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Katsuhiko Mikoshiba - One of the best experts on this subject based on the ideXlab platform.
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synaptotagmin like protein 1 3 a novel family of c terminal type tandem c2 proteins
2001Co-Authors: Mitsunori Fukuda, Katsuhiko MikoshibaAbstract:Synaptotagmins (Syt), rabphilin-3A, and Doc2 belong to a family of carboxyl terminal type (C-type) tandem C2 proteins and are thought to be involved in vesicular trafficking. We have cloned and characterized a novel family of C-type tandem C2 proteins, designated Slp1-3 (synaptotagmin-like protein 1-3). The Slp1-3 C2 domains show high homology to granuphilin-a C2 domains, but the amino-terminal domain of Slp1-3 does not contain any known protein motifs or a transmembrane domain. A subcellular fractionation study indicated that Slp1-3 proteins are peripheral membrane proteins. Phospholipid binding experiments indicated that Slp3 is a Ca(2+)-dependent isoform, but Slp1 and Slp2 are Ca(2+)-independent isoforms, because only the Slp3 C2A domain showed Ca(2+)-dependent phospholipid binding activity. The C-terminus of Slp1-3 also bound neurexin Ialpha in vitro, in the same manner as Syt family proteins, which may be important for the membrane association of Slp1-3. In addition, Slp family proteins are differentially distributed in different mouse tissues and at different developmental stages.
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conserved n terminal cysteine motif is essential for homo and heterodimer formation of Synaptotagmins iii v vi and x
1999Co-Authors: Mitsunori Fukuda, Eiko Kanno, Katsuhiko MikoshibaAbstract:The Synaptotagmins now constitute a large family of membrane proteins characterized by one transmembrane region and two C2 domains. Dimerization of synaptotagmin (Syt) I, a putative low affinity Ca(2+) sensor for neurotransmitter release, is thought to be important for expression of function during exocytosis of synaptic vesicles. However, little is known about the self-dimerization properties of other isoforms. In this study, we demonstrate that a subclass of Synaptotagmins (III, V, VI, and X) (Ibata, K., Fukuda, M., and Mikoshiba, K. (1998) J. Biol. Chem. 273, 12267-12273) forms beta-mercaptoethanol-sensitive homodimers and identify three evolutionarily conserved cysteine residues at the N terminus (N-terminal cysteine motif, at amino acids 10, 21, and 33 of mouse Syt III) that are not conserved in other isoforms. Site-directed mutagenesis of these cysteine residues and co-immunoprecipitation experiments clearly indicate that the first cysteine residue is essential for the stable homodimer formation of Syt III, V, or VI, and heterodimer formation between Syts III, V, VI, and X. We also show that native Syt III from mouse brain forms a beta-mercaptoethanol-sensitive homodimer. Our results suggest that the cysteine-based heterodimerization between Syt III and Syt V, VI, or X, which have different biochemical properties, may modulate the proposed function of Syt III as a putative high affinity Ca(2+) sensor for neurotransmitter release.
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inositol 1 3 4 5 tetrakisphosphate binding activities of neuronal and non neuronal Synaptotagmins identification of conserved amino acid substitutions that abolish inositol 1 3 4 5 tetrakisphosphate binding to Synaptotagmins iii v and x
1998Co-Authors: Keiji Ibata, Mitsunori Fukuda, Katsuhiko MikoshibaAbstract:Abstract Synaptotagmins I and II are essential for Ca2+-regulated exocytosis of synaptic vesicles from neurons, probably serving as Ca2+ sensors. This Ca2+-sensing function is thought to be disrupted by binding of an inositol 1,3,4,5-tetrakisphosphate (IP4) to the C2B domain of synaptotagmin I or II (Fukuda, M., Moreira, J. E., Lewis, F. M. T., Sugimori, M., Niinobe, M., Mikoshiba, K., and Llinas, R. (1995) Proc. Natl. Acad. Sci. U. S. A.92, 10708–10712). Recently, several synaptotagmin isoforms, expressed outside the nervous system, have been identified in rats and proposed to be involved in constitutive vesicle traffic. To test whether the inositol high polyphosphates also regulate constitutive vesicle traffic by binding to the non-neuronal Synaptotagmins, we examined the IP4 binding properties of the recombinant C2 domains of both neuronal (III, V, X, and XI) and non-neuronal (VI–VIII and IX) Synaptotagmins. The C2B domains of Synaptotagmins VII–IX and XI had strong IP4 binding activity, but the C2B domain of synaptotagmin VI showed very weak IP4 binding activity. In contrast, there was no significant IP4 binding activity of the C2B domains of Synaptotagmins III, V, and X or any of the C2A domains. A phylogenetic tree of the C2 domains of 11 isoforms revealed that Synaptotagmins III, V, VI, and X (IP4-insensitive or very weak IP4-binding isoforms) belong to the same branch. Based on the sequence comparison between the IP4-sensitive and -insensitive isoforms, we performed site-directed mutagenesis of synaptotagmin III and identified several amino acid substitutions that abolish IP4 binding activity. Our data suggest that the inositol high polyphosphates might also regulate constitutive vesicle traffic via binding to the IP4-sensitive non-neuronal Synaptotagmins.
