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Thomas C Sudhof - One of the best experts on this subject based on the ideXlab platform.
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Synaptotagmin 1 and 7 are redundantly essential for maintaining the capacity of the readily releasable pool of synaptic vesicles
PLOS Biology, 2015Co-Authors: Taulant Bacaj, Xinran Liu, Robert C Malenka, Jacqueline Burre, Thomas C SudhofAbstract:In forebrain neurons, Ca2+ triggers exocytosis of readily releasable vesicles by binding to Synaptotagmin-1 and -7, thereby inducing fast and slow vesicle exocytosis, respectively. Loss-of-function of Synaptotagmin-1 or -7 selectively impairs the fast and slow phase of release, respectively, but does not change the size of the readily-releasable pool (RRP) of vesicles as measured by stimulation of release with hypertonic sucrose, or alter the rate of vesicle priming into the RRP. Here we show, however, that simultaneous loss-of-function of both Synaptotagmin-1 and -7 dramatically decreased the capacity of the RRP, again without altering the rate of vesicle priming into the RRP. Either Synaptotagmin-1 or -7 was sufficient to rescue the RRP size in neurons lacking both Synaptotagmin-1 and -7. Although maintenance of RRP size was Ca2+-independent, mutations in Ca2+-binding sequences of Synaptotagmin-1 or Synaptotagmin-7—which are contained in flexible top-loop sequences of their C2 domains—blocked the ability of these Synaptotagmins to maintain the RRP size. Both Synaptotagmins bound to SNARE complexes; SNARE complex binding was reduced by the top-loop mutations that impaired RRP maintenance. Thus, Synaptotagmin-1 and -7 perform redundant functions in maintaining the capacity of the RRP in addition to nonredundant functions in the Ca2+ triggering of different phases of release.
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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 SudhofAbstract: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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Synaptotagmin 1 and Synaptotagmin 7 trigger synchronous and asynchronous phases of neurotransmitter release
Neuron, 2013Co-Authors: Taulant Bacaj, Xiaofei Yang, Dick Wu, Wade Morishita, Peng Zhou, Wei Xu, Robert C Malenka, Thomas C SudhofAbstract:Summary In forebrain neurons, knockout of Synaptotagmin-1 blocks fast Ca 2+ -triggered synchronous neurotransmitter release but enables manifestation of slow Ca 2+ -triggered asynchronous release. Here, we show using single-cell PCR that individual hippocampal neurons abundantly coexpress two Ca 2+ -binding Synaptotagmin isoforms, Synaptotagmin-1 and Synaptotagmin-7. In Synaptotagmin-1-deficient synapses of excitatory and inhibitory neurons, loss of function of Synaptotagmin-7 suppressed asynchronous release. This phenotype was rescued by wild-type but not mutant Synaptotagmin-7 lacking functional Ca 2+ -binding sites. Even in Synaptotagmin-1-containing neurons, Synaptotagmin-7 ablation partly impaired asynchronous release induced by extended high-frequency stimulus trains. Synaptotagmins bind Ca 2+ via two C2 domains, the C2A and C2B domains. Surprisingly, Synaptotagmin-7 function selectively required its C2A domain Ca 2+ -binding sites, whereas Synaptotagmin-1 function required its C2B domain Ca 2+ -binding sites. Our data show that nearly all Ca 2+ -triggered release at a synapse is due to Synaptotagmins, with Synaptotagmin-7 mediating a slower form of Ca 2+ -triggered release that is normally occluded by faster Synaptotagmin-1-induced release but becomes manifest upon Synaptotagmin-1 deletion.
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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 SudhofAbstract: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.
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calcium control of neurotransmitter release
Cold Spring Harbor Perspectives in Biology, 2012Co-Authors: Thomas C SudhofAbstract:Upon entering a presynaptic terminal, an action potential opens Ca(2+) channels, and transiently increases the local Ca(2+) concentration at the presynaptic active zone. Ca(2+) then triggers neurotransmitter release within a few hundred microseconds by activating Synaptotagmins Ca(2+). Synaptotagmins bind Ca(2+) via two C2-domains, and transduce the Ca(2+) signal into a nanomechanical activation of the membrane fusion machinery; this activation is mediated by the Ca(2+)-dependent interaction of the Synaptotagmin C2-domains with phospholipids and SNARE proteins. In triggering exocytosis, Synaptotagmins do not act alone, but require an obligatory cofactor called complexin, a small protein that binds to SNARE complexes and simultaneously activates and clamps the SNARE complexes, thereby positioning the SNARE complexes for subsequent Synaptotagmin action. The conserved function of Synaptotagmins and complexins operates generally in most, if not all, Ca(2+)-regulated forms of exocytosis throughout the body in addition to synaptic vesicle exocytosis, including in the degranulation of mast cells, acrosome exocytosis in sperm cells, hormone secretion from endocrine cells, and neuropeptide release.
