Synaptotagmin oligomers are necessary and can be sufficient to form a Ca2+ -sensitive fusion clamp.
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ABSTRACT:
SUBMITTER: Ramakrishnan S
PROVIDER: S-EPMC6349546 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:The synaptic vesicle protein synaptotagmin-1 (SYT) is required to couple calcium influx to the membrane fusion machinery. However, the structural mechanism underlying this process is unclear. Here we report an unexpected circular arrangement (ring) of SYT's cytosolic domain (C2AB) formed on lipid monolayers in the absence of free calcium ions as revealed by electron microscopy. Rings vary in diameter from 18-43 nm, corresponding to 11-26 molecules of SYT. Continuous stacking of the SYT rings occasionally converts both lipid monolayers and bilayers into protein-coated tubes. Helical reconstruction of the SYT tubes shows that one of the C2 domains (most likely C2B, based on its biochemical properties) interacts with the membrane and is involved in ring formation, and the other C2 domain points radially outward. SYT rings are disrupted rapidly by physiological concentrations of free calcium but not by magnesium. Assuming that calcium-free SYT rings are physiologically relevant, these results suggest a simple and novel mechanism by which SYT regulates neurotransmitter release: The ring acts as a spacer to prevent the completion of the soluble N-ethylmaleimide-sensitive factor activating protein receptor (SNARE) complex assembly, thereby clamping fusion in the absence of calcium. When the ring disassembles in the presence of calcium, fusion proceeds unimpeded.
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Project description:Fusion pore formation and expansion, crucial steps for neurotransmitter release and vesicle recycling in soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE)-dependent vesicle fusion, have not been well studied in vitro due to the lack of a reliable content-mixing fusion assay. Using methods detecting the intervesicular mixing of small and large cargoes at a single-vesicle level, we found that the neuronal SNARE complexes have the capacity to drive membrane hemifusion. However, efficient fusion pore formation and expansion require synaptotagmin 1 and Ca(2+). Real-time measurements show that pore expansion detected by content mixing of large DNA cargoes occurs much slower than initial pore formation that transmits small cargoes. Slow pore expansion perhaps provides a time window for vesicles to escape the full collapse fusion pathway via alternative mechanisms such as kiss-and-run. The results also show that complexin 1 stimulates pore expansion significantly, which could put bias between two pathways of vesicle recycling.
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Project description:Ca(2+)-activated Cl(-) channels (CaCCs) are key regulators of numerous physiological functions, ranging from electrolyte secretion in airway epithelia to cellular excitability in sensory neurons and muscle fibers. Recently, TMEM16A (ANO1) and -B were shown to be critical components of CaCCs. It is still unknown whether they are also sufficient to form functional CaCCs, or whether association with other subunits is required. Recent reports suggest that the Ca(2+) sensitivity of TMEM16A is mediated by its association with calmodulin, suggesting that functional CaCCs are heteromultimers. To test whether TMEM16A is necessary and sufficient to form functional CaCCs, we expressed, purified, and reconstituted human TMEM16A. The purified protein mediates Ca(2+)-dependent Cl(-) transport with submicromolar sensitivity to Ca(2+), consistent with what is seen in patch-clamp experiments. The channel is synergistically gated by Ca(2+) and voltage, so that opening is promoted by depolarizing potentials. Mutating two conserved glutamates in the TM6-7 intracellular loop selectively abolishes the Ca(2+) dependence of reconstituted TMEM16A, in a manner similar to what was reported for the heterologously expressed channel. Well-characterized CaCC blockers inhibit Cl(-) transport with Kis comparable to those measured for native and heterologously expressed CaCCs. Finally, direct physical interactions between calmodulin and TMEM16A could not be detected in copurification experiments or in functional assays. Our results demonstrate that purified TMEM16A is necessary and sufficient to recapitulate the biophysical and pharmacological properties of native and heterologously expressed CaCCs. Our results also show that association of TMEM16A with other proteins, such as calmodulin, is not required for function.
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Project description:In neurons, synaptotagmin 1 (Syt1) is thought to mediate the fusion of synaptic vesicles with the plasma membrane when presynaptic Ca2+ levels rise. However, in vitro reconstitution experiments have failed to recapitulate key characteristics of Ca2+-triggered membrane fusion. Using an in vitro single-vesicle fusion assay, we found that membrane-anchored Syt1 enhanced Ca2+ sensitivity and fusion speed. This stimulatory activity of membrane-anchored Syt1 dropped as the Ca2+ level rose beyond physiological levels. Thus, Syt1 requires the membrane anchor to stimulate vesicle fusion at physiological Ca2+ levels and may function as a dynamic presynaptic Ca2+ sensor to control the probability of neurotransmitter release.
