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PubMed1998

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.

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 Llinás, 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.

Amino Acid Sequence↗

PubMed1994

Synaptotagmin III is a novel isoform of rat synaptotagmin expressed in endocrine and neuronal cells.

Synaptotagmin (p65), an integral membrane protein of synaptic vesicles, is thought to be involved in calcium-dependent exocytosis of synaptic vesicles. Here, we report the cloning and tissue distribution of a novel isoform of synaptotagmin, designated synaptotagmin III. The cDNA clones encoding synaptotagmin III have been isolated from a rat brain cDNA library. Rat synaptotagmin III is a protein of 588 amino acids having 40.5, 38.3, and 64.0% identity with rat synaptotagmin I, rat synaptotagmin II, and o-p65-C, a third synaptotagmin isoform of marine ray Discopyge ommata, respectively. The region of the two internal repeats homologous to the regulatory domain (C2 domain) of protein kinase C is highly conserved among synaptotagmin I, II, and III. RNA blotting studies reveal that synaptotagmin III mRNA is expressed in brain, various endocrine tissues, and hormone-secreting clonal cells. These results suggest that rat synaptotagmin III is a mammalian homolog of o-p65-C and is involved in Ca(2+)-dependent exocytosis of secretory vesicles in endocrine cells, as well as in neurons.

Amino Acid Sequence↗

PubMed1991

Synaptotagmin II. A novel differentially distributed form of synaptotagmin.

Synaptotagmin is a synaptic vesicle membrane protein with properties suggestive of a role in synaptic vesicle exocytosis (Perin, M. S., Fried, V. A., Mignery, G. A., Jahn, R., and Südhof, T. C. (1990) Nature 345, 260-263). Here, we report the structure of a novel form of synaptotagmin named synaptotagmin II that is highly homologous to the originally described synaptotagmin, now referred to as synaptotagmin I. Synaptotagmins I and II exhibit the same overall structure, containing a small intravesicular sequence that is glycosylated, a single transmembrane region, and a large carboxyl-terminal cytoplasmic sequence that includes two copies of an internal repeat homologous to the regulatory region of protein kinase C. The homology between synaptotagmins I and II is not uniformly distributed across the molecule but is highest in their carboxyl-terminal regulatory repeats (88% sequence identity) and lowest in their amino-terminal intravesicular sequences (46% sequence identity). RNA blots demonstrate complementary patterns of expression for synaptotagmins I and II, with synaptotagmin I preferentially expressed in rostral, phylogenetically younger brain regions, and synaptotagmin II predominantly expressed in caudal, phylogenetically older brain regions. With this description of two forms of synaptotagmin, all major synaptic vesicle proteins implicated in membrane traffic have now been shown to be present in several isoforms with differential distributions, suggesting that this is a general organizational principle of the mammalian brain.

Amino Acid Sequence↗

PubMed1995

Synaptotagmin I and 1B4 are identical: implications for synaptotagmin distribution in the primate brain.

We have determined that the human cDNA sequence of the previously described primate brain mRNA species 1B4 is nearly identical (99.95% similarity) to that of human Synaptotagmin I. The apparent identity of Synaptotagmin I with 1B4, whose distribution in the brain of the monkey Cynomolgous was determined previously by Northern blot and in situ hybridization (ISH) analyses, reveals the Synaptotagmin I is differentially expressed in the primate brain. Primate Synaptotagmin I mRNA is enriched in hindbrain structures relative to forebrain structures by Northern blot analysis. By ISH analysis, primate Synaptotagmin I mRNA is highly expressed in occipital cortex and lateral geniculate (visual system components) and differentially expressed across topographic cortical boundaries between inferior and superior temporal gyrus (a polymodal zone with visual, auditory and somatosensory inputs) and between areas 17 and 18 of the visual cortex (primary and secondary visual areas). Cortical expression is also enriched in layers V and VI, which contain large pyramidal projection neurons. Synaptotagmin I's greater association with large projection neurons and with some components of visual sensory transduction could reflect a requirement of these neural components for greater synaptic activity. Synaptotagmin I expression in the primate brain is also dissimilar to Synaptotagmin I expression in rodents. Thus, variation of Synaptotagmin I expression has occurred during mammalian evolution, perhaps as a consequence of the larger size and neurotransmitter requirements of primate neurons.

Base Sequence↗

PubMed1997

The first C2 domain of synaptotagmin is required for exocytosis of insulin from pancreatic beta-cells: action of synaptotagmin at low micromolar calcium.

