Calcium-triggered Membrane Fusion Proceeds Independently of Specific Presynaptic Proteins (original) (raw)
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Journal of the American Chemical Society, 2016
Membrane fusion is mediated by the SNARE complex which is formed through a zippering process. Here, we developed a chemical controller for the progress of membrane fusion. A hemifusion state was arrested by a polyphenol myricetin which binds to the SNARE complex. The arrest of membrane fusion was rescued by an enzyme laccase that removes myricetin from the SNARE complex. The rescued hemifusion state was metastable and long-lived with a decay constant of 39 min. This membrane fusion controller was applied to delineate how Ca(2+) stimulates fusion-pore formation in a millisecond time scale. We found, using a single-vesicle fusion assay, that such myricetin-primed vesicles with synaptotagmin 1 respond synchronously to physiological concentrations of Ca(2+). When 10 μM Ca(2+) was added to the hemifused vesicles, the majority of vesicles rapidly advanced to fusion pores with a time constant of 16.2 ms. Thus, the results demonstrate that a minimal exocytotic membrane fusion machinery comp...
Reconstitution of Ca 2+ -Regulated Membrane Fusion by Synaptotagmin and SNAREs
Science, 2004
We investigated the effect of synaptotagmin I on membrane fusion mediated by neuronal SNARE proteins, SNAP-25, syntaxin, and synaptobrevin, which were reconstituted into vesicles. In the presence of Ca 2+ , the cytoplasmic domain of synaptotagmin I (syt) strongly stimulated membrane fusion when synaptobrevin densities were similar to those found in native synaptic vesicles. The Ca 2+ dependence of syt-stimulated fusion was modulated by changes in lipid composition of the vesicles and by a truncation that mimics cleavage of SNAP-25 by botulinum neurotoxin A. Stimulation of fusion was abolished by disrupting the Ca 2+ -binding activity, or by severing the tandem C2 domains, of syt. Thus, syt and SNAREs are likely to represent the minimal protein complement for Ca 2+ -triggered exocytosis.
Biochimie, 2000
Despite groundbreaking work to identify numerous proteins and to focus attention on molecular interactions, the mechanism of calcium-triggered membrane fusion remains unresolved. A major difficulty in such research has been the many overlapping and interacting membrane trafficking steps in the secretory pathway, including those of membrane retrieval. Identifying the specific role(s) of a given protein, beyond its general involvement in exocytosis, has therefore proven problematic. Furthermore, the power of time-resolved optical and electrophysiological assays can be best applied to testing the function of known proteins rather than to the identification of unknown, critical membrane components. The identification of essential membrane constituents requires combined biochemical (molecular) and functional (physiological) analyses. A fully functional, stage-specific physiological membrane preparation would be one direct approach to dissecting the calcium-triggered fusion steps of regulated exocytosis. Herein we review our use of specific minimal membrane preparations consisting of fully primed and docked secretory vesicles, or the isolated vesicles themselves, and characterize the late events of exocytosis, with an aim towards identification of essential molecular components. We have established a functional definition of the fusion complex and its activation by calcium, based on our kinetic analyses. Together with a variety of biochemical and alternate functional assays, we have tested whether the SNARE core complex that is present in our vesicle membranes satisfies the criteria of the functionally defined fusion complex. Rather than a direct fusogenic role, the SNARE complex may promote the calcium sensitivity of fusion, possibly by defining or delimiting a localized, focal membrane fusion site that ensures rapid and efficient exocytosis in vivo. © 2000 Société française de biochimie et biologie moléculaire / Éditions scientifiques et médicales Elsevier SAS
Journal of Cell Biology, 1998
Cortical vesicles (CV) possess components critical to the mechanism of exocytosis. The homotypic fusion of CV centrifuged or settled into contact has a sigmoidal Ca 2 ϩ activity curve comparable to exocytosis (CV-PM fusion). Here we show that Sr 2 ϩ and Ba 2 ϩ also trigger CV-CV fusion, and agents affecting different steps of exocytotic fusion block Ca 2 ϩ , Sr 2 ϩ , and Ba 2 ϩtriggered CV-CV fusion. The maximal number of active fusion complexes per vesicle, Max , was quantified by NEM inhibition of fusion, showing that CV-CV fusion satisfies many criteria of a mathematical analysis developed for exocytosis. Both Max and the Ca 2 ϩ sensitivity of fusion complex activation were comparable to that determined for CV-PM fusion. Using Ca 2 ϩinduced SNARE complex disruption, we have analyzed the relationship between membrane fusion (CV-CV and CV-PM) and the SNARE complex. Fusion and complex disruption have different sensitivities to Ca 2 ϩ , Sr 2 ϩ , and Ba 2 ϩ , the complex remains Ca 2 ϩ -sensitive on fusion-incompetent CV, and disruption does not correlate with the quantified activation of fusion complexes. Under conditions which disrupt the SNARE complex, CV on the PM remain docked and fusion competent, and isolated CV still dock and fuse, but with a markedly reduced Ca 2 ϩ sensitivity. Thus, in this system, neither the formation, presence, nor disruption of the SNARE complex is essential to the Ca 2 ϩ -triggered fusion of exocytotic membranes. Therefore the SNARE complex alone cannot be the universal minimal fusion machine for intracellular fusion. We suggest that this complex modulates the Ca 2 ϩ sensitivity of fusion.
