia, Kerstin Reim, Olaf Jahn, Holger Taschenberger, Nils BroseAbstract:SNARE-mediated synaptic vesicle (SV) fusion is controlled by multiple regulatory proteins that determine neurotransmitter release efficiency. Complexins are essential SNARE regulators whose mode of action is unclear, as available evidence indicates positive SV fusion facilitation and negative "fusion clamp"-like activities, with the latter occurring only in certain contexts. Because these contradictory findings likely originate in part from different experimental perturbation strategies, we attempted to resolve them by examining a conditional Complexin-knockout mouse line as the most stringent genetic perturbation model available. We found that acute Complexin loss after synaptogenesis in autaptic and mass-cultured hippocampal neurons reduces SV fusion probability and thus abates the rates of spontaneous, synchronous, asynchronous, and delayed transmitter release but does not affect SV priming or cause "unclamping" of spontaneous SV fusion. Thus, Complexins act as facilitators of SV fusion but are dispensable for "fusion clamping" in mammalian forebrain neurons.
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Acute Complexin Knockout Abates Spontaneous and Evoked Transmitter Release
Elsevier, 2019Co-Authors: Francisco José López-murcia, Kerstin Reim, Olaf Jahn, Holger Taschenberger, Nils BroseAbstract:Summary: SNARE-mediated synaptic vesicle (SV) fusion is controlled by multiple regulatory proteins that determine neurotransmitter release efficiency. Complexins are essential SNARE regulators whose mode of action is unclear, as available evidence indicates positive SV fusion facilitation and negative “fusion clamp”-like activities, with the latter occurring only in certain contexts. Because these contradictory findings likely originate in part from different experimental perturbation strategies, we attempted to resolve them by examining a conditional Complexin-knockout mouse line as the most stringent genetic perturbation model available. We found that acute Complexin loss after synaptogenesis in autaptic and mass-cultured hippocampal neurons reduces SV fusion probability and thus abates the rates of spontaneous, synchronous, asynchronous, and delayed transmitter release but does not affect SV priming or cause “unclamping” of spontaneous SV fusion. Thus, Complexins act as facilitators of SV fusion but are dispensable for “fusion clamping” in mammalian forebrain neurons. : Complexins are thought to either promote synaptic vesicle fusion or act as “fusion clamps.” López-Murcia et al. show that acute genetic Complexin deletion reduces the rates of all forms of transmitter release in forebrain neurons without affecting vesicle priming. Thus, Complexins are facilitators of vesicle fusion and dispensable for “fusion clamping.” Keywords: Complexin, synaptic vesicle, fusion, hippocampal neurons, synaptic transmissio
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1Complexin I DEFICIENT SPERM ARE SUBFERTILE DUE TO A DEFECT IN ZONA PELLUCIDA PENETRATION
2015Co-Authors: Longmei Zhao, Kerstin Reim, David J. MillerAbstract:Upon adhesion to the zona pellucida, sperm undergo regulated exocytosis of the acrosome. Although it is necessary for sperm to penetrate the zona pellucida and fertilize an egg, the acrosomal membrane fusion process is poorly understood. Complexin I and II are small, cytosolic proteins that bind to a complex of proteins termed the SNARE complex (soluble N-ethyl-maleimide sensitive factor attachment protein receptor) to regulate synaptic vesicle exocytosis. Complexin II deficient mice are fertile but the fertility of sperm from Complexin I deficient male mice is unclear because the mice have ataxia and cannot mate. Here we show that the genes encoding Complexin I and II are expressed in primary spermatocytes and spermatids. Complexin proteins were found in/near the developing acrosome in spermatids and in or around the acrosome of mature sperm. Cell fractionation demonstrated Complexins I and II were predominantly found in the cytosolic fraction. Furthermore, sperm from Complexin I deficient mice had normal morphology, number and only small differences in motility, as assessed by computer assisted semen analysis. Complexin I deficient sperm capacitated normally and bound to the zona pellucida. But when sperm from Complexin I deficient mice were inseminated into females, a defect in fertility was observed, in concordance with previous data showing that in vitro fertilization rate was also reduced. If the zona pellucida was removed prior to in vitro fertilization, fertility was normal, demonstrating that zona pellucida penetration was defective, a step requiring acrosomal exocytosis. Therefore
