Synaptotagmin IV Modulation of Vesicle Size and Fusion Pores in PC12 Cells

Many synaptotagmins are Ca²⁺-binding membrane proteins with functions in Ca²⁺-triggered exocytosis. Synaptotagmin IV (syt IV) has no Ca²⁺ binding activity, but nevertheless modulates exocytosis. Here, cell-attached capacitance recording was used to study single vesicle fusion and fission in control and syt IV overexpressing PC12 cells. Unitary capacitance steps varied widely in size, indicating that both microvesicles (MVs) and dense-core vesicles (DCVs) undergo fusion. Syt IV overexpression reduced the size of DCVs and endocytotic vesicles but not MVs. Syt IV also reduced the basal rate of Ca²⁺-induced fusion. During kiss-and-run, syt IV increased the conductance and duration of DCV fusion pores but not MV fusion pores. During full-fusion of DCVs syt IV increased the fusion pore conductance but not the duration. Syt IV overexpression increased the duration but not the conductance of fission pores during endocytosis. The effects of syt IV on fusion pores in PC12 cells resembled the effects on fusion pores in peptidergic nerve terminals. However, differences between these and results obtained with amperometry may indicate that amperometry and capacitance detect the fusion of different populations of vesicles. The effects of syt IV on fusion pores are discussed in terms of structural models and kinetic mechanisms.     

Kissing and pinching: synaptotagmin and calcium do more between bilayers

Building on recent findings that synaptotagmin (Syt) participates in synaptic vesicle endocytosis, Poskanzer et al., in this issue of Neuron, show distinct mechanisms by which Syt functions in this process. Most significantly, they show (1) that calcium binding to Syt determines the rate but not fidelity of vesicle recycling and (2) that mutations in a different Syt domain affect the shape but not rate of formation of recycled synaptic vesicles.     

PIP2 increases the speed of response of synaptotagmin and steers its membrane-penetration activity toward the plasma membrane

Synaptotagmin-1 (syt), the putative Ca2+ sensor for exocytosis, is anchored to the membrane of secretory organelles. Its cytoplasmic domain is composed of two Ca2+-sensing modules, C2A and C2B. Syt binds phosphatidylinositol 4,5-bisphosphate (PIP2), a plasma membrane lipid with an essential role in exocytosis and endocytosis. We resolved two modes of PIP2 binding that are mediated by distinct surfaces on the C2B domain of syt. A novel Ca2+-independent mode of binding predisposes syt to penetrate PIP2-harboring target membranes in response to Ca2+ with submillisecond kinetics. Thus, PIP2 increases the speed of response of syt and steers its membrane-penetration activity toward the plasma membrane. We propose that syt-PIP2 interactions are involved in exocytosis by facilitating the close apposition of the vesicle and target membrane on rapid time scales in response to Ca2+.     

Synaptotagmin‐11 inhibits clathrin‐mediated and bulk endocytosis

Precise and efficient endocytosis is essential for vesicle recycling during a sustained neurotransmission. The regulation of endocytosis has been extensively studied, but inhibitors have rarely been found. Here, we show that synaptotagmin‐11 (Syt11), a non‐Ca²⁺‐binding Syt implicated in schizophrenia and Parkinson's disease, inhibits clathrin‐mediated endocytosis (CME) and bulk endocytosis in dorsal root ganglion neurons. The frequency of both types of endocytic event increases in Syt11 knockdown neurons, while the sizes of endocytosed vesicles and the kinetics of individual bulk endocytotic events remain unaffected. Specifically, clathrin‐coated pits and bulk endocytosis‐like structures increase on the plasma membrane in Syt11‐knockdown neurons. Structural–functional analysis reveals distinct domain requirements for Syt11 function in CME and bulk endocytosis. Importantly, Syt11 also inhibits endocytosis in hippocampal neurons, implying a general role of Syt11 in neurons. Taken together, we propose that Syt11 functions to ensure precision in vesicle retrieval, mainly by limiting the sites of membrane invagination at the early stage of endocytosis.     