The Ca2+- and phospholipid-binding protein synaptotagmin is involved in neuroexocytosis. Its precise role and Ca2+-affinity in vivo are unclear. We investigated its putative function in insulin secretion which is maximally stimulated by 10 microM cytosolic free Ca2+. The well-characterized synaptotagmin isoforms I and II are present in pancreatic beta-cell lines RINm5F, INS-1 and HIT-T15 as shown by Northern and Western blots. Subcellular fractionation and confocal microscopy revealed their presence mainly on insulin-containing secretory granules whereas only minor amounts were found on synaptic vesicle-like microvesicles. Antibodies or Fab-fragments directed against the Ca2+-dependent phospholipid binding site of the first C2 domain of synaptotagmin I or II inhibited Ca2+-stimulated, but not GTPgammaS-induced exocytosis from streptolysin-O-permeabilized INS-1 and HIT-T15 cells. Transient expression of wild-type synaptotagmin II did not alter exocytosis in HIT-T15 cells. However, mutations in the Ca2+-dependent phospholipid binding site of the first C2 domain (Delta180-183, D231S) again inhibited only Ca2+-, but not GTPgammaS-evoked exocytosis. In contrast, mutations in the IP4-binding sites of the second C2 domain (Delta325-341; K327,328, 332Q) did not alter exocytosis. Synaptotagmin II mutated in both C2 domains (Delta180-183/K327,328,332Q) induced greater inhibition than mutant Delta180-183, suggesting a discrete requirement for the second C2 domain. Thus, synaptotagmin isoforms regulate exocytotic events occurring at low micromolar Ca2+.

Animals↗

PubMed1995

Synaptotagmin V: a novel synaptotagmin isoform expressed in rat brain.

Regulated Ca(2+)-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.

Amino Acid Sequence↗

PubMed1999

Calcium-dependent oligomerization of synaptotagmins I and II. Synaptotagmins I and II are localized on the same synaptic vesicle and heterodimerize in the presence of calcium.

Synaptotagmins constitute a large family of membrane proteins characterized by their distinct distributions and different biochemical features. Genetic evidence suggests that members of this protein family are likely to function as calcium sensors in calcium-regulated events in neurons, although the precise molecular mechanism remains ill defined. Here we demonstrate that different synaptotagmin isoforms (Syt I, II, and IV) are present in the same synaptic vesicle population from rat brain cortex. In addition, Syt I and II co-localize on the same small synaptic vesicle (SSV), and they heterodimerize in the presence of calcium with a concentration dependence resembling that of the starting phase of SSV exocytosis (EC50 = 6 +/- 4 microM). The association between Syt I and Syt II was demonstrated by immunoprecipitation of the native proteins and the recombinant cytoplasmic domains and by using fluorescence resonance energy transfer (FRET). Although a subpopulation of SSV containing Syt I and IV can be isolated, these two isoforms do not show a calcium-dependent interaction. These results suggest that the self-association of synaptotagmins with different calcium binding features may create a variety of calcium sensors characterized by distinct calcium sensitivities. This combinatorial hypothesis predicts that the probability of a single SSV exocytic event is determined, in addition to the gating properties of the presynaptic calcium channels, by the repertoire and relative abundance of distinct synaptotagmin isoforms present on the SSV surface.

Amino Acid Sequence↗

PubMed1994

Functional properties of multiple synaptotagmins in brain.

At least four forms of synaptotagmin are expressed in neurons. Of these, synaptotagmin I has an essential function in mediating Ca(2+)-triggered neurotransmitter release at hippocampal synapses, but the functional implications of multiple synaptotagmins are unknown. Synaptotagmins I-III exhibit a strikingly differential distribution between synapses, with most neurons coexpressing either synaptotagmins I or II with III. Synaptotagmin IV is present uniformly throughout the brain at low levels. Synaptotagmins III and IV are both coexpressed with synaptotagmin I in hippocampal synapses, suggesting that these synaptotagmins are not functionally redundant. The first C2 domains of synaptotagmins I-III exhibit similar Ca2+ affinities in phospholipid-binding assays, whereas that of synaptotagmin IV is unable to bind Ca2+. All synaptotagmins tested bind the clathrin-adaptor protein AP-2 with high affinity. Our results suggest that different synaptotagmins serve distinct but overlapping functions in neuronal membrane traffic, with synaptotagmins I and II representing alternative Ca2+ sensors in exocytosis and all synaptotagmins functioning as AP-2 receptors in endocytosis.

