SNARE-mediated membrane fusion

SNARE proteins have been proposed to mediate all intracellular membrane fusion events. There are over 30 SNARE family members in mammalian cells and each is found in a distinct subcellular compartment. It is likely that SNAREs encode aspects of membrane transport specificity but the mechanism by which this specificity is achieved remains controversial. Functional studies have provided exciting insights into how SNARE proteins interact with each other to generate the driving force needed to fuse lipid bilayers.     

Hemifusion in SNARE-mediated membrane fusion

SNAREs are essential for intracellular membrane fusion. Using EPR, we determined the structure of the transmembrane domain (TMD) of the vesicle (v)-SNARE Snc2p involved in trafficking in yeast. Structural features of the TMD were used to design a v-SNARE mutant in which about half of the TMD was deleted. Liposomes containing this mutant induced outer leaflet mixing but not inner leaflet mixing when incubated with liposomes containing target membrane (t)-SNAREs. Hemifusion was also detected with wild-type SNAREs when low protein concentrations were reconstituted. Thus, these results show that SNARE-mediated fusion can transit through a hemifusion intermediate.     

Single-molecule FRET study of SNARE-mediated membrane fusion

Membrane fusion is one of the most important cellular processes by which two initially distinct lipid bilayers merge their hydrophobic cores, resulting in one interconnected structure. Proteins, called SNARE (soluble N-ethylmaleimide-sensitive factor-attachment protein receptor), play a central role in the fusion process that is also regulated by several accessory proteins. In order to study the SNARE-mediated membrane fusion, the in vitro protein reconstitution assay involving ensemble FRET (fluorescence resonance energy transfer) has been used over a decade. In this mini-review, we describe several single-molecule-based FRET approaches that have been applied to this field to overcome the shortage of the bulk assay in terms of protein and fusion dynamics.     

ATG14 controls SNARE-mediated autophagosome fusion with a lysosome

Autophagosome fusion with a lysosome constitutes the last barrier for autophagic degradation. It is speculated that this fusion process is precisely and tightly regulated. Recent genetic evidence suggests that a set of SNARE proteins, including STX17, SNAP29, and VAMP8, are essential for the fusion between autophagosomes and lysosomes. However, it remains unclear whether these SNAREs are fusion competent and how their fusogenic activity is specifically regulated during autophagy. Using a combination of biochemical, cell biology, and genetic approaches, we demonstrated that fusogenic activity of the autophagic SNARE complex is temporally and spatially controlled by ATG14/Barkor/Atg14L, an essential autophagy-specific regulator of the class III phosphatidylinositol 3-kinase complex (PtdIns3K). ATG14 directly binds to the STX17-SNAP29 binary complex on autophagosomes and promotes STX17-SNAP29-VAMP8-mediated autophagosome fusion with lysosomes. ATG14 homo-oligomerization is required for SNARE binding and fusion promotion, but is dispensable for PtdIns3K stimulation and autophagosome biogenesis. Consequently, ATG14 homo-oligomerization is required for autophagosome fusion with a lysosome, but is dispensable for autophagosome biogenesis. These data support a key role of ATG14 in controlling autophagosome fusion with a lysosome.     

Munc13-4 reconstitutes calcium-dependent SNARE-mediated membrane fusion

Munc13-4 is a widely expressed member of the CAPS/Munc13 protein family proposed to function in priming secretory granules for exocytosis. Munc13-4 contains N- and C-terminal C2 domains (C2A and C2B) predicted to bind Ca(2+), but Ca(2+)-dependent regulation of Munc13-4 activity has not been described. The C2 domains bracket a predicted SNARE-binding domain, but whether Munc13-4 interacts with SNARE proteins is unknown. We report that Munc13-4 bound Ca(2+) and restored Ca(2+)-dependent granule exocytosis to permeable cells (platelets, mast, and neuroendocrine cells) dependent on putative Ca(2+)-binding residues in C2A and C2B. Munc13-4 exhibited Ca(2+)-stimulated SNARE interactions dependent on C2A and Ca(2+)-dependent membrane binding dependent on C2B. In an apparent coupling of membrane and SNARE binding, Munc13-4 stimulated SNARE-dependent liposome fusion dependent on putative Ca(2+)-binding residues in both C2A and C2B domains. Munc13-4 is the first priming factor shown to promote Ca(2+)-dependent SNARE complex formation and SNARE-mediated liposome fusion. These properties of Munc13-4 suggest its function as a Ca(2+) sensor at rate-limiting priming steps in granule exocytosis.     