Synaptotagmin C2B domain regulates Ca2+-triggered fusion in vitro: critical residues revealed by scanning alanine mutagenesis

Synaptotagmin (syt) 1 is localized to synaptic vesicles, binds Ca2+, and regulates neuronal exocytosis. Syt 1 harbors two Ca2+-binding motifs referred to as C2A and C2B. In this study we examine the function of the isolated C2 domains of Syt 1 using a reconstituted, SNARE (soluble N-ethylmaleimide-sensitive factor attachment receptor)-mediated, fusion assay. We report that inclusion of phosphatidylethanolamine into reconstituted SNARE vesicles enabled isolated C2B, but not C2A, to regulate Ca2+-triggered fusion. The isolated C2B domain had a 6-fold lower EC50 for Ca2+-activated fusion than the intact cytosolic domain of Syt 1 (C2AB). Phosphatidylethanolamine increased both the rate and efficiency of C2AB- and C2B-regulated fusion without affecting their abilities to bind membrane-embedded syntaxin-SNAP-25 (t-SNARE) complexes. At equimolar concentrations, the isolated C2A domain was an effective inhibitor of C2B-, but not C2AB-regulated fusion; hence, C2A has markedly different effects in the fusion assay depending on whether it is tethered to C2B. Finally, scanning alanine mutagenesis of C2AB revealed four distinct groups of mutations within the C2B domain that play roles in the regulation of SNARE-mediated fusion. Surprisingly, substitution of Arg-398 with alanine, which lies on the opposite end of C2B from the Ca2+/membrane-binding loops, decreases C2AB t-SNARE binding and Ca2+-triggered fusion in vitro without affecting Ca2+-triggered interactions with phosphatidylserine or vesicle aggregation. In addition, some mutations uncouple the clamping and stimulatory functions of syt 1, suggesting that these two activities are mediated by distinct structural determinants in C2B.     

Functional and Biochemical Analysis of the C2 Domains of Synaptotagmin IV

Synaptotagmins (Syts) are a family of vesicle proteins that have been implicated in both regulated neurosecretion and general membrane trafficking. Calcium-dependent interactions mediated through their C2 domains are proposed to contribute to the mechanism by which Syts trigger calcium-dependent neurotransmitter release. Syt IV is a novel member of the Syt family that is induced by cell depolarization and has a rapid rate of synthesis and a short half-life. Moreover, the C2A domain of Syt IV does not bind calcium. We have examined the biochemical and functional properties of the C2 domains of Syt IV. Consistent with its non-calcium binding properties, the C2A domain of Syt IV binds syntaxin isoforms in a calcium-independent manner. In neuroendocrine pheochromocytoma (PC12) cells, Syt IV colocalizes with Syt I in the tips of the neurites. Microinjection of the C2A domain reveals that calcium-independent interactions mediated through this domain of Syt IV inhibit calcium-mediated neurotransmitter release from PC12 cells. Conversely, the C2B domain of Syt IV contains calcium binding properties, which permit homo-oligomerization as well as hetero-oligomerization with Syt I. Our observation that different combinatorial interactions exist between Syt and syntaxin isoforms, coupled with the calcium stimulated hetero-oligomerization of Syt isoforms, suggests that the secretory machinery contains a vast repertoire of biochemical properties for sensing calcium and regulating neurotransmitter release accordingly.     

Mechanism of botulinum neurotoxin B and G entry into hippocampal neurons

Botulinum neurotoxins (BoNTs) target presynaptic nerve terminals by recognizing specific neuronal surface receptors. Two homologous synaptic vesicle membrane proteins, synaptotagmins (Syts) I and II, bind toxins BoNT/B and G. However, a direct demonstration that Syts I/II mediate toxin binding and entry into neurons is lacking. We report that BoNT/B and G fail to bind and enter hippocampal neurons cultured from Syt I knockout mice. Wild-type Syts I and II (but not Syt I loss-of-function toxin-binding domain mutants) restored binding and entry of BoNT/B and G in Syt I–null neurons, thus demonstrating that Syts I/II are protein receptors for BoNT/B and G. Furthermore, mice lacking complex gangliosides exhibit reduced sensitivity to BoNT/G, and binding and entry of BoNT/A, B, and G into hippocampal neurons lacking gangliosides is diminished. These data suggest that gangliosides are the shared coreceptor for BoNT/A, B, and G, supporting a double-receptor model for these three BoNTs for which the protein receptors are known.     