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functional diversity of c2 domains of synaptotagmin family mutational analysis of inositol high polyphosphate binding domain
1995Co-Authors: Mitsunori Fukuda, Toshio Kojima, Jun Aruga, Michio Niinobe, Katsuhiko MikoshibaAbstract:Synaptotagmins I and II are inositol high polyphosphate series (inositol 1,3,4,5-tetrakisphosphate (IP4), inositol 1,3,4,5,6-pentakisphosphate, and inositol 1,2,3,4,5,6-hexakisphosphate) binding proteins, which are thought to be essential for Ca(2+)-regulated exocytosis of neurosecretory vesicles. In this study, we analyzed the inositol high polyphosphate series binding site in the C2B domain by site-directed mutagenesis and compared the IP4 binding properties of the C2B domains of multiple Synaptotagmins (II-IV). The IP4 binding domain of synaptotagmin II is characterized by a cluster of highly conserved, positively charged amino acids (321 GKRLKKKKTTVKKK 324). Among these, three lysine residues, at positions 327, 328, and 332 in the middle of the C2B domain, which is not conserved in the C2A domain, were found to be essential for IP4 binding in synaptotagmin II. When these lysine residues were altered to glutamine, the IP4 binding ability was completely abolished. The primary structures of the IP4 binding sites are highly conserved among Synaptotagmins I through IV. However, synaptotagmin III did not show significant binding ability, which may be due to steric hindrance by the C-terminal flanking region. These functional diversities of C2B domains suggest that not all Synaptotagmins function as inositol high polyphosphate sensors at the synaptic vesicle.
Mitsunori Fukuda - One of the best experts on this subject based on the ideXlab platform.
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exome sequencing reveals a homozygous syt14 mutation in adult onset autosomal recessive spinocerebellar ataxia with psychomotor retardation
2011Co-Authors: Kunihiro Yoshida, Mitsunori Fukuda, Takao Yasuda, Yoko Fukuda, Hiroshi Morita, Shuichi Ikeda, Rumiko Kato, Yoshinori Tsurusaki, Noriko Miyake, Hirotomo SaitsuAbstract:Autosomal-recessive cerebellar ataxias (ARCAs) are clinically and genetically heterogeneous disorders associated with diverse neurological and nonneurological features that occur before the age of 20. Currently, mutations in more than 20 genes have been identified, but approximately half of the ARCA patients remain genetically unresolved. In this report, we describe a Japanese family in which two siblings have slow progression of a type of ARCA with psychomotor retardation. Using whole-exome sequencing combined with homozygosity mapping, we identified a homozygous missense mutation in SYT14, encoding synaptotagmin XIV (SYT14). Expression analysis of the mRNA of SYT14 by a TaqMan assay confirmed that SYT14 mRNA was highly expressed in human fetal and adult brain tissue as well as in the mouse brain (especially in the cerebellum). In an in vitro overexpression system, the mutant SYT14 showed intracellular localization different from that of the wild-type. An immunohistochemical analysis clearly showed that SYT14 is specifically localized to Purkinje cells of the cerebellum in humans and mice. Synaptotagmins are associated with exocytosis of secretory vesicles (including synaptic vesicles), indicating that the alteration of the membrane-trafficking machinery by the SYT14 mutation may represent a distinct pathomechanism associated with human neurodegenerative disorders.
