Evaluation of the Heterogeneous Reactivity of the Syntaxin Molecules on the Inner Leaflet of the Plasma Membrane

Abstract

The soluble N-ethylmaleimide-sensitive fusion (NSF) attachment protein (SNAP) receptor (SNARE) protein syntaxin 1A forms nanosized clusters (membrane rafts) on the plasma membrane (PM) that are in equilibrium with freely diffusing syntaxin molecules. SNAREcomplex formation between syntaxin 1A and SNAP-25 (synaptosome-associated protein of 25 kDa) on the PM and synaptobrevin 2 on the vesicles (trans-SNAREs) is crucial for vesicle priming and fusion. This process might be impeded by the spontaneous accumulation of non-fusogenic cis-SNARE complexes formed when all three SNARE proteins reside on the PM. We investigated the kinetics of cis-SNARE complex assembly and disassembly and both exhibited biphasic behavior. The experimental measurements were analyzed through integration of differential rate equations pertinent to the reaction mechanism and through the application of a heuristic search for time constants and concentrations using a genetic algorithm. Reconstruction of the measurements necessitated the partitioning of syntaxin into two phases that might represent the syntaxin clusters and free syntaxin outside the clusters. The analysis suggests that most of the syntaxin in the clusters is concentrated in a nonreactive form. Consequently, cis-SNARE complex assembly in the clusters is substantially slower than outside the rafts. Interestingly, the clusters also mediate efficient disassembly of cis-SNARE complexes possibly attributable to the high local concentration of complexes in the clusters area that allows efficient disassembly by the enzymatic reaction of NSF.

Super-resolution Imaging Reveals the Internal Architecture of Nano-sized Syntaxin Clusters

“…5). The results presented herein together with the model presented in Bar-On et al (20) for the kinetics of cis-SNARE complex formation might explain how syntaxin reactivity is affected by its position within and around clusters. The model suggests partitioning of the syntaxin molecules into three reactive states, depending on their environment.…”

“…Accordingly, the densely packed molecules at the cluster center, being inaccessible to the external surroundings, are less reactive than the single syntaxin molecules that can interact freely with their partners. Moreover, the model indicates the presence of a fraction of clustered molecules that are semi-reactive and can interact with syntaxin partners, albeit with much slower kinetics (20). This fraction was suggested to represent molecules situated in the outer parts of the clusters, which are therefore more accessible to the surroundings than molecules in the center.…”

“…To fill in the crucial gaps and form a complete picture of syntaxin distribution at the PM, more than 60 regions of native membrane layers immunostained for syntaxin (20) and a secondary antibody labeled with Cy5 were randomly selected and imaged at single-molecule sensitivity. dSTORM images were then subjected to an extensive analysis characterizing the distribution of syntaxin molecules inside and outside the nanosized clusters.…”

“…Each SNARE protein was assigned with its appropriate diffusion coefficient, encounter radius, and probability to form a reversible complex with the other SNARE proteins. The rates of reactions between reactants were treated as adjustable parameters or taken from Bar-On et al (20) or Mezer et al, (30,31) and expressed as probabilities of a certain protein to react with another once they encountered each other. By varying the probabilities of the intermediate complexes to form or dissociate, we simulated the SNARE complex assembly either in a regular membrane that includes SNARE clusters (see Fig.…”

Differential Interaction of Tomosyn with Syntaxin and SNAP25 Depends on Domains in the WD40 β-Propeller Core and Determines Its Inhibitory Activity

“…Studies using super-resolution imaging showed that syntaxin and SNAP25 form separate clusters with very little overlap, although they require each other for fusion (11)(12)(13). The clusters of syntaxin and SNAP25 are at equilibrium with free SNARE molecules, which are much more mobile (13,14). These free molecules occasionally form syntaxin-SNAP25 dimers termed "acceptor complex," which can interact with synaptobrevin during the fusion process (Ref.…”