Syntaxin1A Lateral Diffusion Reveals Transient and Local SNARE Interactions

Abstract

At the synapse, vesicles stably dock at the active zone. However, in cellular membranes, proteins undergo a diffusive motion. It is not known how the motion of membrane proteins involved in vesicle exocytosis is compatible with both vesicle docking and the dynamic remodeling of the plasma membrane imposed by cycles of exocytosis and endocytosis. To address this question, we studied the motion of the presynaptic membrane protein syntaxin1A at both the population and single-molecule levels in primary cultures of rat spinal cord neurons. Syntaxin1A was rapidly exchanged between synaptic and extrasynaptic regions. Changes in syntaxin1A mobility were associated with interactions related to the formation of the exocytotic complex. Finally, we propose a reaction-diffusion model reconciling the observed diffusive properties of syntaxin at the population level and at the molecular level. This work allows us to describe the diffusive behavior and kinetics of interactions between syntaxin1A and its partners that lead to its transient stabilization at the synapse.

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

“…Although our measurements of syntaxin diffusion coefficient on the PM (0.03 m 2 /s) resemble the published value (0.06 m 2 /s, weighted average (17), and 0.025 m 2 /s (27)), the slightly slower diffusion of tomosyn may stem from its presence in complexes either with syntaxin or syntaxin-SNAP-25 complexes. A recent paper by Ribrault et al (16) found syntaxin diffuses in the synapse (0.07 m 2 /s) but pauses occasionally to participate in fusion complexes. This finding can also add to the complexity of interpretation of diffusion data and suggest SNARE and SNARE-regulating proteins exhibit several rates of lateral diffusion depending on their presence as single molecules or multiprotein complexes.…”

“…15 for additional view). Syntaxin's diffusive motion was also the interest of Ribrault et al (16), which demonstrated, using FRAP in neurons, that syntaxin was rapidly exchanged between synaptic and extrasynaptic regions and characterized dynamic interactions with its partners. Knowles et al (17) measured the diffusion coefficients of syntaxin and SNAP25 in PC12 cells and demonstrated two populations of syntaxin, with rapid and slow diffusion rates, that might represent the distribution of molecules outside and inside the clusters, respectively.…”

Munc18-1 redistributes in nerve terminals in an activity- and PKC-dependent manner

“…Mean fluorescence function as a co-chaperone complex aiding transport and preventing premature breakdown of its constituents. However, in mature neurons syntaxin-1 is mainly transported via lateral diffusion along the plasma membrane (Mitchell and Ryan, 2004;Ribrault et al, 2011), and it is unknown whether at this stage Munc18-1 depends on syntaxin-1 for targeting to fusion sites.…”

“…Munc18-1 colocalizes with syntaxin-1 throughout the axon, and both proteins may be transported as a binary complex (Garcia et al, 1995;Ribrault et al, 2011). To test this, we imaged hippocampal neurons of Next, we tested whether Munc18-1 dynamics are modulated by activity by stimulating Munc18-1-Venus neurons while bleaching synaptic fluorescence during stimulation.…”

“…Our FRAP measurements revealed that synaptic Munc18-1 is very mobile. Munc18-1-Venus exchange is best described with a fast ( = 4.6 s) and a slow component ( = 60 s), comparable to syntaxin-1 (Ribrault et al, 2011), whereas Bassoon (Tsuriel et al, 2009;Schröder et al, 2013), Munc13-1 (Kalla et al, 2006), liprin-2 (Spangler et al, 2013), and synapsin-1 (Tsuriel et al, 2006) turnover at synapses is much slower, with exchange rates in the order of minutes to hours. Exchange rates of the total synaptic vesicle pool are even slower (Orenbuch et al, 2012b), although individual synaptic vesicle exchange between boutons is relatively fast (Darcy et al, 2006;Staras et al, 2010;Herzog et al, 2011).…”