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distinct developmental expression of synaptotagmin i and ix in the mouse brain
2006Co-Authors: Mitsunori FukudaAbstract:: Synaptotagmin IX has been postulated to function as a major Ca2+ sensor for dense-core vesicle exocytosis in neuroendocrine cells. In this study, we investigated the subcellular localization and developmental expression profile of synaptotagmin IX in the mouse brain and found that it is mainly present in the dense-core vesicle fraction, which is devoid of synaptotagmin I and synaptophysin. We also found that the synaptotagmin IX expression level is constant throughout the postnatal development of the mouse brain, whereas the Synaptotagmins I, II, III, VI, and XII are upregulated in parallel with synaptogenesis. These findings suggest that synaptotagmin IX regulates the transport of certain vesicles in the brain other than synaptic vesicles.
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ca2 dependent synaptotagmin binding to snap 25 is essential for ca2 triggered exocytosis
2002Co-Authors: Xiaodong Zhang, Mitsunori Fukuda, Mindy J Kimmiller, Judith A Kowalchyk, Thomas MartinAbstract:Synaptotagmin is a proposed Ca2+ sensor on the vesicle for regulated exocytosis and exhibits Ca2+-dependent binding to phospholipids, syntaxin, and SNAP-25 in vitro, but the mechanism by which Ca2+ triggers membrane fusion is uncertain. Previous studies suggested that SNAP-25 plays a role in the Ca2+ regulation of secretion. We found that Synaptotagmins I and IX associate with SNAP-25 during Ca2+-dependent exocytosis in PC12 cells, and we identified C-terminal amino acids in SNAP-25 (Asp179, Asp186, Asp193) that are required for Ca2+-dependent synaptotagmin binding. Replacement of SNAP-25 in PC12 cells with SNAP-25 containing C-terminal Asp mutations led to a loss-of-function in regulated exocytosis at the Ca2+-dependent fusion step. These results indicate that the Ca2+-dependent interaction of synaptotagmin with SNAP-25 is essential for the Ca2+-dependent triggering of membrane fusion.
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synaptotagmin like protein 1 3 a novel family of c terminal type tandem c2 proteins
2001Co-Authors: Mitsunori Fukuda, Katsuhiko MikoshibaAbstract:Synaptotagmins (Syt), rabphilin-3A, and Doc2 belong to a family of carboxyl terminal type (C-type) tandem C2 proteins and are thought to be involved in vesicular trafficking. We have cloned and characterized a novel family of C-type tandem C2 proteins, designated Slp1-3 (synaptotagmin-like protein 1-3). The Slp1-3 C2 domains show high homology to granuphilin-a C2 domains, but the amino-terminal domain of Slp1-3 does not contain any known protein motifs or a transmembrane domain. A subcellular fractionation study indicated that Slp1-3 proteins are peripheral membrane proteins. Phospholipid binding experiments indicated that Slp3 is a Ca(2+)-dependent isoform, but Slp1 and Slp2 are Ca(2+)-independent isoforms, because only the Slp3 C2A domain showed Ca(2+)-dependent phospholipid binding activity. The C-terminus of Slp1-3 also bound neurexin Ialpha in vitro, in the same manner as Syt family proteins, which may be important for the membrane association of Slp1-3. In addition, Slp family proteins are differentially distributed in different mouse tissues and at different developmental stages.
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synaptotagmin i and iv define distinct populations of neuronal transport vesicles
2000Co-Authors: Frederique Berton, Cecile Iborra, Benedicte Dargent, Mitsunori Fukuda, Juan José Garrido, Véronique Cornet, Michael Seagar, Beatrice MarquezeAbstract:: Mammalian Synaptotagmins constitute a multigene family of at least 11 membrane proteins. We have characterized synaptotagmin IV using antibodies directed against the C2A domain of the protein. Antibodies reacted specifically with a protein band that migrated as a 41-44 kDa doublet. Synaptotagmin IV expression was regulated throughout development. A strong decrease in the amount detected by Western blotting occurred between postnatal day 5 and adulthood, in agreement with studies on the expression of synaptotagmin IV transcripts. In subcellular fractionation, synaptotagmin IV was not detected in the synaptic vesicle-enriched fraction. Immunofluorescence microscopy was concordant with this finding. In 6-day-old rat cerebellum and cultured hippocampal neurons the subcellular distribution of synaptotagmin IV was clearly different from that of synaptotagmin I. Synaptotagmin IV displayed a punctate non-polarized distribution on neuronal extensions, whereas synaptotagmin I staining was essentially synaptic. Synaptotagmin IV staining was also observed in the soma in strong perinuclear fluorescent puncta superimposed on that of Golgi/TGN markers. Furthermore, synaptotagmin IV was seen in the proximal part of the growth cone domain and not in the microfilament-rich region which includes filopodia. Co-localizations with the adhesion molecules vinculin and zyxin at the proximal part of growth cones were observed. Synaptotagmin IV may thus be involved in the regulation of specific membrane-trafficking pathways during brain development.