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Project description:In neurons, SNAREs, synaptotagmin and other factors catalyze Ca(2+)-triggered fusion of vesicles with the plasma membrane. The molecular mechanism of this process, especially the interaction between synaptotagmin and SNAREs, remains an enigma. Here we characterized this interaction by single-molecule fluorescence microscopy and crystallography. The two rigid Ca(2+)-binding domains of synaptotagmin 3 (Syt3) undergo large relative motions in solution. Interaction with SNARE complex amplifies a particular state of the two domains that is further enhanced by Ca(2+). This state is represented by the first SNARE-induced Ca(2+)-bound crystal structure of a synaptotagmin fragment containing both domains. The arrangement of the Ca(2+)-binding loops of this structure of Syt3 matches that of SNARE-bound Syt1, suggesting a conserved feature of synaptotagmins. The loops resemble the membrane-interacting loops of certain viral fusion proteins in the postfusion state, suggesting unexpected similarities between both fusion systems.
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Project description:Mammalian cochlear inner hair cells (IHCs) are specialized for the dynamic coding of continuous and finely graded sound signals. This ability is largely conferred by the linear Ca(2+) dependence of neurotransmitter release at their synapses, which is also a feature of visual and olfactory systems. The prevailing hypothesis is that linearity in IHCs occurs through a developmental change in the Ca(2+) sensitivity of synaptic vesicle fusion from the nonlinear (high order) Ca(2+) dependence of immature spiking cells. However, the nature of the Ca(2+) sensor(s) of vesicle fusion at hair cell synapses is unknown. We found that synaptotagmin IV was essential for establishing the linear exocytotic Ca(2+) dependence in adult rodent IHCs and immature outer hair cells. Moreover, the expression of the hitherto undetected synaptotagmins I and II correlated with a high-order Ca(2+) dependence in IHCs. We propose that the differential expression of synaptotagmins determines the characteristic Ca(2+) sensitivity of vesicle fusion at hair cell synapses.
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Project description:The synaptic vesicle (SV) protein synaptotagmin-1 (Syt1) is the Ca2+ sensor for fast synchronous release. Biochemical and structural data suggest that Syt1 interacts with phospholipids and SNARE complex, but the manner in which these interactions translate into SV fusion remains poorly understood. Using flash-and-freeze electron microscopy, which triggers action potentials with light and coordinately arrests synaptic structures with rapid freezing, we found that synchronous-release-impairing mutations in the Syt1 C2B domain (K325, 327; R398, 399) also disrupt SV-active-zone plasma-membrane attachment. Single action potential induction rescued membrane attachment in these mutants within less than 10?ms through activation of the Syt1 Ca2+-binding site. The rapid SV membrane translocation temporarily correlates with resynchronization of release and paired pulse facilitation. On the basis of these findings, we redefine the role of Syt1 as part of the Ca2+-dependent vesicle translocation machinery and propose that Syt1 enables fast neurotransmitter release by means of its dynamic membrane attachment activities.
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Project description:Neuronal communication is mediated by Ca(2+)-triggered fusion of transmitter-filled synaptic vesicles with the presynaptic plasma membrane. Synaptotagmin I functions as a Ca(2+) sensor that regulates exocytosis, whereas soluble N-ethylmaleimide-sensitive factor attachment protein (SNAP) receptor (SNARE) proteins in the vesicle and target membrane assemble into complexes that directly catalyze bilayer fusion. Here we report that, before the Ca(2+) trigger, synaptotagmin interacts with SNARE proteins in the target membrane to halt SNARE complex assembly at a step after donor vesicles attach, or dock, to target membranes. This results in fusion complexes that, when subsequently triggered by Ca(2+), drive rapid, highly efficient lipid mixing. Ca(2+)-independent interactions with SNAREs also predispose synaptotagmin to selectively penetrate the target membrane in response to Ca(2+); we demonstrate that Ca(2+)-synaptotagmin must insert into the target membrane to accelerate SNARE-catalyzed fusion. These findings demonstrate that Ca(2+) converts synaptotagmin from a clamp to a trigger for exocytosis.
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Project description: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.
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