Synaptotagmin IX regulates Ca2+-dependent secretion in PC12 cells.

Synaptotagmin (Syt) I-deficient phaeochromocytoma (PC12) cell lines show normal Ca(2+)-dependent norepinephrine (NE) release (Shoji-Kasai, Y., Yoshida, A., Sato, K., Hoshino, T., Ogura, A., Kondo, S., Fujimoto, Y., Kuwahara, R., Kato, R., and Takahashi, M. (1992) Science 256, 1821-1823). To identify an alternative Ca(2+) sensor, we searched for other Syt isoforms in Syt I-deficient PC12 cells and identified Syt IX, an isoform closely related to Syt I, as an abundantly expressed dense-core vesicle protein. Here we show that Syt IX is required for the Ca(2+)-dependent release of NE from PC12 cells. Antibodies directed against the C2A domain of either Syt IX or Syt I inhibited Ca(2+)-dependent NE release in permeable PC12 cells indicating that both Syt proteins function in dense-core vesicle exocytosis. Our results support the idea that Syt family proteins that co-reside on secretory vesicles may function cooperatively and redundantly as potential Ca(2+) sensors for exocytosis.     

Synaptotagmin VII as a plasma membrane Ca(2+) sensor in exocytosis

Synaptotagmins I and II are Ca(2+) binding proteins of synaptic vesicles essential for fast Ca(2+)-triggered neurotransmitter release. However, central synapses and neuroendocrine cells lacking these synaptotagmins still exhibit Ca(2+)-evoked exocytosis. We now propose that synaptotagmin VII functions as a plasma membrane Ca(2+) sensor in synaptic exocytosis complementary to vesicular synaptotagmins. We show that alternatively spliced forms of synaptotagmin VII are expressed in a developmentally regulated pattern in brain and are concentrated in presynaptic active zones of central synapses. In neuroendocrine PC12 cells, the C(2)A and C(2)B domains of synaptotagmin VII are potent inhibitors of Ca(2+)-dependent exocytosis, but only when they bind Ca(2+). Our data suggest that in synaptic vesicle exocytosis, distinct synaptotagmins function as independent Ca(2+) sensors on the two fusion partners, the plasma membrane (synaptotagmin VII) versus synaptic vesicles (synaptotagmins I and II).     

Synaptotagmin VII regulates Ca(2+)-dependent exocytosis of lysosomes in fibroblasts

Synaptotagmins (Syts) are transmembrane proteins with two Ca(2+)-binding C(2) domains in their cytosolic region. Syt I, the most widely studied isoform, has been proposed to function as a Ca(2+) sensor in synaptic vesicle exocytosis. Several of the twelve known Syts are expressed primarily in brain, while a few are ubiquitous (Sudhof, T.C., and J. Rizo. 1996. Neuron. 17: 379-388; Butz, S., R. Fernandez-Chacon, F. Schmitz, R. Jahn, and T.C. Sudhof. 1999. J. Biol. Chem. 274:18290-18296). The ubiquitously expressed Syt VII binds syntaxin at free Ca(2+) concentrations ([Ca(2+)]) below 10 microM, whereas other isoforms require 200-500 microM [Ca(2+)] or show no Ca(2+)-dependent syntaxin binding (Li, C., B. Ullrich, Z. Zhang, R.G.W. Anderson, N. Brose, and T.C. Sudhof. 1995. Nature. 375:594-599). We investigated the involvement of Syt VII in the exocytosis of lysosomes, which is triggered in several cell types at 1-5 microM [Ca(2+)] (Rodríguez, A., P. Webster, J. Ortego, and N.W. Andrews. 1997. J. Cell Biol. 137:93-104). Here, we show that Syt VII is localized on dense lysosomes in normal rat kidney (NRK) fibroblasts, and that GFP-tagged Syt VII is targeted to lysosomes after transfection. Recombinant fragments containing the C(2)A domain of Syt VII inhibit Ca(2+)-triggered secretion of beta-hexosaminidase and surface translocation of Lgp120, whereas the C(2)A domain of the neuronal- specific isoform, Syt I, has no effect. Antibodies against the Syt VII C(2)A domain are also inhibitory in both assays, indicating that Syt VII plays a key role in the regulation of Ca(2+)-dependent lysosome exocytosis.     