Pietro De Camilli - One of the best experts on this subject based on the ideXlab platform.
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In Vitro Assays to Measure the Membrane Tethering and Lipid Transport Activities of the Extended Synaptotagmins.
2019Co-Authors: Xin Bian, Pietro De CamilliAbstract:The three extended Synaptotagmins (E-Syts) are endoplasmic reticulum (ER)-localized membrane proteins that mediate tethering of the ER to the plasma membrane (PM) via C2 domain-dependent interactions regulated by Ca2+ and/or PI(4,5)P2. The E-Syts also contains a Synaptotagmin-like Mitochondrial lipid-binding Protein (SMP) domain, a lipid-harboring module through which they mediate lipid transport between the two adjacent membranes. Here, we describe in vitro liposome-based methods to study the membrane tethering and lipid transport functions of E-Syt1. Its membrane tethering activity is monitored through a turbidity-based assay, and its lipid transport property is analyzed via fluorescence resonance energy transfer (FRET)-based assay. These in vitro methods have enabled us to gain insight into the mechanism of action and regulation of E-Syt1, such as the role of Ca2+ in releasing E-Syt1 from an autoinhibitory conformation. The same methods could be adapted to the study of other lipid transport proteins that function at membrane contact sites.
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The Extended-Synaptotagmins
2017Co-Authors: Yasunori Saheki, Pietro De CamilliAbstract:The extended-Synaptotagmins (tricalbins in yeast) derive their name from their partial domain structure similarity to the Synaptotagmins, which are characterized by an N-terminal membrane anchor and cytosolically exposed C2 domains. However, they differ from the Synaptotagmins in localization and function. The Synaptotagmins tether secretory vesicles, including synaptic vesicles, to the plasma membrane (PM) via their C2 domains and regulate their Ca2+ triggered exocytosis. In contrast, the extended-Synaptotagmins are resident proteins of the endoplasmic reticulum (ER), which tether this organelle to the plasma membrane via their C2 domains, but not as a premise to fusion of the two membranes. They transport glycerolipids between the two bilayers via their lipid-harboring SMP domains and Ca2+ regulates their membrane tethering and lipid transport function. Additionally, the extended-Synaptotagmins are more widely expressed in different organisms, as they are present not only in animal cells, but also in fungi and plants, which do not express the Synaptotagmins. Thus, they have a more general function than the Synaptotagmins, whose appearance in animal species correlated with the occurrence of Ca2+ triggered exocytosis. This article is part of a Special Issue entitled: Membrane Contact Sites edited by Christian Ungermann and Benoit Kornmann.