Adaptor Proteins, Vesicular Transport↗

PubMed1997

The evolutionary pressure to inactivate. A subclass of synaptotagmins with an amino acid substitution that abolishes Ca2+ binding.

Synaptotagmin I is a Ca2+-binding protein of synaptic vesicles that serves as a Ca2+ sensor for neurotransmitter release and was the first member found of a large family of trafficking proteins. We have now identified a novel synaptotagmin, synaptotagmin XI, that is highly expressed in brain and at lower levels in other tissues. Like other synaptotagmins, synaptotagmin XI has a single transmembrane region and two cytoplasmic C2-domains but is most closely related to synaptotagmin IV with which it forms a new subclass of synaptotagmins. The first C2-domain of synaptotagmin I (the C2A-domain) binds phospholipids as a function of Ca2+ and contains a Ca2+-binding site, the C2-motif, that binds at least two Ca2+ ions via five aspartate residues and is conserved in most C2-domains (Shao, X., Davletov, B., Sutton, B., Südhof, T. C., Rizo, J. R. (1996) Science 273, 248-253). In the C2A-domains of synaptotagmins IV and XI, however, one of the five Ca2+-binding aspartates in the C2-motif is substituted for a serine, suggesting that these C2-domains do not bind Ca2+. To test this, we produced recombinant C2A-domains from synaptotagmins IV and XI with either wild type serine or mutant aspartate in the C2-motif. Circular dichroism showed that Ca2+ stabilizes both mutant but not wild type C2-domains against temperature-induced denaturation, indicating that the mutations restore Ca2+-binding to the wild type C2-domains. Furthermore, wild type C2A-domains of synaptotagmins IV and XI exhibited no Ca2+-dependent phospholipid binding, whereas mutant C2A-domains bound phospholipids as a function of Ca2+ similarly to wild type synaptotagmin I. These experiments suggest that a class of synaptotagmins was selected during evolution in which the Ca2+-binding site of the C2A-domain was inactivated by a single point mutation. Thus, synaptotagmins must have Ca2+-independent functions as well as Ca2+-dependent functions that are selectively maintained in distinct members of this gene family.

Amino Acid Sequence↗

PubMed1995

Cellular localization of synaptotagmin I, II, and III mRNAs in the central nervous system and pituitary and adrenal glands of the rat.

Three isoforms of synaptotagmin, a synaptic vesicle protein involved in neurotransmitter release, have been characterized in the rat, although functional differences between these isoforms have not been reported. In situ hybridization was used to define the localization of synaptotagmin I, II, and III transcripts in the rat CNS and pituitary and adrenal glands. Each of the three synaptotagmin genes has a unique expression pattern. The synaptotagmin III gene is expressed in most neurons, but transcripts are much less abundant than the products of the synaptotagmin I and II genes. A majority of neurons in the forebrain expressed both synaptotagmin I and III mRNAs while synaptotagmin II gene expression was confined to subsets of neurons in layers IV-VI of the cerebral cortex, in the dentate granule cell region, the hilus, and the CA1-CA3 areas of the hippocampus. In the cerebellum, all three transcripts were visualized in the granule cell layer. Furthermore, synaptotagmin I probes revealed striking differences between distinct populations of neurons, as in addition to moderate labeling of granule cells, much more prominent hybridization signals were detected on scattered cell bodies likely to be Golgi interneurons. In the most caudal part of the brain, synaptotagmin II transcripts were abundant and were coexpressed with synaptotagmin III mRNAs. This pattern was found in putative motoneurons of the spinal cord, suggesting that the two isoforms might be involved in exocytosis at the neuromuscular junction. Only synaptotagmin I mRNAs were detected in the anterior and intermediate pituitary and in adrenal medullary cells. These data reveal an unexpectedly subtle segregation of the expression of synaptotagmin genes and the existence of multiple combinations of synaptotagmin isoforms which may provide diversity in the regulation of neurosecretion.

Adrenal Glands↗

PubMed1993

Phosphorylation of synaptotagmin I by casein kinase II.