Calcium-dependent and -independent hetero-oligomerization in the synaptotagmin family

Synaptotagmins constitute a family of membrane proteins that are characterized by one transmembrane region and two C2 domains. Recent genetic and biochemical studies have indicated that oligomerization of synaptotagmin (Syt) I is important for expression of function during exocytosis of synaptic vesicles. However, little is known about hetero-oligomerization in the synaptotagmin family. In this study, we showed that the synaptotagmin family is a type I membrane protein (N(lumen)/C(cytoplasm)) by introducing an artificial N-glycosylation site at the N-terminal domain, and systematically examined all the possible combinations of hetero-oligomerization among synaptotagmin family proteins (Syts I-XI). We classified the synaptotagmin family into four distinct groups based on differences in Ca(2+)-dependent and -independent oligomerization activity. Group A Syts (III, V, VI, and X) form strong homo- and hetero-oligomers by disulfide bonds at an N-terminal cysteine motif irrespective of the presence of Ca(2+) [Fukuda, M., Kanno, E., and Mikoshiba, K. (1999) J. Biol. Chem. 274, 31421-31427]. Group B Syts (I, II, VIII, and XI) show moderate homo-oligomerization irrespective of the presence of Ca(2+). Group C synaptotagmins are characterized by weak Ca(2+)-dependent (Syts IX) or no homo-oligomerization activity (Syt IV). Syt VII (Group D) has unique Ca(2+)-dependent homo-oligomerization properties with EC(50) values of about 150 microM Ca(2+) [Fukuda, M., and Mikoshiba, K. (2000) J. Biol. Chem. 275, 28180-28185]. Syts IV, VIII, and XI did not show any apparent hetero-oligomerization activity, but some sets of synaptotagmin isoforms can hetero-oligomerize in a Ca(2+)-dependent and/or -independent manner. Our data suggest that Ca(2+)-dependent and -independent hetero-oligomerization of synaptotagmins may create a variety of Ca(2+)-sensors.     

The calcium-binding loops of the tandem C2 domains of synaptotagmin VII cooperatively mediate calcium-dependent oligomerization

Synaptotagmin VII (Syt VII), a proposed regulator for Ca2+-dependent exocytosis, showed a robust Ca2+-dependent oligomerization property via its two C2 domains (Fukuda, M., and Mikoshiba, K. (2001) J. Biol. Chem. 276, 27670-27676), but little is known about its structure or the critical residues directly involved in the oligomerization interface. In this study, site-directed mutagenesis and chimeric analysis between Syt I and Syt VII showed that three Asp residues in Ca2+-binding loop 1 or 3 (Asp-172, Asp-303, and Asp-357) are crucial to robust Ca(2+)-dependent oligomerization. Unlike Syt I, however, the polybasic sequence in the beta4 strands of the C2 structures (so-called "C2 effector domain") is not involved in the Ca2+-dependent oligomerization of Syt VII. The results also showed that the Ca2+-binding loops of the two C2 domains cooperatively mediate Syt VII oligomerization (i.e. the presence of redundant Ca2+-binding site(s)) as well as the importance of Ca2+-dependent oligomerization of Syt VII in Ca2+-regulated secretion. Expression of wild-type tandem C2 domains of Syt VII in PC12 cells inhibited Ca2+-dependent neuropeptide Y release, whereas mutant fragments lacking Ca2+-dependent oligomerization activity had no effect. Finally, rotary-shadowing electron microscopy showed that the Ca2+-dependent oligomer of Syt VII is "a large linear structure," not an irregular aggregate. By contrast, in the absence of Ca2+ Syt VII molecules were observed to form a globular structure. Based on these results, we suggest that the linear Ca2+-dependent oligomer may be aligned at the fusion site between vesicles and plasma membrane and modulate Ca2+-regulated exocytosis by opening or dilating fusion pores.     