Synaptic Vesicles Are Constitutively Active Fusion Machines that Function Independently of Ca2+
Current Biology, 2008
Background-In neurones, release of neurotransmitter occurs through the fusion of synaptic vesicles with the plasma membrane. Many proteins required for this process have been identified, with the SNAREs syntaxin 1, SNAP-25 and synaptobrevin thought to constitute the core fusion machinery. However, there is still a large gap between our understanding of individual proteinprotein interactions and the functions of these proteins revealed by perturbations in intact synaptic preparations. To bridge this gap, we have used purified synaptic vesicles, together with artificial membranes containing co-reconstituted SNAREs as reaction partners, in fusion assays.
Revisiting the role of SNAREs in exocytosis and membrane fusion
Biochimica Et Biophysica Acta-molecular Cell Research, 2003
For over a decade SNARE hypotheses have been proposed to explain the mechanism of membrane fusion, yet the field still lacks sufficient evidence to conclusively identify the minimal components of native fusion. Consequently, debate concerning the postulated role(s) of SNAREs in membrane fusion continues. The focus of this review is to revisit original literature with a current perspective. Our analysis begins with the earliest studies of clostridial toxins, leading to various cellular and molecular approaches that have been used to test for the roles of SNAREs in exocytosis. We place much emphasis on distinguishing between specific effects on membrane fusion and effects on other critical steps in exocytosis. Although many systems can be used to study exocytosis, few permit selective access to specific steps in the pathway, such as membrane fusion. Thus, while SNARE proteins are essential to the physiology of exocytosis, assay limitations often prevent definitive conclusions concerning the molecular mechanism of membrane fusion. In all, the SNAREs are more likely to function upstream as modulators or priming factors of fusion. D
Regulation of Exocytosis and Fusion Pores by Synaptotagmin-Effector Interactions
Molecular Biology of the Cell, 2010
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...
Fusion Pore Dynamics Are Regulated by Synaptotagmin•t-SNARE Interactions
Neuron, 2004
The secretory vesicle protein, synaptotagmin I (syt), is thought to function as a major Ca 2ϩ sensing protein Madison, Wisconsin 53706 that regulates neurotransmitter release (Augustine, 2001; Chapman, 2002). Syt is anchored to the vesicle membrane via a single membrane-spanning domain near its Summary N terminus (Perin et al., 1990); the cytoplasmic domain is composed of two Ca 2ϩ binding modules called C2 Exocytosis involves the formation of a fusion pore that connects the lumen of secretory vesicles with the ex-domains (Brose et al., 1992; Davletov and Sudhof, 1993; Desai et al., 2000; Fernandez et al., 2001). The N-terminal tracellular space. Exocytosis from neurons and neuroendocrine cells is tightly regulated by intracellular C2 domain (C2A) and C-terminal C2 domain (C2B) are tethered together by a short flexible linker composed [Ca 2؉ ] and occurs rapidly, but the molecular events that mediate the opening and subsequent dilation of of nine residues (Sutton et al., 1999). Genetic and acute perturbation studies revealed that syt is essential for fusion pores remain to be determined. A putative Ca 2؉ sensor for release, synaptotagmin I (syt), binds directly evoked neurotransmitter release (Bommert et al., 1993; DiAntonio et al., 1993; Geppert et al., 1994; Littleton et to syntaxin and SNAP-25, which are components of a conserved membrane fusion complex. Here, we show al., 1993; Nonet et al., 1993) and suggest that the Ca 2ϩ binding abilities of each C2 domain play roles in the that Ca 2؉-triggered syt•SNAP-25 interactions occur rapidly. The tandem C2 domains of syt cooperate to Ca 2ϩ-triggered fusion of docked vesicles (Fernandez-Chacon et al., 2001; Littleton et al., 2001; Mackler et al., mediate binding to syntaxin/SNAP-25; lengthening the linker that connects C2A and C2B selectively disrupts 2002; Robinson et al., 2002; Stevens and Sullivan, 2003; but see also Fernandez-Chacon et al., 2002). this interaction. Expression of the linker mutants in PC12 cells results in graded reductions in the stability Biochemical studies led to the identification of a number of Ca 2ϩ-dependent syt-effector interactions that of fusion pores. Thus, the final step of Ca 2؉-triggered exocytosis is regulated, at least in part, by direct con-might couple Ca 2ϩ influx to fusion (Augustine, 2001; Chapman, 2002), including syt•membrane interactions tacts between syt and SNAP-25/syntaxin. (Bai et al., 2002; Brose et al., 1992), syt•syt oligomerization (Wu et al., 2003), and syt•t-SNARE interactions Introduction (Chapman et al., 1995; Schiavo et al., 1997). While direct interactions between syt and t-SNAREs Neurotransmitter