Synaptotagmin I is an abundant synaptic vesicle protein that binds Ca2+ in a phospholipid-dependent manner and is thought to function in synaptic vesicle exocytosis. We have now studied the phosphorylation of synaptotagmin I. Synaptotagmin I is one of the major substrates in brain for casein kinase II, which phosphorylates synaptotagmin at a single threonine. The phosphorylation site was mapped using recombinant proteins to threonine 128 of synaptotagmin I, which is located in the sequence between the transmembrane region and the C2 domain repeats of synaptotagmin I. The phosphorylation site of synaptotagmin I is also present in synaptotagmin II and is evolutionarily conserved between different species. Preceding the phosphorylation site, synaptotagmins I and II contain a lysine-rich sequence. Casein kinase II phosphorylation of many substrates is strongly stimulated by the addition of polylysine, but phosphorylation of synaptotagmin I by casein kinase II is not. In recombinant proteins, removal of the lysine-rich sequence of synaptotagmin I makes its phosphorylation dependent on exogenous polylysine, suggesting that the lysine-rich sequence in synaptotagmin serves as an endogenous polylysine stimulation signal for casein kinase II. Our data demonstrate that synaptotagmin I is an efficient substrate for casein kinase II at a conserved site with a possible modulatory role in nerve terminal function.

Amino Acid Sequence↗

PubMed1999

Analysis of synaptotagmin I-IV messenger RNA expression and developmental regulation in the rat hypothalamus and pituitary.

Synaptotagmins are a large family of synaptic vesicle membrane proteins, that appear to be involved in neurotransmitter secretion from small secretory vesicles. We have quantitatively analysed the messenger RNA levels of synaptotagmin I-IV isoforms in adult hypothalamic and pituitary tissues in order to determine which of these isoforms dominate in these tissues which mainly secrete peptides from large dense core vesicles. We also studied the expression of these isoforms during prenatal (E15, and E17) and postnatal (P1, P7, P14 and P21) rat hypothalamic development. In order to assay small individual samples (e.g., pituitary and embryonic tissues), we employed quantitative reverse transcription-polymerase chain reaction methods. Our results show that synaptotagmin I messenger RNA is the most abundant isoform in all tissues, and is about 5.4- or 38-fold higher in hypothalamus than in neurointermediate and anterior pituitary lobe, respectively. Synaptotagmin II, which is very abundant in cerebellum, is relatively low in hypothalamus (5% of cerebellum) and virtually absent from the pituitary. Synaptotagmin III is about 10 times greater in the neural tissues versus the pituitary, and synaptotagmin IV was the least abundant isoform in all the tissues. Developmental analyses of the synaptotagmin isoforms in rat hypothalamus shows that all isoforms are at low levels during embryonic stages and increase postnatally. Synaptotagmin I and II have similar patterns and rise to maximum (adult) levels around P14, whereas synaptotagmin III and IV reach their maximum levels considerably earlier, at P1. These data show that synaptotagmin I is the dominant isoform in both predominantly peptide secreting systems (e.g., in pituitary tissues) and in neurotransmitter secreting systems (e.g., in cerebellum). While the developmental expression patterns of synaptotagmin I and II parallels the temporal development of synaptogenesis in the nervous system, the early maximal expression of synaptotagmin III and IV suggests that these isoforms may have other functions during early postnatal development.

Age Factors↗

PubMed1996

Synaptotagmin II expression partially rescues the growth defect of the yeast sec15 secretory mutant.

Synaptotagmins are a family of calcium- and phospholipid-binding proteins implicated in the function of cell exocytosis. Synaptotagmins I and II are neurally expressed proteins thought to be involved in neurotransmitter release from neurons. We have expressed rat synaptotagmin II in several Saccharomyces cerevisiae temperature-sensitive secretory mutants that are defective in Golgi to plasma membrane vesicular transport. Synaptotagmin II expression was able to partially rescue the growth defect in one particular mutant, sec15. No suppression was observed when synaptotagmin II was expressed in sec1, sec2, sec4, sec5, sec6, sec8, sec9, sec14, sec17, or sec18. Two synaptotagmin II deletion mutants were also expressed in sec15 and screened for suppression. The expression of the cytoplasmic domain of synaptotagmin alone was not able to suppress the sec15 growth defect. In addition, the expression of a synaptotagmin II fragment lacking the second half of the cytoplasmic domain including the second C2 domain did not suppress sec15. We have isolated a membrane fraction enriched in post-Golgi vesicles from a sec15 strain expressing synaptotagmin II and found that synaptotagmin II co-purifies with this fraction, suggesting that the rat synaptotagmin II is targeted to membranes in yeast. Sec15p forms a large multisubunit protein complex that includes Sec6p and Sec8p. This protein complex is thought to function in a late stage of exocytosis in yeast. Sec6p and Sec8p homologs have been identified in mammalian cells. Our studies suggest that synaptotagmin may be a part of this complex or regulate its function in mammalian cells.