Post-translational modifications and lipid binding profile of insect cell-expressed full-length mammalian synaptotagmin 1

Synaptotagmin 1 (Syt1) is a Ca(2+) sensor for SNARE-mediated, Ca(2+)-triggered synaptic vesicle fusion in neurons. It is composed of luminal, transmembrane, linker, and two Ca(2+)-binding (C2) domains. Here we describe expression and purification of full-length mammalian Syt1 in insect cells along with an extensive biochemical characterization of the purified protein. The expressed and purified protein is properly folded and has increased α-helical content compared to the C2AB fragment alone. Post-translational modifications of Syt1 were analyzed by mass spectrometry, revealing the same modifications of Syt1 that were previously described for Syt1 purified from brain extract or mammalian cell lines, along with a novel modification of Syt1, tyrosine nitration. A lipid binding screen with both full-length Syt1 and the C2AB fragments of Syt1 and Syt3 isoforms revealed new Syt1-lipid interactions. These results suggest a conserved lipid binding mechanism in which Ca(2+)-independent interactions are mediated via a lysine rich region of the C2B domain while Ca(2+)-dependent interactions are mediated via the Ca(2+)-binding loops.     

Distinct self-oligomerization activities of synaptotagmin family. Unique calcium-dependent oligomerization properties of synaptotagmin VII

Synaptotagmins constitute a large protein family, characterized by one transmembrane region and two C2 domains, and can be classified into several subclasses based on phylogenetic relationships and biochemical activities (Fukuda, M., Kanno, E., and Mikoshiba, K. (1999) J. Biol. Chem. 274, 31421-31427). Synaptotagmin I (Syt I), a possible Ca(2+) sensor for neurotransmitter release, showed both Ca(2+)-dependent (via the C2 domain) and -independent (via the NH(2)-terminal domain) self-oligomerization, which are thought to be important for synaptic vesicle exocytosis. However, little is known about the relationship between these two interactions and the Ca(2+)-dependent oligomerization properties of other synaptotagmin isoforms. In this study, we first examined the Ca(2+)-dependent self-oligomerization properties of synaptotagmin family by co-expression of T7- and FLAG-tagged Syts (full-length or cytoplasmic domain) in COS-7 cells. We found that Syt VII is a unique class of synaptotagmins that only showed robust Ca(2+)-dependent self-oligomerization at the cytoplasmic domain with EC(50) values of about 150 micrometer Ca(2+). In addition, Syt VII preferentially interacted with the previously described subclass of Syts (V, VI, and X) in a Ca(2+)-dependent manner. Co-expression of full-length and cytoplasmic portion of Syts VII (or II) indicate that Syt VII cytoplasmic domain oligomerizes in a Ca(2+)-dependent manner without being tethered at the NH(2)-terminal domain, whereas Ca(2+)-dependent self-oligomerization at the cytoplasmic domain of other isoforms (e.g. Syt II) occurs only when the two molecules are tethered at the NH(2)-terminal domain.     

A complexin/synaptotagmin 1 switch controls fast synaptic vesicle exocytosis

Ca(2+) binding to synaptotagmin 1 triggers fast exocytosis of synaptic vesicles that have been primed for release by SNARE-complex assembly. Besides synaptotagmin 1, fast Ca(2+)-triggered exocytosis requires complexins. Synaptotagmin 1 and complexins both bind to assembled SNARE complexes, but it is unclear how their functions are coupled. Here we propose that complexin binding activates SNARE complexes into a metastable state and that Ca(2+) binding to synaptotagmin 1 triggers fast exocytosis by displacing complexin from metastable SNARE complexes. Specifically, we demonstrate that, biochemically, synaptotagmin 1 competes with complexin for SNARE-complex binding, thereby dislodging complexin from SNARE complexes in a Ca(2+)-dependent manner. Physiologically, increasing the local concentration of complexin selectively impairs fast Ca(2+)-triggered exocytosis but retains other forms of SNARE-dependent fusion. The hypothesis that Ca(2+)-induced displacement of complexins from SNARE complexes triggers fast exocytosis accounts for the loss-of-function and gain-of-function phenotypes of complexins and provides a molecular explanation for the high speed and synchronicity of fast Ca(2+)-triggered neurotransmitter release.     