Weiping Han - One of the best experts on this subject based on the ideXlab platform.
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Neuronal calcium sensor Synaptotagmin-9 is not involved in the regulation of glucose homeostasis or insulin secretion.
PloS one, 2010Co-Authors: Natalia Gustavsson, Thomas C Sudhof, George K Radda, Xiaorui Wang, Yue Wang, T. Seah, Weiping HanAbstract:Background: Insulin secretion is a complex and highly regulated process. It is well established that cytoplasmic calcium is a key regulator of insulin secretion, but how elevated intracellular calcium triggers insulin granule exocytosis remains unclear, and we have only begun to define the identities of proteins that are responsible for sensing calcium changes and for transmitting the calcium signal to release machineries. Synaptotagmins are primarily expressed in brain and endocrine cells and exhibit diverse calcium binding properties. Synaptotagmin-1, -2 and -9 are calcium sensors for fast neurotransmitter release in respective brain regions, while Synaptotagmin-7 is a positive regulator of calcium-dependent insulin release. Unlike the three neuronal calcium sensors, whose deletion abolished fast neurotransmitter release, Synaptotagmin-7 deletion resulted in only partial loss of calcium-dependent insulin secretion, thus suggesting that other calcium-sensors must participate in the regulation of insulin secretion. Of the other Synaptotagmin isoforms that are present in pancreatic islets, the neuronal calcium sensor Synaptotagmin-9 is expressed at the highest level after Synaptotagmin-7. Methodology/Principal Findings: In this study we tested whether Synaptotagmin-9 participates in the regulation of glucose-stimulated insulin release by using pancreas-specific Synaptotagmin-9 knockout (p-S9X) mice. Deletion of Synaptotagmin-9 in the pancreas resulted in no changes in glucose homeostasis or body weight. Glucose tolerance, and insulin secretion in vivo and from isolated islets were not affected in the p-S9X mice. Single-cell capacitance measurements showed no difference in insulin granule exocytosis between p-S9X and control mice. Conclusions: Thus, Synaptotagmin-9, although a major calcium sensor in the brain, is not involved in the regulation of glucose-stimulated insulin release from pancreatic b-cells.
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Calcium-sensing beyond neurotransmitters: functions of Synaptotagmins in neuroendocrine and endocrine secretion.
Bioscience reports, 2009Co-Authors: Natalia Gustavsson, Weiping HanAbstract:Neurotransmitters, neuropeptides and hormones are released through the regulated exocytosis of SVs (synaptic vesicles) and LDCVs (large dense-core vesicles), a process that is controlled by calcium. Synaptotagmins are a family of type 1 membrane proteins that share a common domain structure. Most Synaptotagmins are located in brain and endocrine cells, and some of these Synaptotagmins bind to phospholipids and calcium at levels that trigger regulated exocytosis of SVs and LDCVs. This led to the proposed Synaptotagmin-calcium-sensor paradigm, that is, members of the Synaptotagmin family function as calcium sensors for the regulated exocytosis of neurotransmitters, neuropeptides and hormones. Here, we provide an overview of the Synaptotagmin family, and review the recent mouse genetic studies aimed at understanding the functions of Synaptotagmins in neurotransmission and endocrine-hormone secretion. Also, we discuss potential roles of Synaptotagmins in non-traditional endocrine systems.
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Synaptotagmin 7 is a principal ca2 sensor for ca2 induced glucagon exocytosis in pancreas
The Journal of Physiology, 2009Co-Authors: Natalia Gustavsson, Thomas C Sudhof, Ye Lao, Weiping Han, George K Radda, Shunhui Wei, Dong Nhut Hoang, Quan Zhang, Patrik RorsmanAbstract:Hormones such as glucagon are secreted by Ca2+-induced exocytosis of large dense-core vesicles, but the mechanisms involved have only been partially elucidated. Studies of pancreatic β-cells secreting insulin revealed that Synaptotagmin-7 alone is not sufficient to mediate Ca2+-dependent insulin granule exocytosis, and studies of chromaffin cells secreting neuropeptides and catecholamines showed that Synaptotagmin-1 and -7 collaborate as Ca2+ sensors for exocytosis, and that both are equally involved. As no other peptide secretion was analysed, it remains unclear whether Synaptotagmins generally act as Ca2+ sensors in large dense-core vesicle exocytosis in endocrine cells, and if so, whether Synaptotagmin-7 always functions with a partner in that role. In particular, far less is known about the mechanisms underlying Ca2+-triggered glucagon release from α-cells than insulin secretion from β-cells, even though insulin and glucagon together regulate blood glucose levels. To address these issues, we analysed the role of Synaptotagmins in Ca2+-triggered glucagon exocytosis. Surprisingly, we find that deletion of a single Synaptotagmin isoform, Synaptotagmin-7, nearly abolished Ca2+-triggered glucagon secretion. Moreover, single-cell capacitance measurements confirmed that pancreatic α-cells lacking Synaptotagmin-7 exhibited little Ca2+-induced exocytosis, whereas all other physiological and morphological parameters of the α-cells were normal. Our data thus identify Synaptotagmin-7 as a principal Ca2+ sensor for glucagon secretion, and support the notion that Synaptotagmins perform a universal but selective function as individually acting Ca2+ sensors in neurotransmitter, neuropeptide, and hormone secretion.