Phosphatidylinositol 4,5-bisphosphate alters synaptotagmin 1 membrane docking and drives opposing bilayers closer together

Synaptotagmin 1 (syt1) is a synaptic vesicle-anchored membrane protein that acts as the calcium sensor for the synchronous component of neuronal exocytosis. Using site-directed spin labeling, the position and membrane interactions of a fragment of syt1 containing its two C2 domains (syt1C2AB) were assessed in bilayers containing phosphatidylcholine (PC), phosphatidylserine (PS), and phosphatidylinositol 4,5-bisphosphate (PIP(2)). Addition of 1 mol % PIP(2) to a lipid mixture of PC and PS results in a deeper membrane penetration of the C2A domain and alters the orientation of the C2B domain so that the polybasic face of C2B comes into the proximity of the bilayer interface. The C2B domain is found to contact the membrane interface in two regions, the Ca(2+)-binding loops and a region opposite the Ca(2+)-binding loops. This suggests that syt1C2AB is configured to bridge two bilayers and is consistent with a model generated previously for syt1C2AB bound to membranes of PC and PS. Point-to-plane depth restraints, obtained by progressive power saturation, and interdomain distance restraints, obtained by double electron-electron resonance, were obtained in the presence of PIP(2) and used in a simulated annealing routine to dock syt1C2AB to two membrane interfaces. The results yield an average structure different from what is found in the absence of PIP(2) and indicate that bilayer-bilayer spacing is decreased in the presence of PIP(2). The results indicate that PIP(2), which is necessary for bilayer fusion, alters C2 domain orientation, enhances syt1-membrane electrostatic interactions, and acts to drive vesicle and cytoplasmic membrane surfaces closer together.     

Vesicle-associated membrane protein-2/synaptobrevin binding to synaptotagmin I promotes O-glycosylation of synaptotagmin I

Synaptotagmin I (Syt I), an evolutionarily conserved integral membrane protein of synaptic vesicles, is now known to regulate Ca2+-dependent neurotransmitter release. Syt I protein should undergo several post-translational modifications before maturation and subsequent functioning on synaptic vesicles (e.g. N-glycosylation and fatty acylation in vertebrate Syt I), because the apparent molecular weight of Syt I on synaptic vesicles (mature form, 65,000) was much higher than the calculated molecular weight (47,400) predicted from the cDNA sequences both in vertebrates and invertebrates. Common post-translational modification(s) of Syt I conserved across phylogeny, however, have never been elucidated. In the present study, I discovered that dithreonine residues (Thr-15 and Thr-16) at the intravesicular domain of mouse Syt I are post-translationally modified by a complex form of O-linked sugar (i.e. the addition of sialic acids) in PC12 cells and that the O-glycosylation of Syt I in COS-7 cells depends on the coexpression of vesicle-associated membrane protein-2 (VAMP-2)/synaptobrevin. I also showed that a transmembrane domain of Syt I directly interacts with isolated VAMP-2, but not VAMP-2, in the heterotrimeric SNARE (SNAP receptor) complex (vesicle SNARE, VAMP-2, and two target SNAREs, syntaxin IA and SNAP-25). Since di-Thr or di-Ser residues are often found at the intravesicular domain of invertebrate Syt I, and VAMP-dependent O-glycosylation was also observed in squid Syt expressed in COS-7 cells, I propose that VAMP-dependent O-glycosylation of Syt I is a common modification during evolution and may have important role(s) in synaptic vesicle trafficking.     