Single SNARE-mediated vesicle fusion observed in vitro by polarized TIRFM

Single-vesicle fusion assays in vitro are useful tools for examining mechanisms of membrane fusion at the molecular level mediated by soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs). This approach allows the experimentalist to define the lipid and protein composition of the two fusing membranes and perform experiments under highly controlled conditions. In previous experiments, in which we reconstituted a SNARE acceptor complex into supported membranes and observed the docking and fusion of fluorescently labeled synaptobrevin proteoliposomes by total internal reflection fluorescence microscopy with millisecond time resolution, we were able to determine the optimal number of SNARE complexes needed for fast fusion. Here, we utilize this assay in combination with polarized total internal reflection fluorescence microscopy to investigate topology changes that vesicles undergo after the onset of fusion. The theory that describes the fluorescence intensity during the transformation of a single vesicle from a spherical particle to a flat membrane patch is developed and confirmed by experiments with three different fluorescent probes. Our results show that on average, the fusing vesicles flatten and merge into the planar membrane within 8 ms after fusion starts.     

SNARE-mediated vesicle navigation,vesicle anatomy and exocytotic fusion pore

The focus of this special issue (SI) »Membrane Merger in Conventional and Unconventional Vesicle Secretion« is regulated exocytosis, a universally conserved mechanism, consisting of a merger between the vesicle and the plasma membranes. Although this process evolved with eukaryotic organisms some three billion years ago (Spang et al., 2015), the understanding of physiology and patobiology of this process, especially at elementary vesicle level, remains unclear. Exocytotic fusion consists of several stages, starting by vesicle delivery to the plasma membrane, initially establishing a very narrow and stable fusion pore, that can reversibly open and close several times before it can fully widen. This allows vesicle cargo to be completely discharged from the vesicle lumen and permits vesicle-membrane resident proteins including channels, transporters, receptors and other signalling molecules, to be incorporated into the plasma membrane. The contributions in this SI bring new insights on the complexity of vesicle–based secretion, including discussion that vesicle anatomy appears to modulate exocytotic fusion pore properties and that the soluble N-ethylmaleimide-sensitive-factor attachment protein receptor proteins (SNARE-proteins), not only facilitate pre- and post-fusion stages of exocytosis, but also serve in vesicle navigation within the cytoplasm.     

Sec1p directly stimulates SNARE-mediated membrane fusion in vitro

Sec1 proteins are critical players in membrane trafficking, yet their precise role remains unknown. We have examined the role of Sec1p in the regulation of post-Golgi secretion in Saccharomyces cerevisiae. Indirect immunofluorescence shows that endogenous Sec1p is found primarily at the bud neck in newly budded cells and in patches broadly distributed within the plasma membrane in unbudded cells. Recombinant Sec1p binds strongly to the t-SNARE complex (Sso1p/Sec9c) as well as to the fully assembled ternary SNARE complex (Sso1p/Sec9c;Snc2p), but also binds weakly to free Sso1p. We used recombinant Sec1p to test Sec1p function using a well-characterized SNARE-mediated membrane fusion assay. The addition of Sec1p to a traditional in vitro fusion assay moderately stimulates fusion; however, when Sec1p is allowed to bind to SNAREs before reconstitution, significantly more Sec1p binding is detected and fusion is stimulated in a concentration-dependent manner. These data strongly argue that Sec1p directly stimulates SNARE-mediated membrane fusion.     

Synaptotagmin I and Ca(2+) promote half fusion more than full fusion in SNARE-mediated bilayer fusion

Synaptic membrane fusion, which is necessary for neurotransmitter release, may be mediated by SNAREs and regulated by synaptotagmin (Syt) and Ca(2+). Fusion of liposomes mediated by reconstituted SNAREs produces full fusion and hemifusion, a membrane structure in which outer leaflets are mixed but the inner leaflets remain intact. Here, using the liposome fusion assay, it is shown that Syt promoted both hemifusion and full fusion in a Ca(2+)-dependent manner. Syt.Ca(2+) increased hemifusion more than full fusion, modulating the ratio of hemifusion to full fusion. Unlike the case of neuronal SNAREs, stimulation of fusion by Syt.Ca(2+) was not seen for other SNAREs involved in trafficking in yeast, indicating that the Syt.Ca(2+) stimulation was SNARE-specific. We constructed hybrid SNAREs in which transmembrane domains were swapped between neuronal and yeast SNAREs. With these hybrid SNAREs, we demonstrated that the interaction between the SNARE motifs of neuronal proteins and Syt.Ca(2+) was required for the stimulation of fusion.     