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Structure of a lipid-bound Extended-Synaptotagmin indicates a role in lipid transfer
2016Co-Authors: Curtis M Schauder, Pietro De Camilli, Yasunori Saheki, Pradeep Narayanaswamy, Federico Torta, Markus R Wenk, Karin M ReinischAbstract:Growing evidence suggests that close appositions between the endoplasmic reticulum (ER) and other membranes, including appositions with the plasma membrane (PM), mediate exchange of lipids between the two bilayers. The mechanisms of such exchange, which allows lipid transfer independently of vesicular transport, remain poorly understood. The presence of an SMP (synaptotagmin-like-mitochondrial-lipid binding protein) domain, a proposed lipid binding module, in several proteins localized at membrane contact sites raised the possibility that such domains may be implicated in lipid transport1,2. SMP-containing proteins include components of the ERMES complex, an ER-mitochondrial tether3, and the Extended-Synaptotagmins/tricalbins, which are ER-PM tethers4-6. Here we present at 2.44 Å resolution the crystal structure of a fragment of Extended-Synaptotagmin 2 (E-Syt2), including an SMP domain and two adjacent C2 domains. The SMP domain has a beta-barrel structure like protein modules in the TULIP superfamily. It dimerizes to form a ~90 Å long cylinder traversed by a channel lined entirely with hydrophobic residues, with the two C2A-C2B fragments forming arched structures flexibly linked to the SMP domain. Importantly, structural analysis complemented by mass spectrometry revealed the presence of glycerophospholipids in the E-Syt2 SMP channel, indicating a direct role for E-Syts in lipid transport. These findings provide strong evidence for a role of SMP domain containing proteins in the control of lipid transfer at membrane contact sites and have broad implication beyond the field of ER to PM appositions
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triggered ca2 influx is required for extended synaptotagmin 1 induced er plasma membrane tethering
2015Co-Authors: Olof Idevallhagren, Beichen Xie, Pietro De CamilliAbstract:The extended Synaptotagmins (E-Syts) are ER proteins that act as Ca(2+)-regulated tethers between the ER and the plasma membrane (PM) and have a putative role in lipid transport between the two membranes. Ca(2+) regulation of their tethering function, as well as the interplay of their different domains in such function, remains poorly understood. By exposing semi-intact cells to buffers of variable Ca(2+) concentrations, we found that binding of E-Syt1 to the PI(4,5)P2-rich PM critically requires its C2C and C2E domains and that the EC50 of such binding is in the low micromolar Ca(2+) range. Accordingly, E-Syt1 accumulation at ER-PM contact sites occurred only upon experimental manipulations known to achieve these levels of Ca(2+) via its influx from the extracellular medium, such as store-operated Ca(2+) entry in fibroblasts and membrane depolarization in β-cells. We also show that in spite of their very different physiological functions, membrane tethering by E-Syt1 (ER to PM) and by synaptotagmin (secretory vesicles to PM) undergo a similar regulation by plasma membrane lipids and cytosolic Ca(2+).
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structure of a lipid bound extended synaptotagmin indicates a role in lipid transfer
2014Co-Authors: Curtis M Schauder, Pietro De Camilli, Xudong Wu, Yasunori Saheki, Pradeep Narayanaswamy, Federico Torta, Markus R Wenk, Karin M ReinischAbstract:Several proteins localized at membrane contact sites contain an SMP domain, which has been proposed to act as a lipid-binding module; here, the crystal structure of a fragment of the extended synaptotagmin 2 protein, including its SMP, is presented, and indicates that this protein may have a direct role in lipid transport. Growing evidence suggests that close appositions between the endoplasmic reticulum (ER) and other membranes, including appositions with the plasma membrane (PM), mediate exchange of lipids between these bilayers. The mechanisms of such exchange, which allows lipid transfer independently of vesicular transport, remain poorly understood. The presence of a synaptotagmin-like mitochondrial-lipid-binding protein (SMP) domain, a proposed lipid-binding module, in several proteins localized at membrane contact sites has raised the possibility that such domains may be implicated in lipid transport1,2. SMP-containing proteins include components of the ERMES complex, an ER–mitochondrial tether3, and the extended Synaptotagmins (known as tricalbins in yeast), which are ER–PM tethers4,5,6. Here we present at 2.44 A resolution the crystal structure of a fragment of human extended synaptotagmin 2 (E-SYT2), including an SMP domain and two adjacent C2 domains. The SMP domain has a β-barrel structure like protein modules in the tubular-lipid-binding (TULIP) superfamily. It dimerizes to form an approximately 90-A-long cylinder traversed by a channel lined entirely with hydrophobic residues, with the two C2A–C2B fragments forming arched structures flexibly linked to the SMP domain. Importantly, structural analysis complemented by mass spectrometry revealed the presence of glycerophospholipids in the E-SYT2 SMP channel, indicating a direct role for E-SYTs in lipid transport. These findings provide strong evidence for a role of SMP-domain-containing proteins in the control of lipid transfer at membrane contact sites and have broad implications beyond the field of ER-to-PM appositions.
Deda C. Gillespie - One of the best experts on this subject based on the ideXlab platform.