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impaired insulin secretion and glucose intolerance in Synaptotagmin 7 null mutant mice
Proceedings of the National Academy of Sciences of the United States of America, 2008Co-Authors: Natalia Gustavsson, Thomas C Sudhof, Anton Maximov, Ye Lao, Weiping Han, Jen Chieh Chuang, Elena Kostromina, Joyce J Repa, George K RaddaAbstract:Vertebrates express at least 15 different Synaptotagmins with the same domain structure but diverse localizations and tissue distributions. Synaptotagmin-1,-2, and -9 act as calcium sensors for the fast phrase of neurotransmitter release, and Synaptotagmin-12 acts as a calcium-independent modulator of release. The exact functions of the remaining 11 Synaptotagmins, however, have not been established. By analogy to the role of Synaptotagmin-1, -2, and -9 in neurotransmission, these other Synaptotagmins may serve as Ca2+ transducers regulating other Ca2+-dependent membrane processes, such as insulin secretion in pancreatic β-cells. Of these other Synaptotagmins, Synaptotagmin-7 is one of the most abundant and is present in pancreatic β-cells. To determine whether Synaptotagmin-7 regulates Ca2+-dependent insulin secretion, we analyzed Synaptotagmin-7 null mutant mice for glucose tolerance and insulin release. Here, we show that Synaptotagmin-7 is required for the maintenance of systemic glucose tolerance and glucose-stimulated insulin secretion. Mutant mice have normal insulin sensitivity, insulin production, islet architecture and ultrastructural organization, and metabolic and calcium responses but exhibit impaired glucose-induced insulin secretion, indicating a calcium-sensing defect during insulin-containing secretory granule exocytosis. Taken together, our findings show that Synaptotagmin-7 functions as a positive regulator of insulin secretion and may serve as a calcium sensor controlling insulin secretion in pancreatic β cells.
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C-terminal ECFP fusion impairs Synaptotagmin 1 function: crowding out Synaptotagmin 1.
The Journal of biological chemistry, 2004Co-Authors: Weiping Han, Christian Rosenmund, Anton Maximov, Jeong-seop Rhee, Weichun Lin, Robert E Hammer, Thomas C SudhofAbstract:To allow the monitoring of Synaptotagmin 1 trafficking in vivo, we generated transgenic mice expressing a Synaptotagmin 1-enhanced cyan fluorescent protein (ECFP) fusion protein under control of the Thy1 promoter. Transgenic Synaptotagmin 1-ECFP is expressed throughout the brain where it localizes to synapses and marks synapses in vivo. However, when we crossed transgenic Synaptotagmin 1-ECFP mice with Synaptotagmin 1 knock-out mice, we detected no rescue of survival or function. Furthermore, viral overexpression of Synaptotagmin 1-ECFP in Synaptotagmin 1-deficient neurons failed to restore normal Ca2+-triggered release, whereas overexpression of wild type Synaptotagmin 1 did so efficiently. To determine whether Synaptotagmin 1-ECFP is non-functional because the ECFP-fusion interferes with its biochemical activities, we measured Ca2+-independent binding of Synaptotagmin 1-ECFP to SNARE complexes, and Ca2+-dependent binding of Synaptotagmin 1-ECFP to phospholipids and to itself. Although the apparent Ca2+ affinity of Synaptotagmin 1-ECFP was decreased compared with wild type Synaptotagmin 1, we observed no major changes in Ca2+-dependent or -independent activities, indicating that the non-functionality of the Synaptotagmin 1-ECFP fusion protein was not because of inactivation of its biochemical properties. These data suggest that Synaptotagmin 1-ECFP is suitable for monitoring synaptic vesicle traffic in vivo because the Synaptotagmin 1-ECFP marks synaptic vesicles without participating in exocytosis. In addition, the data demonstrate that Synaptotagmin 1 function requires a free C terminus, possibly because of spatial constraints at the release sites.