Otoferlin is a calcium sensor that directly regulates SNARE-mediated membrane fusion

Otoferlin is a large multi-C2 domain protein proposed to act as a calcium sensor that regulates synaptic vesicle exocytosis in cochlear hair cells. Although mutations in otoferlin have been associated with deafness, its contribution to neurotransmitter release is unresolved. Using recombinant proteins, we demonstrate that five of the six C2 domains of otoferlin sense calcium with apparent dissociation constants that ranged from 13-25 μM; in the presence of membranes, these apparent affinities increase by up to sevenfold. Using a reconstituted membrane fusion assay, we found that five of the six C2 domains of otoferlin stimulate membrane fusion in a calcium-dependent manner. We also demonstrate that a calcium binding-deficient form of the C2C domain is incapable of stimulating membrane fusion, further underscoring the importance of calcium for the protein's function. These results demonstrate for the first time that otoferlin is a calcium sensor that can directly regulate soluble N-ethyl-maleimide sensitive fusion protein attachment protein receptor-mediated membrane fusion reactions.     

SNARE-mediated rapid lysosome fusion in membrane raft clustering and dysfunction of bovine coronary arterial endothelium

The present study attempted to evaluate whether soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) mediate lysosome fusion in response to death receptor activation and contribute to membrane raft (MR) clustering and consequent endothelial dysfunction in coronary arterial endothelial cells. By immunohistochemical analysis, vesicle-associated membrane proteins 2 (VAMP-2, vesicle-SNAREs) were found to be abundantly expressed in the endothelium of bovine coronary arteries. Direct lysosome fusion monitoring by N-(3-triethylammoniumpropyl)-4-[4-(dibutylamino)styryl]pyridinium dibromide (FM1-43) quenching demonstrated that the inhibition of VAMP-2 with tetanus toxin or specific small interfering ribonucleic acid (siRNA) almost completely blocked lysosome fusion to plasma membrane induced by Fas ligand (FasL), a well-known MR clustering stimulator. The involvement of SNAREs was further confirmed by an increased interaction of VAMP-2 with a target-SNARE protein syntaxin-4 after FasL stimulation in coimmunoprecipitation analysis. Also, the inhibition of VAMP-2 with tetanus toxin or VAMP-2 siRNA abolished FasL-induced MR clustering, its colocalization with a NADPH oxidase unit gp91(phox), and increased superoxide production. Finally, FasL-induced impairment of endothelium-dependent vasodilation was reversed by the treatment of bovine coronary arteries with tetanus toxin or VAMP-2 siRNA. VAMP-2 is critical to lysosome fusion in MR clustering, and this VAMP-2-mediated lysosome-MR signalosomes contribute to redox regulation of coronary endothelial function.     

Recruitment and SNARE-mediated fusion of vesicles in furrow membrane remodeling during cytokinesis in zebrafish embryos

Cytokinesis is the final stage in cell division that serves to partition cytoplasm and daughter nuclei into separate cells. Membrane remodeling at the cleavage plane is a required feature of cytokinesis in many species. In animal cells, however, the precise mechanisms and molecular interactions that mediate this process are not yet fully understood. Using real-time imaging in live, early stage zebrafish embryos, we demonstrate that vesicles labeled with the v-SNARE, VAMP-2, are recruited to the cleavage furrow during deepening in a microtubule-dependent manner. These vesicles then fuse with, and transfer their VAMP-2 fluorescent label to, the plasma membrane during both furrow deepening and subsequent apposition. This observation indicates that new membrane is being inserted during these stages of cytokinesis. Inhibition of SNAP-25 (a cognate t-SNARE of VAMP-2), using a monoclonal antibody, blocked VAMP-2 vesicle fusion and furrow apposition. Transient expression of mutant forms of SNAP-25 also produced defects in furrow apposition. SNAP-25 inhibition by either method, however, did not have any significant effect on furrow deepening. Thus, our data clearly indicate that VAMP-2 and SNAP-25 play an essential role in daughter blastomere apposition, possibly via the delivery of components that promote the cell-to-cell adhesion required for the successful completion of cytokinesis. Our results also support the idea that new membrane addition, which occurs during late stage cytokinesis, is not required for furrow deepening that results from contractile band constriction.     

Synaptotagmin isoforms couple distinct ranges of Ca2+, Ba2+, and Sr2+ concentration to SNARE-mediated membrane fusion

Ca2+-triggered exocytosis of synaptic vesicles is controlled by the Ca2+-binding protein synaptotagmin (syt) I. Fifteen additional isoforms of syt have been identified. Here, we compared the abilities of three syt isoforms (I, VII, and IX) to regulate soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE)-mediated membrane fusion in vitro in response to divalent cations. We found that different isoforms of syt couple distinct ranges of Ca2+, Ba2+, and Sr2+ to membrane fusion; syt VII was approximately 400-fold more sensitive to Ca2+ than was syt I. Omission of phosphatidylserine (PS) from both populations of liposomes completely abrogated the ability of all three isoforms of syt to stimulate fusion. Mutations that selectively inhibit syt.target-SNARE (t-SNARE) interactions reduced syt stimulation of fusion. Using Sr2+ and Ba2+, we found that binding of syt to PS and t-SNAREs can be dissociated from activation of fusion, uncovering posteffector-binding functions for syt. Our data demonstrate that different syt isoforms are specialized to sense different ranges of divalent cations and that PS is an essential effector of Ca2+.syt action.     