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Synaptotagmins i and ii in the developing rat auditory brainstem synaptotagmin i is transiently expressed in glutamate releasing immature inhibitory terminals
2011Co-Authors: Alan P. Cooper, Deda C. GillespieAbstract:The lateral superior olive (LSO), a nucleus in the auditory brainstem, computes interaural intensity differences for sound localization by comparing converging excitatory and inhibitory inputs that carry tonotopically matched information from the two ears. Tonotopic refinement in the inhibitory projection pathway from the medial nucleus of the trapezoid body (MNTB) is known to be established during the first postnatal week in rats. During this period, immature MNTB terminals in the LSO contain vesicular transporters for both inhibitory and excitatory amino acids and release glutamate. The primary Ca2+ sensors for vesicular release in the CNS are understood to be Synaptotagmins, and in adult auditory brainstem synaptotagmin 2 is the predominant synaptotagmin. We asked here whether a different Ca2+ sensor might be expressed in the immature auditory brainstem. We have found that synaptotagmin 1 is indeed expressed transiently in the immature auditory brainstem, most highly in those areas that receive glutamate-releasing immature inhibitory inputs from the MNTB, and that during the first postnatal week synaptotagmin 1 co-localizes with the vesicular glutamate transporter VGLUT3, a marker of glutamate-releasing immature inhibitory terminals from the MNTB. We suggest that immature MNTB terminals may contain two populations of synaptic vesicles, one expressing the vesicular inhibitory amino acid transporter together with synaptotagmin 2 and another expressing VGLUT3 together with synaptotagmin 1. Because Ca2+ sensing is an important determinant of release properties for the presynaptic terminal, differential expression of the Synaptotagmins might allow the differential release of excitatory and inhibitory neurotransmitters in response to differing patterns of neural activity. J. Comp. Neurol. 519:2417–2433, 2011. © 2011 Wiley-Liss, Inc.
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Synaptotagmins I and II in the developing rat auditory brainstem: Synaptotagmin I is transiently expressed in glutamate-releasing immature inhibitory terminals.
2011Co-Authors: Alan P. Cooper, Deda C. GillespieAbstract:The lateral superior olive (LSO), a nucleus in the auditory brainstem, computes interaural intensity differences for sound localization by comparing converging excitatory and inhibitory inputs that carry tonotopically matched information from the two ears. Tonotopic refinement in the inhibitory projection pathway from the medial nucleus of the trapezoid body (MNTB) is known to be established during the first postnatal week in rats. During this period, immature MNTB terminals in the LSO contain vesicular transporters for both inhibitory and excitatory amino acids and release glutamate. The primary Ca(2+) sensors for vesicular release in the CNS are understood to be Synaptotagmins, and in adult auditory brainstem synaptotagmin 2 is the predominant synaptotagmin. We asked here whether a different Ca(2+) sensor might be expressed in the immature auditory brainstem. We have found that synaptotagmin 1 is indeed expressed transiently in the immature auditory brainstem, most highly in those areas that receive glutamate-releasing immature inhibitory inputs from the MNTB, and that during the first postnatal week synaptotagmin 1 co-localizes with the vesicular glutamate transporter VGLUT3, a marker of glutamate-releasing immature inhibitory terminals from the MNTB. We suggest that immature MNTB terminals may contain two populations of synaptic vesicles, one expressing the vesicular inhibitory amino acid transporter together with synaptotagmin 2 and another expressing VGLUT3 together with synaptotagmin 1. Because Ca(2+) sensing is an important determinant of release properties for the presynaptic terminal, differential expression of the Synaptotagmins might allow the differential release of excitatory and inhibitory neurotransmitters in response to differing patterns of neural activity.
Jakob B Sorensen - One of the best experts on this subject based on the ideXlab platform.