SNARE protein recycling by αSNAP and βSNAP supports synaptic vesicle priming

Neurotransmitter release proceeds by Ca(2+)-triggered, SNARE-complex-dependent synaptic vesicle fusion. After fusion, the ATPase NSF and its cofactors α- and βSNAP disassemble SNARE complexes, thereby recycling individual SNAREs for subsequent fusion reactions. We examined the effects of genetic perturbation of α- and βSNAP expression on synaptic vesicle exocytosis, employing a new Ca(2+) uncaging protocol to study synaptic vesicle trafficking, priming, and fusion in small glutamatergic synapses of hippocampal neurons. By characterizing this protocol, we show that synchronous and asynchronous transmitter release involve different Ca(2+) sensors and are not caused by distinct releasable vesicle pools, and that tonic transmitter release is due to ongoing priming and fusion of new synaptic vesicles during high synaptic activity. Our analysis of α- and βSNAP deletion mutant neurons shows that the two NSF cofactors support synaptic vesicle priming by determining the availability of free SNARE components, particularly during phases of high synaptic activity.     

Axolemmal repair requires proteins that mediate synaptic vesicle fusion

A damaged cell membrane is repaired by a seal that restricts entry or exit of molecules and ions to that of the level passing through an undamaged membrane. Seal formation requires elevation of intracellular Ca(2+) and, very likely, protein-mediated fusion of membranes. Ca(2+) also regulates the interaction between synaptotagmin (Syt) and syntaxin (Syx), which is thought to mediate fusion of synaptic vesicles with the axolemma, allowing transmitter release at synapses. To determine whether synaptic proteins have a role in sealing axolemmal damage, we injected squid and crayfish giant axons with an antibody that inhibits squid Syt from binding Ca(2+), or with another antibody that inhibits the Ca(2+)-dependent interaction of squid Syx with the Ca(2+)-binding domain of Syt. Axons injected with antibody to Syt did not seal, as assessed at axonal cut ends by the exclusion of extracellular hydrophilic fluorescent dye using confocal microscopy, and by the decay of extracellular injury current compared to levels measured in uninjured axons using a vibrating probe technique. In contrast, axons injected with either denatured antibody to Syt or preimmune IgG did seal. Similarly, axons injected with antibody to Syx did not seal, but did seal when injected with either denatured antibody to Syx or preimmune IgG. These results indicate an essential involvement of Syt and Syx in the repair (sealing) of severed axons. We suggest that vesicles, which accumulate and interact at the injury site, re-establish axolemmal continuity by Ca(2+)-induced fusions mediated by proteins such as those involved in neurotransmitter release.     

There's more to life than neurotransmission: the regulation of exocytosis by synaptotagmin VII

Among the 16 known vertebrate synaptotagmins, only Syt I, IV and VII are also present in C. elegans and Drosophila, suggesting that these isoforms play especially important roles in vivo. Extensive evidence indicates that Syt I is a synaptic vesicle Ca(2+) sensor essential for rapid neurotransmitter release. It has been suggested that the ubiquitously expressed Syt VII also regulates synaptic vesicle exocytosis, despite its presence in several tissues in addition to the brain. Here, we discuss recent genetic and biochemical evidence that does not support this view. Syt VII null mutants do not have a neurological phenotype, and the protein is found on the membrane of lysosomes and some non-synaptic secretory granules, where it regulates Ca(2+)-triggered exocytosis and plasma membrane repair.     

Synaptotagmins form a hierarchy of exocytotic Ca(2+) sensors with distinct Ca(2+) affinities

Synaptotagmins constitute a large family of membrane proteins implicated in Ca(2+)-triggered exocytosis. Structurally similar synaptotagmins are differentially localized either to secretory vesicles or to plasma membranes, suggesting distinct functions. Using measurements of the Ca(2+) affinities of synaptotagmin C2-domains in a complex with phospholipids, we now show that different synaptotagmins exhibit distinct Ca(2+) affinities, with plasma membrane synaptotagmins binding Ca(2+) with a 5- to 10-fold higher affinity than vesicular synaptotagmins. To test whether these differences in Ca(2+) affinities are functionally important, we examined the effects of synaptotagmin C2-domains on Ca(2+)-triggered exocytosis in permeabilized PC12 cells. A precise correlation was observed between the apparent Ca(2+) affinities of synaptotagmins in the presence of phospholipids and their action in PC12 cell exocytosis. This was extended to PC12 cell exocytosis triggered by Sr(2+), which was also selectively affected by high-affinity C2-domains of synaptotagmins. Together, our results suggest that Ca(2+) triggering of exocytosis involves tandem Ca(2+) sensors provided by distinct plasma membrane and vesicular synaptotagmins. According to this hypothesis, plasma membrane synaptotagmins represent high-affinity Ca(2+) sensors involved in slow Ca(2+)-dependent exocytosis, whereas vesicular synaptotagmins function as low-affinity Ca(2+) sensors specialized for fast Ca(2+)-dependent exocytosis.     