Josep Rizo - One of the best experts on this subject based on the ideXlab platform.
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Structural and mutational analysis of functional differentiation between Synaptotagmins-1 and -7.
PLOS ONE, 2010Co-Authors: Timothy K. Craig, Ok Ho Shin, Diana R Tomchick, Christian Rosenmund, Liyi Li, Chad A. Brautigam, Thomas C Sudhof, Josep RizoAbstract:Synaptotagmins are known to mediate diverse forms of Ca2+-triggered exocytosis through their C2 domains, but the principles underlying functional differentiation among them are unclear. Synaptotagmin-1 functions as a Ca2+ sensor in neurotransmitter release at central nervous system synapses, but Synaptotagmin-7 does not, and yet both isoforms act as Ca2+ sensors in chromaffin cells. To shed light into this apparent paradox, we have performed rescue experiments in neurons from Synaptotagmin-1 knockout mice using a chimera that contains the Synaptotagmin-1 sequence with its C2B domain replaced by the Synaptotagmin-7 C2B domain (Syt1/7). Rescue was not achieved either with the WT Syt1/7 chimera or with nine mutants where residues that are distinct in Synaptotagmin-7 were restored to those present in Synaptotagmin-1. To investigate whether these results arise because of unique conformational features of the Synaptotagmin-7 C2B domain, we determined its crystal structure at 1.44 A resolution. The Synaptotagmin-7 C2B domain structure is very similar to that of the Synaptotagmin-1 C2B domain and contains three Ca2+-binding sites. Two of the Ca2+-binding sites of the Synaptotagmin-7 C2B domain are also present in the Synaptotagmin-1 C2B domain and have analogous ligands to those determined for the latter by NMR spectroscopy, suggesting that a discrepancy observed in a crystal structure of the Synaptotagmin-1 C2B domain arose from crystal contacts. Overall, our results suggest that functional differentiation in Synaptotagmins arises in part from subtle sequence changes that yield dramatic functional differences.
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Synaptotagmin-SNARE coupling enlightened
Nature Structural & Molecular Biology, 2010Co-Authors: Josep RizoAbstract:Synaptotagmins and SNAREs are known to couple Ca^2+ sensing to membrane fusion during Ca^2+-triggered exocytosis, but unraveling the mechanism of this coupling has proven extremely difficult. Two studies in this issue now provide crucial insights into the nature of Synaptotagmin-SNARE interactions and reveal unsuspected similarities between Synaptotagmins and viral fusion proteins.
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Genetic analysis of Synaptotagmin-7 function in synaptic vesicle exocytosis
Proceedings of the National Academy of Sciences of the United States of America, 2008Co-Authors: Anton Maximov, Josep Rizo, Ye Lao, Jakob B Sorensen, Xiaocheng Chen, Thomas C SudhofAbstract:Synaptotagmin-7 is a candidate Ca2+ sensor for exocytosis that is at least partly localized to synapses. Similar to Synaptotagmin-1, which functions as a Ca2+ sensor for fast synaptic vesicle (SV) exocytosis, Synaptotagmin-7 contains C2A and C2B domains that exhibit Ca2+-dependent phospholipid binding. However, Synaptotagmin-7 cannot replace Synaptotagmin-1 as a Ca2+ sensor for fast SV exocytosis, raising questions about the physiological significance of its Ca2+-binding properties. Here, we examine how Synaptotagmin-7 binds Ca2+ and test whether this Ca2+ binding regulates Ca2+-triggered SV exocytosis. We show that the Synaptotagmin-7 C2A domain exhibits a Ca2+-binding mode similar to that of the Synaptotagmin-1 C2A domain, suggesting that the Synaptotagmin-1 and -7 C2 domains generally employ comparable Ca2+-binding mechanisms. We then generated mutant mice that lack Synaptotagmin-7 or contain point mutations inactivating Ca2+ binding either to both C2 domains of Synaptotagmin-7 or only to its C2B domain. Synaptotagmin-7-mutant mice were viable and fertile. Inactivation of Ca2+ binding to both C2 domains caused an ≈70% reduction in Synaptotagmin-7 levels, whereas inactivation of Ca2+ binding to only the C2B domain did not alter Synaptotagmin-7 levels. The Synaptotagmin-7 deletion did not change fast synchronous release, slow asynchronous release, or short-term synaptic plasticity of release of neurotransmitters. Thus, our results show that Ca2+ binding to the Synaptotagmin-7 C2 domains is physiologically important for stabilizing Synaptotagmin-7, but that Ca2+ binding by Synaptotagmin-7 likely does not regulate SV exocytosis, consistent with a role for Synaptotagmin-7 in other forms of Ca2+-dependent synaptic exocytosis.