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Complexin facilitates exocytosis and synchronizes vesicle release in two secretory model systems
The Journal of Physiology, 2013Co-Authors: Mingyi Lin, Kerstin Reim, Joyce G Rohan, Haijiang Cai, Robert H ChowAbstract:Complexins (Cplxs) are small, SNARE-associated proteins believed to regulate fast, calcium-triggered exocytosis. However, studies have pointed to either an inhibitory and/or facilitatory role in exocytosis, and the role of Cplxs in synchronizing exocytosis is relatively unexplored. Here, we compare the function of two types of Complexin, Cplx 1 and 2, in two model systems of calcium-dependent exocytosis. In mouse neuromuscular junctions (NMJs), we find that lack of Cplx 1 significantly reduces and desynchronizes calcium-triggered synaptic transmission; furthermore, high-frequency stimulation elicits synaptic facilitation, instead of normal synaptic depression, and the degree of facilitation is highly sensitive to the amount of cytoplasmic calcium buffering. In Cplx 2-null adrenal chromaffin cells, we also find decreased and desynchronized evoked release, and identify a significant reduction in the vesicle pool close to the calcium channels (immediately releasable pool, IRP). Viral transduction with either Cplx 1 or 2 rescues both the size of the evoked response and the synchronicity of release, and it restores the IRP size. Our findings in two model systems are mutually compatible and indicate a role of Cplx 1 and 2 in facilitating vesicle priming, and also lead to the new hypothesis that Cplxs may synchronize vesicle release by promoting coupling between secretory vesicles and calcium channels.
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Complexin I plays a bilateral role in synaptic transmission during development at the calyx of Held synapse.
Biophysical Journal, 2013Co-Authors: Shuwen Chang, Kerstin Reim, Meike Pedersen, Holger TaschenbergerAbstract:Complexins are small synaptic proteins which cooperate with the SNARE-complex during synaptic transmission. Different roles of Complexins in the regulation of vesicle exocytosis have been proposed. Based on the results of genetic mutation or knock down/out studies, it is generally agreed that Complexins are involved in vesicle priming and exocytosis during fast synchronous release and in clamping vesicles to prevent asynchronous release. However, depending on cell type, organism and experimental approach used, Complexins appear to either facilitate or inhibit vesicle fusion. Here, we study the function of Complexin I at the calyx of Held synapse. By taking advantage of the large size of the calyx terminal, allowing direct patch-clamp recordings, we investigate the consequences of the loss of function of Complexin I. We demonstrate a developmentally aggravating phenotype of reduced EPSC amplitudes and enhanced asynchronous release. We provide evidence for a role of CPX I in recruiting Ca2+ channels to docked vesicles which may determine their release probability. The enhanced asynchronous release in Complexin-deficient mice slowed-down the recovery of synchronous EPSCs after stimulus trains suggesting both, synchronous and asynchronous release events, were fed by a common pool of vesicles.
Christian Rosenmund - One of the best experts on this subject based on the ideXlab platform.
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Complexin suppresses spontaneous exocytosis by capturing the membrane-proximal regions of VAMP2 and SNAP25
2019Co-Authors: Joerg Malsam, Thorsten Trimbuch, Fereshteh Zarebidaki, Andreas F.-p. Sonnen, Klemens Wild, Andrea Scheutzow, Irmgard Sinning, Simon Baerfuss, John A. G. Briggs, Christian RosenmundAbstract:The neuronal protein Complexin contains multiple domains that exert both clamping and facilitatory functions to tune spontaneous and action potential triggered synaptic release. We address the clamping mechanism and show that the accessory helix of Complexin arrests the assembly of the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex that forms the core machinery of intracellular membrane fusion. In a reconstituted fusion assay, site- and stage-specific photo-cross-linking reveals that prior to fusion the Complexin accessory helix laterally binds the membrane-proximal C-terminal ends of SNAP25 and VAMP2. Corresponding Complexin interface mutants selectively increase spontaneous release of neurotransmitter in living neurons, implying that the accessory helix suppresses final zippering/assembly of the SNARE four-helix bundle by restraining VAMP2 and SNAP25.