alpha-Latrotoxin, acting via two Ca2+-dependent pathways, triggers exocytosis of two pools of synaptic vesicles

alpha-Latrotoxin stimulates three types of [(3)H]gamma-aminobutyric acid and [(14)C]glutamate release from synaptosomes. The Ca(2+)-independent component (i) is insensitive to SNAP-25 cleavage or depletion of vesicle contents by bafilomycin A1 and represents transmitter efflux mediated by alpha-latrotoxin pores. Two other components of release are Ca(2+)-dependent and vesicular but rely on distinct mechanisms. The fast receptor-mediated pathway (ii) involves intracellular Ca(2+) stores and acts upon sucrose-sensitive readily releasable vesicles; this mechanism is insensitive to inhibition of phosphatidylinositol 4-kinase (PI 4-kinase). The delayed pore-dependent exocytotic component (iii) is stimulated by Ca(2+) entering through alpha-latrotoxin pores; it requires PI 4-kinase and occurs mainly from depot vesicles. Lanthanum perturbs alpha-latrotoxin pores and blocks the two pore-mediated components (i, iii) but not the receptor-mediated release (ii). alpha-Latrotoxin mutant (LTX(N4C)) cannot form pores and stimulates only the Ca(2+)-dependent receptor-mediated amino acid exocytosis (ii) (detectable biochemically and electrophysiologically). These findings explain experimental data obtained by different laboratories and implicate the toxin receptors in the regulation of the readily releasable pool of synaptic vesicles. Our results also suggest that, similar to noradrenergic vesicles, amino acid-containing vesicles at some point in their cycle require PI 4-kinase.     

JNK phosphorylates synaptotagmin-4 and enhances Ca2+-evoked release

Ca2+ influx induced by membrane depolarization triggers the exocytosis of secretory vesicles in various cell types such as endocrine cells and neurons. Peptidyl growth factors enhance Ca2+-evoked release, an effect that may underlie important adaptive responses such as the long-term potentiation of synaptic transmission induced by growth factors. Here, we show that activation of the c-Jun N-terminal kinase (JNK) plays an essential role in nerve growth factor (NGF) enhancement of Ca2+-evoked release in PC12 neuroendocrine cells. Moreover, JNK associated with phosphorylated synaptotagmin-4 (Syt 4), a key mediator of NGF enhancement of Ca2+-evoked release in this system. NGF treatment led to phosphorylation of endogenous Syt 4 at Ser135 and translocation of Syt 4 from immature to mature secretory vesicles in a JNK-dependent manner. Furthermore, mutation of Ser135 abrogated enhancement of Ca2+-evoked release by Syt 4. These results provide a molecular basis for the effect of growth factors on Ca2+-mediated secretion.     

Synaptotagmin V is targeted to dense-core vesicles that undergo calcium-dependent exocytosis in PC12 cells

Synaptotagmins (Syts) III, V, VI, and X are classified as a subclass of Syt, based on their sequence similarities and biochemical properties (Ibata, K., Fukuda, M., and Mikoshiba, K. (1998) J. Biol. Chem. 273, 12267-12273; Fukuda, M., Kanno, E., and Mikoshiba, K. (1999) J. Biol. Chem. 274, 31421-31427). Although they have been suggested to be involved in vesicular trafficking, as in the role of the Syt I isoform in synaptic vesicle exocytosis, their exact functions remain to be clarified, and even their precise subcellular localization is still a matter of controversy. In this study, we established rat pheochromocytoma (PC12) cell lines that stably express Syts III-, V-, VI-, and X-GFP (green fluorescence protein) fusion proteins, respectively, to determine their precise subcellular localizations. Surprisingly, Syts III-, V-, VI-, and X-GFP proteins were found to be targeted to specific organelles: Syt III-GFP to near the plasma membrane, Syt V-GFP to dense-core vesicles, Syt VI-GFP to endoplasmic reticulum-like structures, and Syt X-GFP to vesicles (other than dense-core vesicles) present in cytoplasm. We showed that Syt V-containing vesicles at the neurites of PC12 cells were processed to exocytosis in a Ca2+-dependent manner. Immunohistochemical analysis further showed that endogenous Syt V was also localized on dense-core vesicles in the mouse brain and specifically expressed in glucagon-positive alpha-cells in mouse pancreatic islets, but not in beta- or delta-cells. Based on these results, we propose that Syt V is a dense-core vesicle-specific Syt isoform that controls a specific type of Ca2+-regulated secretion.     