release from nerve terminals is initiated by Ca 2ϩ (Katz, 1969) and is mediated by the fusion provides a compelling connection between the putative Ca 2ϩ sensor and the fusion apparatus, the physiological of synaptic vesicles with the presynaptic plasma membrane. The entire process of release is completed in an relevance of this interaction is unclear. Reagents that block the interaction of syt with syntaxin and SNAP-25 extremely short time frame (i.e., micro-to milliseconds) (Llinas et al., 1981; Sabatini and Regehr, 1996). Thus, (Earles et al., 2001; Tucker et al., 2003) or mutations in SNAP-25 that disrupt interactions with syt (Zhang et al., Ca 2ϩ-triggered exocytotic membrane fusion can involve only a handful of molecular rearrangements. These re-2002) reduce secretion. Using purified SNAREs reconstituted into proteoliposomes, syt stimulated membrane strictions imply direct interactions between the Ca 2ϩ sensor(s) and the membrane fusion machinery at a late fusion by facilitating SNARE complex formation (Mahal et al., 2002). These findings support a model in which step in secretion (Augustine, 2001; Chapman, 2002). A set of proteins, termed soluble NSF (N-ethylmalei-syt functions during fusion at least in part by interacting mide-sensitive fusion factor) attachment protein recepwith SNARE proteins. However, there are no data to tors (SNAREs), play essential roles in most, and perhaps indicate whether syt•t-SNARE interactions function durall, intracellular membrane fusion events (Ferro-Novick ing the final steps of fusion, i.e., the opening and dilation and Jahn, 1994; Rothman, 1994). These proteins can be of fusion pores. Indeed, in vitro assays suggest that divided into two categories: target membrane SNAREs syt•t-SNARE interactions may function to mediate vesi-(t-SNAREs), including syntaxin and synaptosome-assocle docking prior to fusion (Chieregatti et al., 2002). ciated protein of 25 kDa (SNAP-25); and the vesicle Here, we have addressed the question of whether SNARE (v-SNARE) synaptobrevin (also known as vesi-Ca 2ϩ-triggered binding of syt to SNAP-25 is fast enough cle-associated membrane protein, VAMP). The cytoto mediate excitation-secretion coupling. Moreover, we plasmic domains of SNARE proteins assemble into a have generated a series of mutant syts with selective four-helix bundle (Sutton et al., 1998) that can mediate and graded losses in SNAP-25/syntaxin binding activity membrane fusion by pulling the vesicle and target memand assessed their impact on the kinetic properties of branes together (Weber et al., 1998). However, in order fusion pores. Our data suggest that rapid Ca 2ϩ-triggered syt•t-SNARE interactions control fusion pore dynamics during the final steps of exocytosis.
Fusion Pores and Fusion Machines in CA2+-TRIGGERED Exocytosis
Annual Review of Biophysics and Biomolecular Structure, 2006
Exocytosis is initiated within a highly localized region of contact between two biological membranes. Small areas of these membranes draw close, molecules on the two surfaces interact, and structural transformations take place. Membrane fusion requires the action of proteins specialized for this task, and these proteins act as a fusion machine. At a critical point in this process, a fusion pore forms within the membrane contact site and then expands as the spherical vesicle merges with the flat target membrane. Hence, the operation of a fusion machine must be realized through the formation and expansion of a fusion pore. Delineating the relation between the fusion machine and the fusion pore thus emerges as a central goal in elucidating the mechanisms of membrane fusion. We summarize present knowledge of fusion machines and fusion pores studied in vitro, in neurons, and in neuroendocrine cells, and synthesize this knowledge into some specific and detailed hypotheses for exocytosis.
Structural Basis for the Clamping and Ca2+Activation of SNARE-mediated Fusion by Synaptotagmin
2019
Synapotagmin-1 (Syt1) interacts with both SNARE proteins and lipid membranes to synchronize neurotransmitter release to Ca2+-influx. To understand the underlying molecular mechanism, we determined the structure of the Syt1-SNARE complex on lipid membranes using cryo-electron microscopy. Under resting conditions, the Syt1 C2 domains adopt a novel membrane orientation with a Mg2+-mediated partial insertion of the aliphatic loops, alongside weak interactions with the anionic lipid headgroups. The C2B domain concurrently binds the SNARE bundle via the ‘primary’ interface and is positioned between the SNAREpins and the membrane. In this configuration, Syt1 is projected to sterically delay the complete assembly of the associated SNAREpins and thus, contribute to clamping fusion. This Syt1-SNARE organization is disrupted upon Ca2+-influx as Syt1 reorients into the membrane, allowing the attached SNAREpins to complete zippering and drive fusion. Overall, we find cation (Mg2+/Ca2+) dependent...