Animals↗

PubMed1996

Synaptotagmin, a synaptic vesicle protein, is present in human cerebrospinal fluid: a new biochemical marker for synaptic pathology in Alzheimer disease?

Using a novel approach, including affinity chromatography, reversed-phase chromatography, and chemiluminescence immunoblotting, we have for the first time been able to demonstrate one of the small synaptic vesicle proteins, synaptotagmin I, in cerebrospinal fluid (CSF). Two other small synaptic vesicle proteins, rab3a and synaptophysin, were not detectable. The approximate molecular weight of CSF-synaptotagmin was 65 kDa, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Further characterization of CSF synaptotagmin by high-performance capillary electrophoresis (HPCE) showed a single peak. These findings support that the whole synaptotagmin molecule is present in CSF, without significant proteolytic degradation. After high-speed centrifugation of CSF, synaptotagmin was exclusively found in the supernatant, suggesting that synaptotagmin is present in CSF as a free protein, and not as a constituent of synaptic vesicles. In a preliminary study, we found a marked reduction of CSF synaptotagmin in patients with early onset Alzheimer disease (EAD) as compared with age-matched healthy individuals. To elucidate the biological relevance of this finding, we also quantified synaptotagmin in brain tissue. A marked reduction in synaptotagmin was found both in the hippocampus and frontal cortex of EAD, suggesting that a decrease in synaptotagmin in the brain is followed by a concomitant decrease in the CSF. Analysis of CSF synaptotagmin might provide a tool to study synaptic function and pathology in the human brain.

Aged↗

PubMed1991

Structural and functional conservation of synaptotagmin (p65) in Drosophila and humans.

Synaptotagmin (p65) is an abundant synaptic vesicle protein that contains two copies of a sequence that is homologous to the regulatory region of protein kinase C. Full length cDNAs encoding human and Drosophila synaptotagmins were characterized to study its structural and functional conservation in evolution. The deduced amino acid sequences for human and rat synaptotagmins show 97% identity, whereas Drosophila and rat synaptotagmins are only 57% identical but exhibit a selective conservation of the two internal repeats that are homologous to the regulatory region of protein kinase C (78% invariant residues in all three species). The two internal repeats of synaptotagmin are only slightly more homologous to each other than to protein kinase C, and the differences between the repeats are conserved in evolution, suggesting that they might not be functionally equivalent. The cytoplasmic domains of human and Drosophila synaptotagmins produced as recombinant proteins in Escherichia coli specifically bound phosphatidylserine similar to rat synaptotagmin. They also hemagglutinated trypsinized erythrocytes at nanomolar concentrations. Hemagglutination was inhibited both by negatively charged phospholipids and by a recombinant fragment from rat synaptotagmin that contained only a single copy of the two internal repeats. Together these results demonstrate that synaptotagmin is highly conserved in evolution compatible with a function in the trafficking of synaptic vesicles at the active zone. The similarity of the phospholipid binding properties of the cytoplasmic domains of rat, human, and Drosophila synaptotagmins and the selective conservation of the sequences that are homologous to protein kinase C suggest that these are instrumental in phospholipid binding. The human gene for synaptotagmin was mapped by Southern blot analysis of DNA from somatic cell hybrids to chromosome 12 region cen-q21, and the Drosophila gene by in situ hybridization to 23B.

Amino Acid Sequence↗

PubMed1993

Expression of synaptotagmin in Drosophila reveals transport and localization of synaptic vesicles to the synapse.

