A fast, single-vesicle fusion assay mimics physiological SNARE requirements

Abstract

Almost all known intracellular fusion reactions are driven by formation of trans-SNARE complexes through pairing of vesicleassociated v-SNAREs with complementary t-SNAREs on target membranes. However, the number of SNARE complexes required for fusion is unknown, and there is controversy about whether additional proteins are required to explain the fast fusion which can occur in cells. Here we show that single vesicles containing the synaptic/exocytic v-SNAREs VAMP/synaptobrevin fuse rapidly with planar, supported bilayers containing the synaptic/exocytic t-SNAREs syntaxin-SNAP25. Fusion rates decreased dramatically when the number of externally oriented v-SNAREs per vesicle was reduced below 5-10, directly establishing this as the minimum number required for rapid fusion. Docking-to-fusion delay time distributions were consistent with a requirement that 5-11 t-SNAREs be recruited to achieve fusion, closely matching the v-SNARE requirement.lipid bilayer | membrane fusion | SNARE mechanisms | supported bilayer T rafficking of proteins in the cell-as well as secretion of physiological mediators such as hormones and neurotransmitters-depends on intracellular membrane fusion. With few exceptions, intracellular fusion reactions are driven by pairing of vesicle-associated v-SNAREs (soluble N-ethylmaleimide-sensitive factor attachment protein receptors) with cognate t-SNAREs on the target membrane, resulting in a four-helix bundle (SNAREpin) that brings bilayers into close proximity (1-3). In cells, the action of SNAREs is regulated by auxiliary proteins, some of which, such as the members of the Sec1/Munc18-like (SM) family, are universally required components of the eukaryotic fusion machinery (4). Whether SNAREs alone can catalyze fusion at physiologically meaningful rates in the absence of modulating proteins or peptides (5-9) remains controversial. In addition, it is unknown how many SNAREpins are required to produce fusion. Here, using an in vitro assay that can resolve single docking and fusion events, we show 5-10 SNAREpins mediate fast fusion in the absence of any auxiliary proteins.Reconstituted fusion assays have played a key role in elucidating mechanisms of SNARE-mediated membrane fusion (1, 2, 5, 6, 10, 11). SNARE proteins reconstituted into small unilamellar vesicles (SUVs) fused bilayers in a bulk fusion assay, albeit with slow kinetics (1, 2). More recently, single SUVs containing the synaptic/exocytic v-SNAREs VAMP/synaptobrevin were shown to fuse rapidly with planar, supported bilayers (SBLs) containing the synaptic/exocytic t-SNAREs syntaxin 1-SNAP25, with single fusion events occurring in ∼10-100 ms (7, 9) to seconds (8, 12). However, the SNAP25 subunit of the t-SNARE was not required (8, 9), or an artificial peptide was needed (7), raising questions about the physiological relevance of these results. These, and other studies of SNARE-mediated membrane fusion, used lipid bilayers where the active fusion catalysts were the only proteins present. By contrast, natural intracellular membranes are popula...

Titration of Syntaxin1 in Mammalian Synapses Reveals Multiple Roles in Vesicle Docking, Priming, and Release Probability

“…Removal of SNAP25 and Syb2 impaired evoked neurotransmitter release and severely reduced the readily releasable pool (RRP) of primed vesicles (Schoch et al, 2001;Washbourne et al, 2002;Delgado-Martínez et al, 2007). Knock-out mutants of the isoform Stx1A did not show significant differences in evoked neurotransmission (Fujiwara et al, 2006;Gerber et al, 2008), but there may be functional redundancy with another isoform, Stx1B (Bennett et al, 1992). Interestingly, endogenous expression of an "open form" mutant of Stx1B (Dulubova et al, 1999), in the absence of Stx1A, decreased RRP but increased vesicular release probability (P vr ;Gerber et al, 2008).…”

Adhesion energy can regulate vesicle fusion and stabilize partially fused states

“…However, it is still unclear whether this occurs in vivo. Importantly, it was demonstrated that the fusion process can be dramatically accelerated when a larger number of SNARE complexes (5 -10) hold the membrane -vesicle complex together [86,87]. Thus, although fusion can be potentially triggered by only a few SNARE complexes, it is likely that action potential release in vivo is mediated by a larger number of SNARE complexes per vesicle -membrane complex, that is, in the order of 5 -10.…”

Robust polarity establishment occurs via an endocytosis-based cortical corralling mechanism

“…Other parameters (e.g., k coat , k growth , and K ex ) were chosen to yield overall endoand exocytic rates and dynamics in accordance with previous studies (Kaksonen et al, 2003(Kaksonen et al, , 2005Layton et al, 2011). The maximal load of v-SNARE exocytic cargo was set to eight molecules per vesicle (Domanska et al, 2009;Karatekin et al, 2010). In the absence of similar data for Cdc42 cargo, we used the same value for Cdc42-GTP and Cdc42-GDP.…”

“…In addition to coat proteins, the cargos of this endocytic pathway include Cdc42 (Liu et al, 2005;Domanska et al, 2009;Karatekin et al, 2010) Scission is not rate limiting in WT cells (Liu et al, 2009;Carroll et al, 2012 ( Fig. 2 B and Video 3;McCusker et al, 2012).…”

“…Subsequently, vesicles are docked to the membrane via assembly of the SNARE complex with rate n v k snare , where n v is the number of v-SNARES carried by the vesicle. Exocytosis concludes with the fusion of vesicles with the plasma membrane at the rate k fus (Liu et al, 2005;Domanska et al, 2009;Karatekin et al, 2010). After fusion, the exocytic cargo is released on the plasma membrane, where v-SNARES diffuse slowly (D mslow = 0.0025 µm 2 s 1 [Valdez-Taubas and Pelham, 2003]).…”