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genetic analysis of Synaptotagmin 2 in spontaneous and ca2 triggered neurotransmitter release
The EMBO Journal, 2006Co-Authors: Zhiping P. Pang, Josep Rizo, Anton Maximov, Jianyuan Sun, Thomas C SudhofAbstract:Synaptotagmin 2 resembles Synaptotagmin 1, the Ca2+ sensor for fast neurotransmitter release in forebrain synapses, but little is known about Synaptotagmin 2 function. Here, we describe a severely ataxic mouse strain that harbors a single, destabilizing amino-acid substitution (I377N) in Synaptotagmin 2. In Calyx of Held synapses, this mutation causes a delay and a decrease in Ca2+-induced but not in hypertonic sucrose-induced release, suggesting that Synaptotagmin 2 mediates Ca2+ triggering of evoked release in brainstem synapses. Unexpectedly, we additionally observed in Synaptotagmin 2 mutant synapses a dramatic increase in spontaneous release. Synaptotagmin 1-deficient excitatory and inhibitory cortical synapses also displayed a large increase in spontaneous release, demonstrating that this effect was shared among Synaptotagmins 1 and 2. Our data suggest that Synaptotagmin 1 and 2 perform equivalent functions in the Ca2+ triggering of action potential-induced release and in the restriction of spontaneous release, consistent with a general role of Synaptotagmins in controlling ‘release slots' for synaptic vesicles at the active zone.
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Genetic analysis of Synaptotagmin 2 in spontaneous and Ca2+‐triggered neurotransmitter release
The EMBO journal, 2006Co-Authors: Zhiping P. Pang, Josep Rizo, Anton Maximov, Jianyuan Sun, Thomas C SudhofAbstract:Synaptotagmin 2 resembles Synaptotagmin 1, the Ca2+ sensor for fast neurotransmitter release in forebrain synapses, but little is known about Synaptotagmin 2 function. Here, we describe a severely ataxic mouse strain that harbors a single, destabilizing amino-acid substitution (I377N) in Synaptotagmin 2. In Calyx of Held synapses, this mutation causes a delay and a decrease in Ca2+-induced but not in hypertonic sucrose-induced release, suggesting that Synaptotagmin 2 mediates Ca2+ triggering of evoked release in brainstem synapses. Unexpectedly, we additionally observed in Synaptotagmin 2 mutant synapses a dramatic increase in spontaneous release. Synaptotagmin 1-deficient excitatory and inhibitory cortical synapses also displayed a large increase in spontaneous release, demonstrating that this effect was shared among Synaptotagmins 1 and 2. Our data suggest that Synaptotagmin 1 and 2 perform equivalent functions in the Ca2+ triggering of action potential-induced release and in the restriction of spontaneous release, consistent with a general role of Synaptotagmins in controlling ‘release slots' for synaptic vesicles at the active zone.
Molly Craxton - One of the best experts on this subject based on the ideXlab platform.
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A manual collection of Syt, Esyt, Rph3a, Rph3al, Doc2, and Dblc2 genes from 46 metazoan genomes - an open access resource for neuroscience and evolutionary biology
BMC Genomics, 2010Co-Authors: Molly CraxtonAbstract:Background Synaptotagmin proteins were first identified in nervous tissue, residing in synaptic vesicles. Synaptotagmins were subsequently found to form a large family, some members of which play important roles in calcium triggered exocytic events. These members have been investigated intensively, but other family members are not well understood, making it difficult to grasp the meaning of family membership in functional terms. Further difficulty arises as families are defined quite legitimately in different ways: by common descent or by common possession of distinguishing features. One definition does not necessarily imply the other. The evolutionary range of genome sequences now available, can shed more light on Synaptotagmin gene phylogeny and clarify family relationships. The aim of compiling this open access collection of Synaptotagmin and Synaptotagmin-like sequences, is that its use may lead to greater understanding of the biological function of these proteins in an evolutionary context. Results 46 metazoan genomes were examined and their complement of Syt , Esyt , Rph3a , Rph3al , Doc2 and Dblc2 genes identified. All of the sequences were compared, named, then examined in detail. Esyt genes were formerly named Fam62 . The species in this collection are Trichoplax , Nematostella , Capitella , Helobdella , Lottia , Ciona , Strongylocentrotus , Branchiostoma , Ixodes , Daphnia , Acyrthosiphon , Tribolium , Nasonia , Apis , Anopheles , Drosophila , Caenorhabditis , Takifugu , Tetraodon , Gasterosteus , Oryzias , Danio , Xenopus , Anolis , Gallus , Taeniopygia , Ornithorhynchus , Monodelphis , Mus and Homo . All of the data described in this paper is available as additional files. Conclusions Only a subset of Synaptotagmin proteins appear able to function as calcium triggers. Syt1, Syt7 and Syt9 are ancient conserved Synaptotagmins of this type. Some animals carry extensive repertoires of Synaptotagmin genes. Other animals of no less complexity, carry only a small repertoire. Current understanding does not explain why this is so. The biological roles of many Synaptotagmins remain to be understood. This collection of genes offers prospects for fruitful speculation about the functional roles of the Synaptotagmin repertoires of different animals and includes a great range of biological complexity. With reference to this gene collection, functional relationships among Syt , Esyt , Rph3a , Rph3al , Doc2 and Dblc2 genes, which encode similar proteins, can better be assessed in future.