Mutations in the effector binding loops in the C2A and C2B domains of synaptotagmin I disrupt exocytosis in a nonadditive manner

The secretory vesicle protein synaptotagmin I (syt) plays a critical role in Ca2+-triggered exocytosis. Its cytoplasmic domain is composed of tandem C2 domains, C2A and C2B; each C2 domain binds Ca2+. Upon binding Ca2+, positively charged residues within the Ca2+-binding loops are thought to interact with negatively charged phospholipids in the target membrane to mediate docking of the cytoplasmic domain of syt onto lipid bilayers. The C2 domains of syt also interact with syntaxin and SNAP-25, two components of a conserved membrane fusion complex. Here, we have neutralized single positively charged residues at the membrane-binding interface of C2A (R233Q) and C2B (K366Q). Either of these mutations shifted the Ca2+ requirements for syt-liposome interactions from approximately 20 to approximately 40 microm Ca2+. Kinetic analysis revealed that the reduction in Ca2+-sensing activity was associated with a decrease in affinity for membranes. These mutations did not affect sytsyntaxin interactions but resulted in an approximately 50% loss in SNAP-25 binding activity, suggesting that these residues lie at an interface between membranes and SNAP-25. Expression of full-length versions of syt that harbored these mutations reduced the rate of exocytosis in PC12 cells. In both biochemical and functional assays, effects of the R233Q and K366Q mutations were not additive, indicating that mutations in one domain affect the activity of the adjacent domain. These findings indicate that the tandem C2 domains of syt cooperate with one another to trigger release via loop-mediated electrostatic interactions with effector molecules.     

Synaptotagmin‐1 promotes the formation of axonal filopodia and branches along the developing axons of forebrain neurons

Synaptotagmin‐1 (syt1) is a Ca²⁺‐binding protein that functions in regulation of synaptic vesicle exocytosis at the synapse. Syt1 is expressed in many types of neurons well before synaptogenesis begins both in vivo and in vitro. To determine if expression of syt1 has a functional role in neuronal development before synapse formation, we examined the effects of syt1 overexpression and knockdown on the growth and branching of the axons of cultured primary embryonic day 8 chicken forebrain neurons. In vivo these neurons express syt1, and most have not yet extended axons. We present evidence that syt1 plays a role in regulating axon branching, while not regulating overall axon length. To study the effects of overexpression of syt1, we used adenovirus‐mediated infection to introduce a syt1‐YFP construct, or control GFP construct, into neurons. Syt1 levels were reduced using RNA interference. Overexpression of syt1 increased the formation of axonal filopodia and branches. Conversely, knockdown of syt1 decreased the number of axonal filopodia and branches. Time‐lapse analysis of filopodial dynamics in syt1‐overexpressing cells demonstrated that elevation of syt1 levels increased both the frequency of filopodial initiation and their lifespan. Taken together these data indicate that syt1 regulates the formation of axonal filopodia and branches before engaging in its conventional functions at the synapse. © 2011 Wiley Periodicals, Inc. Develop Neurobiol, 2013     

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 I and II mediate entry of botulinum neurotoxin B into cells

Botulinum neurotoxins (BoNTs) cause botulism by entering neurons and cleaving proteins that mediate neurotransmitter release; disruption of exocytosis results in paralysis and death. The receptors for BoNTs are thought to be composed of both proteins and gangliosides; however, protein components that mediate toxin entry have not been identified. Using gain-of-function and loss-of-function approaches, we report here that the secretory vesicle proteins, synaptotagmins (syts) I and II, mediate the entry of BoNT/B (but not BoNT/A or E) into PC12 cells. Further, we demonstrate that BoNT/B entry into PC12 cells and rat diaphragm motor nerve terminals was activity dependent and can be blocked using fragments of syt II that contain the BoNT/B-binding domain. Finally, we show that syt II fragments, in conjunction with gangliosides, neutralized BoNT/B in intact mice. These findings establish that syts I and II can function as protein receptors for BoNT/B.     