Roles of SNARE proteins and synaptotagmin I in synaptic transmission: studies at the Drosophila neuromuscular synapse

The roles of SNARE proteins, i.e. neuronal Synaptobrevin (n-Syb), SNAP-25 and Syntaxin 1A (Syx 1A), and Synaptotagmin I (Syt I) in synaptic transmission have been studied in situ using mutant embryos or larvae that lack these molecules or have alterations in them. Because of the ease of genetic manipulation, the Drosophila neuromuscular synapse is widely used for these studies. The functional properties of synaptic transmission have been studied in mutant embryos using the patch-clamp technique, and in larvae by recording with microelectrodes. A major vesicular membrane protein, n-Syb, is indispensable for nerve-evoked synaptic transmission. Spontaneous synaptic currents (minis), however, are present even in embryos totally lacking n-Syb (N-SYB). Furthermore, Ca(2+)-independent enhancement of mini frequency induced by hypertonic sucrose solutions (hypertonicity response) is totally absent in N-SYB. Embryos that have defects in SNAP-25 (SNAP-25) have similar but milder phenotypes than N-SYB. The phenotype in synaptic transmission was most severe in the synapse lacking Syx 1A. Neither nerve-evoked synaptic currents nor minis occur in embryos lacking Syx 1A (SYX 1A). No hypertonicity response was observed in them. Syt I binds Ca(2+) in vitro and probably serves as a Ca(2+) sensor for nerve-evoked synaptic transmission, since nerve-evoked synaptic currents were greatly reduced in embryos lacking Syt I (SYT I). Also, Syt I has a role in vesicle recycling. Interestingly, the Ca(2+)-independent hypertonicity response is also greatly reduced in SYT I. Minis persist in mutant embryos lacking any of these proteins (n-Syb, SNAP-25 and Syt I), except Syx, suggesting that minis have a distinct fusion mechanism from that for fast and synchronized release. It appears that these SNARE proteins and Syt I are coordinated for fast vesicle fusion. Minis, on the other hand, do not require SNARE complex nor Syt I, but Syx is absolutely required for vesicle fusion. The SNARE complex and Syt I are indispensable for the hypertonicity response. None of these molecules seem to serve for selective docking of synaptic vesicles to the release site. For further studies on synaptic transmission, the Drosophila neuromuscular synapse will continue to be a useful model.     

Neurotoxic phospholipases directly affect synaptic vesicle function

Snake neurotoxic phospholipases (SPAN) exclusively affect pre-synaptic nerve terminals where they lead to a block of neurotransmission by not fully understood mechanisms. Here, we report that the SPANs, taipoxin and paradoxin, in nanomolar concentrations directly dissociate the synaptophysin/synaptobrevin (Syp/Syb) complex on isolated synaptic vesicles in the presence of synaptosomal cytosol. The phospholipase activity of SPANs depends on Ca(2+) but the dissociation of the Syp/Syb complex does not require Ca(2+). Ca(2+) (100 μM free) alone also dissociates the Syp/Syb complex in the presence of cytosol. Treatment with SPANs disturbs the lipid raft association of synaptophysin and synaptobrevin comparable to cholesterol depletion by β-methyl-cyclodextrin while Ca(2+) alone has no effect. SPANs but not Ca(2+) directly inhibit vesicular uptake of serotonin and glutamate. It is concluded that SPANs directly affect vesicular properties independent from their Ca(2+) -dependent phospholipase activity. SPANs and Ca(2+) dissociate the Syp/Syb complex as a prerequisite for exocytosis. SPANs also prevent the filling of synaptic vesicles thereby adding to the inhibition of neurotransmission.     