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A manual collection of Syt, Esyt, Rph3a, Rph3al, Doc2, and Dblc2 genes from 46 metazoan genomes - an open access resource for neuroscience and evolutionary biology
BMC genomics, 2010Co-Authors: Molly CraxtonAbstract:Background Synaptotagmin proteins were first identified in nervous tissue, residing in synaptic vesicles. Synaptotagmins were subsequently found to form a large family, some members of which play important roles in calcium triggered exocytic events. These members have been investigated intensively, but other family members are not well understood, making it difficult to grasp the meaning of family membership in functional terms. Further difficulty arises as families are defined quite legitimately in different ways: by common descent or by common possession of distinguishing features. One definition does not necessarily imply the other. The evolutionary range of genome sequences now available, can shed more light on Synaptotagmin gene phylogeny and clarify family relationships. The aim of compiling this open access collection of Synaptotagmin and Synaptotagmin-like sequences, is that its use may lead to greater understanding of the biological function of these proteins in an evolutionary context.
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Synaptotagmin gene content of the sequenced genomes
BMC genomics, 2004Co-Authors: Molly CraxtonAbstract:Synaptotagmins exist as a large gene family in mammals. There is much interest in the function of certain family members which act crucially in the regulated synaptic vesicle exocytosis required for efficient neurotransmission. Knowledge of the functions of other family members is relatively poor and the presence of Synaptotagmin genes in plants indicates a role for the family as a whole which is wider than neurotransmission. Identification of the Synaptotagmin genes within completely sequenced genomes can provide the entire Synaptotagmin gene complement of each sequenced organism. Defining the detailed structures of all the Synaptotagmin genes and their encoded products can provide a useful resource for functional studies and a deeper understanding of the evolution of the gene family. The current rapid increase in the number of sequenced genomes from different branches of the tree of life, together with the public deposition of evolutionarily diverse transcript sequences make such studies worthwhile. I have compiled a detailed list of the Synaptotagmin genes of Caenorhabditis, Anopheles, Drosophila, Ciona, Danio, Fugu, Mus, Homo, Arabidopsis and Oryza by examining genomic and transcript sequences from public sequence databases together with some transcript sequences obtained by cDNA library screening and RT-PCR. I have compared all of the genes and investigated the relationship between plant Synaptotagmins and their non-Synaptotagmin counterparts. I have identified and compared 98 Synaptotagmin genes from 10 sequenced genomes. Detailed comparison of transcript sequences reveals abundant and complex variation in Synaptotagmin gene expression and indicates the presence of Synaptotagmin genes in all animals and land plants. Amino acid sequence comparisons indicate patterns of conservation and diversity in function. Phylogenetic analysis shows the origin of Synaptotagmins in multicellular eukaryotes and their great diversification in animals. Synaptotagmins occur in land plants and animals in combinations of 4–16 in different species. The detailed delineation of the Synaptotagmin genes presented here, will allow easier identification of Synaptotagmins in future. Since the functional roles of many of these genes are unknown, this gene collection provides a useful resource for future studies.
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Alternative splicing of Synaptotagmins involving transmembrane exon skipping
FEBS letters, 1999Co-Authors: Molly Craxton, Michel GoedertAbstract:The Synaptotagmin gene family currently includes 12 members. Analysis of the three known genomic Synaptotagmin sequences reveals conserved exon-intron patterns which delineate the Synaptotagmin structural domains. We used expressed sequence tag, reverse transcription PCR and RNAse protection assay analysis of Synaptotagmin messenger RNAs to demonstrate the occurrence of alternative splicing events involving a number of exons. Exon-skipped messages where transmembrane sequences have been removed or altered were found to be abundantly expressed by Synaptotagmins 1, 4, 6 and 7. Although the expression of most Synaptotagmins predominates in neural tissue, we find that by contrast, Synaptotagmin 6 is more abundant in thymus.