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should i stop or should i go the role of Complexin in neurotransmitter release
Nature Reviews Neuroscience, 2016Co-Authors: Thorsten Trimbuch, Christian RosenmundAbstract:When it comes to fusion with the neuronal cell membrane, does a synaptic vesicle have a choice whether to stop or to go? Recent work suggests that Complexin, a tiny protein found within the synaptic terminal, contributes to the mechanism through which this choice is made. How Complexin plays this consulting part and which synaptic vesicle proteins it interacts with remain open questions. Indeed, studies in mice and flies have led to the proposal of different models of Complexin function. We suggest that understanding the modular nature of Complexin will help us to unpick its role in synaptic vesicle release.
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Should I stop or should I go? The role of Complexin in neurotransmitter release
Nature reviews. Neuroscience, 2016Co-Authors: Thorsten Trimbuch, Christian RosenmundAbstract:When it comes to fusion with the neuronal cell membrane, does a synaptic vesicle have a choice whether to stop or to go? Recent work suggests that Complexin, a tiny protein found within the synaptic terminal, contributes to the mechanism through which this choice is made. How Complexin plays this consulting part and which synaptic vesicle proteins it interacts with remain open questions. Indeed, studies in mice and flies have led to the proposal of different models of Complexin function. We suggest that understanding the modular nature of Complexin will help us to unpick its role in synaptic vesicle release.
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Re-examining how Complexin inhibits neurotransmitter release
eLife, 2014Co-Authors: Thorsten Trimbuch, Josep Rizo, David K. Flaherty, Diana R. Tomchick, Christian RosenmundAbstract:Complexins play activating and inhibitory functions in neurotransmitter release. The Complexin accessory helix inhibits release and was proposed to insert into SNARE complexes to prevent their full assembly. This model was supported by ‘superclamp’ and ‘poor-clamp’ mutations that enhanced or decreased the Complexin-I inhibitory activity in cell–cell fusion assays, and by the crystal structure of a superclamp mutant bound to a synaptobrevin-truncated SNARE complex. NMR studies now show that the Complexin-I accessory helix does not insert into synaptobrevin-truncated SNARE complexes in solution, and electrophysiological data reveal that superclamp mutants have slightly stimulatory or no effects on neurotransmitter release, whereas a poor-clamp mutant inhibits release. Importantly, increasing or decreasing the negative charge of the Complexin-I accessory helix inhibits or stimulates release, respectively. These results suggest a new model whereby the Complexin accessory helix inhibits release through electrostatic (and perhaps steric) repulsion enabled by its location between the vesicle and plasma membranes. DOI: http://dx.doi.org/10.7554/eLife.02391.001
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Binding of the Complexin N terminus to the SNARE complex potentiates synaptic-vesicle fusogenicity
Nature structural & molecular biology, 2010Co-Authors: Mingshan Xue, Hsiaotuan Chao, Josep Rizo, Timothy K. Craig, Christian RosenmundAbstract:Complexins facilitate and inhibit neurotransmitter release through distinct domains, and their function was proposed to be coupled to the Ca(2+) sensor synaptotagmin-1 (Syt1). However, the mechanisms underlying Complexin function remain unclear. We now uncover an interaction between the Complexin N terminus and the SNARE complex C terminus, and we show that disrupting this interaction abolishes the facilitatory function of Complexins in mouse neurons. Analyses of hypertonically induced exocytosis show that Complexins enhance synaptic-vesicle fusogenicity. Genetic experiments crossing Complexin- and Syt1-null mice indicate a functional interaction between these proteins but also show that Complexins can promote Ca(2+)-triggered release in the absence of Syt1. We propose that the Complexin N terminus stabilizes the SNARE complex C terminus and/or helps release the inhibitory function of Complexins, thereby activating the fusion machinery in a manner that may cooperate with Syt1 but does not require Syt1.