Synaptotagmin is a synaptic vesicle-specific integral membrane protein that has been suggested to play a key role in synaptic vesicle docking and fusion. By monitoring Synaptotagmin's cellular and subcellular distribution during development, it is possible to study synaptic vesicle localization and transport, and synapse formation. We have initiated the study of Synaptotagmin's expression during Drosophila neurogenesis in order to follow synaptic vesicle movement prior to and during synapse formation, as well as to localize synaptic sites in Drosophila. In situ hybridizations to whole-mount embryos show that synaptotagmin (syt) message is present in the cell bodies of all peripheral nervous system neurons and many, if not all, central nervous system neurons during neurite outgrowth and synapse formation, and in mature neurons. Immunocytochemical staining with antisera specific to Synaptotagmin indicates that the protein is present at all stages of the Drosophila life cycle following germ band retraction. In embryos, Synaptotagmin is only transiently localized to the cell body of neurons and is transported rapidly along axons during axonogenesis. After synapse formation, Synaptotagmin accumulates in a punctate pattern at all identifiable synaptic contact sites, suggesting a general role for Synaptotagmin in synapse function. In embryos and larvae, the most intense staining is found along two broad longitudinal tracts on the dorsal side of the ventral nerve cord and the brain, and at neuromuscular junctions in the periphery. In the adult head, Synaptotagmin localizes the discrete regions of the neurophil where synapses are predicted to occur. These data indicate that synaptic vesicles are present in axons before synapse formation, and become restricted to synaptic contact sites after synapses are formed. Since a similar expression pattern of Synaptotagmin has been reported in mammals, we propose that the function of Synaptotagmin and the mechanisms governing localization of the synaptic vesicle before and after synapse formation are conserved in invertebrate and vertebrate species. The ability to mark synapses in Drosophila should facilitate the study of synapse formation and function, providing a new tool to dissect the molecular mechanisms underlying these processes.

Animals↗

PubMed1997

Developmental regulation of synaptotagmin I, II, III, and IV mRNAs in the rat CNS.

Synaptotagmin I is an abundant synaptic vesicle protein that has an essential function in mediating Ca2+-triggered neurotransmitter release. We have analyzed the distribution of four neural synaptotagmin isoforms during postnatal development of the rat CNS by in situ hybridization. Synaptotagmin I, II, III, and IV genes have distinct patterns of spatiotemporal expression except in cerebellum granule cells, where the four transcripts were detected during the formation of parallel fiber/Purkinje cell synapses. Throughout development synaptotagmin I mRNAs were widely expressed in brain, whereas synaptotagmin II transcripts were predominant in spinal cord. At all stages synaptotagmin III mRNAs were expressed uniformly in most neurons examined, although at a low level. Synaptotagmin I, II, and III gene expressions mainly increased during development and persisted in adulthood, mirroring neuronal differentiation. Conversely, synaptotagmin IV transcripts were predominant during perinatal development in a heterogeneous population of neurons and subsequently were expressed uniformly at a low level. Intense labeling was observed in the hippocampal CA3 field and in the subiculum, but not in the CA1 field, of the newborn rat. In cerebral cortex, lamina-specific labeling was detected with a high expression in cell layer V. Only a small number of Purkinje cell clusters were labeled in the flocculus and paraflocculus of the cerebellum. Heterogeneous sets of neurons expressing synaptotagmin IV gene also were observed in spinal cord. We thus speculate that synaptotagmin IV may a play a role in the development of the mammalian nervous system.

Aging↗

PubMed1995

Distinct Ca2+ and Sr2+ binding properties of synaptotagmins. Definition of candidate Ca2+ sensors for the fast and slow components of neurotransmitter release.

Ca(2+)-dependent neurotransmitter release consists of at least two components: a major fast component that is insensitive to Sr2+ and a minor slow component that is potentiated by Sr2+ (Goda, Y., and Stevens, C. F. (1994) Proc. Natl. Acad. U. S. A. 91, 12942-12946). These results suggest that at least two Ca2+ sensors act in synaptic vesicle fusion with distinct Ca2+ and Sr2+ binding properties. We have now investigated the relative Ca2+ and Sr2+ binding activities of synaptotagmins to evaluate their potential roles as Ca2+ sensors for the fast and slow components. Our results demonstrate that the first C2 domains of synaptotagmins I, II, III, V, and VII have very similar Ca2+ requirements for phospholipid binding (range of EC50 = 2.6 microM to 5.0 microM), but distinct Sr2+ requirements (EC50 range = 23 microM to 133 microM); synaptotagmins I and II had the lowest Sr2+ affinity, and synaptotagmin III the highest Sr2+ affinity. Purified synaptotagmin I from bovine brain exhibited similar properties as its recombinant first C2 domain, suggesting that the first C2 domain fully accounts for its Ca(2+)-dependent phospholipid binding properties. Sr2+ was unable to trigger syntaxin binding by synaptotagmin I at all concentrations tested, whereas it was effective for synaptotagmin III. These results suggest that different C2 domains have distinct Sr2+ binding properties. They support the hypothesis that synaptotagmins localized on the same vesicle perform distinct functions, with synaptotagmins I and II serving as candidate Ca2+ sensors for the fast component in release and synaptotagmin III for the slow component.

Animals↗