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Synaptotagmin V: a novel Synaptotagmin isoform expressed in rat brain
FEBS letters, 1995Co-Authors: Molly Craxton, Michel GoedertAbstract:Regulated Ca2+-dependent release of transmitters from synaptic vesicles is an important characteristic of chemical neurotransmission. Synaptotagmins are abundant synaptic vesicle transmembrane proteins that probably function as Ca2+ sensors. Molecular cloning has identified four different Synaptotagmin isoforms in mammals. We report here the cloning and sequencing of a novel isoform of 386 amino acids. Synaptotagmin V is 54% identical in sequence to Synaptotagmin I and possesses all the domains that characterise this multigene family. It is expressed at high levels in rat brain, but not in spinal cord or a number of peripheral non-neuronal tissues.
Anton Maximov - One of the best experts on this subject based on the ideXlab platform.
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Push-and-pull regulation of the fusion pore by Synaptotagmin-7
Proceedings of the National Academy of Sciences of the United States of America, 2010Co-Authors: Margarita Segovia, Thomas C Sudhof, Anton Maximov, Eva Alés, María Ángeles Montes, Imelda Bonifas, Imane Jemal, Manfred Lindau, Guillermo Alvarez De ToledoAbstract:In chromaffin cells, Ca2+ binding to Synaptotagmin-1 and -7 triggers exocytosis by promoting fusion pore opening and fusion pore expansion. Synaptotagmins contain two C2 domains that both bind Ca2+ and contribute to exocytosis; however, it remains unknown whether the C2 domains act similarly or differentially to promote opening and expansion of fusion pores. Here, we use patch amperometry measurements in WT and Synaptotagmin-7–mutant chromaffin cells to analyze the role of Ca2+ binding to the two Synaptotagmin-7 C2 domains in exocytosis. We show that, surprisingly, Ca2+ binding to the C2A domain suffices to trigger fusion pore opening but that the resulting fusion pores are unstable and collapse, causing a dramatic increase in kiss-and-run fusion events. Thus, Synaptotagmin-7 controls fusion pore dynamics during exocytosis via a push-and-pull mechanism in which Ca2+ binding to both C2 domains promotes fusion pore opening, but the C2B domain is selectively essential for continuous expansion of an otherwise unstable fusion pore.
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Synaptotagmin 1 and 7 are functionally overlapping ca2 sensors for exocytosis in adrenal chromaffin cells
Proceedings of the National Academy of Sciences of the United States of America, 2008Co-Authors: Jeansebastien Schonn, Thomas C Sudhof, Anton Maximov, Ye Lao, Jakob B SorensenAbstract:Synaptotagmin-1, the canonical isoform of the Synaptotagmin family, is a Ca2+ sensor for fast synchronous neurotransmitter release in forebrain neurons and chromaffin cells. Even though deletion of Synaptotagmin-1 abolishes fast exocytosis in chromaffin cells, it reduces overall secretion by only 20% because of the persistence of slow exocytosis. Therefore, another Ca2+ sensor dominates release in these cells. Synaptotagmin-7 has a higher Ca2+ affinity and slower binding kinetics than Synaptotagmin-1, matching the proposed properties for the second, slower Ca2+ sensor. Here, we examined Ca2+-triggered exocytosis in chromaffin cells from KO mice lacking Synaptotagmin-7, and from knockin mice containing normal levels of a mutant Synaptotagmin-7 whose C2B domain does not bind Ca2+. In both types of mutant chromaffin cells, Ca2+-triggered exocytosis was decreased dramatically. Moreover, in chromaffin cells lacking both Synaptotagmin-1 and -7, only a very slow release component, accounting for ≈30% of WT exocytosis, persisted. These data establish Synaptotagmin-7 as a major Ca2+ sensor for exocytosis in chromaffin cells, which, together with Synaptotagmin-1, mediates almost all of the Ca2+ triggering of exocytosis in these cells, a surprising result, considering the lack of a role of Synaptotagmin-7 in synaptic vesicle exocytosis.
