During neurotransmitter release at the synapse, influx of calcium ions stimulates the release of neurotransmitter. However, the mechanism by which synaptic vesicle fusion is coupled to calcium has been unclear, despite the identification of both the core fusion machinery [soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE)] and the principal calcium sensor (synaptotagmin). Here, we describe what may represent a basic principle of the coupling mechanism: a reversible clamping protein (complexin) that can freeze the SNAREpin, an assembled fusion-competent intermediate en route to fusion. When calcium binds to the calcium sensor synaptotagmin, the clamp would then be released. SNARE proteins, and key regulators like synaptotagmin and complexin, can be ectopically expressed on the cell surface. Cells expressing such "flipped" synaptic SNAREs fuse constitutively, but when we coexpressed complexin, fusion was blocked. Adding back calcium triggered fusion from this intermediate in the presence of synaptotagmin.

SummaryComplexin prevents SNAREs from releasing neurotransmitters until an action potential arrives at the synapse. To understand the mechanism for this inhibition, we determined the structure of complexin bound to a mimetic of a pre-fusion SNAREpin lacking the portion of the v-SNARE which zippers last to trigger fusion. The “central helix” of complexin is anchored to one SNARE complex while its “accessory helix” extends away at ~45° and bridges to a second complex, occupying the vacant v-SNARE binding site to inhibit fusion. That the accessory helix competes with the v-SNARE for t-SNARE binding was expected, but surprisingly, the interaction occurs inter-molecularly. Thus complexin organizes the SNAREs into a zig-zag topology which, when interposed between the vesicle and plasma membranes, is incompatible with fusion.

Membrane proteins exit the endoplasmic reticulum (ER) in COPII-transport vesicles. ER export is a selective process in which transport signals present in the cytoplasmic tail (CT) of cargo membrane proteins must be recognized by coatomer proteins for incorporation in COPII vesicles. Two classes of ER export signals have been described for type I membrane proteins, the diacidic and the dihydrophobic motifs. Both motifs participate in the Sar1-dependent binding of Sec23p-Sec24p complex to the CTs during early steps of cargo selection. However, information concerning the amino acids in the CTs that interact with Sar1 is lacking. Herein, we describe a third class of ER export motif, [RK](X)[RK], at the CT of Golgi resident glycosyltransferases that is required for these type II membrane proteins to exit the ER. The dibasic motif is located proximal to the transmembrane border, and experiments of cross-linking in microsomal membranes and of binding to immobilized peptides showed that it directly interacts with the COPII component Sar1. Sar1GTP-bound to immobilized peptides binds Sec23p. Collectively, the present data suggest that interaction of the dibasic motif with Sar1 participates in early steps of selection of Golgi resident glycosyltransferases for transport in COPII vesicles.

Membrane fusion between vesicles and target membranes involves the zippering of a four-helix bundle generated by constituent helices derived from t- and v-SNAREs found on the target and vesicular membranes. In neurons the protein complexin clamps otherwise spontaneous fusion by SNARE proteins, allowing neurotransmitters and other mediators to be secreted when and where they are needed as this clamp is released. The membrane-proximal accessory helix of complexin is necessary for clamping, but its mechanism of action is unknown. Here, we present experiments using a reconstituted fusion system that suggest a simple model in which the complexin accessory helix forms an alternative four-helix bundle with the t-SNARE near the membrane, preventing the v-SNARE from completing its zippering.

Summary Background Class II myosins generate contractile forces in cells by polymerizing into bipolar filaments and pulling on anchored actin filaments. Nonmuscle myosin II (NMII) plays central roles during cell adhesion, migration, cytokinesis, and tissue morphogenesis. NMII is present in virtually all mammalian cell types as tissue-specific combinations of NMIIA, NMIIB, and NMIIC isoforms. It remains poorly understood how the highly dynamic NMII-actin contractile system begins to assemble at new cellular locations during cell migration and how incorporation of different NMII isoforms into this system is coordinated. Results Using platinum replica electron microscopy in combination with immunogold labeling, we demonstrate that individual activated (phosphorylated on the regulatory light chain and unfolded) NMIIA and NMIIB molecules represent a functional form of NMII in motile cells and that NMIIA and NMIIB copolymerize into nascent bipolar filaments during contractile system assembly. Using subdiffraction STED microscopy together with a pharmacological block-and-release approach, we report that NMIIA and NMIIB simultaneously incorporate into the cytoskeleton during initiation of contractile system assembly, whereas the characteristic rearward shift of NMIIB relative to NMIIA is established later in the course of NMII turnover. Conclusions We show existence of activated NMII monomers in cells, copolymerization of endogenous NMIIA and NMIIB molecules, and contribution of both isoforms, rather than only NMIIA, to early stages of the contractile system assembly. These data change the current paradigms about dynamics and functions of NMII and provide new conceptual insights into organization and dynamics of the ubiquitous cellular machinery for contraction that acts in multiple cellular contexts.

Mesenchymal stem cells (MSCs) are capable of secreting exosomes, extracellular vesicles, and cytokines to regulate cell and tissue homeostasis. However, it is unknown whether MSCs use a specific exocytotic fusion mechanism to secrete exosomes and cytokines. We show that Fas binds with Fas-associated phosphatase-1 (Fap-1) and caveolin-1 (Cav-1) to activate a common soluble -ethylmaleimide-sensitive factor (NSF) attachment protein receptor (SNARE)-mediated membrane fusion mechanism to release small extracellular vesicles (sEVs) in MSCs. Moreover, we reveal that MSCs produce and secrete interleukin-1 receptor antagonist (IL-1RA) associated with sEVs to maintain rapid wound healing in the gingiva via the Fas/Fap-1/Cav-1 cascade. Tumor necrosis factor-α (TNF-α) serves as an activator to up-regulate Fas and Fap-1 expression via the nuclear factor κB pathway to promote IL-1RA release. This study identifies a previously unknown Fas/Fap-1/Cav-1 axis that regulates SNARE-mediated sEV and IL-1RA secretion in stem cells, which contributes to accelerated wound healing.

SUMMARY The crystal structure of Complexin bound to a pre-fusion SNAREpin mimetic shows that the accessory helix extends away from the SNAREpin in an “open” conformation, binding another SNAREpin, and inhibiting its assembly to clamp fusion. In contrast, the accessory helix in the post-fusion complex parallels the SNARE complex in a “closed” conformation. Here we use targeted mutations, FRET spectroscopy, and a functional assay that reconstitutes Ca2+-triggered exocytosis to show that the conformational switch from open to closed in Complexin is needed for Synaptotagmin-Ca2+ to trigger fusion. Triggering fusion requires the zippering of three key Asp residues in the “switch” region (64–68) of v-SNARE. Conformational switching in Complexin is integral to clamp release and is likely triggered when its accessory helix is released from its trans-binding to the neighboring SNAREpin, allowing the v-SNARE to complete zippering and open a fusion pore.

Using a cell fusion assay, we show here that in addition to complete fusion SNAREs also promote hemifusion as an alternative outcome. Approximately 65% of events resulted in full fusion, and the remaining 35% in hemifusion; of those, approximately two thirds were permanent and approximately one third were reversible. We predict that this relatively close balance among outcomes could be tipped by binding of regulatory proteins to the SNAREs, allowing for dynamic physiological regulation between full fusion and reversible kiss-and-run–like events.