Synaptotagmin VIII is localized to the mouse sperm head and may function in acrosomal exocytosis.

The acrosome is a large secretory granule that undergoes exocytosis when receptors on the sperm surface bind ligands in the egg extracellular matrix. Acrosomal exocytosis resembles stimulated secretion in neurons in that it is triggered by a rise in intracellular Ca(2+). Synaptotagmins (Syt) comprise proteins thought to transduce this Ca(2+) signal to the fusion machinery. In this study, we showed that Syt VIII is present in spermatogenic cDNA libraries. Antiserum raised against a Syt VIII-specific peptide, which recognizes Syt VIII but does not cross-react with other Syt isoforms, labeled a single prominent band on Western immunoblots of mouse sperm homogenate. Syt VIII was restricted to the sperm membrane fraction enriched in markers associated with the mouse sperm head. Fluorescent immunocytochemistry on intact mouse sperm showed that Syt VIII is localized to the acrosomal crescent and is lost upon acrosome reaction. Moreover, the amount of Syt VIII remaining with the sperm decreased proportionately with the extent of acrosome-reacted sperm. Thus, Syt VIII is a candidate for the Ca(2+) sensor that regulates acrosomal exocytosis in mammalian sperm.     

Synaptotagmin VII is targeted to dense-core vesicles and regulates their Ca2+ -dependent exocytosis in PC12 cells

It has recently been proposed that synaptotagmin (Syt) VII functions as a plasma membrane Ca2+ sensor for dense-core vesicle exocytosis in PC12 cells based on the results of transient overexpression studies using green fluorescent protein (GFP)-tagged Syt VII; however, the precise subcellular localization of Syt VII is still a matter of controversy (plasma membrane versus secretory granules). In this study we established a PC12 cell line "stably expressing" the Syt VII-GFP molecule and demonstrated by immunocytochemical and immunoelectron microscopic analyses that the Syt VII-GFP protein is localized on dense-core vesicles as well as in other intracellular membranous structures, such as the trans-Golgi network and lysosomes. Syt VII-GFP forms a complex with endogenous Syts I and IX, but not with Syt IV, and it colocalize well with Syts I and IX in the cellular processes (where dense-core vesicles are accumulated) in the PC12 cell line. We further demonstrated by an N-terminal antibody-uptake experiment that Syt VII-GFP-containing dense-core vesicles undergo Ca2+ -dependent exocytosis, the same as endogenous Syt IX-containing vesicles. Moreover, silencing of Syt VII-GFP with specific small interfering RNA dramatically reduced high KCl-dependent neuropeptide Y secretion from the stable PC12 cell line (approximately 60% of the control cells), whereas the same small interfering RNA had little effect on neuropeptide Y secretion from the wild-type PC12 cells (approximately 85-90% of the control cells), indicating that the level of endogenous expression of Syt VII molecules must be low. Our results indicate that the targeting of Syt VII-GFP molecules to specific membrane compartment(s) is affected by the transfection method (transient expression versus stable expression) and suggested that Syt VII molecule on dense-core vesicles functions as a vesicular Ca2+ sensor for exocytosis in endocrine cells.     

Otoferlin, defective in a human deafness form, is essential for exocytosis at the auditory ribbon synapse

The auditory inner hair cell (IHC) ribbon synapse operates with an exceptional temporal precision and maintains a high level of neurotransmitter release. However, the molecular mechanisms underlying IHC synaptic exocytosis are largely unknown. We studied otoferlin, a predicted C2-domain transmembrane protein, which is defective in a recessive form of human deafness. We show that otoferlin expression in the hair cells correlates with afferent synaptogenesis and find that otoferlin localizes to ribbon-associated synaptic vesicles. Otoferlin binds Ca(2+) and displays Ca(2+)-dependent interactions with the SNARE proteins syntaxin1 and SNAP25. Otoferlin deficient mice (Otof(-/-)) are profoundly deaf. Exocytosis in Otof(-/-) IHCs is almost completely abolished, despite normal ribbon synapse morphogenesis and Ca(2+) current. Thus, otoferlin is essential for a late step of synaptic vesicle exocytosis and may act as the major Ca(2+) sensor triggering membrane fusion at the IHC ribbon synapse.