The mesoscale organization of syntaxin 1A and SNAP25 is determined by SNARE–SNARE interactions

Abstract

SNARE proteins have been described as the effectors of fusion events in the secretory pathway more than two decades ago. The strong interactions between SNARE domains are clearly important in membrane fusion, but it is unclear whether they are involved in any other cellular processes. Here, we analyzed two classical SNARE proteins, syntaxin 1A and SNAP25. Although they are supposed to be engaged in tight complexes, we surprisingly find them largely segregated in the plasma membrane. Syntaxin 1A only occupies a small fraction of the plasma membrane area. Yet, we find it is able to redistribute the far more abundant SNAP25 on the mesoscale by gathering crowds of SNAP25 molecules onto syntaxin clusters in a SNARE-domain-dependent manner. Our data suggest that SNARE domain interactions are not only involved in driving membrane fusion on the nanoscale, but also play an important role in controlling the general organization of proteins on the mesoscale. Further, we propose these mechanisms preserve active syntaxin 1A–SNAP25 complexes at the plasma membrane.

Mass spectrometry uncovers intermediates and off-pathway complexes for SNARE complex assembly

“…An equilibrium between the 2:1 dead end acceptor complex and the 1:1 active acceptor complex, which were both observed in our mass spectra, might accelerate complex’ rearrangements or disassembly-reassembly processes. Similar assumptions were made previously when studying the mesoscale organisation of the plasma membrane in PC12 cells 56 . This study revealed co-existing tight clusters of alpha-helical Syntaxin-1A bundles and diffuse clusters of SNAP25, both interacting only at their peripheries.…”

“…Diffuse SNAP25 clusters are recruited to Syntaxin-1A clusters through alpha-helical interactions of the N-terminal SNARE motif of SNAP25 showing Syntaxin-1A:SNAP25 complexes only at the interfaces of the clusters. Higher abundance of SNAP25 in the membranes, as observed in PC12 cells, prevents formation of the dead end acceptor complex and drives formation of 1:1 active acceptor complexes 56 . However, the abundance of SNAP25 differs between species and additional regulators such as Munc18 and Munc13 regulate formation of the acceptor complex 12 – 14 .…”

“…The question whether the N- or the C-terminal SNARE motif of SNAP25 is engaged in the helical arrangement can, however, not be answered with the experimental set-up employed in this study. However, a recent study showed that Syntaxin-1A and SNAP25 clusters interact at their peripheries through the N-terminal motif of SNAP25 38 , 56 . We, therefore, speculate that this SNARE motif is also involved in formation of this binary complex.…”

Microscopic clusters feature the composition of biochemical tetraspanin-assemblies and constitute building-blocks of tetraspanin enriched domains

“…3 ). However, the resolution limit of our STED microscope ranges between 65 and 100 nm 28 . As a result, we largely overestimate the size and may not resolve, if there is any, relationship between size and R 2 .…”

Influence of metabolic stress and metformin on synaptic protein profile in SH‐SY5Y‐derived neurons

“…Synaptophysin (Cell Signaling #5461 (Wu et al, 2018)), NeuN (Cell Signaling #24307 (Tang et al, 2021)), PSD-95 (post-synaptic marker, Cell Signaling #36233 (Shui et al, 2022)), Homer-1 (post-synaptic marker, SC-136358 (Wang et al, 2014) (Tang et al, 2019)), mTOR S2448 (Cell signaling #2971 (Luo et al, 2022)), total mTOR (Cell signaling #2972 (Luo et al, 2022)), Akt T308 (Cell signaling #9275 (Mathieu et al, 2019)), Akt S473 (Cell signaling #4058 (Marko et al, 2020)), and total Akt (Cell signaling #4685 (Marko et al, 2020)) were measured as markers of insulin signaling. Ponceau S, α-tubulin (Cell signaling #2144 (Mertins et al, 2021)), GAPDH (Abcam ab8245 (Luo et al, 2023)) or β-Actin (Abcam ab8227 (Moll et al, 2023)) were utilized as loading controls.…”