Regulation of Exocytosis and Fusion Pores by Synaptotagmin-Effector Interactions

Synaptotagmin (syt) serves as a Ca²⁺ sensor in the release of neurotransmitters and hormones. This function depends on the ability of syt to interact with other molecules. Syt binds to phosphatidylserine (PS)-containing lipid bilayers as well as to soluble N-ethylmaleimide sensitive factor receptors (SNAREs) and promotes SNARE assembly. All these interactions are regulated by Ca²⁺, but their specific roles in distinct kinetic steps of exocytosis are not well understood. To explore these questions we used amperometry recording from PC12 cells to investigate the kinetics of exocytosis. Syt isoforms and syt I mutants were overexpressed to perturb syt-PS and syt-SNARE interactions to varying degrees and evaluate the effects on fusion event frequency and the rates of fusion pore transitions. Syt I produced more rapid dilation of fusion pores than syt VII or syt IX, consistent with its role in synchronous synaptic release. Stronger syt-PS interactions were accompanied by a higher frequency of fusion events and more stable fusion pores. By contrast, syt-SNARE interactions and syt-induced SNARE assembly were uncorrelated with rates of exocytosis. This associates the syt-PS interaction with two distinct kinetic steps in Ca²⁺ triggered exocytosis and supports a role for the syt-PS interaction in stabilizing open fusion pores.     

High metal concentrations are required for self-association of synaptotagmin II

Several members of the synaptotagmin (syt) family of vesicle proteins have been proposed to act as Ca2+ sensors on synaptic vesicles. The mechanism by which calcium activates this class of proteins has been the subject of controversy, yet relatively few detailed biophysical studies have been reported on how isoforms other than syt I respond to divalent metal ions. Here, we report a series of studies on the response of syt II to a wide range of metal ions. Analytical ultracentrifugation studies demonstrate that Ca2+ induces protein dimerization upon exposure to 5 mM Ca2+. Whereas Ba2+, Mg2+, or Sr2+ do not potentiate self-association as strongly as Ca2+, Pb2+ triggers self-association of syt II at concentrations as low as 10 microM. Partial proteolysis studies suggest that the various divalent metals cause different changes in the conformation of the protein. The high calcium concentrations required for self-association of syt II suggest that the oligomerized state of this protein is not a critical intermediate in vesicle fusion; however, low-affinity calcium sites on syt II may play a critical role in buffering calcium at the presynaptic active zone. In addition, the high propensity of lead to oligomerize syt II offers a possible molecular explanation for how lead interferes with calcium-evoked neurotransmitter release.     

C2B polylysine motif of synaptotagmin facilitates a Ca2+-independent stage of synaptic vesicle priming in vivo

Synaptotagmin I, a synaptic vesicle protein required for efficient synaptic transmission, contains a highly conserved polylysine motif necessary for function. Using Drosophila, we examined in which step of the synaptic vesicle cycle this motif functions. Polylysine motif mutants exhibited an apparent decreased Ca2+ affinity of release, and, at low Ca2+, an increased failure rate, increased facilitation, and increased augmentation, indicative of a decreased release probability. Disruption of Ca2+ binding, however, cannot account for all of the deficits in the mutants; rather, the decreased release probability is probably due to a disruption in the coupling of synaptotagmin to the release machinery. Mutants exhibited a major slowing of recovery from synaptic depression, which suggests that membrane trafficking before fusion is disrupted. The disrupted process is not endocytosis because the rate of FM 1-43 uptake was unchanged in the mutants, and the polylysine motif mutant synaptotagmin was able to rescue the synaptic vesicle depletion normally found in syt(null) mutants. Thus, the polylysine motif functions after endocytosis and before fusion. Finally, mutation of the polylysine motif inhibits the Ca2+-independent ability of synaptotagmin to accelerate SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor)-mediated fusion. Together, our results demonstrate that the polylysine motif is required for efficient Ca2+-independent docking and/or priming of synaptic vesicles in vivo.     