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impaired insulin secretion and glucose intolerance in Synaptotagmin 7 null mutant mice
Proceedings of the National Academy of Sciences of the United States of America, 2008Co-Authors: Natalia Gustavsson, Thomas C Sudhof, Anton Maximov, Ye Lao, Weiping Han, Jen Chieh Chuang, Elena Kostromina, Joyce J Repa, George K RaddaAbstract:Vertebrates express at least 15 different Synaptotagmins with the same domain structure but diverse localizations and tissue distributions. Synaptotagmin-1,-2, and -9 act as calcium sensors for the fast phrase of neurotransmitter release, and Synaptotagmin-12 acts as a calcium-independent modulator of release. The exact functions of the remaining 11 Synaptotagmins, however, have not been established. By analogy to the role of Synaptotagmin-1, -2, and -9 in neurotransmission, these other Synaptotagmins may serve as Ca2+ transducers regulating other Ca2+-dependent membrane processes, such as insulin secretion in pancreatic β-cells. Of these other Synaptotagmins, Synaptotagmin-7 is one of the most abundant and is present in pancreatic β-cells. To determine whether Synaptotagmin-7 regulates Ca2+-dependent insulin secretion, we analyzed Synaptotagmin-7 null mutant mice for glucose tolerance and insulin release. Here, we show that Synaptotagmin-7 is required for the maintenance of systemic glucose tolerance and glucose-stimulated insulin secretion. Mutant mice have normal insulin sensitivity, insulin production, islet architecture and ultrastructural organization, and metabolic and calcium responses but exhibit impaired glucose-induced insulin secretion, indicating a calcium-sensing defect during insulin-containing secretory granule exocytosis. Taken together, our findings show that Synaptotagmin-7 functions as a positive regulator of insulin secretion and may serve as a calcium sensor controlling insulin secretion in pancreatic β cells.
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Genetic analysis of Synaptotagmin-7 function in synaptic vesicle exocytosis
Proceedings of the National Academy of Sciences of the United States of America, 2008Co-Authors: Anton Maximov, Josep Rizo, Ye Lao, Jakob B Sorensen, Xiaocheng Chen, Thomas C SudhofAbstract:Synaptotagmin-7 is a candidate Ca2+ sensor for exocytosis that is at least partly localized to synapses. Similar to Synaptotagmin-1, which functions as a Ca2+ sensor for fast synaptic vesicle (SV) exocytosis, Synaptotagmin-7 contains C2A and C2B domains that exhibit Ca2+-dependent phospholipid binding. However, Synaptotagmin-7 cannot replace Synaptotagmin-1 as a Ca2+ sensor for fast SV exocytosis, raising questions about the physiological significance of its Ca2+-binding properties. Here, we examine how Synaptotagmin-7 binds Ca2+ and test whether this Ca2+ binding regulates Ca2+-triggered SV exocytosis. We show that the Synaptotagmin-7 C2A domain exhibits a Ca2+-binding mode similar to that of the Synaptotagmin-1 C2A domain, suggesting that the Synaptotagmin-1 and -7 C2 domains generally employ comparable Ca2+-binding mechanisms. We then generated mutant mice that lack Synaptotagmin-7 or contain point mutations inactivating Ca2+ binding either to both C2 domains of Synaptotagmin-7 or only to its C2B domain. Synaptotagmin-7-mutant mice were viable and fertile. Inactivation of Ca2+ binding to both C2 domains caused an ≈70% reduction in Synaptotagmin-7 levels, whereas inactivation of Ca2+ binding to only the C2B domain did not alter Synaptotagmin-7 levels. The Synaptotagmin-7 deletion did not change fast synchronous release, slow asynchronous release, or short-term synaptic plasticity of release of neurotransmitters. Thus, our results show that Ca2+ binding to the Synaptotagmin-7 C2 domains is physiologically important for stabilizing Synaptotagmin-7, but that Ca2+ binding by Synaptotagmin-7 likely does not regulate SV exocytosis, consistent with a role for Synaptotagmin-7 in other forms of Ca2+-dependent synaptic exocytosis.
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Synaptotagmin-12, a synaptic vesicle phosphoprotein that modulates spontaneous neurotransmitter release.
The Journal of cell biology, 2006Co-Authors: Anton Maximov, Ok Ho Shin, Xinran Liu, Thomas C SudhofAbstract:Central synapses exhibit spontaneous neurotransmitter release that is selectively regulated by cAMP-dependent protein kinase A (PKA). We now show that synaptic vesicles contain Synaptotagmin-12, a Synaptotagmin isoform that differs from classical Synaptotagmins in that it does not bind Ca2+. In synaptic vesicles, Synaptotagmin-12 forms a complex with Synaptotagmin-1 that prevents Synaptotagmin-1 from interacting with SNARE complexes. We demonstrate that Synaptotagmin-12 is phosphorylated by cAMP-dependent PKA on serine97, and show that expression of Synaptotagmin-12 in neurons increases spontaneous neurotransmitter release by approximately threefold, but has no effect on evoked release. Replacing serine97 by alanine abolishes Synaptotagmin-12 phosphorylation and blocks its effect on spontaneous release. Our data suggest that spontaneous synaptic-vesicle exocytosis is selectively modulated by a Ca2+-independent Synaptotagmin isoform, Synaptotagmin-12, which is controlled by cAMP-dependent phosphorylation.