Calcium-dependent Regulation of SNARE-mediated Membrane Fusion by Calmodulin

Neuroexocytosis requires SNARE proteins, which assemble into trans complexes at the synaptic vesicle/plasma membrane interface and mediate bilayer fusion. Ca²⁺ sensitivity is thought to be conferred by synaptotagmin, although the ubiquitous Ca²⁺-effector calmodulin has also been implicated in SNARE-dependent membrane fusion. To examine the molecular mechanisms involved, we examined the direct action of calmodulin and synaptotagmin in vitro, using fluorescence resonance energy transfer to assay lipid mixing between target- and vesicle-SNARE liposomes. Ca²⁺/calmodulin inhibited SNARE assembly and membrane fusion by binding to two distinct motifs located in the membrane-proximal regions of VAMP2 (K_D = 500 nm) and syntaxin 1 (K_D = 2 μm). In contrast, fusion was increased by full-length synaptotagmin 1 anchored in vesicle-SNARE liposomes. When synaptotagmin and calmodulin were combined, synaptotagmin overcame the inhibitory effects of calmodulin. Furthermore, synaptotagmin displaced calmodulin binding to target-SNAREs. These findings suggest that two distinct Ca²⁺ sensors act antagonistically in SNARE-mediated fusion.     

Crystallizing Transmembrane Peptides in Lipidic Mesophases

Structure determination of membrane proteins by crystallographic means has been facilitated by crystallization in lipidic mesophases. It has been suggested, however, that this so-called in meso method, as originally implemented, would not apply to small protein targets having ≤4 transmembrane crossings. In our study, the hypothesis that the inherent flexibility of the mesophase would enable crystallogenesis of small proteins was tested using a transmembrane pentadecapeptide, linear gramicidin, which produced structure-grade crystals. This result suggests that the in meso method should be considered as a viable means for high-resolution structure determination of integral membrane peptides, many of which are predicted to be coded for in the human genome.     

SNARE-mediated membrane traffic modulates RhoA-regulated focal adhesion formation

In the present study, we examined the role of soluble NSF attachment protein receptor (SNARE)-mediated membrane traffic in the formation of focal adhesions during cell spreading. CHO-K1 cells expressing a dominant-negative form of N-ethylmaleimide-sensitive factor (E329Q-NSF) were unable to spread as well as control cells and they formed focal adhesions (FAs) that were larger than those in control cells. FA formation was impaired in cells transfected with a dominant-negative form of RhoA, but, significantly, not in cells simultaneously expressing dominant-negative NSF. Treatment of E329Q-NSF-expressing cells with the ROCK inhibitor Y-27632 did inhibit FA formation. The results are consistent with a model of cell adhesion in which SNARE-mediated membrane traffic is required for both the elaboration of lamellipodia and the modulation of biochemical signals that control RhoA-mediated FA assembly.     

Inhibition of SNARE-mediated membrane traffic impairs cell migration

Cell migration occurs as a highly-regulated cycle of cell polarization, membrane extension at the leading edge, adhesion, contraction of the cell body, and release from the extracellular matrix at the trailing edge. In this study, we investigated the involvement of SNARE-mediated membrane trafficking in cell migration. Using a dominant-negative form of the enzyme N-ethylmaleimide-sensitive factor as a general inhibitor of SNARE-mediated membrane traffic and tetanus toxin as a specific inhibitor of VAMP3/cellubrevin, we conducted transwell migration assays and determined that serum-induced migration of CHO-K1 cells is dependant upon SNARE function. Both VAMP3-mediated and VAMP3-independent traffic were involved in regulating this cell migration. Inhibition of SNARE-mediated membrane traffic led to a decrease in the protrusion of lamellipodia at the leading edge of migrating cells. Additionally, the reduction in cell migration resulting from the inhibition of SNARE function was accompanied by perturbation of a Rab11-containing alpha(5)beta(1) integrin compartment and a decrease in cell surface alpha(5)beta(1) without alteration to total cellular integrin levels. Together, these observations suggest that inhibition of SNARE-mediated traffic interferes with the intracellular distribution of integrins and with the membrane remodeling that contributes to lamellipodial extension during cell migration.