Dynamin GTPase domain mutants block endocytic vesicle formation at morphologically distinct stages

Abundant evidence has shown that the GTPase dynamin is required for receptor-mediated endocytosis, but its exact role in endocytic clathrin-coated vesicle formation remains to be established. Whereas dynamin GTPase domain mutants that are defective in GTP binding and hydrolysis are potent dominant-negative inhibitors of receptor-mediated endocytosis, overexpression of dynamin GTPase effector domain (GED) mutants that are selectively defective in assembly-stimulated GTPase-activating protein activity can stimulate the formation of constricted coated pits and receptor-mediated endocytosis. These apparently conflicting results suggest that a complex relationship exists between dynamin's GTPase cycle of binding and hydrolysis and its role in endocytic coated vesicle formation. We sought to explore this complex relationship by generating dynamin GTPase mutants predicted to be defective at distinct stages of its GTPase cycle and examining the structural intermediates that accumulate in cells overexpressing these mutants. We report that the effects of nucleotide-binding domain mutants on dynamin's GTPase cycle in vitro are not as predicted by comparison to other GTPase superfamily members. Specifically, GTP and GDP association was destabilized for each of the GTPase domain mutants we analyzed. Nonetheless, we find that overexpression of dynamin mutants with subtle differences in their GTPase properties can lead to the accumulation of distinct intermediates in endocytic coated vesicle formation.     

Mechanism of the calcium-dependent multimerization of synaptotagmin VII mediated by its first and second C2 domains

The Ca(2+)-dependent oligomerization activity of the second C2 (C2B) domain of synaptotagmin I (Syt I) has been hypothesized to regulate neurotransmitter release. We previously showed that the cytoplasmic domains of several other Syt isoforms also show Ca(2+)-dependent oligomerization activity (Fukuda, M., and Mikoshiba, K. (2000) J. Biol. Chem. 275, 28180-28185), but little is known about the involvement of their C2 domains in Ca(2+)-dependent oligomerization. In this study, we analyzed the Ca(2+)-dependent oligomerization properties of the first (C2A) and the second C2 (C2B) domains of Syt VII. Unlike Syt I, both C2 domains of Syt VII contribute to Ca(2+)-dependent homo- and hetero-oligomerization with other isoforms. For instance, the Syt VII C2A domain Ca(2+)-dependently binds itself and the C2A domain of Syt VI but not its C2B domain, whereas the Syt VII C2B domain Ca(2+)-dependently binds itself and the C2B domain of Syt II but not its C2A domain. In addition, we showed by gel filtration that a single Syt VII C2 domain is sufficient to form a Ca(2+)-dependent multimer of very high molecular weight. Because of this "two handed" structure, the Syt VII cytoplasmic domain has been found to show the strongest Ca(2+)-dependent multimerization activity in the Syt family. We also identified Asn-328 in the C2B domain as a crucial residue for the efficient Ca(2+)-dependent switch for multimerization by site-directed mutagenesis. Our results suggest that Syt VII is a specific isoform that can cluster different Syt isoforms with two hands in response to Ca(2+).     

Distinct roles of the C2A and the C2B domain of the vesicular Ca2+ sensor synaptotagmin 9 in endocrine beta-cells

Synaptotagmins form a family of calcium-sensor proteins implicated in exocytosis, and these vesicular transmembrane proteins are endowed with two cytosolic calcium-binding C2 domains, C2A and C2B. Whereas the isoforms syt1 and syt2 have been studied in detail, less is known about syt9, the calcium sensor involved in endocrine secretion such as insulin release from large dense core vesicles in pancreatic beta-cells. Using cell-based assays to closely mimic physiological conditions, we observed SNARE (soluble N-ethylmaleimide-sensitive fusion protein-attachment protein receptor)-independent translocation of syt9C2AB to the plasma membrane at calcium levels corresponding to endocrine exocytosis, followed by internalization to endosomes. The use of point mutants and truncations revealed that initial translocation required only the C2A domain, whereas the C2B domain ensured partial pre-binding of syt9C2AB to the membrane and post-stimulatory localization to endosomes. In contrast with the known properties of neuronal and neuroendocrine syt1 or syt2, the C2B domain of syt9 did not undergo calcium-dependent membrane binding despite a high degree of structural homology as observed through molecular modelling. The present study demonstrates distinct intracellular properties of syt9 with different roles for each C2 domain in endocrine cells.