t-SNARE Protein Conformations Patterned by the Lipid Microenvironment

Abstract

The spatial distribution of the target (t-)SNARE proteins (syntaxin and SNAP-25) on the plasma membrane has been extensively characterized. However, the protein conformations and interactions of the two t-SNAREs in situ remain poorly defined. By using super-resolution optical techniques and fluorescence lifetime imaging microscopy, we observed that within the t-SNARE clusters syntaxin and SNAP-25 molecules interact, forming two distinct conformations of the t-SNARE binary intermediate. These are spatially segregated on the plasma membrane with each cluster exhibiting predominantly one of the two conformations, representing the two- and three-helical forms previously observed in vitro. We sought to explain why these two t-SNARE intermediate conformations exist in spatially distinct clusters on the plasma membrane. By disrupting plasma membrane lipid order, we found that all of the t-SNARE clusters now adopted a single conformational state corresponding to the three helical t-SNARE intermediates. Together, our results define spatially distinct t-SNARE intermediate states on the plasma membrane and how the conformation adopted can be patterned by the underlying lipid environment.

Nanoscale Landscape of Phosphoinositides Revealed by Specific Pleckstrin Homology (PH) Domains Using Single-molecule Superresolution Imaging in the Plasma Membrane

“…Syntaxin1A clusters had variable sizes, with a median FWHM of ϳ68 nm (Fig. 1i), which is comparable with the size estimated in previous studies in PC-12 cells using STED (5), dSTORM (41,67), and PALM (68). The molecular density in syntaxin1A clusters in the PM of INS-1 cells was 20.5 Ϯ 6.7 per cluster (n ϭ 95 from 7 cells), equivalent to 41.0 Ϯ 13.4 per cluster after considering 50% photoactivation efficiency for PAmCherry1 (66).…”

Contact-induced clustering of syntaxin and munc18 docks secretory granules at the exocytosis site

“…6c), which showed clustered syntaxin-EGFP with an average width (FHWM) of 103±4 nm (n ¼ 74). This is slightly larger than previous estimates of syntaxin cluster size (60-90 nm; refs 28,43,45,46), possibly due to the use of live rather than fixed cells or higher precision of the earlier measurements. Visually, two types of syntaxin behaviour were apparent: one of apparently random diffusion and another where molecules were temporarily confined to a small area, often beneath a granule (Fig.…”

“…We observed striking differences in the amount of SNAP25 or munc13 between clusters, likely as a consequence of slow recruitment. This may in part explain why the interaction between syntaxin and SNAP25 (measured as FRET) varies from cluster to cluster 45 .…”

The Munc18-1 domain 3a loop is essential for neuroexocytosis but not for syntaxin-1A transport to the plasma membrane

“…The effect of mutating the domain 3a loop of Munc18-1 on these other interactions warrants further investigation. Furthermore, Sx1a is known to form 40-70 nm nanoclusters at the cell surface (Bar-On et al, 2012;Rickman et al, 2010), which correlate with sites of SG exocytosis (Barg et al, 2010). While our study has shown that Sx1a is delivered to the cell surface by Munc18-1 D317-333 , possible effects on the distribution of Sx1a into microdomains, and the underlying nanoarchitecture of these domains, remains unknown.…”