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synaptotagmin 7 places dense core vesicles at the cell membrane to promote munc13 2 and ca2 dependent priming
2021Co-Authors: Bassam Tawfik, Joana S Martins, Sebastien Houy, Cordelia Imig, Paulo S Pinheiro, Sonja M Wojcik, Nils Brose, Benjamin H Cooper, Jakob B SorensenAbstract:Synaptotagmins confer calcium-dependence to the exocytosis of secretory vesicles, but how coexpressed Synaptotagmins interact remains unclear. We find that synaptotagmin-1 and synaptotagmin-7 when present alone act as standalone fast and slow Ca2+-sensors for vesicle fusion in mouse chromaffin cells. When present together, synaptotagmin-1 and synaptotagmin-7 are found in largely non-overlapping clusters on dense-core vesicles. Synaptotagmin-7 stimulates Ca2+-dependent vesicle priming and inhibits depriming, and it promotes ubMunc13-2- and phorbolester-dependent priming, especially at low resting calcium concentrations. The priming effect of synaptotagmin-7 increases the number of vesicles fusing via synaptotagmin-1, while negatively affecting their fusion speed, indicating both synergistic and competitive interactions between Synaptotagmins. Synaptotagmin-7 places vesicles in close membrane apposition (<6 nm); without it, vesicles accumulate out of reach of the fusion complex (20-40 nm). We suggest that a synaptotagmin-7-dependent movement toward the membrane is involved in Munc13-2/phorbolester/Ca2+-dependent priming as a prelude to fast and slow exocytosis triggering.
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doc2b acts as a calcium sensor for vesicle priming requiring synaptotagmin 1 munc13 2 and snares
2017Co-Authors: Sebastien Houy, Paulo S Pinheiro, Matthijs Verhage, Alexander J Groffen, Iwona Ziomkiewicz, Jakob B SorensenAbstract:Doc2B is a cytosolic protein with binding sites for Munc13 and Tctex-1 (dynein light chain), and two C2-domains that bind to phospholipids, Ca2+ and SNAREs. Whether Doc2B functions as a calcium sensor akin to Synaptotagmins, or in other calcium-independent or calcium-dependent capacities is debated. We here show by mutation and overexpression that Doc2B plays distinct roles in two sequential priming steps in mouse adrenal chromaffin cells. Mutating Ca2+-coordinating aspartates in the C2A-domain localizes Doc2B permanently at the plasma membrane, and renders an upstream priming step Ca2+-independent, whereas a separate function in downstream priming depends on SNARE-binding, Ca2+-binding to the C2B-domain of Doc2B, interaction with ubMunc13-2 and the presence of synaptotagmin-1. Another function of Doc2B - inhibition of release during sustained calcium elevations - depends on an overlapping protein domain (the MID-domain), but is separate from its Ca2+-dependent priming function. We conclude that Doc2B acts as a vesicle priming protein.
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synaptotagmin 1 and 7 are functionally overlapping ca2 sensors for exocytosis in adrenal chromaffin cells
2008Co-Authors: Jeansebastien Schonn, 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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different effects on fast exocytosis induced by synaptotagmin 1 and 2 isoforms and abundance but not by phosphorylation
2006Co-Authors: Gabor Nagy, Zhiping P. Pang, Jun Hee Kim, Ulf Matti, Jens Rettig, Jakob B SorensenAbstract:Synaptotagmins comprise a large protein family, of which synaptotagmin 1 (Syt1) is a Ca2+ sensor for fast exocytosis, and its close relative, synaptotagmin 2 (Syt2), is assumed to serve similar functions. Chromaffin cells express Syt1 but not Syt2. We compared secretion from chromaffin cells from Syt1 null mice overexpressing either Syt isoform. High time-resolution capacitance measurement showed that Syt1 null cells lack the exocytotic phase corresponding to the readily-releasable pool (RRP) of vesicles. Comparison with the amperometric signal confirmed that the missing phase of exocytosis consists of catecholamine-containing vesicles. Overexpression of Syt1 rescued the RRP and increased its size above wild-type values, whereas the size of the slowly releasable pool decreased, indicating that the availability of Syt1 regulates the relative size of the two releasable pools. The RRP was also rescued by Syt2 overexpression, but the kinetics of fusion was slightly slower than in cells expressing Syt1. Biochemical experiments showed that Syt2 has a slightly lower Ca2+ affinity for phospholipid binding than Syt1 because of a difference in the C2A domain. These data constitute evidence for the function of Syt1 and Syt2 as alternative, but not identical, calcium-sensors for RRP fusion. By overexpression of Syt1 mutated in the shared PKC/calcium/calmodulin-dependent kinase phosphorylation site, we show that phorbol esters act independently and upstream of Syt1 to regulate the size of the releasable pools. We conclude that exocytosis from mouse chromaffin cells can be modified by the differential expression of Syt isoforms and by Syt abundance but not by phosphorylation of Syt1.
